A co-crystal mixture for improving stability and antibacterial activity of hydroxytyrosol and a preparation method and application thereof

Abstract

The application provides a co-crystal mixture for improving stability and antibacterial activity of hydroxytyrosol and a preparation method and application thereof, and belongs to the technical field of cosmetics and biological medicine. The co-crystal mixture is composed of the following chemical components in mass fraction: high-melting-point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, and hydroxytyrosol: 5-60%; wherein the high-melting-point hydrogen bond acceptor includes high-melting-point amino acid or amino acid derivative. The co-crystal mixture can significantly improve the melting point of hydroxytyrosol, prevent or slow down the moisture absorption and deterioration of hydroxytyrosol, thereby reducing the transportation cost and storage cost of hydroxytyrosol. In addition, the co-crystal mixture can be configured into an aqueous solution preparation with different proportions, can be stored for a long time without discoloration in room temperature, refrigeration and high-temperature environments, and significantly improves the storage stability and biological activity of hydroxytyrosol.

Description

Technical Field

[0001] This invention belongs to the fields of cosmetics and biomedicine, and specifically relates to a eutectic mixture that enhances the stability and antibacterial activity of hydroxytyrosol, 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 exhibits 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. It is considered one of the strongest antioxidants and also possesses various biological activities such as antibacterial, anti-inflammatory, lipid-regulating, anti-atherosclerotic, anti-pathogenic microorganism, bone protection, and osteoporosis prevention. It is 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, and is prone to oxidation and discoloration during use, gradually losing its activity, which limits its application in food, beverages, health products, pharmaceuticals, and food additives. In addition, hydroxytyrosol has a low melting point (less than 60°C) and high hygroscopicity. At room temperature and certain humidity, it appears as a waxy solid or a viscous oily liquid, making it unsuitable for direct use in the preparation of hard capsules or tablets. Furthermore, it is inconvenient to store and transport, often requiring low-temperature storage to prevent further moisture absorption and oxidation, which increases the application cost of this raw material.

[0003] To address the issues of low melting point, poor stability, and hygroscopicity of hydroxytyrosol, co-crystallization methods are commonly used to improve its stability, transport convenience, and antioxidant activity. Chinese Patent Application No. CN202110244109, "Hydroxytyrosol-Betaine Co-crystallization, Preparation Method and Composition Thereof," discloses a method for preparing hydroxytyrosol-betaine co-crystallization and its application. This invention produces a hydroxytyrosol-betaine co-crystallization with a high melting point, non-hygroscopicity, and good stability, significantly improving the ease of use and chemical stability of hydroxytyrosol. Chinese Patent Application No. CN202110243777, "Hydroxytyrosol-Nicotinamide Co-crystallization, Preparation Method and Composition Thereof," discloses a method for preparing hydroxytyrosol-nicotinamide co-crystallization and its application. This co-crystallization also has a high melting point, is non-hygroscopic, and exhibits good stability. Chinese patent application CN202411483710, entitled "A Hydroxytyrosol-Ecdoline Cocrystal and Its Preparation Method and Application", discloses a hydroxytyrosol-ectoline cocrystal and its preparation method to improve the melting point and stability of hydroxytyrosol and reduce its hygroscopicity.

[0004] Chinese patent application CN202311688837, "A Multiple Antioxidant Composition and Its Preparation Method and Application," discloses a method for preparing and applying a composition containing hydroxytyrosol, ergothioneine, and astaxanthin. The composition, obtained through emulsification encapsulation, exhibits a synergistic effect that enhances the stability of the antioxidant active ingredients. Chinese patent application CN202510559394, "An Antioxidant Composition Based on Reverse Micellar Enhancement Technology and Its Preparation Method and Application," discloses an antioxidant composition based on reverse micelle enhancement technology, comprising a reverse micelle structure and an aqueous solution of antioxidant actives such as hydroxytyrosol, ergothioneine, and diglycerides. This invention encapsulates water-soluble antioxidant actives through a reverse micelle structure, preventing oxidation, degradation, or discoloration. After encapsulation with reverse micelles, stability and efficacy are significantly improved. It is worth noting that Chinese patent application No. 20241032477.X, entitled "A composition, application and preparation method for preventing oxidative discoloration of hydroxytyrosol", discloses a hydroxytyrosol reducing agent and its preparation method and application. The reducing agent, such as sodium metabisulfite, L-cysteine ​​and vitamin C, can improve the stability of hydroxytyrosol.

[0005] Currently, the stability of the eutectic system in aqueous solution remains poor. After dissolving in aqueous solution, hydroxytyrosol solutions oxidize, change color, and become inactive, especially when stored at high temperatures; hydroxytyrosol aqueous solutions will oxidize, change color, and become inactive after just a few days. This reduces the activity of the product and affects its color stability, greatly limiting the further application of hydroxytyrosol. Furthermore, while hydroxytyrosol, as a small polyphenol molecule, possesses certain antibacterial activity, there are no research reports on the antibacterial activity of hydroxytyrosol eutectic mixtures. Summary of the Invention

[0006] To address the issues of low melting point and poor stability of hydroxytyrosol, this invention provides a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol. This co-crystal mixture significantly increases the melting point of hydroxytyrosol, prevents or slows down its moisture absorption and deterioration, thereby reducing transportation and storage costs. Furthermore, this co-crystal mixture can be formulated into aqueous solutions of varying proportions, allowing for long-term storage at room temperature, in frozen, and high-temperature environments without discoloration, significantly improving the storage stability and bioactivity of hydroxytyrosol.

[0007] The present invention also discloses a method for preparing and applying a eutectic mixture that enhances the stability and antibacterial activity of hydroxytyrosol.

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

[0009] This invention provides a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol, wherein the co-crystal mixture comprises the following chemical components by mass fraction:

[0010] High melting point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, hydroxytyrosol: 5-60%;

[0011] The high-melting-point hydrogen bond acceptor includes high-melting-point amino acids or amino acid derivatives, and the melting point of the high-melting-point hydrogen bond acceptor is ≥220℃.

[0012] The sulfur-containing strong reducing agent includes at least one of acetylcysteine, glutathione, acetylglutathione, glycine-cysteine ​​dipeptide, glycine-cysteine-glycine tripeptide, and sodium sulfite.

[0013] Preferably, the sulfur-containing strong reducing agent is acetylcysteine.

[0014] Preferably, the eutectic mixture comprises the following chemical components by mass fraction:

[0015] High melting point hydrogen bond acceptor: 40-60%, sulfur-containing strong reducing agent: 10-30%, hydroxytyrosol: 20-40%.

[0016] Furthermore, the high-melting-point hydrogen bond acceptor includes at least one of glycine, arginine, valine, serine, threonine, histidine, glutamic acid, leucine, acetylglutamine, glutamine, asparagine, and alanine.

[0017] Furthermore, in the eutectic mixture, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-4):(1-3).

[0018] The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-10).

[0019] The molar ratio of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is (1-20):(1-2).

[0020] Preferably, in the eutectic mixture, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-2):(1-2).

[0021] The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-2).

[0022] The molar ratio of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is (1-5):(1-2).

[0023] Based on the same inventive concept, this invention provides a method for preparing a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol, the preparation method comprising:

[0024] A mixed solution was obtained by dissolving a high-melting-point hydrogen bond acceptor, a sulfur-containing strong reducing agent, and hydroxytyrosol together in a solvent.

[0025] The mixed solution is homogenized for 0.5 to 6 hours in a vacuum environment or under inert gas protection at a temperature range of 20 to 80°C to obtain a homogenized solution.

[0026] The homogeneous solution is cooled and crystallized, and the resulting crystals are dried to obtain a eutectic mixture;

[0027] The mass fractions of each chemical component in the eutectic mixture are as follows:

[0028] High melting point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, hydroxytyrosol: 5-60%;

[0029] The high-melting-point hydrogen bond acceptor includes high-melting-point amino acids or amino acid derivatives;

[0030] The sulfur-containing strong reducing agent includes at least one of acetylcysteine, glutathione, acetylglutathione, glycine-cysteine ​​dipeptide, glycine-cysteine-glycine tripeptide, and sodium sulfite.

[0031] Furthermore, the high-melting-point hydrogen bond acceptor includes at least one of glycine, arginine, valine, serine, threonine, histidine, glutamic acid, leucine, acetylglutamine, glutamine, asparagine, and alanine.

[0032] Furthermore, in the eutectic mixture, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-4):(1-3).

[0033] The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-10).

[0034] The molar ratio of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is (1-20):(1-2).

[0035] Furthermore, the solvent includes at least one selected from water, n-propanol, n-butanol, dimethylformamide (DMF), glacial acetic acid, methanol, ethanol, ethyl acetate, dimethyl sulfoxide (DMSO), N-methylpyrrolidone, diethyl ether, and acetone.

[0036] The process of cooling and crystallizing the homogeneous solution, followed by drying the resulting crystals to obtain a eutectic mixture, specifically includes:

[0037] The homogeneous solution was crystallized at -80℃ to 10℃ for 1 to 24 hours, and the resulting crystals were dried to obtain a eutectic mixture.

[0038] The drying method can be any one of freeze drying, vacuum drying, spray drying, and rotary evaporation drying.

[0039] Preferably, the solvent is water, and the drying method is freeze drying.

[0040] Based on the same inventive concept, this invention provides the application of a eutectic mixture that enhances the stability and antibacterial activity of hydroxytyrosol in the preparation of cosmetics or pharmaceuticals.

[0041] Based on the same inventive concept, the present invention provides a cosmetic product, wherein the raw materials for preparing the cosmetic product contain the above-mentioned co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol.

[0042] Furthermore, the dosage form of the cosmetic includes any one of the following: serum, ointment, orally disintegrating tablet, gel, foam, tablet, capsule, jelly, oral liquid, and pill.

[0043] Based on the same inventive concept, the present invention also provides the application of a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol in the preparation of an aqueous solution formulation, wherein the pH of the aqueous solution formulation is 4.5 to 8.

[0044] In the aqueous solution preparation, the mass fraction of the eutectic mixture is 0.1% to 15%.

[0045] Preferably, the pH of the aqueous solution preparation is 5-7;

[0046] In the aqueous solution preparation, the mass fraction of the eutectic mixture is 0.1% to 5%.

[0047] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0048] 1. This invention provides a co-crystal mixture for improving the stability and antibacterial activity of hydroxytyrosol. Through supramolecular co-crystal technology, a supramolecular co-crystal mixture is constructed, comprising hydroxytyrosol, high-melting-point amino acids and their derivatives, and a sulfur-containing strong reducing agent. This co-crystal mixture can significantly increase the melting point of the hydroxytyrosol raw material, transforming its viscous state into a powder state, thereby reducing the transportation and storage costs of hydroxytyrosol. More importantly, this co-crystal mixture can be formulated into aqueous solutions of different proportions and can remain unchanged in color for extended periods under weakly acidic, neutral, and weakly alkaline conditions, at room temperature, under freezing, high temperature, and light exposure. This effectively solves the color change problem of hydroxytyrosol during product development and significantly improves its storage stability, biological activity, and antibacterial activity.

[0049] 2. This invention provides a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol. It utilizes high-melting-point amino acids and their derivatives as hydrogen bond acceptors, forming a supramolecular co-crystal system with hydroxytyrosol, which possesses hydrogen bond donor properties. The amino acids and their derivatives interact with the hydroxytyrosol molecules through dipole-dipole attraction, dipole-induced dipole attraction, hydrogen bonds, π-π bonds, or van der Waals forces between polar molecules, significantly improving the melting point and storage stability of hydroxytyrosol. Furthermore, the amino acids and their derivatives exhibit good biocompatibility and certain antioxidant regulatory capabilities. Combined with the strong hydrogen bond forces between the hydrogen bond acceptors and hydrogen bond donors in the supramolecular system, even when both exist independently, it can greatly improve the discoloration problem of hydroxytyrosol during long-term storage in aqueous solutions.

[0050] 3. This invention provides a eutectic mixture for improving the stability and antibacterial activity of hydroxytyrosol. The sulfur-containing strong reducing agent in the eutectic mixture has high antioxidant activity, which can greatly improve the stability of hydroxytyrosol in aqueous solution. The high-melting-point amino acids and their derivatives work synergistically with the sulfur-containing strong reducing agent to greatly increase the melting point of the hydroxytyrosol eutectic mixture and synergistically improve the long-term stability and antioxidant activity of hydroxytyrosol in aqueous solution. In addition, the sulfur-containing strong reducing agent is an antioxidant compound containing sulfur, and its large-scale use will emit irritating gas. The addition of high-melting-point amino acids can minimize the amount of sulfur-containing strong reducing agent used, and at the same time effectively mask the irritating odor emitted by the sulfur-containing strong reducing agent.

[0051] 4. The present invention provides a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol. First, the co-crystal mixture can maintain its transparent color in aqueous systems with weak acid to weak alkaline environments, and the texture and color of samples placed at room temperature and high temperature environments for a long time remain unchanged. Second, the co-crystal mixture also has specific biological activities and unique functions in the fields of antibacterial, repair, and anti-inflammatory properties, which can enhance the therapeutic efficacy of polyphenolic small molecules in specific scenarios. In addition, the components selected in this system have good biocompatibility and can be used in the cosmetic field. Attached Figure Description

[0052] 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.

[0053] Figure 1 Powder photographs of hydroxytyrosol-valine-N-acetylcysteine ​​from Example 1 (left image) and hydroxytyrosol-valine-N-acetylcysteine ​​from Example 2 (right image).

[0054] Figure 2DSC (left) and TG-DTG spectra (right) of Comparative Example 1 (mixture of hydroxytyrosol and N-acetylcysteine).

[0055] Figure 3 DSC (left) and TG-DTG spectra (right) of Comparative Example 3 (hydroxytyrosol-valine mixture).

[0056] Figure 4 DSC (left) and TG-DTG spectra (right) of Comparative Example 4 (hydroxytyrosol-glycine mixture).

[0057] Figure 5 DSC (left) and TG-DTG spectra (right) of Example 1 (hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture).

[0058] Figure 6 DSC (left) and TG-DTG spectra (right) of Example 2 (hydroxytyrosol-glycine-acetylcysteine ​​cocrystal mixture).

[0059] Figure 7 XRD pattern of Example 1 (hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture).

[0060] Figure 8 XRD pattern of Example 2 (hydroxytyrosol-glycine-N-acetylcysteine ​​cocrystal mixture).

[0061] Figure 9 Fourier transform infrared spectral characteristics of hydroxytyrosol.

[0062] Figure 10 Fourier transform infrared spectral characteristics of glycine.

[0063] Figure 11 Fourier transform infrared spectral characteristics of valine.

[0064] Figure 12 Fourier transform infrared spectral characteristics of Comparative Example 3 (hydroxytyrosol-valine mixture).

[0065] Figure 13 Fourier transform infrared spectral characteristics of Comparative Example 4 (hydroxytyrosol-glycine mixture).

[0066] Figure 14 Fourier transform infrared spectral characteristics of Comparative Example 1 (mixture of hydroxytyrosol and N-acetylcysteine).

[0067] Figure 15 Fourier transform infrared spectral characteristics of Example 1 (hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture).

[0068] Figure 16 Fourier transform infrared spectral characteristics of Example 2 (hydroxytyrosol-glycine-N-acetylcysteine ​​cocrystal mixture).

[0069] Figure 17 The stability of freeze-dried powders from Examples 1 (hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture), 2 (hydroxytyrosol-glycine-N-acetylcysteine ​​cocrystal mixture), and 3 (hydroxytyrosol-arginine-N-acetylcysteine ​​cocrystal mixture) prepared into 2.5% and 5% aqueous solutions was observed after being placed at room temperature (20°C) and at high temperature (45°C) for two months.

[0070] Figure 18 Stability of 2.5% aqueous solutions prepared by drying different proportions was observed after 15 days in ambient temperature (20℃) and high temperature (45℃) environments.

[0071] Figure 19 Example 1 and analysis of the antibacterial effects of hydroxytyrosol on Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0072] 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.

[0073] 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.

[0074] 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.

[0075] The overall concept of this invention is as follows:

[0076] Currently, the application of hydroxytyrosol faces the following challenges: 1) It has a low melting point and high hygroscopicity, exhibiting a waxy solid or viscous oily liquid state at room temperature and certain humidity levels. Low-temperature storage is often required to prevent further moisture absorption and oxidation, increasing the storage and transportation costs of this raw material. 2) While high-melting-point hydroxytyrosol cocrystals can be effectively obtained through co-crystallization, solving the transportation and storage problems, in practical applications, especially in the development of aqueous solution formulations, water molecules and oxygen can still continuously oxidize hydroxytyrosol molecules after contact with water molecules in weakly acidic or neutral environments. Prolonged exposure to high temperatures and room temperature still presents the problem of oxidation, discoloration, and inactivation. Most cosmetics or foods used on the human body have aqueous solutions with a pH that is weakly acidic, neutral, or weakly alkaline. Too low or too high a pH can cause significant irritation and physiological toxicity. Therefore, developing a hydroxytyrosol formulation that can exist stably in weakly acidic, neutral, or weakly alkaline aqueous solutions is of great significance and urgently needed for the application of hydroxytyrosol in the pharmaceutical and health fields. 3) There are few reports on the antibacterial and biofunctional effects of hydroxytyrosol cocrystal mixtures, and expanding the specific application scenarios of hydroxytyrosol is another key issue that needs to be addressed. In summary, there is an urgent need to develop a green, simple, and economical method to increase the melting point of hydroxytyrosol and reduce its hygroscopic properties. More importantly, it is crucial to address the stability of hydroxytyrosol in weakly acidic, neutral, or weakly alkaline aqueous solutions to maintain its long-term bioactivity and enhance its efficacy in cosmetics.

[0077] As a means to solve the above-mentioned technical problems, this invention utilizes supramolecular co-crystal technology to disclose a co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol, its preparation method, and its applications. Firstly, to improve the stability of hydroxytyrosol itself, increase the melting point of the hydroxytyrosol raw material, reduce the problem of hygroscopic deterioration of hydroxytyrosol, and simultaneously reduce the transportation and storage costs of hydroxytyrosol, this invention, through extensive preliminary experimental screening and optimization, discovered that high-melting-point amino acids and their derivatives can act as ideal hydrogen bond acceptors to form a supramolecular co-crystal system with hydroxytyrosol, which possesses hydrogen bond donor properties. The supramolecular system, composed of hydrogen bond donor and acceptor pairs, can instantaneously 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 structure can act as a barrier to oxygen diffusion, effectively protecting the encapsulated drug from oxidation. This invention, by forming a supramolecular co-crystal system with high-melting-point amino acids and their derivatives and hydroxytyrosol, not only significantly improves the melting point and storage stability of hydroxytyrosol but also enhances its transdermal absorption performance and the stability of its aqueous solution.

[0078] In addition, amino acids and their derivatives, as hydrogen bond acceptors, have good biocompatibility and the following unique advantages, such as participating in the body's metabolic cycle, playing a key role in the occurrence, development and treatment of various diseases, being economical, and having certain antioxidant regulatory capabilities. These properties make amino acids and their derivatives show great application potential in the development of biomedical cocrystal mixture materials.

[0079] Secondly, to address the issue of hydroxytyrosol's susceptibility to oxidation and discoloration in weakly acidic, neutral, or weakly alkaline aqueous solutions, and to improve the long-term storage stability of hydroxytyrosol in aqueous formulations, this invention, through extensive screening, discovered that strong sulfur-containing reducing agents (cysteine ​​derivatives, small molecule peptides containing cysteine, and sodium sulfite) can significantly improve the stability of hydroxytyrosol in aqueous solutions, providing a guarantee for the development of hydroxytyrosol solution formulations. More importantly, the high-melting-point amino acids and their derivatives optimized and screened in this invention can also significantly improve the discoloration problem of hydroxytyrosol during long-term storage in aqueous solutions through strong hydrogen bonding, even when existing independently. Therefore, the synergistic effect of high-melting-point amino acids and their derivatives with strong sulfur-containing reducing agents greatly improves the long-term stability and antioxidant activity of hydroxytyrosol in aqueous solutions, further reducing the application concentration and economic cost of hydroxytyrosol cocrystal mixtures, and expanding the application scope of hydroxytyrosol in pharmaceutical and health products.

[0080] Currently, Chinese Patent Application No. 201910315938.5, "A Hydroxytyrosol Inclusion Complex, Its Preparation Method and Uses," discloses a hydroxytyrosol inclusion complex, its preparation method, and its uses for improving the stability of hydroxytyrosol powder and its aqueous solutions. The hydroxytyrosol inclusion complex comprises hydroxytyrosol, hydroxypropyl-β-cyclodextrin, and a protective agent. The protective agent includes any one or more of the following: vitamin C, vitamin C ethyl ether, vitamin C glucoside, vitamin E, vitamin E acetate, vitamin E succinate, vitamin E linoleate, p-hydroxyacetophenone, glutathione, glutathione ethyl ester, N-acetylcysteine, lipoic acid, zinc pyrrolidone carboxylate, ubiquinone, panthenol, sodium bisulfite, and sodium sulfite. However, this patent primarily studies the inclusion effect of cyclodextrin and sulfur-containing strong reducing agents on hydroxytyrosol. The effects of these agents on the eutectic structure, melting point, and thermal stability of hydroxytyrosol are not investigated. Furthermore, optimized methods are needed for the transportation and storage of hydroxytyrosol raw materials. Additionally, most of the protective agents disclosed in this patent, such as vitamins, lipoic acid, and panthenol, have no significant impact on the color stability of hydroxytyrosol aqueous solutions. Even after the inclusion of the sulfur-containing strong reducing agent, hydroxytyrosol, and cyclodextrin, further dissolution in a weakly acidic or neutral aqueous solution does not ultimately solve the problem of hydroxytyrosol gradually darkening in color during long-term storage at high temperatures.

[0081] Furthermore, Chinese patent application number 202110747906.X, entitled "A Mitochondrial Nutrient Combination for Improving Cardiovascular Health," discloses a mitochondrial nutrient composition and its application for improving cardiovascular health. This invention's mitochondrial nutrient composition includes a first composition and a second composition. The first composition includes one or more of coenzyme Q10, lipoic acid, acetylcysteine, acetylcarnitine, creatine, B vitamins, chromium niacin, magnesium glycine, zinc sulfate, and hydroxytyrosol. While the composition mentions combining acetylcysteine ​​and hydroxytyrosol, it does not address the effect of this combination on the stability, eutectic structure, and thermal stability of hydroxytyrosol.

[0082] In summary, the hydroxytyrosol cocrystal raw materials developed using supramolecular technology have significantly improved the melting point of hydroxytyrosol, enhancing its storage stability and ease of use. However, several issues still need to be addressed: 1) The stability of the prepared hydroxytyrosol cocrystal system in weakly acidic or neutral aqueous solutions is not ideal. Water molecules and oxygen, when combined with the cocrystal, further oxidize the hydroxytyrosol. Especially when exposed to high temperatures (45°C) or light for extended periods, discoloration still occurs, with the hydroxytyrosol gradually oxidizing, changing from transparent and colorless to light yellow, brownish-yellow, and dark red, significantly weakening its antioxidant activity. 2) Most currently developed hydroxytyrosol cocrystals are prepared in organic solvents, resulting in melting points ranging from 120-170°C. Developing hydroxytyrosol cocrystals that can operate in pure water systems can prevent organic solvent residue, resulting in safer cocrystal raw materials. Furthermore, developing hydroxytyrosol cocrystals with higher melting points is beneficial for improving its thermal stability. 3) Hydroxytyrosol is the core phenolic active substance in olives, which has both broad-spectrum antibacterial activity and low cytotoxicity. It is widely used in food preservation, biomedicine, and daily antibacterial applications. However, there are no reports on the application and systematic comparison of hydroxytyrosol cocrystal system and hydroxytyrosol in antibacterial and bactericidal aspects.

[0083] The applicant focused on improving the melting point and stability of hydroxytyrosol. Through systematic experiments and analysis, they discovered that high-melting-point amino acids and their derivatives can form supramolecular forces with hydroxytyrosol in weakly acidic or neutral environments, significantly improving the discoloration problem of hydroxytyrosol in aqueous solutions. More importantly, the co-crystallization technique between high-melting-point amino acids and hydroxytyrosol can significantly increase the melting point of hydroxytyrosol, transforming viscous hydroxytyrosol into a uniform powder state, improving its stability, and greatly reducing its moisture absorption capacity, thus facilitating transportation, storage, and use. However, using high-melting-point amino acids alone in combination with hydroxytyrosol also faces the following problems: 1) When the prepared high-melting-point amino acid / hydroxytyrosol co-crystallized mixture is further dissolved in weakly acidic or neutral aqueous solutions, a high-melting-point amino acid mass fraction greater than 2% is often required to stabilize hydroxytyrosol with a mass fraction of 0.25% or less, greatly limiting the application range of hydroxytyrosol and increasing the amount of amino acid used. 2) In weakly acidic or neutral environments, if the amount of hydroxytyrosol added to the aqueous solution exceeds 0.25%, especially above 0.5%, even with an increased proportion of amino acids, the solution color will gradually darken after being left at 45°C for more than one month, affecting product stability. 3) While the eutectic mixture formed by high-melting-point amino acids and hydroxytyrosol can significantly increase the melting point of hydroxytyrosol, its melting point is mostly around 100-150°C. Developing a more stable high-melting-point hydroxytyrosol eutectic has significant application value.

[0084] To address the aforementioned issues with the co-crystallization of high-melting-point amino acids and hydroxytyrosol, the applicant further discovered that incorporating cysteine ​​derivatives, small molecule peptides containing cysteine, or sulfur-containing strong reducing agents such as sodium sulfite into the above system can effectively solve these problems. This can improve the melting point of the amino acid-hydroxytyrosol, enhance the stability of hydroxytyrosol in aqueous solutions, and reduce the amount of amino acids required.

[0085] Furthermore, the above-mentioned strong sulfur-containing reducing agents, when combined alone with hydroxytyrosol to form a eutectic mixture, face the following problems: 1) These strong sulfur-containing reducing agents are all small-molecule sulfur-containing compounds. If they can form a eutectic with hydroxytyrosol, a large amount will be used. The sulfur-containing strong reducing agent-hydroxytyrosol eutectic is highly irritating, has a pungent taste, and the irritating gases emitted will also affect the product's user experience. 2) More importantly, the above-mentioned strong sulfur-containing reducing agents are all highly hygroscopic. The mixture formed when they react with hydroxytyrosol alone will exacerbate the hygroscopic capacity of hydroxytyrosol, increasing the difficulty of storing and transporting hydroxytyrosol. 3) Except for sodium sulfite, the other strong sulfur-containing reducing agents have low melting points, making it difficult to form a eutectic with hydroxytyrosol alone. Even if a eutectic can be formed, the melting point of the prepared product is very low. 4) The above-mentioned strong sulfur-containing reducing agents all have strong reducing power. The complexes formed by them alone with hydroxytyrosol are very unstable and easily oxidized and deteriorated under the influence of air and water.

[0086] Therefore, as a means to solve the above-mentioned problems, the present invention provides a co-crystal mixture that improves the stability and antibacterial activity of hydroxytyrosol, wherein the co-crystal mixture comprises the following chemical components by mass fraction:

[0087] High melting point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, hydroxytyrosol: 5-60%;

[0088] The high-melting-point hydrogen bond acceptor includes high-melting-point amino acids or amino acid derivatives.

[0089] The sulfur-containing strong reducing agent includes at least one of acetylcysteine, glutathione, acetylglutathione, glycine-cysteine ​​dipeptide, glycine-cysteine-glycine tripeptide, and sodium sulfite.

[0090] In this invention, the advantage of using a high-melting-point hydrogen bond acceptor mass fraction of 20-95% is that it can effectively obtain a powdered eutectic mixture with high melting point properties, which is beneficial for reducing the hygroscopic properties of hydroxytyrosol. An excessively high mass fraction of high-melting-point hydrogen bond acceptors will increase the content of amino acids and derivatives in the entire system, while an excessively low mass fraction will prevent the effective acquisition of a powdered eutectic mixture.

[0091] The advantage of using a sulfur-containing strong reducing agent with a mass fraction of 4-35% is that it can effectively improve the stability of the eutectic mixture in pure aqueous solution and significantly increase the melting point of the eutectic mixture. Excessive mass fraction of the sulfur-containing strong reducing agent will cause the eutectic mixture to emit a pungent odor, and more importantly, it will greatly reduce the melting point and thermal stability of the eutectic mixture. Conversely, excessively low mass fraction of the reducing agent will significantly decrease the stability of the eutectic mixture in aqueous solution.

[0092] The advantage of a hydroxytyrosol mass fraction of 5-60% is that it maximizes the proportion of hydroxytyrosol in the eutectic mixture while also ensuring a high melting point and stability. Excessive hydroxytyrosol mass fraction significantly lowers the melting point of the eutectic mixture, increases its hygroscopicity, and causes the raw material to transition from a powdery to a viscous state. Insufficient hydroxytyrosol mass fraction reduces drying efficiency, increases production costs, and significantly increases the effective amount added to the final product, which is detrimental to transportation and cost control.

[0093] Furthermore, the high-melting-point hydrogen bond acceptor includes at least one of glycine, arginine, valine, serine, threonine, histidine, glutamic acid, leucine, threonine, glutamine, asparagine, alanine, and ergothioneine.

[0094] Furthermore, in the eutectic mixture, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-4):(1-3).

[0095] The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-10).

[0096] The molar ratio of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is (1-20):(1-2).

[0097] In this invention, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-4):(1-3). Under this ratio, the resulting eutectic mixture has a high melting point and good stability. An excessively high proportion of high-melting-point hydrogen bond acceptors will significantly increase the number of free hydrogen bond acceptors in the system, reducing the system's reaction efficiency; conversely, an excessively low proportion of high-melting-point hydrogen bond acceptors will significantly decrease the melting point of the eutectic mixture, making drying difficult and resulting in a viscous eutectic mixture.

[0098] The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-10). Under this ratio, the eutectic mixture can be diluted into an aqueous solution system in any proportion, while ensuring that the system does not change color under high temperature, room temperature, and light conditions. Excessive proportion of the sulfur-containing strong reducing agent will increase the pungent odor of the system and increase the hygroscopic properties of the eutectic mixture; insufficient proportion of the sulfur-containing strong reducing agent will reduce the stability of the eutectic mixture in an aqueous solution environment, and will easily cause color changes and deterioration of the aqueous solution system under high temperature conditions.

[0099] The advantage of a molar ratio of (1-20):(1-2) of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is that the resulting eutectic mixture has the highest melting point and the most stable system. If the proportion of high-melting-point hydrogen bond acceptor is too high, the color stability of the resulting eutectic mixture will decrease when dissolved in aqueous solution, and discoloration will occur after prolonged standing. If the proportion of high-melting-point hydrogen bond acceptor is too low, the melting point of the entire eutectic system will decrease significantly, and the hygroscopicity will increase.

[0100] This invention also provides a method for preparing a eutectic mixture that enhances the stability and antibacterial activity of hydroxytyrosol, the method comprising:

[0101] A mixed solution was obtained by dissolving a high-melting-point hydrogen bond acceptor, a sulfur-containing strong reducing agent, and hydroxytyrosol together in a solvent.

[0102] The mixed solution is homogenized for 0.5 to 6 hours in a vacuum environment or under inert gas protection at a temperature range of 20 to 80°C to obtain a homogenized solution.

[0103] The homogeneous solution is cooled and crystallized, and the resulting crystals are dried to obtain a eutectic mixture;

[0104] The mass fractions of each chemical component in the eutectic mixture are as follows:

[0105] High melting point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, hydroxytyrosol: 5-60%;

[0106] The high-melting-point hydrogen bond acceptor includes high-melting-point amino acids or amino acid derivatives;

[0107] The sulfur-containing strong reducing agent includes at least one of acetylcysteine, glutathione, acetylglutathione, glycine-cysteine ​​dipeptide, glycine-cysteine-glycine tripeptide, and sodium sulfite.

[0108] The eutectic mixture prepared by this invention exhibits strong antibacterial activity against both Gram-positive and Gram-negative bacteria.

[0109] The following will provide a detailed description of a eutectic mixture for improving the stability and antibacterial activity of hydroxytyrosol, its preparation method, and its application, in conjunction with embodiments and experimental data.

[0110] In the following examples and experimental cases, glycine, valine, arginine, serine, threonine, glutamine, acetylglutamine, asparagine, alanine, histidine, glutamic acid, leucine, and NAC (N-acetylcysteine) were purchased from Shantou Jiahe Biotechnology Co., Ltd.; glutathione, sulfurous acid, L-cysteine, and vitamin C were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and hydroxytyrosol (purity > 98%) was purchased from Hangzhou Xizhenglin Biotechnology Co., Ltd. All reagents and raw materials not specifically mentioned were commercially available products.

[0111] I. Implementation Examples

[0112] The chemical composition, weight parts, solvent, lyophilization method, and drying state of Examples 1-12 are shown in Table 1 below. The detailed preparation process is as follows:

[0113] The detailed preparation steps for the hydroxytyrosol eutectic mixture of Example 1 are as follows:

[0114] S1. Prepare a solution according to a 100 parts by weight system. Take 87.5 parts by weight of deionized water (87.5 g) and raise the temperature of the pure aqueous solution to 50°C under nitrogen protection. Weigh 5 parts by weight of valine (5 g, 42.7 mM), 2.5 parts by weight of NAC (2.5 g, 15.3 mM), and 5 parts by weight of hydroxytyrosol (5 g, 32.4 mM) and slowly dissolve them in the above deionized aqueous solution under nitrogen protection. Stir magnetically (600 r / min) at 60°C for 4 h.

[0115] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0116] S3. Place the reactants that form crystals in a freeze dryer and dry for 48 hours to obtain a hydroxytyrosol / valine / NAC eutectic mixture. Based on 100 parts by weight of the eutectic mixture, the weight fraction of valine is (5 / 12.5)*100, which is 40 parts by weight, hydroxytyrosol is 40 parts by weight, and NAC is 20 parts by weight.

[0117] The preparation method of the hydroxytyrosol eutectic mixtures in Examples 2-3 is the same as that in Example 1, except that the high-melting-point valine in Example 1 is replaced with glycine and arginine, respectively. The specific components, contents, drying process, solvents, and powder states are shown in Table 1.

[0118] The detailed steps for preparing the hydroxytyrosol eutectic mixture of Example 4 are as follows:

[0119] S1. Prepare a solution according to a system of 100 parts by weight. Take 85 parts by weight (85g) of deionized water and raise the temperature of the pure aqueous solution to 40℃ under nitrogen protection. Weigh 5 parts by weight of threonine (5g, 41.9mM), 5 parts by weight of NAC (5g, 30.6mM) and 5 parts by weight of hydroxytyrosol (32.4mM) and slowly dissolve them in the pure aqueous solution under nitrogen protection. Stir magnetically (800r / min) at 40℃ for 2h.

[0120] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0121] S3. Place the reactants that form crystals in a freeze dryer and dry for 48 hours to obtain a hydroxytyrosol / threonine / NAC eutectic mixture. Based on 100 parts by weight of the eutectic mixture, the weight of threonine is (5 / 15)*100, which is 33.3 parts by weight, hydroxytyrosol is 33.3 parts by weight, and NAC is 33.3 parts by weight.

[0122] The preparation method of the hydroxytyrosol cocrystal mixture in Example 5 was the same as that in Example 4, except that the high-melting-point amino acid threonine in Example 4 was replaced with valine, and NAC was replaced with glutathione. The specific components, contents, drying process, solvents, and powder states are shown in Table 1.

[0123] The detailed preparation steps for the hydroxytyrosol cocrystal mixture of Example 6 are as follows:

[0124] S1. Prepare a solution according to 100 parts by weight of the system. Prepare a pure water and methanol solution at a volume ratio of 1:1. Weigh 95 parts by weight (95g) of the above mixed solvent. Under nitrogen protection, slowly dissolve 2 parts by weight of glycine (2g, 26.6 mM), 1 part by weight of NAC (1g, 6.1 mM), and 2 parts by weight of hydroxytyrosol (2g, 13 mM) in the above composite solvent. Stir magnetically (600r / min) at room temperature for 4h.

[0125] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0126] S3. Place the reactants that form crystals in a vacuum drying apparatus (vacuum degree -0.08 to -0.095 MPa, temperature 60℃) and dry for 24 hours to obtain a hydroxytyrosol / glycine / NAC eutectic mixture. Based on 100 parts by weight of the eutectic mixture, the weight of glycine is (2 / 5)*100, which is 40 parts by weight, hydroxytyrosol is 40 parts by weight, and NAC is 20 parts by weight.

[0127] The detailed preparation steps for the hydroxytyrosol eutectic mixture of Example 7 are as follows:

[0128] S1. Prepare a solution according to 100 parts by weight of the system. Prepare a pure water and ethanol solution at a volume ratio of 1:1. Weigh 92.5 parts by weight of the above mixed solvent (92.5 g). Under nitrogen protection, slowly dissolve 3 parts by weight of valine (3 g, 25.6 mM), 1.5 parts by weight of NAC (1.5 g, 9.2 mM), and 3 parts by weight of hydroxytyrosol (3 g, 19.5 mM) in the above composite solvent. Stir magnetically (600 r / min) for 4 h at room temperature.

[0129] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0130] S3. Place the reactants that form crystals in a vacuum drying apparatus (vacuum degree -0.08 to -0.095 MPa, temperature 60℃) and dry for 24 hours to obtain a hydroxytyrosol / valine / NAC eutectic mixture. Based on 100 parts by weight of the eutectic mixture, the weight parts of valine are (3 / 7.5)*100, which is 40 parts by weight, hydroxytyrosol is 40 parts by weight, and NAC is 20 parts by weight.

[0131] The detailed preparation steps for the hydroxytyrosol eutectic mixture of Example 8 are as follows:

[0132] S1. Prepare a solution according to 100 parts by weight of the system. Prepare pure water and DMF solution at a volume ratio of 1:1. Weigh 90 parts by weight of the above mixed solvent (90g). Under nitrogen protection, slowly dissolve 4 parts by weight of acetylglutamine (4g, 21.3mM), 2 parts by weight of NAC (2g, 12.3mM), and 4 parts by weight of hydroxytyrosol (4g, 25.9mM) in the above composite solvent. Stir magnetically (600r / min) at room temperature for 4h.

[0133] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0134] S3. Place the reactants that form crystals in a vacuum drying apparatus (vacuum degree -0.08 to -0.095 MPa, temperature 60℃) and dry for 24 hours to obtain a hydroxytyrosol / acetylglutamine / NAC eutectic mixture. Based on 100 parts by weight of the eutectic mixture, the weight percentage of acetylglutamine is (4 / 10)*100, which is 40 parts by weight; hydroxytyrosol is 40 parts by weight; and NAC is 20 parts by weight.

[0135] The preparation method of the hydroxytyrosol eutectic mixture in Example 9 was the same as that in Example 8, except that the high-melting-point acetylglutamine in Example 8 was replaced with alanine. Specific components, contents, drying processes, solvents, and powder states are shown in Table 1.

[0136] Table 1. Description of the composition ratio of each raw material and solvent, drying process and sample state in different embodiments

[0137]

[0138] II. Comparative Example

[0139] The chemical composition, solvent type, component ratio, drying process, and sample condition of Comparative Examples 1-13 are shown in Table 2.

[0140] The detailed preparation steps for the hydroxytyrosol mixture in Comparative Example 1 are as follows:

[0141] S1. Prepare a solution according to a 100 parts by weight system. Take 92 parts by weight of deionized water (92 g) and raise the temperature of the pure aqueous solution to 40°C under nitrogen protection. Weigh 4 parts by weight of NAC (4 g, 24.5 mM) and 4 parts by weight of hydroxytyrosol (4 g, 25.9 mM), and slowly dissolve them in the above pure aqueous solution under nitrogen protection. Stir magnetically (600 r / min) at 40°C for 4 h.

[0142] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours.

[0143] S3. Place the reactants in a freeze dryer and dry for 48 hours to obtain a hydroxytyrosol / NAC mixture. Based on 100 parts by weight of the mixture, the weight of NAC is (4 / 8)*100, which is 50 parts by weight, and the weight of hydroxytyrosol is 50 parts by weight.

[0144] The preparation method of Comparative Example 2 was the same as that of Comparative Example 1, except that the amount of hydroxytyrosol added in Comparative Example 1 was changed from 4 parts by weight to 2 parts by weight. The specific components, contents, drying process, solvents and sample states are shown in Table 2.

[0145] The detailed steps for preparing the hydroxytyrosol mixture of Comparative Example 3 are as follows:

[0146] S1. Prepare a solution according to a 100 parts by weight system. Take 90 parts by weight (90 g) of deionized water and raise the temperature of the pure aqueous solution to 50°C under nitrogen protection. Weigh 5 parts by weight of valine (5 g, 42.7 mM) and 5 parts by weight of hydroxytyrosol (5 g, 32.4 mM), and slowly dissolve them in the above pure aqueous solution under nitrogen protection. Stir magnetically (600 r / min) at 50°C for 4 h.

[0147] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours.

[0148] S3. Place the reactants in a freeze dryer and dry for 48 hours to obtain a hydroxytyrosol / valine mixture. Based on 100 parts by weight of the mixture, the weight of valine is 5 / 10, which is 50 parts by weight, and the weight of hydroxytyrosol is 50 parts by weight.

[0149] The experimental process parameters for Comparative Examples 4-9 were the same as those for Comparative Example 3, and the specific components, proportions, and processes are shown in Table 2.

[0150] The detailed steps for preparing the hydroxytyrosol mixture of Comparative Example 10 are as follows:

[0151] S1. Prepare a solution according to 100 parts by weight of the system. Prepare pure water and DMF solution at a volume ratio of 1:1. Weigh 80 parts by weight of the above mixed solvent (80g). Under nitrogen protection, slowly dissolve 10 parts by weight of betaine (10g, 85.4mM) and 10 parts by weight of hydroxytyrosol (10g, 64.9mM) in the above composite solvent. Stir magnetically (600r / min) at room temperature for 4h.

[0152] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0153] S3. Place the reactants that form crystals in a vacuum drying apparatus (vacuum degree -0.08 to -0.095 MPa, temperature 60℃) and dry for 24 hours to obtain a hydroxytyrosol betaine eutectic mixture. Based on 100 parts by weight of the eutectic mixture, the weight of betaine is (10 / 20)*100, which is 50 parts by weight, and the weight of hydroxytyrosol is 50 parts by weight.

[0154] The detailed preparation steps for the hydroxytyrosol eutectic mixture of Comparative Example 11 are as follows:

[0155] S1. Prepare a solution according to a 100 parts by weight system. Take 80 parts by weight of deionized water (80 g) and raise the temperature of the pure aqueous solution to 50°C under nitrogen protection. Weigh 8 parts by weight of glycine (8 g, 106.6 mM), 4 parts by weight of vitamin C (4 g, 22.8 mM), and 8 parts by weight of hydroxytyrosol (8 g, 51.9 mM) and slowly dissolve them in the above pure aqueous solution under nitrogen protection. Stir magnetically (600 r / min) at 60°C for 4 h.

[0156] S2. Slowly cool the reaction system to -40℃ and maintain this temperature for 12 hours to gradually form crystals.

[0157] S3. Place the reactants that form crystals in a freeze dryer and dry for 48 hours to obtain a hydroxytyrosol / glycine / vitamin C co-crystal mixture. Based on 100 parts by weight of the co-crystal mixture, the weight of glycine is (8 / 20)*100, which is 40 parts by weight, hydroxytyrosol is 40 parts by weight, and vitamin C is 20 parts by weight.

[0158] The experimental process parameters for Comparative Examples 12 and 13 were the same as those for Comparative Example 11, and the specific components, proportions, and processes are shown in Table 2.

[0159] Table 2. Composition ratios of raw materials and solvents, drying processes, and sample conditions in different comparative examples.

[0160]

[0161] III. Effect Verification

[0162] Fourier transform infrared spectroscopy (FIR) was used to determine the absorbing functional groups of amino acids, sulfur-containing strong reducing agents, and their synthesized eutectic mixtures. Infrared spectral scanning was performed using potassium bromide pellet mode. The test wavelengths were all from 400 to 4000 cm⁻¹. -1 .

[0163] X-ray detection: The internal crystal structure of DES was detected using an X-ray whole-rock quantitative analysis instrument (Rigaku Ultima IV, Japan). A copper target was selected for wide-angle diffraction, with a scanning range of 3-60° and a scanning rate of 5° / min.

[0164] Thermogravimetric analysis: The thermal stability and thermal properties of amino acids, hydroxytyrosol, and the synthesized eutectic mixture were determined using a simultaneous thermal analyzer (NETZS STA449 F3). The tests were conducted under nitrogen atmosphere, with the temperature gradually increased from room temperature to 800°C at a heating rate of 10°C / min.

[0165] Differential scanning calorimetry (DSC) was used to measure the heat absorbed or released by the eutectic mixture and its components during temperature changes using a Hitachi STA200 calorimeter. The measurements were performed under nitrogen atmosphere. The sample was heated from -70°C to 100°C at a rate of 10°C / min, with a nitrogen flow rate of 50 mL / min. Heating was also performed from 30°C to 250°C at a rate of 10°C / min, with a nitrogen flow rate of 50 mL / min.

[0166] Antibacterial experiment: 1) Prepare liquid culture medium, sterilize culture medium, pipette tips, centrifuge tubes, and other experimental equipment at high temperature; 2) After sterilization, pour plates to prepare solid culture medium, which needs to be dried in a clean bench for 30 minutes; 3) Dilute the bacterial solution 1*103 4) Material preparation: Control group: Hydroxytyrosol was dissolved in pure water at room temperature, stirred until dissolved, and prepared into a solution of the corresponding concentration. Experimental group: Hydroxytyrosol was dissolved in Example 1 at room temperature, stirred until dissolved, and prepared into a solution of the corresponding concentration; 5) Co-incubation of materials and bacterial suspension: 1 mL of each of the different concentrations of hydroxytyrosol solution was mixed with 1 mL of bacterial suspension at a volume ratio of 1:1, shaken well, and co-incubated for 30 min. The same procedure was followed for both the control and experimental groups. 6) Dilution before coating: The incubated mixture was diluted 1*10 2 6) Plate coating: Take 100 μL of each concentration of the mixture and coat it onto a plate. Place the plate in a 37°C incubator and take a picture and count the samples after 24 hours. The final concentrations of the bacterial culture in Example 1 and the hydroxytyrosol aqueous solution group were set to 0, 100, 200, 400, and 800 μg / mL, respectively, with n=3.

[0167] IV. Results Analysis:

[0168] from Figure 1 It can be seen that after freeze-drying, Examples 1 and 2 yielded white powder samples with fine particles. These samples could be stored for a long time after sealing without significant changes. Examples 3-9 all formed eutectic powder forms after drying. Comparative Examples 1 and 2, due to the low melting points of their two components, remained viscous and lumpy during drying, failing to form a eutectic powder state regardless of the drying time. Therefore, these two groups were not further investigated.

[0169] Depend on Figure 2 It can be seen that N-acetylcysteine ​​has an endothermic peak at around 200℃, while hydroxytyrosol has an endothermic peak at around 58.9℃. And if... Figure 2 As shown on the left, the DSC spectrum of N-acetylcysteine-hydroxytyrosol is completely different from that of the two monomers. This is because the removal of adsorbed water in the sample exhibits a weak, continuous endothermic process from 0-200℃, consistent with the results of the sample freeze-drying at that time. Combined with the TG-DTG spectrum analysis of N-acetylcysteine-hydroxytyrosol (…), Figure 2 (Right) A broad and gentle endothermic peak appears in the DSC spectrum between 200-350℃, with a peak temperature of approximately 284.7℃, which completely coincides with the TG weight loss peak, indicating that it is a pyrolysis peak of the mixed system. This suggests that the introduction of N-acetylcysteine ​​may form a covalent compound.

[0170] Depend on Figure 3 It is known that valine has an endothermic peak at around 200℃, while hydroxytyrosol has an endothermic peak at around 58.9℃. And if... Figure 3 As shown on the left, the DSC chromatogram of valine-hydroxytyrosol is completely different from the spectra of the two monomers, showing an endothermic peak at around 52.4℃. Combined with the TG-DTG chromatogram analysis of valine-hydroxytyrosol (…), Figure 3(Right) The endothermic peak appearing at around 52.4℃ is the eutectic melting peak. Between 0 and 200℃, the weight of valine-hydroxytyrosol remains relatively stable (>90%), indicating that valine-hydroxytyrosol does not decompose significantly in the molten state, while the main decomposition stage occurs between 200 and 350℃. The relatively large side chain of valine may explain the two-stage decomposition observed at 235.5℃ and 306.3℃ in this system.

[0171] Depend on Figure 4 It is known that glycine has an endothermic peak at around 200℃, while hydroxytyrosol has an endothermic peak at around 58.9℃. And... Figure 4 As shown on the left, the DSC chromatogram of glycine-hydroxytyrosol is completely different from the spectra of the two monomers, showing an endothermic peak at around 102.9℃. Combined with the TG-DTG chromatogram analysis of glycine-hydroxytyrosol (…), Figure 4 (Right) The endothermic peak appearing at around 102.9℃ is the eutectic melting peak, and the TG curve shows no weight loss in this stage, indicating that physical melting of the eutectic occurs at this stage. 200-400℃ is the main decomposition stage of glycine-hydroxytyrosol, reaching the maximum weight loss rate at 243.3℃.

[0172] And such Figure 5 As shown on the left, the DSC analysis chromatogram of the hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture in Example 1 is completely different from the spectra of the three monomers. The first endothermic peak appears at approximately 240.6℃, and the second endothermic peak appears at approximately 299.4℃. This is in conjunction with the TG-DTG chromatogram analysis of Example 1 (…). Figure 5 (Right) The temperature slowly decreases from 0-150℃ as adsorbed water is removed, and rapid weight loss occurs from 150-300℃, reaching its maximum rate around 238.6℃. This is the main decomposition stage of the ternary mixture system, representing the thermal degradation peak of Example 1. A second, smaller weight loss peak appears at 299.4℃, corresponding to further decomposition of intermediate products or removal of residual groups after thermal degradation. This ternary system does not exhibit a melting point of 240.6℃; combined with the TG-DTG curve, it can be seen that Example 1 begins to decompose at its melting point.

[0173] Depend on Figure 6 It can be seen that glycine and N-acetylcysteine ​​have endothermic peaks at around 200℃ and 210℃, respectively, while hydroxytyrosol has an endothermic peak at around 58.9℃. And... Figure 6 As shown on the left, the DSC analysis spectrum of the hydroxytyrosol-glycine-N-acetylcysteine ​​cocrystal mixture in Example 2 is completely different from the spectra of the three monomers, showing an endothermic peak at around 244.2℃. Combined with the TG-DTG spectrum analysis in Example 2 ( Figure 6(Right) An endothermic peak appears at around 244.2℃, and the position of the peak completely coincides with the main weight loss peak of the TG curve, which is a decomposition endothermic peak, corresponding to the process of absorbing a large amount of heat when the main chain of the sample breaks. Example 2 melts and begins to decompose at 244.2℃.

[0174] The melting point, degradation temperature, and thermal properties of Examples 1, 2, 1, 3, and 4 are shown in Table 3.

[0175] Figure 7 The results show that in the ternary mixed system composed of valine, N-acetylcysteine ​​and hydroxytyrosol, the X-ray powder diffraction pattern, expressed in 2θ angles, has characteristic absorption peaks at diffraction angles of 14.72, 18.32, 19.6, 22.22, 29.76, 37.44 and 45.34.

[0176] Figure 8 The results show that in the ternary mixed system composed of glycine, N-acetylcysteine ​​and hydroxytyrosol, the X-ray powder diffraction pattern, expressed in 2θ angles, has characteristic absorption peaks at diffraction angles of 14.28, 17.96, 23.78, 26.88, 28.46, 30.28, 31.08, 33.72, 36.38, 39.16, 53.16 and 57.82.

[0177] Figure 9 The infrared spectral characteristics of hydroxytyrosol are shown. Hydroxytyrosol is spectral at 3392 cm⁻¹. -1 3228 cm -1 2939 cm -1 2882 ​​cm -1 1601 cm -1 1526cm -1 1423 cm -1 1378 cm -1 1281cm -1 1248 cm -1 1181 cm -1 1105 cm -1 1055 cm -1 1006 cm -1 909 cm -1 804 cm -1 714cm -1 631 cm -1 576 cm -1 456 cm -1 It exhibits an absorption peak at 3392 cm⁻¹. -1 and 3228 cm -1The broad and strong absorption peak at 1601 cm⁻¹ is attributed to the OH stretching vibrations of the phenolic hydroxyl (-OH) and terminal primary alcohol hydroxyl groups in the structure, and its broadening characteristics indicate the presence of strong intermolecular hydrogen bonding. -1 1526 cm -1 and 1423 cm -1 The absorption peak at 1281 cm⁻¹ corresponds to the C=C stretching vibration of the benzene ring skeleton and is a characteristic signal of this aromatic structure. -1 and 1248 cm -1 The strong peak at 1105 cm⁻¹ is due to the CO stretching vibration of the phenolic hydroxyl group. -1 and 1055 cm -1 The absorption at 804 cm⁻¹ is attributed to the CO stretching vibration of the primary alcohol (-CH₂OH). -1 The absorption peak at that point is a characteristic fingerprint of the out-of-plane bending vibration of the CH group of the benzene ring.

[0178] Figure 10 The infrared spectral characteristics of glycine are shown. Glycine at 3151 cm⁻¹ -1 2606 cm -1 1591 cm -1 1498 cm -1 1399 cm -1 1322 cm -1 1122 cm -1 1024 cm -1 1004cm -1 688 cm -1 603 cm -1 491cm -1 It exhibits an absorption peak at 3151 cm⁻¹. -1 The broad and strong absorption peak at 2606 cm⁻¹ is attributed to the OH stretching vibration in the carboxyl group, which is significantly broadened due to strong hydrogen bonding. -1 The absorption peak in the vicinity is related to the stretching vibration of NH in the zwitterionic form of glycine. (1591 cm⁻¹) -1 The strong peak at 1399 cm⁻¹ is a superposition of the in-plane bending vibration of NH and the asymmetric stretching vibration of COO. -1 The absorption peak at 1498 cm⁻¹ is attributed to the symmetric stretching vibration of COO⁻, indicating the presence of a carboxylate ion in the molecule. Furthermore, the absorption peak at 1498 cm⁻¹... -1 The absorption at 1122 cm⁻¹ is related to the CH₂ shear vibration, while the absorption at 1122 cm⁻¹ is related to the CH₂ shear vibration. -1 With 1024 cm -1 The multiple absorption peaks at these locations mainly correspond to the stretching vibrations of CN and CO, as well as the skeletal vibrations.

[0179] Figure 11The infrared spectral characteristics of valine are shown. Valine is at 3148 cm⁻¹. -1 2923 cm -1 2093 cm -1 1562 cm -1 1499 cm -1 1418 cm -1 1387 cm -1 1322 cm -1 1266 cm -1 1170 cm -1 1138 cm -1 1058 cm -1 1025 cm -1 947 cm -1 849 cm -1 818 cm -1 711 cm -1 661 cm -1 539 cm -1 431cm -1 It exhibits an absorption peak at 3148 cm⁻¹. -1 The broad and strong absorption band at 2923 cm⁻¹ corresponds to the NH stretching vibration of the ammonium group in the zwitterionic form of valine. This absorption peak is significantly broadened due to strong intermolecular hydrogen bonding. -1 The absorption peak in the vicinity is attributed to the stretching vibration of the CH bond in the isopropyl group of the side chain. (1562 cm⁻¹) -1 The strong peak at 1418 cm⁻¹ is attributed to the coupling of the in-plane bending vibration of the ammonium NH group and the asymmetric stretching vibration of the carboxylate group (COO⁻). -1 The absorption peak at 1387 cm⁻¹ is attributed to the symmetric stretching vibration of COO⁻, and the two pairs of characteristic peaks of the carboxylate group mentioned above together confirm that it exists mainly in the form of a zwitterion. -1 and 1170 cm -1 The absorption peak corresponds to the CH bending vibration and CC skeleton vibration of the isopropyl side chain.

[0180] Figure 12 The image shows the characteristic infrared spectrum of Comparative Example 3 (a mixture of hydroxytyrosol and valine). The combined sample is located at 3155 cm⁻¹. -1 2984 cm -1 2099 cm -1 1695 cm -1 1561 cm -1 1515 cm -1 1444 cm -1 1385 cm -11278 cm -1 1191 cm -1 1111 cm -1 1026 cm -1 947 cm -1 812 cm -1 780 cm -1 713 cm -1 660 cm -1 cm -1 633 cm -1 537 cm -1 It exhibits an absorption peak. When the two combine, the peak is at 3155 cm⁻¹. -1 A strong and broad characteristic peak appears at 3392 cm⁻¹, corresponding to hydroxytyrosol. -1 Compared with the absorption peaks at 1562 cm⁻¹, the characteristic peak of the phenolic hydroxyl group in the hydroxytyrosol-valine cocrystal shifts to a lower wavenumber and its peak shape broadens significantly. This is attributed to the formation of new and stronger intermolecular hydrogen bonds between the -OH group of hydroxytyrosol and the carboxyl group (-COOH) or amino group (-NH₂) of valine. The carboxyl ion pair peak of valine is also observed at 1562 cm⁻¹. -1 and 1418 cm -1 The particles were displaced to 1561 cm in the composite. -1 and 1444 cm -1 Furthermore, the peak shape and intensity changed significantly, indicating that these groups participated in hydrogen bond interactions with hydroxytyrosol. These results demonstrate that new intermolecular hydrogen bond interactions (OH···O and / or OH···N) form a stable eutectic or supramolecular complex with novel physicochemical properties.

[0181] Figure 13 The image shows the characteristic infrared spectrum of Comparative Example 4 (a mixture of hydroxytyrosol and glycine). The combined sample reaches a wavelength of 3177 cm⁻¹. -1 2519 cm -1 1598 cm -1 1511 cm -1 1408 cm -1 1329 cm -1 1115 cm -1 1039 cm -1 912 cm -1 889 cm -1 852 cm -1 806 cm -1 757 cm -1 701 cm -1 605 cm -1 519 cm-1 476 cm -1 It exhibits an absorption peak. In the spectrum, the characteristic peak of the glycine carboxylate ion (1591 cm⁻¹) is present. -1 and 1399 cm -1 The stretching vibration peak of the phenolic hydroxyl group (OH) of hydroxytyrosol (3392 cm⁻¹) -1 and 3228 cm -1 All showed changes in position and intensity. The hydroxytyrosol-glycine eutectic at 3177 cm⁻¹... -1 A broadened and strongly red-shifted absorption peak appears at [value missing], which is attributed to the superposition and merging of intermolecular hydrogen bonds formed between the ammonium ion and OH group of glycine and the phenolic hydroxyl OH group of hydroxytyrosol, and the shift to lower frequencies. Hydroxytyrosol ( Figure 1 In the study, the CO stretching vibration peak of the phenolic hydroxyl group appeared at 1248 cm⁻¹. -1 At wavenumber, the benzene ring skeletal vibration is at 1601 cm⁻¹. -1 and 1526 cm -1 In the composite, 1248 cm -1 The nearby phenol CO peak disappeared, while the benzene ring skeletal vibration peak shifted to 1598 cm⁻¹. -1 and 1511 cm -1 This indicates that the phenolic hydroxyl group participates in the formation of intermolecular hydrogen bonds (acting as a hydrogen bond donor OH··· or acceptor Ar-O⁻···H). These results demonstrate that hydroxytyrosol and glycine combine to form a stable cocrystal or supramolecular complex through the formation of an intermolecular hydrogen bond network.

[0182] Figure 14 The Fourier transform infrared (FTIR) spectrum of Comparative Example 1 (a mixture of hydroxytyrosol and N-acetylcysteine) is shown. The combined sample is at 3273 cm⁻¹. -1 2947 cm -1 1717 cm -1 1603 cm -1 1518 cm -1 1441 cm -1 1370 cm -1 1281 cm -1 1248 cm -1 1192 cm -1 1112 cm -1 1009 cm -1 953 cm -1 917 cm -1 808 cm -1 781cm -1 751 cm -1 629 cm-1 535 cm -1 It has an absorption peak. The absorption peak is related to the stretching vibration of the OH group in hydroxytyrosol (3392 cm⁻¹). -1 Compared to hydroxytyrosol-N-acetylcysteine ​​conjugates, the phenolic hydroxyl group in the hydroxytyrosol-N-acetylcysteine ​​conjugate is at 3273 cm⁻¹. -1 The presence of a significantly broadened characteristic peak, marked low-wavenumber shift, and peak broadening indicates the formation of a new hydrogen bond network in the complex. This is attributed to the formation of stronger intermolecular hydrogen bonds (such as OH···O, OH···N) between the phenolic hydroxyl group (-OH) of hydroxytyrosol and the carboxyl group (-COOH) and amino group (-NH2) of N-acetylcysteine. Hydroxytyrosol reaches a peak at 1601 cm⁻¹. -1 1526 cm -1 A vibrational peak of the benzene ring skeleton (C=C) appears at the wavenumber, while in the complex, a peak appears at 1603 cm⁻¹. -1 1518 cm -1 The positions and intensities of the two absorption peaks changed. In the complex spectrum, 1717 cm⁻¹ -1 A new characteristic peak appears at the wavenumber, which is attributed to the C=O stretching vibration of the carboxylic acid (-COOH) in N-acetylcysteine. These results indicate that N-acetylcysteine ​​has been integrated into the complex structure and forms a stable molecular complex through a hydrogen bond network with hydroxytyrosol.

[0183] Figure 15 The Fourier transform infrared (FTIR) spectrum of Example 1 (hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture) is shown. The combined three components are visualized at 3145 cm⁻¹. -1 2933 cm -1 2116 cm -1 1724 cm -1 1583 cm -1 1506 cm -1 1442 cm -1 1392 cm -1 1327 cm -1 1280 cm -1 1190 cm -1 1139 cm -1 1111 cm -1 1026 cm -1 948 cm -1 902 cm -1 808 cm -1 776 cm -1 715 cm -1 665 cm -1 539 cm-1 It has an absorption peak. Figure 15 The ternary complex at 3145 cm -1 and 2933 cm -1 A broad and strong absorption peak exists at this point, which is attributed to the N-terminus of valine. + The stretching vibrations of various hydrogen bond donors / acceptors, such as -H, OH of hydroxytyrosol, and NH of N-acetylcysteine, form an intermolecular hydrogen bond network that superimposes, merges, and broadens, interacting with hydroxytyrosol-valine (3155 cm⁻¹). -1 ) and hydroxytyrosol-N-acetylcysteine ​​(3273cm) -1 Compared to the characteristic hydrogen bond peaks of binary compounds, the peak shape of the ternary complex is broader and shifts to lower wavenumbers, indicating stronger hydrogen bond interactions in the ternary complex. The skeletal vibrations of the hydroxytyrosol benzene ring (e.g., 1601 cm⁻¹) are also observed. -1 1526 cm -1 )exist Figure 8 The center moved to 1583cm -1 1506 cm -1 The proximity suggests the presence of hydrogen bonds or electrostatic interactions. 1724 cm -1 A stretching vibration peak of the carboxylic acid carbonyl group (C=O) was observed, which is attributed to the acetyl / carboxyl group of N-acetylcysteine. These results indicate that the three molecules form a stable complex molecule through intermolecular hydrogen bonding.

[0184] Figure 16 The Fourier transform infrared (FTIR) spectrum of Example 2 (hydroxytyrosol-glycine-N-acetylcysteine ​​cocrystal mixture) is shown. The combined three components are visualized at 3375 cm⁻¹. -1 3174 cm -1 2546 cm -1 2127 cm -1 1716 cm -1 1660 cm -1 1590 cm -1 1511 cm -1 1412 cm -1 1114 cm -1 1037 cm -1 914 cm -1 892 cm -1 797 cm -1 701cm -1 605 cm -1 514 cm -1 It exhibits an absorption peak at 3174 cm⁻¹. -1 and 2546 cm -1A broad and strong complex absorption peak is observed at this point, which is attributed to the superposition, merging, and significant red shift of the hydrogen bond network between the OH group of hydroxytyrosol, the NH group of glycine, and the NH group of N-acetylcysteine, compared to approximately 3273 cm⁻¹ in the binary complex. Figure 6 The hydrogen bond peak of tyrosol is further broadened and shifted to a lower wavenumber compared to tyrosol, indicating that hydrogen bonding is further enhanced in the ternary system. In addition, the characteristic aromatic ring CO stretching vibration peak of orthohydroxytyrosol (approximately 1248 cm⁻¹) is also observed. -1 , Figure 1 It almost completely disappears in the complex, while at approximately 1590 cm -1 and 1511 cm -1 At locations such as 1716 cm⁻¹, the characteristic absorptions of aromatic ring skeletal vibration (C=C) are retained, but the peak position and intensity are altered. -1 A stretching vibration peak of the carboxylic acid carbonyl group (C=O) was observed, which is attributed to the acetyl / carboxyl group of N-acetylcysteine. These results indicate that the three molecules form a stable complex through an intermolecular hydrogen bond network.

[0185] Figure 17 It can be seen that the lyophilized powders of Examples 1 (hydroxytyrosol-valine-N-acetylcysteine ​​cocrystal mixture), 2 (hydroxytyrosol-glycine-N-acetylcysteine ​​cocrystal mixture), and 3 (hydroxytyrosol-arginine-N-acetylcysteine ​​cocrystal mixture) were prepared into 2.5% and 5% aqueous solutions (pH 6.0) and placed at room temperature (20°C) and high temperature (45°C) for two months, respectively. The stability observation confirmed that the cocrystal mixtures obtained in this invention exhibit good stability even in a weakly acidic environment when reconstituted in pure water. However, the stability observation results of the comparative sample prepared into a 2.5% aqueous solution with pH 6.0 after drying and placed at room temperature (20°C) and high temperature (45°C) for 15 days are as follows: Figure 18 As shown in the table. The results showed that Comparative Examples 3-11 exhibited significant color changes within 15 days, indicating decreased sample stability. Comparative Examples 12 and 13 showed no significant color change within 15 days, but produced substantial precipitation, also indicating decreased sample stability. Table 4 further analyzes the stability of samples prepared by dissolving the dried powder from different examples in pure aqueous solution at pH 6.0 and a concentration of 2.5% (mass fraction). The results further verify that the solution color did not change significantly even after being placed at a high temperature of 45°C for two months. In summary, the eutectic mixture of the present invention can remain stable in pure aqueous solution for a long time without color deepening, providing a solution for the further application of hydroxytyrosol.

[0186] Figure 19The results showed that both Example 1 and the hydroxytyrosol group exhibited good antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*, confirming that the co-crystal mixture did not affect the antibacterial activity of hydroxytyrosol. Specifically, for *Escherichia coli*, Example 1 showed better antibacterial activity than the hydroxytyrosol group alone (the top row in the figure is Example 1, and the bottom row is the hydroxytyrosol aqueous solution group). The hydroxytyrosol in Example 1 showed complete inhibition at a concentration of 400 μg / mL, while the hydroxytyrosol group alone could not achieve complete inhibition at this concentration. This confirms that the co-crystal mixture in this invention not only improves the melting point, thermal stability, and stability in water of hydroxytyrosol, but also enhances its antibacterial effect against *Escherichia coli*. For *Staphylococcus aureus*, the antibacterial effects of Example 1 and the hydroxytyrosol group alone were similar, both showing complete inhibition at 400 μg / mL.

[0187] Table 3. Comparison of melting point, degradation temperature and thermal properties of samples from different embodiments

[0188]

[0189] Table 4. Stability analysis of the dried powder dissolved in pure water in different examples

[0190]

[0191] The samples in Table 4 were prepared with a mass fraction of 2.5%, pH set to 6.0, refrigeration temperature set to 4-8℃, room temperature set to 20℃, high temperature set to 45℃, and the sample storage time was 2 months.

[0192] 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.

[0193] 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.

[0194] 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 co-crystal mixture that enhances the stability and antibacterial activity of hydroxytyrosol, characterized in that, The eutectic mixture, by mass fraction, comprises the following chemical components: High melting point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, hydroxytyrosol: 5-60%; The high-melting-point hydrogen bond acceptor includes one of glycine, arginine, valine, threonine, acetylglutamine, and alanine. The sulfur-containing strong reducing agent includes acetylcysteine ​​or glutathione.

2. The co-crystal mixture for improving the stability and antibacterial activity of hydroxytyrosol according to claim 1, characterized in that, In the eutectic mixture, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-4):(1-3). The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-10). The molar ratio of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is (1-20):(1-2).

3. A method for preparing a eutectic mixture that enhances the stability and antibacterial activity of hydroxytyrosol as described in claim 1 or 2, characterized in that, The preparation method includes: A mixed solution was obtained by dissolving a high-melting-point hydrogen bond acceptor, a sulfur-containing strong reducing agent, and hydroxytyrosol together in a solvent. The mixed solution is homogenized for 0.5 to 6 hours in a vacuum environment or under inert gas protection at a temperature range of 20 to 80°C to obtain a homogenized solution. The homogeneous solution is cooled and crystallized, and the resulting crystals are dried to obtain a eutectic mixture; The mass fractions of each chemical component in the eutectic mixture are as follows: High melting point hydrogen bond acceptor: 20-95%, sulfur-containing strong reducing agent: 4-35%, hydroxytyrosol: 5-60%; The high-melting-point hydrogen bond acceptor includes one of glycine, arginine, valine, threonine, acetylglutamine, and alanine; The sulfur-containing strong reducing agent includes acetylcysteine ​​or glutathione; The solvent is water or a mixed solvent, wherein the mixed solvent is a mixture of water and an organic solvent in a volume ratio of 1:1, and the organic solvent is one of methanol, ethanol and DMF; The drying process can be selected from freeze drying, vacuum drying, and spray drying.

4. The method for preparing a eutectic mixture that enhances the stability and antibacterial activity of hydroxytyrosol according to claim 3, characterized in that, In the eutectic mixture, the molar ratio of the high-melting-point hydrogen bond acceptor to hydroxytyrosol is (1-4):(1-3). The molar ratio of the sulfur-containing strong reducing agent to hydroxytyrosol is (1-2):(1-10). The molar ratio of the high-melting-point hydrogen bond acceptor to the sulfur-containing strong reducing agent is (1-20):(1-2).

5. The method for preparing a eutectic mixture for improving the stability and antibacterial activity of hydroxytyrosol according to claim 3, characterized in that, The process of cooling and crystallizing the homogeneous solution, followed by drying the resulting crystals to obtain a eutectic mixture, specifically includes: The homogeneous solution is crystallized at -80℃ to 10℃ for 1 to 24 hours, and the resulting crystals are dried to obtain a eutectic mixture.

6. The use of the eutectic mixture as described in claim 1 or 2, which enhances the stability and antibacterial activity of hydroxytyrosol, in the preparation of cosmetics or pharmaceuticals.

7. A cosmetic product, characterized in that, The cosmetic preparation materials contain a eutectic mixture as described in claim 1 or 2 that enhances the stability and antibacterial activity of hydroxytyrosol.

8. A cosmetic product according to claim 7, characterized in that, The dosage form of the cosmetic includes any one of cream, gel, and foam.

9. The application of the co-crystal mixture for improving the stability and antibacterial activity of hydroxytyrosol as described in claim 1 or 2 in the preparation of aqueous solutions, characterized in that, The pH of the aqueous solution preparation is 4.5–8; In the aqueous solution preparation, the mass fraction of the eutectic mixture is 0.1% to 15%.

Images