Propolis polyphenol extract, hydrogel and preparation method of propolis polyphenol extract

Abstract

The invention discloses a propolis polyphenol extract, hydrogel and preparation methods of the propolis polyphenol extract and the hydrogel, and the preparation method of the propolis polyphenol extract comprises the following steps: (1) crushing a propolis sample; (2) degreasing the propolis powder by using petroleum ether; (3) mixing 65%-70% ethanol with the degreased propolis raw material; (4) ultrasonic-assisted extraction; (5) extracting with ethyl acetate; (6) purifying the extract liquid by using HPD-100 macroporous resin column chromatography, and collecting a fraction containing polyphenol components; (7) concentrating the collected liquid in vacuum; and (8) carrying out vacuum drying on the concentrated solution to obtain the propolis polyphenol extract. The invention provides a method for efficiently extracting propolis polyphenols. The polyphenol content in an extract is remarkably increased (greater than or equal to 80%); based on the extracted polyphenol, the propolis polyphenol hydrogel which is stable in structure and definite in function is developed, and the application of the propolis polyphenol hydrogel in the fields of antibacterial wound nursing, antioxidant skin care and the like is expanded.

Description

Technical Field

[0001] The invention relates to the technical field of extraction of active ingredients of natural products, and in particular to a propolis polyphenol extract, a hydrogel, and a preparation method thereof. Background Art

[0002] Propolis is a resinous substance formed by bees from plant resins, young shoots and their own secretions. It has attracted much attention due to its complex chemical composition and significant biological activity. Propolis contains a variety of functional ingredients, including polyphenols, flavonoids, terpenes, phenolic acids and their esters, which have been shown to have antioxidant, antibacterial, anti-inflammatory, anti-cancer and other biological activities. Therefore, propolis has broad application prospects in the fields of medicine, cosmetics and functional foods.

[0003] However, due to the complex composition and strong heterogeneity of propolis, and the coexistence of polyphenols with other components (such as lipids and waxes) in propolis, the extraction and purification of polyphenols with high biological activity is a technical challenge.

[0004] In addition, the development of functional materials based on propolis polyphenols (such as hydrogels) is still in its early stages. There are problems such as unstable gel structure and loss of active ingredients during the preparation process, which makes it difficult to meet the demand for high-performance materials in the medical and beauty fields.

[0005] In general, the existing technologies have the following main shortcomings in the extraction of propolis polyphenols and the development of their functional materials:

[0006] 1. Deficiencies in the extraction of propolis polyphenols:

[0007] (1) Low purity: The existing organic solvent extraction method, ultrasonic-assisted extraction method and enzymatic extraction method all have the problem of low polyphenol purity. For example, ethanol extraction easily dissolves lipids and waxes at the same time, resulting in low polyphenol content and high purification cost.

[0008] (2) Loss of active ingredients: Ultrasonic and high temperature conditions may lead to the degradation of active ingredients such as polyphenols, reducing their biological activity;

[0009] (3) The process is complex and not environmentally friendly: it involves multi-step liquid-liquid extraction or high-cost column chromatography separation, and some solvent residues will affect the safety of the extract.

[0010] 2. Deficiencies in the development of functional hydrogels:

[0011] (1) Poor stability: The mechanical properties of natural polysaccharide matrices (such as gelatin and chitosan) are weak, and the hydrogel structure is easily degraded or swelled, which limits its use in complex application scenarios;

[0012] (2) Biocompatibility and toxicity risks: Although synthetic polymers (such as polyethylene glycol and polyacrylic acid) have good mechanical properties, their potential biotoxicity and cross-linking agent (such as glutaraldehyde) residues may cause safety issues. Summary of the invention

[0013] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a propolis polyphenol extract, a hydrogel, and a preparation method thereof. The present invention provides a method for efficiently extracting propolis polyphenols, which significantly increases the polyphenol content in the extract (≥80%); and based on the extracted polyphenols, a propolis polyphenol hydrogel with a stable structure and clear functions is developed to expand its application in the fields of antibacterial wound care, antioxidant skin care, etc.

[0014] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0015] A method for preparing a propolis polyphenol extract comprises the following steps:

[0016] (1) crushing: crushing the propolis sample to obtain propolis powder;

[0017] (2) Defatting: Use petroleum ether to defatt the propolis powder to remove fat-soluble components;

[0018] (3) Alcohol extraction: using 65% to 75% (v / v) ethanol solution to mix with the defatted propolis raw material, allowing full contact to obtain an alcohol extract;

[0019] (4) Ultrasonic-assisted extraction: subjecting the alcohol extract to ultrasonic-assisted extraction to obtain an extract;

[0020] (5) Liquid-liquid extraction: extracting the extract with ethyl acetate, separating the upper ethyl acetate phase, and obtaining the extract;

[0021] (6) Column chromatography purification: The extract was purified by HPD-100 macroporous resin column chromatography, and gradient elution was performed in three steps using 10% (v / v), 25% (v / v), and 45% (v / v) ethanol solutions, respectively; after each elution, the fraction containing the polyphenol components was collected to obtain a collected solution;

[0022] (7) Concentration: vacuum concentrating the collected solution to remove the ethanol solvent to obtain a concentrated solution;

[0023] (8) Drying: The concentrated solution is vacuum dried to obtain the propolis polyphenol extract.

[0024] Preferably, in step (1), the particle size of the crushed propolis powder is controlled to be 0.5 to 1 mm.

[0025] Preferably, in step (2), the volume mass ratio of petroleum ether to propolis powder is 1:4.5-5.5, that is, 1 ml of petroleum ether is used for 4.5-5.5 g of propolis powder.

[0026] Preferably, in step (2), the degreasing treatment time is 12 hours.

[0027] Preferably, in step (3), the mass volume ratio of the defatted propolis raw material to the ethanol solution is 1:10, that is, 10 ml of ethanol solution is used for 1 gram of propolis.

[0028] Preferably, in step (4), the power of the ultrasonic-assisted extraction is 100 W, the temperature is 50° C., the time is 30 min, and the frequency is 40 kHz.

[0029] Preferably, in step (5), the volume ratio of the extract to ethyl acetate is 1:1.

[0030] Preferably, in step (5), the extraction is performed twice, and the time for each extraction is 30 minutes.

[0031] Preferably, in step (7), the vacuum concentration is performed under the conditions of 0.1 mbar and 50°C.

[0032] Preferably, in step (7), the vacuum drying conditions are 0.1 mbar and -30°C.

[0033] A propolis polyphenol extract is prepared by using the above-mentioned preparation method of a propolis polyphenol extract.

[0034] A method for preparing a hydrogel comprises the following steps:

[0035] (1) preparing a hydrogel matrix: using 1 wt% to 2 wt% of sodium alginate, dissolving it in deionized water, and heating it to completely dissolve it to obtain a sodium alginate solution; using 2 wt% to 3 wt% of gelatin, dissolving it in deionized water, and heating it to completely dissolve it to obtain a gelatin solution; mixing the gelatin solution and the sodium alginate solution in a volume ratio of 1:1 to prepare a uniform matrix solution;

[0036] (2) preparing a propolis polyphenol solution: dissolving the propolis polyphenol extract in deionized water and stirring uniformly to obtain a propolis polyphenol solution;

[0037] (3) mixing: mixing the propolis polyphenol solution and the matrix solution, stirring evenly to obtain a mixed solution;

[0038] (4) Cross-linking: Slowly add CaCl2 solution to the mixture and continue stirring to ensure that the cross-linking agent fully acts to form a stable three-dimensional network structure;

[0039] (5) Molding: pouring the mixed solution into a mold to form a hydrogel film or particles;

[0040] (6) Drying: The product is subjected to vacuum freeze drying to remove moisture and obtain a stable propolis polyphenol hydrogel.

[0041] Preferably, in step (1), the heating temperature of the sodium alginate is 70°C.

[0042] Preferably, in step (1), the heating temperature of the gelatin is 60°C.

[0043] Preferably, in step (2), the concentration of the propolis polyphenol solution is 2 wt %.

[0044] Preferably, in step (3), the propolis polyphenol solution and the matrix solution are mixed in a volume ratio of 1:4.

[0045] Preferably, in step (4), the concentration of the CaCl2 solution is 1% w / v.

[0046] Preferably, in step (4), the stirring time is 30 min.

[0047] A hydrogel is prepared by using the above-mentioned method for preparing a hydrogel.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) Efficient extraction: The present invention adopts an optimized polyphenol extraction process, which significantly increases the polyphenol content in the product (≥80%) and retains the biological activity of the polyphenol;

[0050] (2) Diversified functions: The hydrogel prepared by the present invention has excellent antibacterial activity (antibacterial rate>90%), antioxidant performance (DPPH free radical scavenging rate>85%), moisturizing performance (24h moisturizing rate≥85%) and mechanical properties (compression strength≥1.5Mpa), and is suitable for a variety of application scenarios, such as medicine (such as wound care), cosmetics (such as skin care products) and other fields;

[0051] (3) The process is simple and controllable: the preparation process of the hydrogel prepared by the present invention is simple and easy to produce on a large scale;

[0052] (4) Environmental friendliness: The entire process of extraction and hydrogel preparation of the present invention avoids the use of toxic solvents, which complies with the green chemistry principles of sustainability and environmental friendliness;

[0053] (5) Long-term stability: The extract and hydrogel prepared in the present invention exhibit good stability under storage conditions. DETAILED DESCRIPTION

[0054] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments. It should be understood that these embodiments are only used to further illustrate the present invention, and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0055] Example 1

[0056] A method for preparing a propolis polyphenol extract, comprising the following steps:

[0057] (1) crushing: crushing the propolis sample to obtain propolis powder, wherein the particle size of the crushed propolis powder is controlled to be 0.5 to 1 mm;

[0058] (2) Defatting: Use petroleum ether (volume-to-mass ratio of 1:4.5) to defatted propolis powder for 12 h to remove fat-soluble components such as wax and fatty acids;

[0059] (3) Alcohol extraction: 70% (v / v) ethanol is used to mix with the defatted propolis raw material, and the mass volume ratio of propolis powder to ethanol solution is 1:10, that is, 10 ml of ethanol solution is used for 1 g of propolis, ensuring that the defatted propolis raw material is fully in contact with the ethanol to obtain an alcohol extract;

[0060] (4) Ultrasonic-assisted extraction: The alcohol extract was placed in an ultrasonic extractor, the power was set to 100 W, the temperature was set to 50°C, and ultrasonic extraction was performed for 30 min under this condition; the ultrasonic frequency was 40 kHz, which helped the solvent to penetrate the propolis cell wall and promote the release of polyphenols to obtain the extract;

[0061] (5) Liquid-liquid extraction: The extract was extracted twice with ethyl acetate (volume ratio of 1:1), each extraction time was 30 min, to increase the enrichment of polyphenols; the extract was separated into layers through a separatory funnel, the upper ethyl acetate phase was separated, and the upper ethyl acetate phase was collected to obtain the extract;

[0062] (6) Column chromatography purification: The extract was further purified using HPD-100 macroporous resin column chromatography, and gradient elution was performed in three steps using 10% (v / v), 25% (v / v), and 45% (v / v) ethanol solutions, respectively; after each elution, the fraction containing the polyphenol components was collected to obtain a collected solution;

[0063] (7) Concentration: The collected solution is concentrated under vacuum at 0.1 mbar and 50° C. to remove the ethanol solvent and obtain a concentrated solution;

[0064] (8) Drying: The concentrated solution is placed in a vacuum freeze dryer and vacuum dried at 0.1 mbar and -30°C to finally obtain a propolis polyphenol extract; after testing, the content of polyphenols in the propolis polyphenol extract is 85%.

[0065] A method for preparing a hydrogel, comprising the following steps:

[0066] (1) Preparation of hydrogel matrix:

[0067] Sodium alginate: 1 wt% sodium alginate was dissolved in deionized water and heated at 70°C to completely dissolve the alginate solution;

[0068] Gelatin: Use 2 wt% gelatin, dissolve it in deionized water, heat it to 60°C with warm water, and obtain a gelatin solution after it is completely dissolved;

[0069] The gelatin solution and the sodium alginate solution were mixed in a volume ratio of 1:1 to prepare a uniform matrix solution;

[0070] (2) Preparation of propolis polyphenol solution:

[0071] Dissolving the propolis polyphenol extract in deionized water at a concentration of 2 wt %, stirring with a stirrer to ensure that the polyphenol is evenly dissolved, to obtain a propolis polyphenol solution;

[0072] (3) Mixing:

[0073] The propolis polyphenol solution and the dissolved matrix solution are mixed in a volume ratio of 1:4, and stirred until uniform to obtain a mixed solution;

[0074] (4) Cross-linking:

[0075] Slowly add 1% w / v CaCl2 solution to the mixture and continue stirring for 30 min to ensure that the crosslinking agent fully acts to form a stable three-dimensional network structure;

[0076] (5) Molding:

[0077] The mixed solution is poured into a mold to form a hydrogel film or particles; the volume of each mold is 10 ml;

[0078] (6) Drying:

[0079] The hydrogel is subjected to vacuum freeze drying to remove moisture, thereby obtaining a stable propolis polyphenol hydrogel.

[0080] The propolis polyphenol hydrogel of this embodiment can be used in the fields of wound care, anti-aging skin care products, functional foods, wound dressings, etc., and its performance test results are as follows:

[0081] (1) Antibacterial activity: The antibacterial rate of the hydrogel against Staphylococcus aureus was 94%, and the antibacterial rate against Escherichia coli was 91%;

[0082] (2) Moisture retention: 24h moisturizing rate is 85%;

[0083] (3) Antioxidant properties: The DPPH free radical scavenging rate is 90%, with significant antioxidant effect;

[0084] (4) Mechanical properties: Compression strength is 1.5 MPa.

[0085] Example 2

[0086] A method for preparing a propolis polyphenol extract, comprising the following steps:

[0087] (1) crushing: crushing the propolis sample to obtain propolis powder, wherein the particle size of the crushed propolis powder is controlled to be 0.5 to 1 mm;

[0088] (2) Defatting: Use petroleum ether (volume-to-mass ratio of 1:5) to defatted propolis powder for 12 h to remove fat-soluble components such as wax and fatty acids;

[0089] (3) Alcohol extraction: 65% (v / v) ethanol is used to mix with the defatted propolis raw material, and the mass volume ratio of propolis powder to ethanol solution is 1:10, that is, 10 ml of ethanol solution is used for 1 g of propolis, ensuring that the defatted propolis raw material is fully in contact with the ethanol to obtain an alcohol extract;

[0090] (4) Ultrasonic-assisted extraction: The alcohol extract was placed in an ultrasonic extractor, the power was set to 100 W, the temperature was set to 50°C, and ultrasonic extraction was performed for 30 min under this condition; the ultrasonic frequency was 40 kHz, which helped the solvent to penetrate the propolis cell wall and promote the release of polyphenols to obtain the extract;

[0091] (5) Liquid-liquid extraction: The extract was extracted twice with ethyl acetate (volume ratio of 1:1), each extraction time was 30 min, to increase the enrichment of polyphenols; the extract was separated into layers through a separatory funnel, the upper ethyl acetate phase was separated, and the upper ethyl acetate phase was collected to obtain the extract;

[0092] (6) Column chromatography purification: The extract was further purified using HPD-100 macroporous resin column chromatography, and gradient elution was performed in three steps using 10% (v / v), 25% (v / v), and 45% (v / v) ethanol solutions, respectively; after each elution, the fraction containing the polyphenol components was collected to obtain a collected solution;

[0093] (7) Concentration: The collected solution is concentrated under vacuum at 0.1 mbar and 50° C. to remove the ethanol solvent and obtain a concentrated solution;

[0094] (8) Drying: The concentrated solution is placed in a vacuum freeze dryer and vacuum dried at 0.1 mbar and -30°C to finally obtain a propolis polyphenol extract; after testing, the content of polyphenols in the propolis polyphenol extract is 80%.

[0095] A method for preparing a hydrogel, comprising the following steps:

[0096] (1) Preparation of hydrogel matrix:

[0097] Sodium alginate: 2 wt% sodium alginate was dissolved in deionized water and heated at 70°C to completely dissolve the alginate solution;

[0098] Gelatin: 3 wt% gelatin was dissolved in deionized water, heated to 60°C with warm water, and the gelatin solution was obtained after the gelatin was completely dissolved;

[0099] The gelatin solution and the sodium alginate solution were mixed in a volume ratio of 1:1 to prepare a uniform matrix solution;

[0100] (2) Preparation of propolis polyphenol solution:

[0101] Dissolving the propolis polyphenol extract in deionized water at a concentration of 2 wt %, stirring with a stirrer to ensure that the polyphenol is evenly dissolved, to obtain a propolis polyphenol solution;

[0102] (3) Mixing:

[0103] The propolis polyphenol solution and the dissolved matrix solution are mixed in a volume ratio of 1:4, and stirred until uniform to obtain a mixed solution;

[0104] (4) Cross-linking:

[0105] Slowly add 1% w / v CaCl2 solution to the mixture and continue stirring for 30 min to ensure that the crosslinking agent fully acts to form a stable three-dimensional network structure;

[0106] (5) Molding:

[0107] The mixed solution is poured into a mold to form a hydrogel film or particles; the volume of each mold is 10 ml;

[0108] (6) Drying:

[0109] The hydrogel is subjected to vacuum freeze drying to remove moisture, thereby obtaining a stable propolis polyphenol hydrogel.

[0110] The propolis polyphenol hydrogel of this embodiment can be used in the fields of wound care, anti-aging skin care products, functional foods, wound dressings, etc., and its performance test results are as follows:

[0111] (1) Antibacterial activity: The antibacterial rate of the hydrogel against Staphylococcus aureus was 92%, and the antibacterial rate against Escherichia coli was 93%;

[0112] (2) Moisture retention: 24h moisturizing rate is 86%;

[0113] (3) Antioxidant properties: The DPPH free radical scavenging rate was 85.4%, with significant antioxidant effect;

[0114] (4) Mechanical properties: Compression strength is 1.5Mpa.

[0115] Example 3

[0116] A method for preparing a propolis polyphenol extract, comprising the following steps:

[0117] (1) crushing: crushing the propolis sample to obtain propolis powder, wherein the particle size of the crushed propolis powder is controlled to be 0.5 to 1 mm;

[0118] (2) Defatting: Use petroleum ether (volume-to-mass ratio of 1:5.5) to defatted propolis powder for 12 h to remove fat-soluble components such as wax and fatty acids;

[0119] (3) Alcohol extraction: 75% (v / v) ethanol is used to mix with the defatted propolis raw material, and the mass volume ratio of propolis powder to ethanol solution is 1:10, that is, 10 ml of ethanol solution is used for 1 g of propolis, to ensure that the defatted propolis raw material is fully in contact with the ethanol, and an alcohol extract is obtained;

[0120] (4) Ultrasonic-assisted extraction: The alcohol extract was placed in an ultrasonic extractor, the power was set to 100 W, the temperature was set to 50°C, and ultrasonic extraction was performed for 30 min under this condition; the ultrasonic frequency was 40 kHz, which helped the solvent to penetrate the propolis cell wall and promote the release of polyphenols to obtain the extract;

[0121] (5) Liquid-liquid extraction: The extract was extracted twice with ethyl acetate (volume ratio of 1:1), each extraction time was 30 min, to increase the enrichment of polyphenols; the extract was separated into layers through a separatory funnel, the upper ethyl acetate phase was separated, and the upper ethyl acetate phase was collected to obtain the extract;

[0122] (6) Column chromatography purification: The extract was further purified using HPD-100 macroporous resin column chromatography, and gradient elution was performed in three steps using 10% (v / v), 25% (v / v), and 45% (v / v) ethanol solutions, respectively; after each elution, the fraction containing the polyphenol components was collected to obtain a collected solution;

[0123] (7) Concentration: The collected solution is concentrated under vacuum at 0.1 mbar and 50° C. to remove the ethanol solvent and obtain a concentrated solution;

[0124] (8) Drying: The concentrated solution is placed in a vacuum freeze dryer and vacuum dried at 0.1 mbar and -30°C to finally obtain a propolis polyphenol extract; after testing, the polyphenol content in the propolis polyphenol extract is 82%.

[0125] A method for preparing a hydrogel, comprising the following steps:

[0126] (1) Preparation of hydrogel matrix:

[0127] Sodium alginate: 1 wt% sodium alginate was dissolved in deionized water and heated at 70°C to completely dissolve the alginate solution;

[0128] Gelatin: 3 wt% gelatin was dissolved in deionized water, heated to 60°C with warm water, and the gelatin solution was obtained after the gelatin was completely dissolved;

[0129] The gelatin solution and the sodium alginate solution were mixed in a volume ratio of 1:1 to prepare a uniform matrix solution;

[0130] (2) Preparation of propolis polyphenol solution:

[0131] Dissolving the propolis polyphenol extract in deionized water at a concentration of 2 wt %, stirring with a stirrer to ensure that the polyphenol is evenly dissolved, to obtain a propolis polyphenol solution;

[0132] (3) Mixing:

[0133] The propolis polyphenol solution and the dissolved matrix solution are mixed in a volume ratio of 1:4, and stirred until uniform to obtain a mixed solution;

[0134] (4) Cross-linking:

[0135] Slowly add 1% w / v CaCl2 solution to the mixture and continue stirring for 30 min to ensure that the crosslinking agent fully acts to form a stable three-dimensional network structure;

[0136] (5) Molding:

[0137] The mixed solution is poured into a mold to form a hydrogel film or particles; the volume of each mold is 10 ml;

[0138] (6) Drying:

[0139] The hydrogel is subjected to vacuum freeze drying to remove moisture, thereby obtaining a stable propolis polyphenol hydrogel.

[0140] The propolis polyphenol hydrogel of this embodiment can be used in the fields of wound care, anti-aging skin care products, functional foods, wound dressings, etc., and its performance test results are as follows:

[0141] (1) Antibacterial activity: The antibacterial rate of the hydrogel against Staphylococcus aureus was 93%, and the antibacterial rate against Escherichia coli was 91%;

[0142] (2) Moisture retention: 24h moisturizing rate is 85%;

[0143] (3) Antioxidant properties: The DPPH free radical scavenging rate was 87.8%, with significant antioxidant effect;

[0144] (4) Mechanical properties: Compression strength is 1.5Mpa.

[0145] Comparative Example 1

[0146] Compared with Example 1, the only difference is that in the method for preparing the propolis polyphenol extract, in step (5), the extract is extracted twice with 70% methanol (volume ratio of 1:1); the content of polyphenols in the prepared propolis polyphenol extract is 80%.

[0147] The performance test results of the hydrogel prepared using it as raw material are as follows:

[0148] (1) Antibacterial activity: The antibacterial rate of the hydrogel against Staphylococcus aureus was 85%, and the antibacterial rate against Escherichia coli was 82%;

[0149] (2) Moisture retention: 24h moisturizing rate is 75%;

[0150] (3) Antioxidant properties: DPPH free radical scavenging rate was 76.5%;

[0151] (4) Mechanical properties: Compression strength is 1.3Mpa.

[0152] Comparative Example 2

[0153] Compared with Example 1, the only difference is that in the method for preparing the propolis polyphenol extract, in step (6), the extract is further purified using XAD-7 macroporous resin column chromatography; the content of polyphenols in the prepared propolis polyphenol extract is 75%.

[0154] The performance test results of the hydrogel prepared using it as raw material are as follows:

[0155] (1) Antibacterial activity: The antibacterial rate of the hydrogel against Staphylococcus aureus was 86%, and the antibacterial rate against Escherichia coli was 85%;

[0156] (2) Moisture retention: 24h moisturizing rate is 78%;

[0157] (3) Antioxidant properties: DPPH free radical scavenging rate was 75.8%;

[0158] (4) Mechanical properties: Compression strength is 1.4Mpa.

[0159] Comparative Example 3

[0160] Compared with Example 1, the only difference is that in the method for preparing the hydrogel, in step (1), the gelatin solution and the sodium alginate solution are mixed in a volume ratio of 2:1; the performance test results of the prepared hydrogel are as follows:

[0161] (1) Antibacterial activity: The antibacterial rate of the hydrogel against Staphylococcus aureus was 75%, and the antibacterial rate against Escherichia coli was 73%;

[0162] (2) Moisture retention: 24h moisturizing rate is 77%;

[0163] (3) Antioxidant properties: DPPH free radical scavenging rate was 72.6%;

[0164] (4) Mechanical properties: Compression strength is 1.2Mpa.

[0165] The content of polyphenols in the propolis polyphenol extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was determined by high performance liquid chromatography (HPLC):

[0166] (1) separating the propolis polyphenol extract by HPLC equipment and selecting an appropriate reverse phase chromatographic column (such as a C18 column);

[0167] (2) Set a suitable mobile phase (e.g., water / acetonitrile containing 0.1% phosphoric acid), set the flow rate to 1.0 ml / min, and set the column temperature to 30°C;

[0168] (3) separating the extract, detecting the retention time of the main polyphenol components, comparing with the standard, and determining the types of polyphenols;

[0169] (4) Calculate the peak area of ​​each polyphenol component and estimate the specific content of polyphenols.

[0170] Testing methods for various properties of the hydrogels prepared in Examples 1 to 3 and Comparative Examples 1 to 3:

[0171] 1. Antibacterial activity test (inhibition zone method):

[0172] (1) Evenly spread a suspension of target bacteria (such as Staphylococcus aureus and Escherichia coli) on nutrient agar medium;

[0173] (2) preparing the hydrogel sample into a certain size and placing it on the inoculated culture medium;

[0174] (3) After 24 h of incubation, the diameter of the inhibition zone was measured and the antibacterial effect was calculated;

[0175] (4) The control group used solvent or basic hydrogel for comparison.

[0176] 2. Moisturizing performance test (skin hydration tester):

[0177] (1) Applying the hydrogel evenly on the artificial skin model or the hands of volunteers;

[0178] (2) Use a skin hydration tester (such as Corneometer) to measure skin hydration values ​​before and after application;

[0179] (3) Record the changes in hydration within 24 hours after application and derive the moisturizing properties.

[0180] 3. Antioxidant activity detection (DPPH free radical scavenging method):

[0181] (1) Prepare DPPH solution with a concentration of 0.1 mM and dissolve it in ethanol;

[0182] (2) taking an appropriate amount of propolis polyphenol extract solution (e.g., a concentration of 10 μg / ml to 100 μg / ml) and mixing it with an equal volume of DPPH solution;

[0183] (3) After reacting for 30 min at room temperature, the color of DPPH changed from purple to yellow;

[0184] (4) using a spectrophotometer to measure the absorbance (OD value) after the reaction at a wavelength of 517 nm;

[0185] (5) Calculate the scavenging rate of the sample on DPPH free radicals:

[0186]

[0187] A0: absorbance of control group;

[0188] At: absorbance of the sample set.

[0189] 4. Mechanical properties test (compression test):

[0190] (1) Performing a compression test using a universal material testing machine to compress the hydrogel sample to a certain ratio;

[0191] (2) Record the compression force and deformation of the sample and calculate the compressive strength.

[0192] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for preparing a propolis polyphenol extract, characterized in that: The steps include: (1) crushing: crushing the propolis sample to obtain propolis powder; (2) Defatting: Use petroleum ether to defatt the propolis powder to remove fat-soluble components; (3) Alcohol extraction: Use 65% to 75% ethanol solution to mix with the defatted propolis raw material, and fully contact to obtain an alcohol extract; (4) Ultrasonic-assisted extraction: subjecting the alcohol extract to ultrasonic-assisted extraction to obtain an extract; (5) Liquid-liquid extraction: extracting the extract with ethyl acetate, separating the upper ethyl acetate phase, and obtaining the extract; (6) Column chromatography purification: The extract was purified using HPD-100 macroporous resin column chromatography, with gradient elution using 10%, 25%, and 45% ethanol solutions in three steps; After each elution, the fractions containing polyphenol components are collected to obtain a collection solution; (7) Concentration: vacuum concentrating the collected solution to remove the ethanol solvent to obtain a concentrated solution; (8) Drying: The concentrated solution is vacuum dried to obtain the propolis polyphenol extract.

2. The method for preparing a propolis polyphenol extract according to claim 1, characterized in that: In step (2), the volume mass ratio of petroleum ether to propolis powder is 1:4.5-5.

5.

3. The method for preparing a propolis polyphenol extract according to claim 1, characterized in that: In step (3), the mass volume ratio of the defatted propolis raw material to the ethanol solution is 1:

10.

4. The method for preparing a propolis polyphenol extract according to claim 1, characterized in that: In step (4), the power of the ultrasonic-assisted extraction is 100 W, the temperature is 50° C., the time is 30 min, and the frequency is 40 kHz.

5. The method for preparing a propolis polyphenol extract according to claim 1, characterized in that: In step (5), the volume ratio of the extract to ethyl acetate is 1:

1.

6. A propolis polyphenol extract, characterized in that: The propolis polyphenol extract is prepared by the preparation method of any one of claims 1 to 5.

7. A method for preparing a hydrogel, characterized in that: The steps include: (1) preparing a hydrogel matrix: using 1% to 2% sodium alginate, dissolving it in deionized water, and heating it to completely dissolve it to obtain a sodium alginate solution; using 2% to 3% gelatin, dissolving it in deionized water, and heating it to completely dissolve it to obtain a gelatin solution; mixing the gelatin solution and the sodium alginate solution in a volume ratio of 1:1 to prepare a uniform matrix solution; (2) Preparing a propolis polyphenol solution: dissolving the propolis polyphenol extract according to claim 6 in deionized water and stirring uniformly to obtain a propolis polyphenol solution; (3) mixing: mixing the propolis polyphenol solution and the matrix solution, stirring evenly to obtain a mixed solution; (4) Cross-linking: Slowly add CaCl2 solution to the mixture and continue stirring to ensure that the cross-linking agent fully acts to form a stable three-dimensional network structure; (5) Molding: pouring the mixed solution into a mold to form a hydrogel film or particles; (6) Drying: The product is subjected to vacuum freeze drying to remove moisture and obtain a stable propolis polyphenol hydrogel.

8. The method for preparing a hydrogel according to claim 7, characterized in that: In step (3), the propolis polyphenol solution and the matrix solution are mixed in a volume ratio of 1:

4.

9. The method for preparing a hydrogel according to claim 7, characterized in that: In step (4), the concentration of the CaCl2 solution is 1%.

10. A hydrogel, characterized in that: The hydrogel is prepared by the method for preparing the hydrogel as described in any one of claims 7 to 9.

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