Eutectic resveratrol as well as liposome, preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines and cosmetics, and particularly relates to eutectic resveratrol, a liposome thereof, a preparation method and application of the eutectic resveratrol. The eutectic resveratrol is prepared from the following raw materials: resveratrol, amino acid and glycerol; the amino acid is selected from one of proline, lysine and arginine. The eutecticevaporate resveratrol liposome is prepared from the following raw materials: soybean phospholipid, cholesterol and eutecticevaporate resveratrol, the mass ratio of the soybean phospholipid to the cholesterol is (1-9): 1, and the mass ratio of the sum of the mass of the soybean phospholipid and the cholesterol to the mass of the eutecticevaporate resveratrol is 1: (0.5-2). A method capable of improving the solubility and stability of resveratrol is found, the eutectic resveratrol liposome which is stable in particle size and PDI and excellent in zeta potential and has certain anti-oxidation and antibacterial performance is prepared, the solubility, stability and bioavailability of resveratrol (RES) are improved, and the resveratrol liposome is suitable for being used as an anti-inflammatory drug. And a new thought is provided for further development and application research of resveratrol (RES).

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine and cosmetics, and particularly relates to a eutectic resveratrol, a liposome thereof, and a preparation method and application thereof. Background Art

[0002] In recent years, deep eutectic solvents (DES) have been reported as new natural solvents with potential applications. They are tunable, economical, environmentally friendly, easy to synthesize, non-toxic, and biocompatible. As ionic liquids, DESs are composed of two- or three-component eutectic mixtures of hydrogen bond donors (HBDs) (such as amides, urea, and glycerol) and hydrogen bond acceptors (HBAs) (such as choline chloride and amino acids) in a specific stoichiometric ratio, formed through hydrogen bonding. The properties of DESs are largely determined by the HBA and HBD components. By adjusting the composition and ratio of DESs, they can be used to extract, separate, and solubilize a variety of natural bioactive compounds, including phenols, flavonoids, terpenoids, and alkaloids.

[0003] Liposomes are microscopic vesicles formed by a bilayer of monolayer phospholipids or multilayer soluble substances, with particle sizes typically ranging from 20 to 1000 nm. The unique amphiphilic structure of liposomes enables them to encapsulate both hydrophilic and hydrophobic substances, a property that significantly expands the scope of drug delivery systems. As a novel drug carrier, liposomes offer multiple advantages. On the one hand, they can slow the oxidation and degradation of encapsulated drugs, extending their shelf life and improving drug stability. On the other hand, the structure of liposomes enables them to slowly release drugs, further improving bioavailability and reducing drug toxicity. Therefore, liposomes have become a research hotspot in the medical field in recent years.

[0004] Resveratrol (RES) is a natural polyphenolic stilbene compound isolated from plants. It is a phytoalexin produced spontaneously by plants in response to external stimuli. It has both cis and trans isomers, with the trans structure being the most prevalent. This directly leads to its poor physicochemical stability, sensitivity to light and heat, and rapid degradation. Research has also shown that resveratrol is extremely unstable to both light and heat, with some trans forms converting to cis forms under illumination. RES exhibits anti-tumor, antibacterial, anti-inflammatory, antipyretic and analgesic properties, and is used to treat cardiovascular disease, tumors, diabetes, mechanical burns, and injuries, with high medicinal value. In the cosmetics field, resveratrol is widely used in a variety of product formulations, including creams, lotions, and serums, for its moisturizing, skin-firming, whitening, and anti-wrinkle properties. However, its poor water solubility, low bioavailability, and rapid metabolism significantly limit its application. Therefore, addressing the solubility and stability issues of resveratrol is a key research priority.

[0005] Chinese invention patent application CN 113521005 A discloses a nanolipid particle carrier for delivering resveratrol, its preparation method, and applications. The nanolipid particle carrier uses phospholipids as lipid carriers and cholesterol as a stabilizer to encapsulate resveratrol, creating nanoliposome particles that improve the drug's water solubility and stability, thereby achieving an oral resveratrol drug with ideal bioavailability. Phospholipids, as the main component of the cell membrane, enable effective encapsulation of both water-soluble and fat-soluble active drugs, providing protection, transport, and sustained release of the active drugs. This significantly improves the solubility of resveratrol, achieves a high encapsulation efficiency, and is easy to obtain and store. Summary of the Invention In view of the deficiencies in the prior art, the present invention provides a eutectic resveratrol, liposomes thereof and a preparation method thereof.

[0006] In order to achieve the purpose of the present invention, the technical solutions adopted are as follows: A low eutectic resveratrol, wherein the raw materials of the low eutectic resveratrol include resveratrol, amino acid and glycerol; the amino acid is selected from one of proline, lysine and arginine.

[0007] Preferably, the molar ratio of the amino acid to glycerol is 1-2:5.

[0008] Preferably, the mass ratio of resveratrol to amino acids is 0.3-0.7:1.

[0009] The second object of the present invention is to provide a method for preparing the eutectic resveratrol, comprising the following steps: (1) Mix amino acids and glycerol, heat and stir in a water bath; (2) Add resveratrol and stir until the liquid becomes uniform.

[0010] Preferably, the temperature of the water bath heating in step (1) is 70-90°C.

[0011] The third object of the present invention is to provide a low eutectic resveratrol liposome, wherein the raw materials of the low eutectic resveratrol liposome include: soybean lecithin, cholesterol and the low eutectic resveratrol, wherein the mass ratio of soybean lecithin to cholesterol is 1-9:1, and the mass ratio of the sum of the mass of the soybean lecithin and cholesterol to the low eutectic resveratrol is 1:0.5-2.

[0012] The fourth object of the present invention is to provide a method for preparing the eutectic resveratrol liposomes, comprising the following steps: (1) Dissolve soybean lecithin and cholesterol in ethanol to obtain solution A; (2) Dissolve eutectic resveratrol in ethanol to obtain solution B; (3) Mix solution A and solution B, evaporate to dryness to form a film, add water, stir, and filter.

[0013] Preferably, the volume ratio of the sum of the mass of soybean lecithin and cholesterol to ethanol in step (1) is 0.05-0.2 g:5 mL.

[0014] Preferably, the mass ratio of the low eutectic resveratrol to the volume ratio of ethanol in step (2) is 0.1-1 g:5 mL.

[0015] Preferably, the evaporation to dryness in step (3) is to evaporate to dryness on a rotary evaporator at 50-70°C for 10-20 min, and after adding water, the mixture is placed in an ice-water bath and stirred for 20-40 min, and then filtered through a 0.22 μm membrane.

[0016] Another object of the present invention is to provide the use of the above-mentioned eutectic resveratrol or the above-mentioned eutectic resveratrol liposomes in the preparation of resveratrol medicines or cosmetics.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention has found a method to improve the solubility and stability of resveratrol, and prepared low-melting resveratrol liposomes with stable particle size, PDI and excellent zeta potential, and certain antioxidant and antibacterial properties, thereby improving the solubility, stability and bioavailability of RES, providing new ideas for the further development and application research of RES.

[0018] (2) The method of the present invention is simple to prepare. The obtained liposomes have excellent particle size, PDI and zeta potential, and show good stability within 30 days at 4°C. They also have certain antioxidant and antibacterial properties, which significantly improve the solubility and bioavailability of RES.

[0019] (3) The present invention has other advantages over the prior art: On the solution: The present invention prepares a low-melting eutectic system and uses ethanol for dissolution. Almost all of the ethanol will evaporate during the spin-drying process and ethanol is a low-toxic solvent in organic solutions, while other solvents such as chloroform and methanol are toxic and it is difficult to assess whether there are any residues.

[0020] In terms of physical properties: the zeta potential of the liposomes prepared by the present invention is above ±40 mV, the liposomes are not easy to aggregate, and have better stability.

[0021] Preparation Method: The present invention prepares resveratrol, a deep eutectic solvent, without ultrasound. Instead, it disperses the liposomes in an ice-water bath with stirring before passing through a membrane. Ultrasound can disrupt the encapsulated liposomes, resulting in a weak zeta potential and ultimately prone to liposome aggregation. Stirring in an ice-water bath avoids this problem, resulting in high zeta potential liposomes. Passing through a membrane removes unencapsulated liposomes, resulting in excellent PDI and particle size values. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagrams of Examples 1-3 and Comparative Examples 1-2.

[0023] Figure 2 Temperature-viscosity change diagram of Example 1-Example 3.

[0024] Figure 3 Temperature-conductivity variation diagram of Example 2-Example 3.

[0025] Figure 4 Infrared change diagrams of Example 1-Example 3.

[0026] Figure 5 This is the H NMR spectrum of resveratrol (RES).

[0027] Figure 6 This is the hydrogen nuclear magnetic resonance spectrum of the eutectic resveratrol in Example 1.

[0028] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the eutectic resveratrol in Example 2.

[0029] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of the eutectic resveratrol in Example 3.

[0030] Figure 9The full-wavelength scan of UV absorbance of resveratrol (RES) and the standard curve of absorbance-RES concentration are shown; (a) is the full-wavelength UV image of RES raw material, and (b) is the standard curve of absorbance-RES concentration.

[0031] Figure 10 This is a graph showing the degradation rate of the eutectic resveratrol under the influence of ultraviolet light in Examples 1 to 3.

[0032] Figure 11 This is a graph showing the DPPH clearance rate of the eutectic resveratrol of Examples 1 to 3.

[0033] Figure 12 Schematic diagram of the antibacterial plate of resveratrol (RES) and the eutectic resveratrol of Examples 1-3.

[0034] Figure 13 This is a schematic diagram of the eutectic resveratrol liposomes of Example 4-8.

[0035] Figure 14 This is a microscopic image of the eutectic resveratrol liposomes of Example 7.

[0036] Figure 15 This is a graph showing the stability of physical properties of the eutectic resveratrol liposomes in Example 7 after 30 days of storage.

[0037] Figure 16 This is a graph showing the DPPH clearance rate of the eutectic resveratrol liposomes in Example 7.

[0038] Figure 17 This is the solubility diagram of the eutectic resveratrol liposomes in water in Example 7.

[0039] The unclear parts in the above figures do not affect the understanding of the technical solution of the present invention by those skilled in the art. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific embodiments. The following raw materials are all commercially available conventional raw materials.

[0041] 1. Eutectic Resveratrol and Its Preparation Example 1: 1 g of proline and 2.107 g of glycerol (molar ratio 2:5) were weighed into a 50 mL round-bottom flask and dissolved in water at 80°C with stirring to obtain a homogeneous clear solution (i.e., Pro-DES). 0.472 g of RES was then added in small batches and stirred continuously until a clear, transparent, homogeneous liquid was obtained, i.e., the proline eutectic resveratrol (Pro-DES-RES). The solution was stored at room temperature in the dark for future use.

[0042] Example 2: 1 g of lysine and 2.079 g of glycerol (molar ratio 2:5) were weighed into a 50 mL round-bottom flask and dissolved in water at 80°C with stirring to obtain a homogeneous clear solution (i.e., Lys-DES). 0.587 g of RES was then added in small batches and stirred continuously until a clear, transparent, homogeneous liquid was obtained. This was the lysine eutectic resveratrol (Lys-DES-RES). The solution was stored at room temperature in the dark for later use.

[0043] Example 3: 1 g of arginine and 2.628 g of glycerol (molar ratio 1:5) were weighed into a round-bottom flask and dissolved in water at 80°C with stirring to obtain a homogeneous clear solution (i.e., Arg-DES). 0.602 g of RES was then added in small batches and stirred continuously until a clear, transparent, homogeneous liquid was obtained. This was the arginine eutectic resveratrol (Arg-DES-RES). The mixture was stored at room temperature in the dark for later use.

[0044] Comparative Example 1 Weigh 1 g of xylitol and 4.949 g of glycerol (molar ratio 1:5) into a 50 mL round-bottom flask, dissolve in water at 80°C and stir to obtain a homogeneous clear solution (i.e., Xylitol-DES). Add 0.037 g of RES in small batches and continue stirring until a clear, transparent, homogeneous liquid is obtained. This is the xylitol eutectic resveratrol (Xylitol--DES-RES). Store at room temperature away from light for future use.

[0045] Comparative Example 2 Weigh 1 g of arginine and 5.111 g of lactic acid (molar ratio 1:5) into a 50 mL round-bottom flask. Dissolve in water at 80°C and stir to obtain a homogeneous clear solution (i.e., Lac-DES). Add 0.096 g of RES in small batches and continue stirring until a clear, transparent, homogeneous liquid is obtained. This is the lactic acid eutectic resveratrol (Lac-DES-RES). Store at room temperature in the dark for later use.

[0046] 2. Preparation of eutectic resveratrol liposomes Example 4: Weigh 0.09 g of soybean lecithin and 0.01 g of cholesterol and dissolve them in 5 mL of ethanol to obtain solution A; weigh 0.7352 g of Arg-DES-RES (prepared in Example 3) and dissolve it in 5 mL of ethanol to obtain solution B; then draw 1 mL from solution A and 1 mL from solution B, respectively, and add 8 mL of ethanol, place them together in a 250 mL round-bottom flask, shake evenly, and evaporate to dryness on a rotary evaporator at 60°C to form a film (15 min), add 10 mL of deionized water to the round-bottom flask, place it in an ice-water bath (about 4°C) and stir for 30 min, and finally pass it through a 0.22 μm film and place it in a refrigerator for 24 h.

[0047] Example 5: Weigh 0.0095 g of soybean lecithin and 0.005 g of cholesterol and dissolve them in 5 mL of ethanol to obtain solution A; weigh 0.7352 g of Arg-DES-RES (prepared in Example 3) and dissolve it in 5 mL of ethanol to obtain solution B; then draw 1 mL from solution A and 1 mL from solution B, respectively, and add 8 mL of ethanol, place them together in a 250 mL round-bottom flask, shake evenly, and evaporate to dryness on a rotary evaporator at 60°C to form a film (15 min), add 10 mL of deionized water to the round-bottom flask, place it in an ice-water bath (about 4°C) and stir for 30 min, and finally pass it through a 0.22 μm film and place it in a refrigerator for 24 h.

[0048] Example 6: Weigh 0.085 g of soybean lecithin and 0.015 g of cholesterol and dissolve them in 5 mL of ethanol to obtain solution A; weigh 0.7352 g of Arg-DES-RES (prepared in Example 3) and dissolve it in 5 mL of ethanol to obtain solution B; then draw 1 mL from solution A and 1 mL from solution B, respectively, and add 8 mL of ethanol, place them together in a 250 mL round-bottom flask, shake evenly, and evaporate to dryness on a rotary evaporator at 60°C to form a film (15 min), add 10 mL of deionized water to the round-bottom flask, put it in an ice-water bath (about 4°C) and stir for 30 min, and finally pass it through a 0.22 μm film and put it in a refrigerator for 24 h.

[0049] Example 7: Weigh 0.09 g of soybean lecithin and 0.01 g of cholesterol and dissolve them in 5 mL of ethanol to obtain solution A; weigh 0.7352 g of Arg-DES-RES (prepared in Example 3) and dissolve it in 5 mL of ethanol to obtain solution B; then draw 2 mL from solution A and 1 mL from solution B, respectively, and add 7 mL of ethanol, place them together in a 250 mL round-bottom flask, shake evenly, and evaporate to dryness on a rotary evaporator at 60°C to form a film (15 min), add 10 mL of deionized water to the round-bottom flask, put it in an ice-water bath (about 4°C) and stir for 30 min, and finally pass it through a 0.22 μm film and put it in a refrigerator for 24 h.

[0050] Example 8: Weigh 0.09 g of soybean lecithin and 0.01 g of cholesterol and dissolve them in 5 mL of ethanol to obtain solution A; weigh 0.7352 g of Arg-DES-RES (prepared in Example 3) and dissolve it in 5 mL of ethanol to obtain solution B; then draw 1 mL from solution A and 2 mL from solution B, respectively, and add 7 mL of ethanol, place them together in a 250 mL round-bottom flask, shake evenly, and evaporate to dryness on a rotary evaporator at 60°C to form a film (15 min), add 10 mL of deionized water to the round-bottom flask, put it in an ice-water bath (about 4°C) and stir for 30 min, and finally pass it through a 0.22 μm film and put it in a refrigerator for 24 h.

[0051] 3. Test Experiment Test Example 1: Comparison of HPLC determination of eutectic resveratrol content (1) Preparation of standard solution and sample solution Accurately weigh 25 mg of RES standard, dilute to a 50 mL volumetric flask with methanol, shake well, take 2 mL of it and dilute to a 20 mL volumetric flask, shake well to obtain a mother solution with a concentration of 50 μg / mL.

[0052] The sample concentration was controlled at 20-500 μg / mL by calculation. The sample was weighed and dissolved, and the volume was added to a 20 mL volumetric flask. 1 mL was taken from the sample and the volume was added to a 10 mL volumetric flask. The sample solution was obtained after shaking.

[0053] (2) Establishment of standard curve The standard stock solution was gradually prepared into standard solutions of varying concentrations (2, 5, 10, 20, 25, and 50 μg / mL). The solution was filtered through a 0.22 μm microporous membrane, and samples were collected for analysis by liquid chromatography to establish a linear working curve for the standard solution. A linear regression was performed using peak area (Y) versus standard concentration (X) to plot the standard curve.

[0054] (3) Analysis of resveratrol The RES content in DES-RES was determined by HPLC. The sample was filtered with a 0.22 μm filter membrane before injection. RES was separated by C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm, Acclaim TM 120, Thermo). The mobile phase consisted of acetonitrile (A) and water (B) in a volume ratio of 70:30. The flow rate was 1.0 mL / min, the injection volume was 10 μL, and the detection wavelength was 306 nm.

[0055] The results are shown in Tables 1 and 2. RES solution has good linearity in the range of 2-50 μg / mL, and the standard curve is: , where Y is the peak area and X is the concentration of RES (μg / mL). When testing the content of RES in each group of Examples 1-3, the content of RES in the Pro-DES-RES sample solution of Example 1 was calculated based on the peak area as (33.06×10 (expansion factor)×20 (dissolution factor)) / (1000 (unit)×0.0917 (sample amount, g))=72.10 mg / g; Figure 1As shown, the eutectic resveratrol prepared in the comparative example suffers from low solubility, poor stability, and prone to delamination. However, Examples 1-3 (amino acid-glycerol eutectic system) yield eutectic resveratrol with high solubility and good stability. Research has found that combining amino acids with glycerol significantly improves the solubility of resveratrol.

[0056] Table 1 Resveratrol HPLC standard curve

[0057] Table 2 RES content in various examples and comparative examples

[0058] Test Example 2: Study on the Solubility of Eutectic Resveratrol in Water Excessive amounts of Examples 1-3 and Comparative Examples 1-2 were weighed into 2 mL of deionized water, stirred, ultrasonicated for 20 min, centrifuged at 4000 rpm for 10 min, and the absorbance was measured at 306 nm using an ultraviolet spectrophotometer.

[0059] The results are shown in Table 3. The solubility of RES in water is only 0.0417 mg / mL. Using the deep eutectic solvent (glycerol-amino acid system) of the present invention in Examples 1-3 can significantly improve the solubility of RES in water. Among them, Example 3 increases the solubility of RES in water by approximately 187 times. Although the comparative example can improve the solubility of RES to a certain extent, it has problems with instability and stratification.

[0060] Table 3 Comparison of solubility of RES, comparative examples and examples

[0061] Test Example 3: Temperature-Viscosity and Conductivity Analysis The viscosity of Examples 1-3 was measured from 20°C to 60°C using a temperature program, with the temperature increased at 2°C / 30s. Furthermore, the conductivity of Examples 1-3 was measured from 20°C to 60°C using a temperature program, with the temperature increased at 5°C / 2min to 60°C.

[0062] The viscosity of DES is a basic physical property that can explain the formation of stable liquid DES and reflect the flow characteristics of the sample. Figure 2 As shown in (a) and (b), the viscosity of the embodiments 1-3 all showed a decreasing trend with increasing temperature, and the decreasing rate was faster in the low temperature region and gradually slowed down with increasing temperature. Figure 2The curve of Example 1 (Pro-DES-RES) in (a) is not smooth but uneven, showing the characteristics of a semi-solid and poor fluidity. Figure 3 As shown, the conductivity of both Lys-DES-RES (Example 2) and Arg-DES-RES (Example 3) gradually increases with increasing temperature, confirming that ions are dissociated and can move independently in the liquid. Notably, the conductivity of Arg-DES-RES (Example 3) is lower than that of Lys-DES-RES (Example 2), indicating a greater degree of ion dissociation. This is speculated to slow the hydrolysis of resveratrol and extend its storage life. In contrast, Pro-DES-RES (Example 1) becomes a white paste upon cooling, making its conductivity difficult to measure.

[0063] Test Example 4: Fourier Transform Infrared Spectroscopy (FTIR) Analysis Record the various raw materials of Examples 1-3, proline, lysine, arginine, and glycerol at 500-4000 cm -1 The infrared spectrum at 370 nm was used to observe the changes in its functional groups.

[0064] like Figure 4 As shown, the RES spectrum shows 3220 cm -1 There is a characteristic absorption peak at 1588, 1144, 965 and 828 cm -1 The bands at 965 cm are related to aromatic C=C stretching vibration, olefin C=C bending vibration, CO bending vibration and trans olefin C=C deformation vibration. In the spectra of Examples 1-3, since the test concentration of RES is not high, the 965 cm -1 The characteristic peak of is masked by the glycerol intensity peak. However, the peaks of Examples 1-3 at 3220 cm -1 、1588cm -1、 1144cm -1 and 828 cm -1 The characteristic peak of RES can still be observed.

[0065] Test Example 5: H-NMR Spectrum Analysis A deuterated solvent was selected based on the polarity of the sample. The samples of Examples 1-3 were dissolved in the deuterated reagent and transferred to a 5 mm NMR tube with a liquid column height of approximately 4 cm. The H NMR spectra of the samples were detected and compared with the standard NMR spectra to observe whether the sample structures had changed.

[0066] The H NMR spectra of RES raw materials and Examples 1-3 are as follows: Figure 5-Figure 8 As shown. Figure 5 It can be seen that the main characteristic peak area ratio of RES is 2:1:1:3:3:2. Figure 6-Figure 8 After integration of the hydrogen spectrum peaks, it can be seen that the peak area ratios of Examples 1-3 are all 2:1:1:3:3:2, which are the same as the peak area ratios of the characteristic peaks of RES and have similar chemical shifts, indicating that RES is indeed stably present in the DES system.

[0067] Test Example 6 Effect of Ultraviolet Light on Eutectic Resveratrol Depend on Figure 9 (a) shows that the maximum absorption wavelength of resveratrol is 306 nm. Figure 9 From (b) in the figure, it is known that the RES solution has good linearity in the range of 2-10 μg / mL, and the standard curve is: , where Y is the absorbance and X is the RES concentration (μg / mL).

[0068] To investigate the effect of ultraviolet light at 254 nm on DES-RES samples, an 8 μg / mL RES solution was prepared as a control, and 8 μg / mL Pro-DES-RES (Example 1), Lys-DES-RES (Example 2), and Arg-DES-RES (Example 3) sample solutions were prepared, respectively. The samples were irradiated with ultraviolet light at 0, 5, 10, 20, 30, 60, 120, and 240 min, and the changes in absorbance and concentration were observed.

[0069] like Figure 10 As shown, it can be seen that the degradation rates of RES and the samples of Examples 1-3 increased significantly in the first 30 minutes under ultraviolet irradiation. This is because RES belongs to a polyphenol structure, so it is easy to accelerate its degradation efficiency under ultraviolet catalysis. The degradation rate slowed down from 30 to 120 minutes, which may be because some groups changed, causing its structure to become relatively stable, slowing down its degradation process. From 120 min to 240 min, the stable compound structure was further degraded after long-term continuous irradiation. The structure of RES itself is extremely unstable, and it also lacks the protection of a low eutectic solvent. Therefore, after 120 minutes of ultraviolet irradiation, its degradation rate showed a significant upward trend compared with Examples 1-3. Due to the protection of the low eutectic solvent, the degradation rates of Examples 1-3 showed a slow growth trend after 30 minutes. The degradation rate of Example 1 is lower than that of Examples 2-3 because the system generates 2,4,6-trihydroxyphenanthrene (THP) under ultraviolet light irradiation, which is a photochemically toxic derivative that can have harmful effects on human health; the degradation rates of Examples 2-3 are similar and lower than the degradation rate of RES, indicating that the low eutectic solvent can act as a protective barrier for RES and reduce the degradation of RES.

[0070] Test Example 7 Determination of Antioxidant Properties of Eutectic Resveratrol The antioxidant activity of Examples 1-3 was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. DPPH was weighed to prepare a 0.04 mg / mL DPPH solution, stored in the dark, and used immediately after preparation. RES and Examples 1-3 were prepared to 2.5-17.5 μg / mL, respectively. 2 mL of DPPH and 2 mL of RES and Examples 1-3 were placed in a 10 mL centrifuge tube, shaken thoroughly, and reacted in the dark for 30 min at room temperature. The absorbance A was measured at a wavelength of 517 nm. i , where the absorbance measured after mixing 2 mL of anhydrous ethanol solution and 2 mL of DPPH solution is A0. The DPPH clearance rate is calculated using formula (1).

[0071] (1) Where: A0 is the absorbance of the blank control group, A i is the absorbance of samples of different concentrations after reaction with DPPH.

[0072] like Figure 11 As shown in Figure 2, as the sample concentration increased from 2.5 μg / mL to 17.5 μg / mL, the DPPH free radical scavenging ability of each sample gradually increased. When the sample concentration reached 17.5 μg / mL, the DPPH scavenging rates of RES, Pro-DES-RES (Example 1), Lys-DES-RES (Example 2), and Arg-DES-RES (Example 3) were 63.19%, 65.13%, 61.42%, and 61.06%, respectively. The half-scavenging efficiency (IC) of DPPH free radicals was calculated by linear fitting. 50 ) shows that the IC of RES 50 The IC of Example 1 was 11.26 μg / mL. 50 The IC of Example 2 was 11.62 μg / mL. 50 The IC of Example 3 was 11.14 μg / mL. 50 The concentration of resveratrol in the eutectic solvent was 11.10 μg / mL. This indicates that the eutectic resveratrol system still maintains good antioxidant properties. This also shows that the eutectic solvent system does not affect the efficacy of RES.

[0073] Test Example 8: Determination of the antibacterial properties of eutectic resveratrol using the pore method 40 μL of 1.0×10 8The bacterial suspensions of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were evenly spread on sterile agar plates, and holes were punched on the plates using a hole puncher. 20 μL of the sample was carefully added to the holes. After incubation in a 37 °C incubator for 10 h, the diameter of the inhibition zone was measured.

[0074] From Table 4 and Figure 12 As can be seen, inhibition zone experiments using DMSO as the solvent for dissolving Examples 1-3 showed that DMSO had no significant antibacterial effect against the three strains. When the samples of Examples 1-3 were all at a concentration of 10 mg / mL, they all exhibited significant antibacterial activity against all three strains. The inhibition zone diameters against Staphylococcus aureus were all ≥2.0 cm, indicating the highest inhibitory effect, indicating that RES had a more pronounced antibacterial effect against them. Lys-DES-RES (Example 2) exhibited the largest inhibition zone diameter because lysine itself possesses antibacterial activity. When it comes into contact with bacterial cell membranes, it increases cell membrane permeability, triggering cytoplasm leakage and ultimately leading to bacterial death. Therefore, the inherent antibacterial activity of Example 2, prepared using lysine, further expanded the inhibition zone. Arg-DES-RES (Example 3) exhibited slightly stronger inhibition against Pseudomonas aeruginosa and Escherichia coli than RES and Pro-DES-RES (Example 1), and also demonstrated significant antibacterial activity against Staphylococcus aureus. This demonstrates that RES retains its antibacterial activity even after dissolving it in a deep eutectic solvent.

[0075] Table 4 Comparison of the size of the inhibition zones of three bacteria by RES and Examples 1-3

[0076] Note: Bacterial concentration: 1.5×10 8 ; Effect: “-” (≤0.6cm), no antibacterial effect; “- +” (>0.6cm,≤1.0cm), general antibacterial effect; “+” (>1.0cm,≤2.0cm), good antibacterial effect; “++” (>2.0cm), better antibacterial effect.

[0077] Test Example 9: Determination of Particle Size, Polydispersity Index (PDI), Zeta Potential, and Encapsulation Efficiency of Examples 4-8 The particle size distribution, polydispersity index (PDI) and zeta potential of the examples were measured using a Malvern laser particle size analyzer.

[0078] 0.6 mL of Example 4-8 was injected into a 10 mL centrifuge tube and centrifuged at 3000 r / min for 30 min. The supernatant was separated and dissolved in methanol. The absorbance at 306 nm was measured as A2. The uncentrifuged Example 4-8 was dissolved in methanol. The absorbance at 306 nm was measured as A1. The encapsulation efficiency was calculated according to formula (2): (2) Liposome formulations are optimized to achieve smaller particle size, lower PDI, and higher surface charge. Particle size is a critical attribute of liposomes, which affects properties such as stability, encapsulation efficiency, and biodistribution. PDI is used to describe the uniformity of particle size distribution. In drug delivery applications using liposomes, a PDI of 0.3 and below is considered acceptable, indicating a uniform distribution of liposome vesicles. Zeta potential is an important indicator of the stability of the colloidal dispersion coefficient. A high zeta potential value prevents aggregation of particles with similar charges due to electrostatic repulsion, thereby conferring stability to the colloidal dispersion. Generally, a zeta potential above the threshold of +30 mV or below -30 mV is considered to be less prone to aggregation and has good stability.

[0079] The results are shown in Table 5. Examples 4-8 all have good particle size, PDI and excellent zeta potential (less than -30mV) properties. These systems are not easy to agglomerate or settle, have excellent stability, and have high bioavailability. Among them, the Example 7 group has the best physical and chemical properties, with an average particle size of 184.07 nm, a PDI of 0.182, and a zeta potential of -56.40 mV, which will generate sufficient electrostatic repulsion to prevent it from aggregating. In addition, the encapsulation efficiency of Example 7 is 82.90%, and the liposomes of this system can simultaneously encapsulate more low-eutectic resveratrol. Therefore, Example 7 will be selected as the sample for the following test. The schematic diagram of the actual objects of Examples 4-8 prepared by the present invention is shown in FIG. Figure 13 .

[0080] Table 5 Physical properties of particle size, PDI, zeta potential and encapsulation efficiency of Examples 4-8

[0081] Test Example 10: Liposome structure of eutectic resveratrol liposomes (Example 7) under a microscope The microstructure of liposomes was observed under a mechanical microscope. The prepared liposomes were dipped into the center of a glass slide with a capillary tube, the liquid was evenly pressed with a coverslip, and then observed under a mechanical microscope.

[0082] like Figure 14As shown, under a microscope, it can be observed that the eutectic resveratrol liposomes (Example 7) have a clear bilayer structure, in which RES is encapsulated, generally presenting a spherical or ellipsoidal shape with clear edges and no obvious deformation, and are relatively evenly distributed in the liposome suspension.

[0083] Test Example 11 Stability Test of Eutectic Resveratrol Liposomes (Example 7) Example 7 was stored at 4° C. after preparation, and the changes in particle size distribution, PDI, zeta potential, and encapsulation efficiency were measured using a Malvern laser particle size analyzer at 0, 3, 7, 21, and 30 days.

[0084] like Figure 15 As shown, over time, the particle size of the eutectic resveratrol liposomes prepared in Example 7 remained relatively stable, ranging from 184 nm to 210 nm, with a PDI between 0.18 and 0.24, a zeta potential between -48 mV and -55 mV, and an encapsulation efficiency between 71% and 83%. This indicates that the liposome structure did not undergo significant aggregation or polymerization during this period. Furthermore, the resveratrol content in the sample did not change significantly. This demonstrates that the liposomes maintain good stability and exhibit good storage performance.

[0085] Test Example 12 DPPH Free Radical Scavenging Ability of Eutectic Resveratrol Liposomes (Example 7) The antioxidant activity of Example 7 was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method. DPPH was weighed to prepare a 0.04 mg / mL DPPH solution, which was stored in the dark and used immediately after preparation. Example 7 was prepared into a solution of 18.38-64.33 μg / mL. 2 mL of DPPH and the sample solution of Example 7 were placed in a 10 mL centrifuge tube, shaken thoroughly, and reacted in the dark for 30 min at room temperature. The absorbance A was measured at a wavelength of 517 nm. i , where the absorbance measured after mixing 2 mL of anhydrous ethanol solution and 2 mL of DPPH solution is A0. The DPPH clearance rate is calculated using formula (1).

[0086] The free radical scavenging ability of Example 7 (as Figure 16 As shown). As the sample concentration increases from 18.38 μg / mL to 64.33 μg / mL, the DPPH free radical scavenging ability gradually increases. When the sample concentration reaches 64.33 μg / mL, the DPPH scavenging rate of Example 7 is 68.79%. (Y: clearance rate, X: concentration (μg / mL)). Calculation of the half clearance rate (IC 50) was 42.397 μg / mL. This indicates that the synthesis of liposomes does not affect the efficacy of resveratrol. The eutectic resveratrol liposomes (Example 7) can be used as an antioxidant (whitening agent) in skin care products, cosmetics, and other fields.

[0087] Test Example 13 Minimum Inhibitory Concentration (MIC) of Eutectic Resveratrol Liposomes (Example 7) The minimum inhibitory concentration of (Example 7) was determined by the 96-well plate microdilution method. 4 mL of the liposome sample of Example 7 and the nutrient broth culture medium were respectively aspirated to prepare a mother solution concentration of 1838 μg / mL. 200 μL of the mother solution was added to the first column of the 96-well plate, and the working concentrations were 919 μg / mL, 459.5 μg / mL, 229.75 μg / mL, 114.875 μg / mL, 57.4375 μg / mL, 28.71875 μg / mL, and 14.359375 μg / mL using the culture medium half-dilution method. 100 μL of culture medium and 100 μL of bacterial solution were added to the control group. 100 μL of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa solutions (1.5×10 5 After preparing the gradient concentration, seal the 96-well plate and place it in a 37°C constant temperature incubator for 12 h to observe the bacterial growth.

[0088] The results are shown in Table 6. The minimum MIC for Staphylococcus aureus was 459.5 μg / mL; the minimum MIC for Escherichia coli was 919 μg / mL; and the minimum MIC for Pseudomonas aeruginosa was 919 μg / mL. This indicates that the eutectic resveratrol liposomes (Example 7) exhibited moderate antibacterial activity and were more sensitive to S. aureus. These results indicate that liposome synthesis does not affect the antibacterial properties of RES, and that eutectic resveratrol liposomes can be used in cosmetics and other fields for their antibacterial effects. Table 6 Minimum inhibitory concentration of Example 7 for three bacteria

[0089] Note: “-” indicates that no bacteria were generated at this concentration, and the growth of bacteria was successfully inhibited; “+” indicates that bacteria were generated at this concentration.

[0090] Test Example 14: Water Solubility Test of Eutectic Resveratrol Liposomes (Example 7) 0.5, 1, 1.5, and 2 mL of Example 7 were respectively added to 3 mL of water, stirred for 6 h, sonicated for 30 min, and centrifuged at 4000 rpm for 10 min. The dissolution in water was observed and the RES content was determined using an ultraviolet spectrophotometer at 306 nm after centrifugation of each sample.

[0091] like Figure 17 As shown, the centrifuged liposomes are uniformly dispersed in water without agglomeration, precipitation, etc. As can be seen from Test Example 2, the solubility of resveratrol in water is only 0.0417 mg / mL.

[0092] As shown in Table 7, sample ④-2mL increased the solubility by a factor of 9.217 / 0.0417 ≈ 221. This suggests that the eutectic resveratrol liposomes significantly increased the solubility of RES in water. This is because the strong hydrogen bonding in the eutectic system enhances the solubility of RES, and the liposome encapsulation further enhances the solubility of RES in water.

[0093] In addition, the eutectic system provides thickening, moisturizing and other effects. Combined with the above-mentioned antioxidant and antibacterial experimental studies, this can further illustrate that using functional eutectic resveratrol liposomes directly as raw materials for emulsions and essence skin care products is more convenient and stable.

[0094] Table 7 Solubility of eutectic resveratrol liposomes in water

[0095] Summarize The present invention utilizes a deep eutectic solvent to increase the solubility of resveratrol, addressing its poor water solubility. Using amino acids as variables, the study revealed that the solubility of RES in the arginine-glycerol DES system (Arg-DES-RES, Example 3) increased by 10.88%. Furthermore, DES acts as a protective barrier, enhancing the stability of RES and preventing its rapid oxidation. Furthermore, the antioxidant and antibacterial properties of Arg-DES-RES (Example 3) are retained, making it suitable for use as an antioxidant and antibacterial agent in cosmetics. Furthermore, through continuous optimization of the formulation, deep eutectic resveratrol liposomes (Example 7) were prepared with an average particle size of 184.07 nm, a PDI of 0.182, a zeta potential of -56.40 mV, and an encapsulation efficiency of 82.90%. After 30 days of stability testing, the particle size, PDI, zeta potential, and encapsulation efficiency remained unchanged, demonstrating that these liposomes can improve the stability and bioavailability of RES, providing valuable insights for the development of new RES formulations.

[0096] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A eutectic resveratrol, characterized in that: The raw materials of the eutectic resveratrol include resveratrol, amino acid and glycerol; the amino acid is selected from one of proline, lysine and arginine.

2. The eutectic resveratrol according to claim 1, characterized in that The molar ratio of the amino acid to glycerol is 1-2:

5.

3. The eutectic resveratrol according to claim 1, characterized in that The mass ratio of resveratrol to amino acids is 0.3-0.7:

1.

4. A method for preparing the eutectic resveratrol according to any one of claims 1 to 3, comprising the following steps: (1) Mix amino acids and glycerol, heat and stir in a water bath; (2) Add resveratrol and stir until the liquid becomes uniform.

5. The preparation method according to claim 4, characterized in that The temperature of the water bath heating in step (1) is 70-90°C.

6. A eutectic resveratrol liposome, characterized in that: The raw materials of the eutectic resveratrol liposomes include: soybean lecithin, cholesterol and the eutectic resveratrol according to any one of claims 1 to 3, wherein the mass ratio of soybean lecithin to cholesterol is 1-9:1, and the mass ratio of the sum of the mass of the soybean lecithin and cholesterol to the eutectic resveratrol is 1:0.5-2.

7. A method for preparing the eutectic resveratrol liposome according to claim 6, characterized in that: The steps include: (1) Dissolve soybean lecithin and cholesterol in ethanol to obtain solution A; (2) Dissolve eutectic resveratrol in ethanol to obtain solution B; (3) Mix solution A and solution B, evaporate to dryness to form a film, add water and stir to form a film.

8. The preparation method according to claim 7, characterized in that: The volume ratio of the sum of the mass of soybean lecithin and cholesterol to ethanol in step (1) is 0.05-0.2 g:5 mL, and the volume ratio of the mass of the low eutectic resveratrol to ethanol in step (2) is 0.1-1 g:5 mL.

9. The preparation method according to claim 7, characterized in that: The evaporation to dryness in step (3) is to evaporate to dryness on a rotary evaporator at 50-70°C for 10-20 minutes, add water, place in an ice water bath and stir for 20-40 minutes, and filter through a 0.22 μm filter membrane to obtain the product.

10. Use of the eutectic resveratrol according to any one of claims 1 to 3 or the eutectic resveratrol liposome according to claim 6 in the preparation of resveratrol medicines or cosmetics.

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