A hydroxytyrosol nanoparticle with high antioxidant capacity and stability and a preparation method thereof

Abstract

This invention relates to hydroxytyrosol nanoparticles with high antioxidant capacity and stability, and a method for preparing the same. The method includes the following steps: (1) preparing a cyclic γ-polyglutamic acid solution; (2) preparing an ε-polylysine solution; (3) preparing a hydroxytyrosol solution; (4) adding the hydroxytyrosol solution to the ε-polylysine solution, stirring until homogeneous, and obtaining a mixed solution; then adding the cyclic γ-polyglutamic acid solution dropwise to the mixed solution, stirring the reaction mixture, and dialysis of the reaction solution to obtain hydroxytyrosol nanoparticles with high antioxidant capacity and stability. This invention utilizes the characteristics of cyclic γ-polyglutamic acid side chains containing a large number of negatively charged free carboxyl groups and ε-polylysine side chains containing a large number of positively charged free amino groups. By dissolving cyclic γ-polyglutamic acid, hydroxytyrosol, and ε-polylysine in water and mixing them, the antioxidant capacity and stability of hydroxytyrosol are effectively improved, solving the problem of poor stability and easy oxidation and decomposition of hydroxytyrosol under light conditions.

Description

Technical Field

[0001] This invention relates to hydroxytyrosol nanoparticles with high antioxidant capacity and stability, and their preparation method, belonging to the field of polyphenol compound technology. Background Technology

[0002] Hydroxytyrosol (HT) is a natural polyphenolic compound extracted from olive oil. Its antioxidant and free radical scavenging abilities are higher than some other synthetic or natural compounds, effectively scavenging both endogenous and exogenous free radicals and oxidants, making it a potent antioxidant. Studies have found that hydroxytyrosol possesses various physiological activities, such as antioxidant, anti-aging, antibacterial, and anticancer effects. Currently, crude extracts or high-purity products of hydroxytyrosol are widely used in food, health products, and cosmetics. However, hydroxytyrosol is photosensitive and easily oxidized and decomposed under light conditions, and it also has low thermal stability. Therefore, there is an urgent need to develop a method to enhance the antioxidant activity of hydroxytyrosol.

[0003] Polymer nanoparticles (PNPs) are prepared from biocompatible and biodegradable polymers ranging from 10 to 1000 nm. Drugs are dissolved, embedded, encapsulated, or attached to a nanoparticle matrix. Depending on the method used to prepare the nanoparticles, nanospheres or nanocapsules can be obtained. Ionogelation is a method based on hydrophilic ionopolymers, typically involving the complexation of polyelectrolytes with oppositely charged polyelectrolytes for encapsulation.

[0004] Chinese patent document CN108157942A discloses a method for spray-coating hydroxytyrosol with ginkgo starch gelatinization. This method uses starch encapsulation to protect hydroxytyrosol from oxidation. However, starch absorbs moisture from the air, leading to a deterioration in the physical state of the patented product. Chinese patent document CN114557963A discloses a nano-hydroxytyrosol liposome and its preparation method. It utilizes lecithin and cholesterol to obtain a lecithin film, which is then used to prepare the hydroxytyrosol liposome. However, this patent suffers from boiling over during the evaporation of organic solvents, and the lecithin film is unstable with a low encapsulation rate.

[0005] ε-Polylysine is a homomonomer polymer composed of lysine residues. Rich in cations, it exhibits strong electrostatic interactions with anionic substances and demonstrates excellent adhesion and penetration into biofilms. Based on this property, polylysine has become a focus of research in the biomedical field. Furthermore, polylysine can be broken down into essential lysine in the human body, which can be absorbed and provides nutrients without toxicity or harm. Therefore, ε-polylysine possesses excellent biocompatibility. More importantly, ε-polylysine exhibits a broad antibacterial spectrum and strong antibacterial effect, showing strong inhibition against Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli. Therefore, using polylysine as a matrix, polymer nanoparticles are endowed with multiple biological properties, including antibacterial and bacteriostatic effects.

[0006] Polyglutamic acid typically has a large molecular weight, and its long, straight-chain polymer structure makes it prone to cross-linking in solution, forming a gel. Cyclic γ-polyglutamic acid, on the other hand, has a smaller molecular weight and readily forms uniform nanoparticles in reactions with cationic molecules, exhibiting better fluidity and avoiding hydrogel formation. Cyclic γ-polyglutamic acid lacks free N- and C-termini, significantly reducing its sensitivity to aminopeptidase and carboxypeptidase, making it more stable. The connection of the proximal and distal amide rings provides a more stable skeletal structure. Degraded cyclic γ-polyglutamic acid, due to its smaller molecular weight, more easily penetrates cells, thereby inhibiting tyrosinase activity and exerting a highly effective whitening effect. Furthermore, cyclic γ-polyglutamic acid can promote the accumulation of NMF in the dermis, thus improving the skin's moisturizing ability.

[0007] Chinese patent document CN115531296A discloses a method for preparing drug-loaded polyglutamic acid-based hydrogels. The hydrogel prepared by this patent is a condensed matter with a three-dimensional network structure. The gel exhibits solid-like rheological properties, but its high viscosity and mechanical strength result in poor flowability, making it unsuitable for use in skincare products such as lotions, creams, and serums. Chinese patent document CN107163263A discloses a method for preparing and applying uniformly porous hydrogels. However, this patent involves adding N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as stabilizers and activators during the preparation process. The resulting hydrogel has a certain degree of toxicity, increasing the safety risks associated with the hydrogel. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides hydroxytyrosol nanoparticles with high antioxidant capacity and stability, and a method for preparing the same.

[0009] The technical solution of the present invention is as follows:

[0010] A method for preparing hydroxytyrosol nanoparticles with high antioxidant capacity and stability includes the following steps:

[0011] (1) Dissolve cyclic γ-polyglutamic acid in water to obtain a cyclic γ-polyglutamic acid solution;

[0012] (2) Dissolve ε-polylysine in water to obtain an ε-polylysine solution;

[0013] (3) Dissolve hydroxytyrosol in water under light-protected conditions to obtain a hydroxytyrosol solution;

[0014] (4) Add the hydroxytyrosol solution to the ε-polylysine solution and stir until homogeneous to obtain a mixed solution; then add the cyclic γ-polyglutamic acid solution dropwise to the mixed solution and stir at 200-300 rpm for 30-90 min. After dialysis to remove free hydroxytyrosol, hydroxytyrosol nanoparticles with high antioxidant capacity and stability are obtained.

[0015] According to a preferred embodiment of the present invention, in step (1), the cyclic γ-polyglutamic acid is a cyclic molecule composed of 4 to 30 glutamic acid residues with a molecular weight of 500 to 3900 Da.

[0016] According to a preferred embodiment of the present invention, in step (1), the concentration of the cyclic γ-polyglutamic acid solution is 2.5 to 3.5 g / L.

[0017] More preferably, the concentration of the cyclic γ-polyglutamic acid solution is 3 g / L.

[0018] According to a preferred embodiment of the present invention, in step (2), the molecular weight of the ε-polylysine is 3500 to 4500 Da.

[0019] According to a preferred embodiment of the present invention, in step (2), the concentration of the ε-polylysine solution is 2.5 to 10.5 g / L.

[0020] More preferably, the concentration of the ε-polylysine solution is 6 g / L.

[0021] According to a preferred embodiment of the present invention, in step (3), the concentration of the hydroxytyrosol solution is 2-5 g / L.

[0022] More preferably, the concentration of the hydroxytyrosol solution is 3 g / L.

[0023] According to a preferred embodiment of the present invention, in step (4), the mass ratio of cyclic γ-polyglutamic acid, hydroxytyrosol and polylysine is (2-3):1:5.

[0024] More preferably, the mass ratio of the cyclic γ-polyglutamic acid, hydroxytyrosol, and polylysine is 2.5:1:5.

[0025] According to a preferred embodiment of the present invention, in step (4), the dropping rate is 5 to 20 mL / h.

[0026] More preferably, the dropping rate is 10 mL / h.

[0027] According to a preferred embodiment of the present invention, in step (4), the stirring rate of the stirring reaction is 200-300 rpm and the reaction time is 45-60 min.

[0028] The present invention also provides hydroxytyrosol nanoparticles with high antioxidant capacity and stability, which are prepared by the preparation method described in the above technical solution.

[0029] This invention also provides the application of the above-mentioned hydroxytyrosol nanoparticles with high antioxidant capacity and stability in the preparation of cosmetics and medical aesthetic products.

[0030] Beneficial effects:

[0031] 1. This invention utilizes the characteristics of cyclic γ-polyglutamic acid side chains containing a large number of negatively charged free carboxyl groups and ε-polylysine side chains containing a large number of positively charged free amino groups. Cyclic γ-polyglutamic acid, hydroxytyrosol, and ε-polylysine are dissolved in water and then blended. Through the adsorption capacity of negatively charged cyclic γ-polyglutamic acid and positively charged ε-polylysine, hydroxytyrosol is encapsulated and preserved, which effectively improves the antioxidant capacity and stability of hydroxytyrosol and solves the problem of poor stability and easy oxidation and decomposition of hydroxytyrosol under light conditions.

[0032] 2. The hydroxytyrosol rice granules provided by this invention have good antibacterial properties against Escherichia coli and Staphylococcus aureus. They are also highly water-soluble in cell membranes, release slowly, are easily absorbed by cells, are biodegradable, and are biocompatible and non-toxic. They can be used to prepare skin care products such as lotions, creams, and serums.

[0033] 3. The preparation method provided by this invention has mild reaction conditions, simple steps, no need for further chemical modification of raw materials, and the encapsulation rate reaches more than 80%, making it suitable for large-scale industrial production. Attached Figure Description

[0034] Figure 1 The image shows an electron microscope image of the hydroxytyrosol nanoparticles prepared in Example 1.

[0035] Figure 2 The liquid phase diagram shows the hydroxytyrosol content in the hydroxytyrosol nanoparticles prepared in Example 1.

[0036] Figure 3 Liquid phase diagram showing the hydroxytyrosol content in the hydroxytyrosol nanoparticles prepared for Comparative Example 1.

[0037] Figure 4 An antibacterial image of the hydroxytyrosol nanoparticles prepared in Example 1;

[0038] In the figure: a, the antibacterial effect of hydroxytyrosol aqueous solution on Staphylococcus aureus; b, the antibacterial effect of hydroxytyrosol aqueous solution on Escherichia coli; c, the antibacterial effect of hydroxytyrosol nanoparticles on Staphylococcus aureus; d, the antibacterial effect of hydroxytyrosol nanoparticles on Escherichia coli. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] The specific synthesis method of cyclic γ-polyglutamic acid used in the following examples is as follows:

[0041] (1) Preparation of crude enzyme solution: The nucleotide sequence of gamma-glutamyl hydrolase PgdS (GenBank: API45102.1) from Bacillus subtilis was artificially synthesized by BGI Genomics Co., Ltd., and the restriction enzymes Nhe I and Xho were used. I. The PgdS sequence of gamma glutamyl hydrolase was ligated into the expression vector pET28A to obtain the recombinant plasmid pET28A-PgdS. Then, the recombinant plasmid pET28A-PgdS was introduced into Escherichia coli BL21 competent cells to obtain engineered bacteria BL21-pET28A-PgdS containing gamma glutamyl hydrolase PgdS. Escherichia coli BL21 containing the recombinant vector pET28A-PgdS was inoculated at a 1% inoculum (V / V) into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37℃ with shaking at 200 r / min. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mmol / L and the cells were induced to grow at 25℃ for 20 h. The cells were then collected by centrifugation, sonicated under ice bath conditions, and the supernatant was collected by centrifugation to obtain crude gamma glutamyl hydrolase PgdS enzyme solution.

[0042] (2) Preparation of oligopolyglutamic acid: Using 50 mmol / L phosphate buffer with pH 7.5 as solvent, prepare 1 L of 10 g / L γ-polyglutamic acid solution (molecular weight 100 kDa), adjust the temperature to 37℃, and then add 20 mL of crude glutamyl hydrolase PgdS obtained in step (1) to the polyglutamic acid solution. Incubate for 8 h, and after the reaction, incubate at 90℃ for 30 min to inactivate the enzyme. Filter the enzyme solution through a 0.45 μm filter membrane, add 3 times the volume of ethanol to the enzyme solution for precipitation, collect the precipitate, wash with ethanol, dehydrate, and vacuum dry to obtain oligopolyglutamic acid, the structural formula of which is shown in Formula I below:

[0043]

[0044] In Equation I, 4 ≤ n ≤ 30, and n is an integer.

[0045] (3) Activation of resin: Weigh 0.5g of Wang resin (Sigma, catalog number: 17095) and add it to a round bottom flask. Then add 10mL of DMF solution and soak at 40℃ for 30min to allow the resin to fully swell and obtain activated Wang resin.

[0046] (4) Protection of amino-terminal and terminal carboxyl groups: 50 mL of 1,4-dioxane and 32 g of oligopolyglutamic acid obtained in step (2) were added sequentially to a 250 mL three-necked flask. Then, 4 mL of 98% concentrated sulfuric acid was added dropwise under stirring and low temperature of 0-5 °C. Isobutylene was introduced at 5 °C and TLC was used for detection (developing solvent: methanol: ethyl acetate = 1:1; color reagent: ninhydrin) until the terminal carboxyl group in the oligopolyglutamic acid was completely reacted. The above reaction solution was cooled to 0 °C in an ice-water bath. A 20% NaOH solution was added to adjust the pH to 7. Then, a 0.1 mol / L Na2CO3 solution was added to adjust the pH to 9.5. The mixture was stirred for 30 min until the pH value did not change. At 0–5°C, 25 mL of a 1,4-dioxane solution containing 9-fluorenylmethyl-N-succinimide carbonate (Fmoc-Osu, 15 g) was added dropwise to the pH-adjusted reaction solution over 15 min. After the addition was complete, the cooling was removed, the mixture was brought to room temperature, and the reaction was stirred until the amino terminus of the oligomeric polyglutamic acid was completely reacted (detection method: TLC, developing solvent: pure ethyl acetate, UV light development, ninhydrin development). The mixture was then filtered, and the filtrate was extracted with diethyl ether. The aqueous phase was cooled to 0–5°C, and a dilute hydrochloric acid solution (2 mol / L) was added to adjust the pH to 5.5. After extraction with ethyl acetate, the organic phase was washed with water, and anhydrous magnesium sulfate was added to the organic phase for drying. The solvent was removed by rotary evaporation, and the mixture was cooled to obtain a white crude product. The white crude product was recrystallized from the crude product using dichloromethane and petroleum ether to obtain Fmoc-(Glu). n -OtBu, recrystallization process is as follows: add dichloromethane of equal volume to the crude product to dissolve, then add petroleum ether of equal volume to the dichloromethane, refrigerate at 4℃ for 1 hour to crystallize, filter, wash 3 times with anhydrous ethanol, and dry at 50℃ for 2 hours to obtain Fmoc-(Glu). n -OtBu;

[0047] (5) Fmoc-(Glu) n - Connecting OtBu to Wang resin: Weigh 0.5g of coupling agent HBTU and 2.4g of Fmoc-(Glu). n-OtBu was added sequentially to the Wang resin that had swollen in step (3), and stirred at 40°C for 3 hours. After the reaction was completed, the reaction solution was filtered, and the resin was washed three times each with dichloromethane and DMF solution, and 15 mL of anhydrous methanol was added each time to shrink the resin twice, for 5 minutes each time. The solution was then dried to obtain Fmoc-(Glu). n -(Solid-phase synthetic resin)-OtBu, place in a vacuum dryer for later use;

[0048] (6) Removal of tert-butyl group: Fmoc-(Glu) obtained in step (5) n -(solid-phase synthetic resin)-OtBu was added to 15 mL of TFA-DCM mixed solution at a volume ratio of 1:1 and reacted at room temperature for 2 h. Simultaneously, 0.1 g of triethylsilane was added as a positive ion scavenger. After filtration, Fmoc-(Glu) was obtained. n -(solid-phase synthetic resin)-OH;

[0049] (7) Removal of Fmoc: Fmoc-(Glu) obtained in step (6) is removed. n -(solid-phase synthetic resin)-OH was added to 20 mL of DMF solution containing 20% ​​(v / v) piperidine, and reacted at room temperature for 10 min. Then, another 10 mL of DMF solution containing 20% ​​(v / v) piperidine was added, and the reaction was continued for another 20 min. The resulting reaction solution was filtered to remove the solvent, washed twice with DMF to remove residual piperidine, and dried at 50 °C for 2 h to obtain NH2-(Glu). n -(Solid-phase synthetic resin)-OH; Use ninhydrin to test whether the amino terminus has been deprotected: Filter the reaction solution to remove the solvent, wash the obtained solid twice with DMF to remove residual piperidine, then take a small amount of the reaction mixture in a test tube, add a small amount of 6% ninhydrin solution, and heat in boiling water for 5 minutes. Observe whether the color changes. If the color turns blue-purple, it indicates that the amino terminus of the amino acid has been deprotected.

[0050] (8) Cyclocyclization: Weigh 0.2g of EDCI, 0.4g of HBTU, and 1.8g of NH2-(Glu). n The reaction mixture (-(solid-phase synthetic resin)-OH) was stirred at room temperature for 2 hours to induce cyclization. The resulting reaction solution was then filtered, and the solid was washed alternately with dichloromethane and DMF solution 5 times each. The solid was then dried at 50°C for 2 hours to obtain the peptide resin. The reaction process was monitored using ninhydrin: the reaction solution was filtered, and the resulting solid was washed alternately with dichloromethane and DMF solution. A small amount of 6% ninhydrin solution was added to the solid in a test tube, and the tube was incubated in boiling water for 5 minutes. A color change was observed; a colorless result indicated that the amino terminus of the amino acid had completely reacted.

[0051] (9) Peptide resin lysis: The peptide resin was placed in an eggplant-shaped flask and 30 mL of the prepared lysis buffer (trifluoroacetic acid: triisopropylsilane: water volume ratio of 90:5:5) was added under ice bath conditions of 0-5℃. The mixture was stirred for 30 min and then brought to room temperature. The reaction was continued to be stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was filtered through a G4 sintered glass funnel to remove the resin. The solution was washed with 10 mL of trifluoroacetic acid and the trifluoroacetic acid was removed by vacuum distillation. Three times the volume of ethanol was added to the remaining solution to precipitate the precipitate. The precipitate was collected by filtration and then washed three times with ethanol. The solution was dried under vacuum at 50℃ for 6 h to obtain cyclic γ-polyglutamic acid.

[0052] The molecular weight of the cyclic γ-polyglutamic acid prepared by this method is 500–3900 Da.

[0053] Example 1

[0054] A method for preparing hydroxytyrosol nanoparticles with high antioxidant capacity and stability includes the following steps:

[0055] (1) A cyclic γ-polyglutamic acid with a molecular weight of 2500 Da was dissolved in water to obtain a cyclic γ-polyglutamic acid solution with a concentration of 3 g / L.

[0056] (2) Dissolve ε-polylysine with a molecular weight of 4000 Da in water to obtain an ε-polylysine solution with a concentration of 6 g / L.

[0057] (3) Dissolve hydroxytyrosol in water under light-protected conditions to obtain a hydroxytyrosol solution with a concentration of 3 g / L.

[0058] (4) Add 20 mL of hydroxytyrosol solution to 50 mL of ε-polylysine solution and stir at 250 rpm for 50 min to obtain a mixed solution; then slowly add 50 mL of cyclic γ-polyglutamic acid solution to the mixed solution at a rate of 10 mL / h. After the addition is complete, stir the reaction at 250 rpm for 60 min. Then transfer the reaction solution to a dialysis bag with a molecular weight of 1000 Da and remove free hydroxytyrosol by dialysis to obtain a hydroxytyrosol nanoparticle solution with high antioxidant capacity and stability.

[0059] Electron microscopy images of the hydroxytyrosol nanoparticles prepared in this embodiment are shown below. Figure 1 As shown. From Figure 1 The electron micrographs show that the hydroxytyrosol nanoparticles are spherical with a pore size of 50–150 nm.

[0060] Example 2

[0061] A method for preparing hydroxytyrosol nanoparticles with high antioxidant capacity and stability includes the following steps:

[0062] (1) A cyclic γ-polyglutamic acid with a molecular weight of 500 Da was dissolved in water to obtain a cyclic γ-polyglutamic acid solution with a concentration of 2.5 g / L.

[0063] (2) Dissolve ε-polylysine with a molecular weight of 3500 Da in water to obtain an ε-polylysine solution with a concentration of 2.5 g / L;

[0064] (3) Dissolve hydroxytyrosol in water under light-protected conditions to obtain a hydroxytyrosol solution with a concentration of 2 g / L;

[0065] (4) Add 25 mL of hydroxytyrosol solution to 100 mL of ε-polylysine solution and stir at 200 rpm for 60 min to obtain a mixed solution; then slowly add 50 mL of cyclic γ-polyglutamic acid solution to the mixed solution at a rate of 5 mL / h. After the addition is complete, stir the reaction at 300 rpm for 30 min. Then transfer the reaction solution to a dialysis bag with a molecular weight of 1000 Da and remove free hydroxytyrosol by dialysis to obtain a hydroxytyrosol nanoparticle solution with high antioxidant capacity and stability.

[0066] Example 3

[0067] A method for preparing hydroxytyrosol nanoparticles with high antioxidant capacity and stability includes the following steps:

[0068] (1) A cyclic γ-polyglutamic acid with a molecular weight of 3900 Da was dissolved in water to obtain a cyclic γ-polyglutamic acid solution with a concentration of 3.5 g / L.

[0069] (2) Dissolve ε-polylysine with a molecular weight of 4500 Da in water to obtain an ε-polylysine solution with a concentration of 10.5 g / L;

[0070] (3) Dissolve hydroxytyrosol in water under light-protected conditions to obtain a hydroxytyrosol solution with a concentration of 5 g / L.

[0071] (4) Add 21 mL of hydroxytyrosol solution to 50 mL of ε-polylysine solution and stir at 300 rpm for 45 min to obtain a mixed solution; then slowly add 60 mL of cyclic γ-polyglutamic acid solution to the mixed solution at a rate of 10 mL / h. After the addition is complete, stir the reaction at 200 rpm for 90 min. Then transfer the reaction solution to a dialysis bag with a molecular weight of 1000 Da and remove free hydroxytyrosol by dialysis to obtain a hydroxytyrosol nanoparticle solution with high antioxidant capacity and stability.

[0072] Comparative Example 1

[0073] A method for preparing hydroxytyrosol nanoparticles with high antioxidant capacity and stability, the steps are as described in Example 1, except that the cyclic γ-polyglutamic acid used in step (1) is replaced with chitosan powder.

[0074] Comparative Example 2

[0075] A porous hydrogel was prepared according to the method disclosed in Chinese patent document CN107163263A, and then hydroxytyrosol was loaded onto the porous hydrogel to obtain a hydrogel-hydroxytyrosol polymer.

[0076] Experimental Example 1: Determination of Encapsulation Efficiency of Hydroxytyrosol Nanoparticles

[0077] The hydroxytyrosol content of the hydroxytyrosol nanoparticle solutions prepared in Example 1 and Comparative Example 1 was determined to obtain the encapsulation efficiency of the hydroxytyrosol nanoparticles.

[0078] The specific method is as follows:

[0079] (1) High performance liquid chromatograph: Waters 2489.

[0080] (2) Chromatographic conditions: Column: Waters Spherisorb ODS2 column (150mm×4.6mm, 2.7μm); Column temperature: 30℃; Mobile phase: 0.12% formic acid aqueous solution: 0.12% formic acid methanol solution = 85:15 (V:V); Flow rate: 0.6mL / min; Detector: UV detector; Detection wavelength: 280nm; Injection volume: 10μL.

[0081] (3) Hydroxytyrosol standard solution: Weigh an appropriate amount of hydroxytyrosol standard (purity ≥99%), add water to dissolve it, prepare a solution of 1.0 mg / mL, and filter it through a 0.22 μm filter membrane.

[0082] (4) Sample preparation: Accurately measure the hydroxytyrosol nanoparticle solutions prepared in Examples 1 and 4, dissolve them in water to prepare a solution of 1.0 mg / mL, and filter it through a 0.22 μm filter membrane.

[0083] (5) Encapsulation efficiency determination: The hydroxytyrosol standard solution and sample solution were injected into the liquid chromatograph, and the peak area was recorded to obtain the hydroxytyrosol content. The results are as follows: Figure 2 and Figure 3 As shown.

[0084] according to Figure 2 , Figure 3 The encapsulation efficiency of hydroxytyrosol nanoparticles in the hydroxytyrosol nanoparticle solution prepared in Example 1 was calculated to be 90.92% using the following formula; the encapsulation efficiency of hydroxytyrosol nanoparticles in the hydroxytyrosol nanoparticle solution prepared in Comparative Example 1 was 63.54%.

[0085] Encapsulation efficiency = Amount of hydroxytyrosol actually contained in the inclusion compound / Amount of hydroxytyrosol added.

[0086] Experimental Example 2: In vitro release test of hydroxytyrosol nanoparticles

[0087] In vitro release tests were conducted on the hydroxytyrosol nanoparticle solution prepared in Example 1 and the hydrogel-hydroxytyrosol polymer prepared in Comparative Example 2.

[0088] The specific method is as follows:

[0089] The residual amounts of the hydroxytyrosol nanoparticle solution prepared in Example 1 and the hydrogel-hydroxytyrosol polymer prepared in Comparative Example 2 in saline medium were determined by dialysis. A hydroxytyrosol aqueous solution was used as a control group. 1.0 mL of the sample was placed in a dialysis bag with a molecular weight of 1000, and then placed in a beaker filled with 200 mL of phosphate buffer. The dialysis bag was incubated in a shaking water bath at 37°C with a magnetic stirrer at 100 rpm. At predetermined time intervals (0 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after the start of incubation), 10 mL of complete culture medium was collected and replaced with the same volume of fresh culture medium (10 mL). The amount of hydroxytyrosol released was determined by HPLC.

[0090] Results of the determination of hydroxytyrosol aqueous solution (control group): Hydroxytyrosol started at 100%; at 10 min, the hydroxytyrosol content decreased to 62.57%; at 20 min, the hydroxytyrosol content decreased to 45.82%; at 30 min, the hydroxytyrosol content decreased to 22.67%; at 1 h, the hydroxytyrosol content decreased to 4.29%; at 2 h, the hydroxytyrosol content decreased to 0.53%; and then slowly decreased to 0.15% over the next 8 h. This indicates that the permeation of the hydroxytyrosol aqueous solution proceeds over time, and by 2 h, the unencapsulated hydroxytyrosol has been completely released.

[0091] The determination results of the hydroxytyrosol nanoparticle solution prepared in Example 1 are as follows: hydroxytyrosol content was initially 100%; after 10 min, the hydroxytyrosol content decreased to 90.21%; after 20 min, the hydroxytyrosol content decreased to 88.42%; after 30 min, the hydroxytyrosol content decreased to 80.72%; after 1 h, the hydroxytyrosol content decreased to 75.81%; after 2 h, the hydroxytyrosol content decreased to 65.69%; after 4 h, the hydroxytyrosol content decreased to 50.29%; after 8 h, the hydroxytyrosol content decreased to 30.29%, and the slow release was completed in about 24 h.

[0092] The results of the hydrogel-hydroxytyrosol polymer prepared in Comparative Example 2 were as follows: hydroxytyrosol content started at 100%; after 10 min, it decreased to 83.42%; after 20 min, it decreased to 70.18%; after 30 min, it decreased to 58.95%; after 1 h, it decreased to 24.36%; after 2 h, it decreased to 5.71%; after 4 h, it decreased to 0.21%; and then slowly decreased to 0.11% over the next 8 h. This indicates that the hydroxytyrosol aqueous solution permeates over time, and by 4 h, the sustained release of hydroxytyrosol from the hydrogel-hydroxytyrosol polymer is complete.

[0093] The experimental results show that the hydroxytyrosol rice particles provided by this invention have a significant sustained-release effect compared with hydrogel-hydroxytyrosol polymer and hydroxytyrosol aqueous solution, and can achieve a significant in vitro sustained-release function.

[0094] Experiment Example 3: Determination of the antibacterial function of hydroxytyrosol nanoparticles

[0095] Hydroxytyrosol aqueous solution of the same concentration and hydroxytyrosol nanoparticle solution prepared in Example 1 were placed in growth medium of Escherichia coli ATCC8739 and Staphylococcus aureus ATCC 6538 with absorbance of 0.4 at 600 nm and cultured at 37℃ with shaking at 110 r / min for about 12 h. The bacterial solution was then diluted and spread onto LB solid medium and cultured at 37℃ for 18 h. The results were then photographed and observed. Figure 4 As shown.

[0096] from Figure 4 The results show that the number of colonies on LB solid medium coated with hydroxytyrosol nanoparticles after treatment is significantly lower than that on aqueous hydroxytyrosol medium, indicating that the hydroxytyrosol rice particles provided by this invention have good antibacterial activity against Escherichia coli and Staphylococcus aureus.

[0097] Experimental Example 4: Determination of the antioxidant properties of hydroxytyrosol nanoparticles

[0098] The hydroxytyrosol nanoparticle solutions prepared in Examples 1-3 were used to prepare a hydroxytyrosol ethanol solution with a concentration of 50 μg / mL, which served as the sample solution. Simultaneously, a hydroxytyrosol ethanol solution of the same concentration was prepared as a control solution. 2.0 mL of each sample solution of different concentrations was accurately transferred to a 10 mL test tube, and 2.0 mL of the prepared DPPH·ethanol solution (1 mg of DPPH dissolved in 20 mL of ethanol) was added. The mixture was shaken and allowed to stand for 30 min. The spectrophotometer was zeroed with anhydrous ethanol, and the absorbance value A1 (optimal between 1.2 and 1.3) was measured at 517 nm. Simultaneously, 2.0 mL of the sample solution was accurately transferred and mixed with 2.0 mL of anhydrous ethanol, and the absorbance value A2 was measured at 517 nm. Similarly, 2.0 mL of the prepared DPPH·ethanol solution was accurately transferred and mixed with 2.0 mL of anhydrous ethanol, and the absorbance value A0 was measured at 517 nm. The DPPH scavenging rate of the test solution was calculated, and the results are shown in Table 1. The formula for calculating the clearance rate is as follows:

[0099]

[0100] Table 1: DPPH Scavenging Rate Data

[0101] name DPPH· clearance rate (%) Example 1 96.10 Example 2 95.29 Example 3 96.38 Hydroxytyrosol ethanol solution 55.16

[0102] As shown in Table 1, the DPPH scavenging rate of the hydroxytyrosol nanoparticles of the present invention is above 95%, indicating that the present invention encapsulates and preserves hydroxytyrosol by adsorbing the negatively charged cyclic γ-polyglutamic acid and the positively charged ε-polylysine, effectively improving the antioxidant capacity and stability of hydroxytyrosol and solving the problem of poor stability and easy oxidation and decomposition of hydroxytyrosol under light conditions.

[0103] Experimental Example 5: Determination of Cell Absorption of Hydroxytyrosol Nanoparticle Solution

[0104] Cell uptake experiments were performed using mouse fibroblasts (L929). L929 cells were resuscitated and cultured in DMEM medium containing 10% fetal bovine serum. L929 cells in the logarithmic growth phase were digested with trypsin and seeded into 96-well plates. The plates were cultured at 37°C and 5% CO2 for 24 h. After cell attachment, a solution of hydroxytyrosol nanoparticles (1.0 mg / mL) prepared in Example 1 and an aqueous solution of hydroxytyrosol were transferred and incubated with the cells at 37°C for 10–60 min. The hydroxytyrosol-containing solution was discarded, and the cell surface was gently washed three times with PBS buffer. Then, 0.5 mL of sterile water was added, and the cells were subjected to three freeze-thaw cycles at -80°C. The cell lysates were collected and centrifuged at 10,000 rpm for 5 min.

[0105] The supernatant was collected, and the concentration of hydroxytyrosol was detected using the method described in Example 1. It was found that L929 cells with the added hydroxytyrosol nanoparticle solution prepared in Example 1 showed an increase in hydroxytyrosol concentration within 10–20 minutes, and after 20 minutes, cell uptake reached saturation, with the concentration remaining essentially unchanged. L929 cells with the added hydroxytyrosol aqueous solution showed an increase in concentration within 10–40 minutes, and after 40 minutes, cell uptake reached saturation, with the concentration remaining essentially unchanged. Therefore, the hydroxytyrosol nanoparticles provided by this invention have better water solubility in cell membranes, are easily absorbed by cells, and possess good biocompatibility and are non-toxic and harmless, making them suitable for preparing skincare products such as lotions, creams, and serums.

Claims

1. A method for preparing hydroxytyrosol nanoparticles having high antioxidant capacity and stability, characterized by, Includes the following steps: (1) Dissolve cyclic γ-polyglutamic acid in water to obtain a cyclic γ-polyglutamic acid solution; Among them, cyclic γ-polyglutamic acid is a cyclic molecule composed of 4 to 30 glutamic acid residues with a molecular weight of 500 to 3900 Da; the concentration of the cyclic γ-polyglutamic acid solution is 2.5 to 3.5 g / L; (2) Dissolve ε-polylysine in water to obtain an ε-polylysine solution; The molecular weight of ε-polylysine is 3500~4500 Da, and the concentration of the ε-polylysine solution is 2.5~10.5 g / L. (3) Dissolve hydroxytyrosol in water under light-protected conditions to obtain a hydroxytyrosol solution; The concentration of the hydroxytyrosol solution is 2-5 g / L; (4) Add the hydroxytyrosol solution to the ε-polylysine solution and stir until homogeneous to obtain a mixed solution; then add the cyclic γ-polyglutamic acid solution dropwise to the mixed solution and stir at 200-300 rpm for 30-90 min. After removing the free hydroxytyrosol by dialysis, hydroxytyrosol nanoparticles with high antioxidant capacity and stability are obtained. The mass ratio of cyclic γ-polyglutamic acid, hydroxytyrosol and polylysine is (2~3):1:5, and the dropping rate is 5~20mL / h.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the cyclic γ-polyglutamic acid solution is 3 g / L.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the ε-polylysine solution is 6 g / L.

4. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the hydroxytyrosol solution is 3 g / L.

5. The preparation method according to claim 1, characterized in that, In step (4), the mass ratio of cyclic γ-polyglutamic acid, hydroxytyrosol and polylysine is 2.5:1:

5.

6. The preparation method according to claim 1, characterized in that, In step (4), the dropping rate is 10 mL / h.

7. The preparation method according to claim 1, characterized in that, In step (4), the stirring rate of the stirring reaction is 250 rpm and the reaction time is 45~60 min.

8. A hydroxytyrosol nanoparticle with high antioxidant capacity and stability, characterized in that, It is prepared according to the preparation method described in any one of claims 1 to 7.

9. The application of the hydroxytyrosol nanoparticles with high antioxidant capacity and stability as described in claim 8 in the preparation of cosmetics and medical aesthetic products.

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