A composite hydrogel for co-encapsulating myricetin and epicatechin and preparation method thereof

Abstract

The invention discloses a composite hydrogel for co-encapsulating myricetin and epicatechin and a preparation method thereof, belonging to the technical field of hydrogel microspheres. A phyllophyte protein solution and a myricetin solution are self-assembled to form a phyllophyte protein-myricetin hydrogel; then the epicatechin solution is added into the prepared composite hydrogel to form a phyllophyte protein-myricetin-epicatechin composite hydrogel; finally, the prepared composite hydrogel is added into an oxidized indica rice starch solution to obtain a composite hydrogel for co-encapsulating myricetin and epicatechin; the formed composite hydrogel has a small particle size, high stability, good core material encapsulation effect, and meets the requirements for the common application of myricetin and epicatechin; the hyaluronic acid in the dispersant keeps a proper distance between particles through electrostatic repulsion, and the polylactic acid segment improves the steric hindrance effect, prevents micelle aggregation and precipitation, and prolongs the shelf life.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel microspheres, and particularly relates to a composite hydrogel for co-encapsulating myricetin and epicatechin and a preparation method thereof. Background Art

[0002] Myricetin belongs to the flavonol class of compounds. Its molecular structure contains multiple phenolic hydroxyl groups, which are important structural foundations for multiple biological activities. Myricetin is extracted from natural plants, such as the bark and vine tea of ​​Myricetaceae. It is rich in sources and naturally pollution-free, with high safety and biocompatibility. Myricetin has multiple pharmacological activities such as antioxidant, anti-tumor, cardiovascular protection, hypoglycemic, liver protection, and neuroprotection. It has multiple uses and has great medicinal value and development potential.

[0003] Epicatechin belongs to the polyphenolic compounds and is widely found in plants, such as tea, cocoa beans, grape seeds, hawthorn, apples, etc. It is rich in sources and natural and pollution-free. Compared with chemically synthesized antioxidants and drugs, it has higher safety and better biocompatibility, and is more in line with people's pursuit of health and environmental protection. Epicatechin has multiple pharmacological activities such as antioxidant, cardiovascular protection, anti-tumor, antibacterial and anti-inflammatory, and neuroprotection. It can play a role in multiple diseases or health problems at the same time and has great development potential.

[0004] However, bioactive compounds such as polyphenols and flavonoids have low solubility, stability and bioavailability. Encapsulating bioactive ingredients in delivery vehicles can effectively avoid the inactivation and degradation of substances, thereby achieving efficient targeted delivery and maximum absorption. However, the existing delivery vehicles have a low embedding rate for these active compounds, especially for active ingredients with different polarities. The co-embedding effect is poor, and it is difficult to meet the requirements for the co-application of myricetin and epicatechin. Summary of the invention

[0005] The purpose of the present invention is to provide a composite hydrogel for co-encapsulating myricetin and epicatechin and a preparation method thereof. Indica rice starch is modified by a TEMPO oxidation method, and the composite hydrogel for co-encapsulating myricetin and epicatechin is obtained by a layer-by-layer self-assembly technology. The formed composite hydrogel has a small particle size, high stability, and good core material encapsulation effect, thereby meeting the requirements for the common application of myricetin and epicatechin.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin comprises the following steps:

[0008] Step 1: Mix chlorophyll protein and phosphate buffer in a dosage ratio of 2 mg: 1 mL to obtain a chlorophyll protein solution, transfer the chlorophyll protein solution to a reactor and heat it to 80-85°C, ultrasonically disperse it at 480-500 W and 40 kHz for 25-30 min, then drop a myricetin solution with a mass concentration of 10 mg / mL into the reactor, stir at 200-300 r / min in the dark for 6 h, and obtain a chlorophyll protein-myricetin hydrogel.

[0009] Step 2: under the stirring condition of 200-300 r / min, add the epicatechin solution with a mass concentration of 8 mg / mL into the phylloxetine-myricetin hydrogel, stir in the dark for 1.8-2.2 hours, and obtain the phylloxetine-myricetin-epicatechin composite hydrogel.

[0010] Step 3: Prepare the oxidized indica rice starch solution with a mass concentration of 7.5 mg / mL by using deionized water, transfer the oxidized indica rice starch solution to a reactor, heat it at 80-85°C for 25-35 min, then add the phyllotoxin-myricetin-epicatechin composite hydrogel to the reactor, stir at 450-480 r / min for 4.5-5.5 h, centrifuge the product at a speed of 3000 r / min for 10 min, collect the supernatant, and evenly mix the supernatant with 1.2-1.5% of its mass of a dispersant to obtain a composite hydrogel for co-encapsulating myricetin and epicatechin.

[0011] Furthermore, in step 1, the volume ratio of the phylloxera protein solution to the myricetin solution is 1:20.

[0012] Furthermore, in step 2, the volume ratio of the epicatechin solution to the phytoplanin-myricetin hydrogel is 1:20.

[0013] Furthermore, in step three, the volume ratio of the oxidized indica rice starch solution to the phylloxera protein-myricetin-epicatechin composite hydrogel is 2:1.

[0014] Furthermore, in step 3, the oxidized indica rice starch is prepared by TEMPO oxidation method:

[0015] Add indica rice starch and deionized water into a reactor, gelatinize at 95° C. for 15 min, cool naturally, add TEMPO, sodium bromide, sodium hypochlorite and a sodium hydroxide solution with a molar concentration of 0.5 mol / L into the reactor, control the oxidation degree of the oxidized indica rice starch by controlling the amount of sodium hydroxide solution added, stir the reaction at 200-300 r / min for 4 h, add a saturated sodium sulfate solution to terminate the reaction, then add sodium borohydride into the reactor under stirring, let it stand for 2 h, transfer the reaction product into anhydrous ethanol for precipitation, filter, wash the filter cake with anhydrous ethanol for 3-5 times, and vacuum dry to obtain oxidized indica rice starch.

[0016] Furthermore, the usage ratio of indica rice starch, deionized water, TEMPO, sodium bromide, sodium hypochlorite and sodium borohydride is 5 g: 200 mL: 0.1 g: 1 g: 1.75 g: 0.1 g.

[0017] Furthermore, the oxidation degree of the oxidized indica rice starch is 25-100%.

[0018] Further, the dispersant is prepared by the following steps:

[0019] Step 1: Add 15% sodium hydroxide aqueous solution and hyaluronic acid into the reactor, stir at 200-300r / min for 1-1.5h, then adjust the pH value to 6.5-7 with L-lactic acid, continue stirring for 1-1.5h, and finally transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 12000Da, dialyze with ultrapure water for 24h, and freeze-dry the dialyzed product to obtain pretreated hyaluronic acid powder.

[0020] Step 2: Add the pretreated hyaluronic acid powder, L-lactide and dimethyl sulfoxide into a reactor and stir to mix, perform vacuum dehydration at 110-120°C, then add stannous isooctanoate into the reactor, keep the reaction warm for 6-8h, add 31% hydrochloric acid to terminate the reaction, filter, wash the filter cake with deionized water until the last washing liquid is neutral, vacuum dry, grind, and pass through a 180-mesh sieve to obtain a dispersant.

[0021] Furthermore, in step 1, the ratio of the sodium hydroxide aqueous solution to the hyaluronic acid powder is 20-25 mL: 1 g.

[0022] Furthermore, in step 2, the dosage ratio of the pretreated hyaluronic acid powder, dimethyl sulfoxide, L-lactide, stannous isooctanoate and 31 wt % hydrochloric acid is 3 g: 30 mL: 1 g: 0.015-0.02 g: 0.15-0.2 mL.

[0023] Beneficial effects of the present invention:

[0024] 1. The present invention adopts TEMPO oxidation method to modify indica rice starch, and obtains a composite hydrogel co-encapsulating myricetin and epicatechin through layer-by-layer self-assembly technology.

[0025] The preparation method provided by the present invention uses chlorophyll protein and oxidized indica rice starch as encapsulation wall materials, and two active substances (myricetin and epicatechin) with different polarities as core materials. In the process of self-assembly of chlorophyll protein and myricetin to form hydrogel, hydrogen bonds and hydrophobic interactions are the main binding forces; the composite hydrogel co-encapsulating myricetin and epicatechin presents an obvious core-shell structure, in which the hydrophobic chlorophyll protein is the core and the hydrophilic oxidized indica rice starch is the shell, and the stability between different components is mainly maintained by hydrogen bonds and hydrophobic interactions. The composite hydrogel finally formed has a small particle size, high stability and good core material encapsulation effect, which can significantly enhance the pH stability, thermal stability and storage stability of myricetin and epicatechin.

[0026] 2. The dispersant in the present invention is based on hyaluronic acid, and polylactic acid is grafted onto the polylactic acid through L-lactide. Both hyaluronic acid and polylactic acid have good biocompatibility. Hyaluronic acid is a natural polysaccharide with a large amount of negative charge. In the dispersion system, it can be adsorbed on the surface of the composite hydrogel micelles that co-embed myricetin and epicatechin. The electrostatic repulsion keeps the particles at an appropriate distance to prevent micelle aggregation and precipitation. The polylactic acid segment increases the spatial volume of the hyaluronic acid molecule, improves the steric hindrance effect, and further enhances the electrostatic repulsion, which helps to stabilize the dispersion of the composite hydrogel micelles that embed myricetin and epicatechin and prolong the shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings.

[0028] Figure 1 are transmission electron microscope images of reference examples 1 and 2 of the present invention;

[0029] Figure 2 are transmission electron microscope images of Examples 1 to 4 of the present invention and Comparative Examples 1 to 2;

[0030] Figure 3 It is a characterization result diagram of corresponding indicators in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1: This example provides a composite hydrogel co-encapsulating myricetin and epicatechin, which is prepared by the following method:

[0033] S1: Add 20L of 15% sodium hydroxide aqueous solution and 1kg of hyaluronic acid into the reactor, stir at 200r / min for 1h, then adjust the pH value to 6.5 with L-lactic acid, continue stirring for 1h, and finally transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 12000Da, dialyze with ultrapure water for 24h, and freeze-dry the dialyzed product to obtain pretreated hyaluronic acid powder.

[0034] Hyaluronic acid with a larger molecular weight is partially degraded under alkaline conditions, thereby breaking the molecular chain and helping to expose more active groups. The added L-lactic acid combines with hyaluronic acid through hydrogen bonding forces between hydrophilic groups, which helps to perform subsequent ring-opening grafting through L-lactide and improve the reaction efficiency.

[0035] S2: Add 300 g of pretreated hyaluronic acid powder, 100 g of L-lactide and 3 L of dimethyl sulfoxide into a reactor, stir and mix, vacuum dehydrate at 110°C, then add 1.5 g of stannous isooctanoate into the reactor, keep the temperature for reaction for 6 hours, dropwise add 15 mL of 31% hydrochloric acid to terminate the reaction, filter, wash the filter cake with deionized water until the last washing liquid is neutral, vacuum dry, grind, and pass through a 180-mesh sieve to obtain a dispersant.

[0036] S3: 500 g of indica rice starch and 20 L of deionized water were added to a reactor, gelatinized at 95° C. for 15 min, and naturally cooled. 10 g of TEMPO, 100 g of sodium bromide, 175 g of sodium hypochlorite and an appropriate amount of sodium hydroxide solution with a molar concentration of 0.5 mol / L were added to the reactor. The oxidation degree of the oxidized indica rice starch was regulated by controlling the amount of sodium hydroxide solution added. The reaction was stirred at 200 r / min for 4 h, and a saturated sodium sulfate solution was added to terminate the reaction. Then, 10 g of sodium borohydride was added to the reactor under stirring, and the mixture was allowed to stand for 2 h. The reaction product was transferred to anhydrous ethanol for precipitation, filtered, and the filter cake was washed 3 times with anhydrous ethanol and dried in vacuo to obtain oxidized indica rice starch with an oxidation degree of 25%.

[0037] S4: Mix chlorophyll protein with phosphate buffer in a dosage ratio of 2 mg:1 mL to obtain chlorophyll protein solution, transfer 10 mL of chlorophyll protein solution to a reactor and heat to 80°C, ultrasonically disperse at 480 W and 40 kHz for 25 min, then drop 200 mL of myricetin solution with a mass concentration of 10 mg / mL into the reactor, stir at 200 r / min in the dark for 6 h to obtain chlorophyll protein-myricetin hydrogel.

[0038] S5: Under the stirring condition of 200 r / min, 10 mL of epicatechin solution with a mass concentration of 8 mg / mL was added to 200 mL of phylloxetine-myricetin hydrogel, and stirred for 1.8 h in the dark to obtain phylloxetine-myricetin-epicatechin composite hydrogel.

[0039] S6: Prepare oxidized indica rice starch solution with a mass concentration of 7.5 mg / mL by using deionized water, transfer 200 mL of oxidized indica rice starch solution to a reactor, heat at 80°C for 25 min, then add 100 mL of phylloxera protein-myricetin-epicatechin composite hydrogel to the reactor, stir at 450 r / min for 4.5 h, centrifuge the product at 3000 r / min for 10 min, collect the supernatant, and evenly mix 1.2% of the mass of dispersant into the supernatant to obtain a composite hydrogel co-encapsulating myricetin and epicatechin.

[0040] Example 2: This example provides a composite hydrogel co-encapsulating myricetin and epicatechin, which is prepared by the following method:

[0041] S1: Add 22L of 15% sodium hydroxide aqueous solution and 1kg of hyaluronic acid into the reactor, stir at 250r / min for 1.2h, then adjust the pH value to 6.8 with L-lactic acid, continue stirring for 1.2h, and finally transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 12000Da, dialyze with ultrapure water for 24h, and freeze-dry the dialyzed product to obtain pretreated hyaluronic acid powder.

[0042] S2: Add 300 g of pretreated hyaluronic acid powder, 100 g of L-lactide and 3 L of dimethyl sulfoxide into a reactor, stir and mix, vacuum dehydrate at 115°C, then add 1.8 g of stannous isooctanoate into the reactor, keep the temperature for reaction for 7 hours, drop 18 mL of 31% hydrochloric acid to terminate the reaction, filter, wash the filter cake with deionized water until the last washing liquid is neutral, vacuum dry, grind, and pass through a 180-mesh sieve to obtain a dispersant.

[0043] S3: 500 g of indica rice starch and 20 L of deionized water were added to a reactor, gelatinized at 95° C. for 15 min, and naturally cooled. 10 g of TEMPO, 100 g of sodium bromide, 175 g of sodium hypochlorite and an appropriate amount of sodium hydroxide solution with a molar concentration of 0.5 mol / L were added to the reactor. The oxidation degree of the oxidized indica rice starch was regulated by controlling the amount of sodium hydroxide solution added. The reaction was stirred at 250 r / min for 4 h, and a saturated sodium sulfate solution was added to terminate the reaction. Then, 10 g of sodium borohydride was added to the reactor under stirring, and the reaction was allowed to stand for 2 h. The reaction product was transferred to anhydrous ethanol for precipitation, filtered, and the filter cake was washed 4 times with anhydrous ethanol and vacuum dried to obtain an oxidized indica rice starch with an oxidation degree of 50%.

[0044] S4: Mix chlorophyll protein and phosphate buffer in a dosage ratio of 2 mg:1 mL to obtain chlorophyll protein solution, transfer 10 mL of chlorophyll protein solution to a reactor and heat to 82°C, ultrasonically disperse for 28 min at 490 W and 40 kHz, then drop 200 mL of myricetin solution with a mass concentration of 10 mg / mL into the reactor, stir at 250 r / min in the dark for 6 h to obtain chlorophyll protein-myricetin hydrogel.

[0045] S5: Under the stirring condition of 250 r / min, 10 mL of epicatechin solution with a mass concentration of 8 mg / mL was added to 200 mL of phylloxetine-myricetin hydrogel, and stirred for 2 h in the dark to obtain phylloxetine-myricetin-epicatechin composite hydrogel.

[0046] S6: Prepare oxidized indica rice starch solution with a mass concentration of 7.5 mg / mL by using deionized water, transfer 200 mL of oxidized indica rice starch solution to a reactor, heat at 82°C for 30 min, then add 100 mL of phylloxera protein-myricetin-epicatechin composite hydrogel to the reactor, stir at 460 r / min for 5 h, centrifuge the product at 3000 r / min for 10 min, collect the supernatant, and evenly mix 1.3% of the mass of dispersant into the supernatant to obtain a composite hydrogel co-encapsulating myricetin and epicatechin.

[0047] Example 3: This example provides a composite hydrogel co-encapsulating myricetin and epicatechin, which is prepared by the following method:

[0048] S1: Add 22L of 15% sodium hydroxide aqueous solution and 1kg of hyaluronic acid into the reactor, stir at 250r / min for 1.2h, then adjust the pH value to 6.8 with L-lactic acid, continue stirring for 1.2h, and finally transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 12000Da, dialyze with ultrapure water for 24h, and freeze-dry the dialyzed product to obtain pretreated hyaluronic acid powder.

[0049] S2: Add 300 g of pretreated hyaluronic acid powder, 100 g of L-lactide and 3 L of dimethyl sulfoxide into a reactor, stir and mix, vacuum dehydrate at 115°C, then add 1.8 g of stannous isooctanoate into the reactor, keep the temperature for reaction for 7 hours, drop 18 mL of 31% hydrochloric acid to terminate the reaction, filter, wash the filter cake with deionized water until the last washing liquid is neutral, vacuum dry, grind, and pass through a 180-mesh sieve to obtain a dispersant.

[0050] S3: 500 g of indica rice starch and 20 L of deionized water were added to a reactor, gelatinized at 95° C. for 15 min, and naturally cooled. 10 g of TEMPO, 100 g of sodium bromide, 175 g of sodium hypochlorite and an appropriate amount of sodium hydroxide solution with a molar concentration of 0.5 mol / L were added to the reactor. The oxidation degree of the oxidized indica rice starch was regulated by controlling the amount of sodium hydroxide solution added. The reaction was stirred at 250 r / min for 4 h, and a saturated sodium sulfate solution was added to terminate the reaction. Then, 10 g of sodium borohydride was added to the reactor under stirring, and the reaction was allowed to stand for 2 h. The reaction product was transferred to anhydrous ethanol for precipitation, filtered, and the filter cake was washed 4 times with anhydrous ethanol and vacuum dried to obtain an oxidized indica rice starch with an oxidation degree of 75%.

[0051] S4: Mix chlorophyll protein and phosphate buffer in a dosage ratio of 2 mg:1 mL to obtain chlorophyll protein solution, transfer 10 mL of chlorophyll protein solution to a reactor and heat to 82°C, ultrasonically disperse for 28 min at 490 W and 40 kHz, then drop 200 mL of myricetin solution with a mass concentration of 10 mg / mL into the reactor, stir at 250 r / min in the dark for 6 h to obtain chlorophyll protein-myricetin hydrogel.

[0052] S5: Under the stirring condition of 250 r / min, 10 mL of epicatechin solution with a mass concentration of 8 mg / mL was added to 200 mL of phylloxetine-myricetin hydrogel, and stirred for 2 h in the dark to obtain phylloxetine-myricetin-epicatechin composite hydrogel.

[0053] S6: Prepare oxidized indica rice starch solution with a mass concentration of 7.5 mg / mL by using deionized water, transfer 200 mL of oxidized indica rice starch solution to a reactor, heat at 82°C for 30 min, then add 100 mL of phylloxera protein-myricetin-epicatechin composite hydrogel to the reactor, stir at 460 r / min for 5 h, centrifuge the product at 3000 r / min for 10 min, collect the supernatant, and evenly mix 1.4% of the mass of the dispersant into the supernatant to obtain a composite hydrogel co-encapsulating myricetin and epicatechin.

[0054] Example 4: This example provides a composite hydrogel co-encapsulating myricetin and epicatechin, which is prepared by the following method:

[0055] S1: Add 25L of 15% sodium hydroxide aqueous solution and 1kg of hyaluronic acid into the reactor, stir at 300r / min for 1.5h, then adjust the pH value to 7 with L-lactic acid, continue stirring for 1.5h, and finally transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 12000Da, dialyze with ultrapure water for 24h, and freeze-dry the dialyzed product to obtain pretreated hyaluronic acid powder.

[0056] S2: Add 300 g of pretreated hyaluronic acid powder, 100 g of L-lactide and 3 L of dimethyl sulfoxide into a reactor, stir and mix, vacuum dehydrate at 120°C, then add 2 g of stannous isooctanoate into the reactor, keep the temperature for reaction for 8 hours, drop 20 mL of 31% hydrochloric acid to terminate the reaction, filter, wash the filter cake with deionized water until the last washing liquid is neutral, vacuum dry, grind, and pass through a 180-mesh sieve to obtain a dispersant.

[0057] S3: 500 g of indica rice starch and 20 L of deionized water were added to a reactor, gelatinized at 95° C. for 15 min, and naturally cooled. 10 g of TEMPO, 100 g of sodium bromide, 175 g of sodium hypochlorite and an appropriate amount of sodium hydroxide solution with a molar concentration of 0.5 mol / L were added to the reactor. The oxidation degree of the oxidized indica rice starch was regulated by controlling the amount of sodium hydroxide solution added. The reaction was stirred at 300 r / min for 4 h, and a saturated sodium sulfate solution was added to terminate the reaction. Then, 10 g of sodium borohydride was added to the reactor under stirring, and the mixture was allowed to stand for 2 h. The reaction product was transferred to anhydrous ethanol for precipitation, filtered, and the filter cake was washed with anhydrous ethanol 5 times, and vacuum dried to obtain an oxidized indica rice starch with an oxidation degree of 100%.

[0058] S4: The phylloprotein was mixed with phosphate buffer at a dosage ratio of 2 mg: 1 mL to obtain a phylloprotein solution. 10 mL of the phylloprotein solution was transferred to a reactor and heated to 85°C. Ultrasonic dispersion was performed at 500 W and 40 kHz for 30 min. Then, 200 mL of a myricetin solution with a mass concentration of 10 mg / mL was added dropwise to the reactor. The mixture was stirred at 300 r / min in the dark for 6 h to obtain a phylloprotein-myricetin hydrogel (the phylloprotein solution and the phylloprotein-myricetin hydrogel were observed by transmission electron microscopy and marked as Reference Example 1 and Reference Example 2, respectively. The transmission electron microscopy images thereof can be found in Figure 1 ).

[0059] S5: Under the stirring condition of 300 r / min, 10 mL of epicatechin solution with a mass concentration of 8 mg / mL was added to 200 mL of phylloxetine-myricetin hydrogel, and stirred for 2.2 h in the dark to obtain phylloxetine-myricetin-epicatechin composite hydrogel.

[0060] S6: Prepare oxidized indica rice starch solution with a mass concentration of 7.5 mg / mL by using deionized water, transfer 200 mL of oxidized indica rice starch solution to a reactor, heat at 85°C for 35 min, then add 100 mL of phylloxera protein-myricetin-epicatechin composite hydrogel to the reactor, stir at 480 r / min for 5.5 h, centrifuge the product at 3000 r / min for 10 min, collect the supernatant, and evenly mix 1.5% of the mass of dispersant into the supernatant to obtain a composite hydrogel co-encapsulating myricetin and epicatechin.

[0061] Comparative Example 1: Based on Example 4, the product in step S5 is directly used as the final composite hydrogel without going through step S6.

[0062] Comparative Example 2: Based on Example 4, in step S6, the oxidized indica rice starch is replaced with indica rice starch with an oxidation degree of 0, and the other steps remain unchanged to prepare a composite hydrogel.

[0063] The composite hydrogels in Examples 1 to 4 and Comparative Examples 1 to 2 were observed by transmission electron microscopy. Figure 2 .

[0064] The herbaceous grass protein solution in Reference Example 1, the herbaceous grass protein-myricetin hydrogel in Reference Example 2, the composite hydrogels in Comparative Examples 1-2 and Examples 1-4 were characterized. The results are shown in Figure 3 .

[0065] Combination Figure 1 , Figure 2 and Figure 3As shown in A, the average particle size of the micelles of the phylloprotein-myricetin hydrogel (EDP-Myr) in Reference Example 2 is 220 nm. When epicatechin (EC) is complexed with the phylloprotein-myricetin hydrogel, the micelle particle size of the phylloprotein-myricetin-epicatechin composite hydrogel (EDP-Myr-EC) is slightly smaller than that of the phylloprotein-myricetin hydrogel (EDP-Myr), which may be because the binding of epicatechin (EC) makes the ternary complex more compact.

[0066] The size of oxidized indica rice starch has a great influence on the particle size of EMEO (composite hydrogel co-embedded with myricetin and epicatechin). When unoxidized indica rice starch is used, the micelle particle size of the composite hydrogel in Comparative Example 2 increases most significantly, reaching 648.4 nm. As the oxidation degree of oxidized indica rice starch increases, the micelle particle size of EMEO in the examples gradually decreases, and the micelle particle size of EMEO in Example 4 (the oxidation degree of oxidized indica rice starch is 100%) is the smallest, which is 276 nm. However, when the oxidation degree of oxidized indica rice starch is greater than 50%, the difference in the micelle particle size of EMEO in Examples 3 and 4 is not significant, indicating that the oxidized indica rice starch attached to the surface of EMEO is saturated.

[0067] like Figure 3 As shown in B, the polydispersity coefficient of the chlorophyll protein solution in Reference Example 1, the chlorophyll protein-myricetin hydrogel in Reference Example 2, the composite hydrogels in Comparative Example 1-Comparative Example 2 and Examples 1-4 is proportional to the micelle particle size therein, and the EMEO micelles in the examples are more evenly distributed.

[0068] The reference examples, comparative examples and examples all showed negative charges for Zeta potential (see Figure 3 C), it can be seen from Reference Example 1 and Comparative Example 1 that the addition of myricetin and epicatechin has little effect on the surface charge of micelles. It can be seen from Comparative Example 2 and Examples 1 to 4 that when indica rice starch or oxidized indica rice starch with different oxidation degrees is complexed with EDP-Myr-EC, the charge on the surface of the micelle is similar to the potential of indica rice starch or oxidized indica rice starch, which proves that the oxidized indica rice starch in Example EMEO participates in the co-encapsulation.

[0069] Figure 3 D shows the embedding rate of myricetin and epicatechin (Myr-EC) in the composite hydrogels of Examples 1 to 4 and Comparative Examples 1 to 2. The specific results are shown in Table 1:

[0070] Table 1 Results of Myr-EC embedding rate in each composite hydrogel

[0071]

[0072] It can be seen from Table 1 that the embedding rate of the composite hydrogel in Examples 1 to 4 increases with the increase in the oxidation degree of the oxidized indica rice starch. Compared with Comparative Example 1 (not embedded by oxidized indica rice starch), Comparative Example 2 directly uses indica rice starch, and its embedding rate is less than that of Comparative Example 1. When the oxidation degree of the oxidized indica rice starch reaches 25%, its embedding rate reaches 53.1% and is greater than the embedding rate in Comparative Example 1, indicating that the composite hydrogel in Examples 1 to 3 has obvious co-encapsulation effect on myricetin and epicatechin.

[0073] Comparative Example 3: Based on Example 4, the dispersant was not mixed into step S6, and the other steps remained unchanged to prepare a composite hydrogel.

[0074] Comparative Example 4: Based on Example 4, hyaluronic acid is directly used as the dispersant in step S6, and the other steps remain unchanged to prepare a composite hydrogel.

[0075] EMEO in Example 1 to Example 4 and the composite hydrogel in Comparative Example 3 to Comparative Example 4 were taken respectively, 20 mL of each was placed in a round-bottom test tube with a volume of 25 mL, vacuum-sealed, and left to stand at 25° C. for 30 days. Whether there was sedimentation at the bottom of the round-bottom test tube was observed to judge the dispersion stability. The results are shown in Table 2:

[0076] Table 2 Dispersion stability results of various composite hydrogels

[0077]

[0078] It can be seen from Table 2 that the dispersants added in Examples 1 to 4 can improve the dispersion stability of EMEO. This may be because the dispersants prepared in the examples contain polylactic acid segments, which help to increase the rigidity of hyaluronic acid and enhance its steric hindrance, thereby improving the dispersion stability of the EMEO micelles in the examples.

[0079] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0080] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin, characterized in that: The steps include: Step 1: Evenly mix chlorophyll protein with phosphate buffer at a dosage ratio of 2 mg: 1 mL to obtain a chlorophyll protein solution, transfer the chlorophyll protein solution to a reactor and heat to 80-85°C, ultrasonically disperse for 25-30 min at 480-500 W and 40 kHz, then dropwise add a 10 mg / mL myricetin solution to the reactor, stir at 200-300 r / min in the dark for 6 h to obtain a chlorophyll protein-myricetin hydrogel; Step 2: under stirring conditions of 200-300 r / min, add an epicatechin solution with a concentration of 8 mg / mL to the phylloxetine-myricetin hydrogel, and stir in the dark for 1.8-2.2 hours to obtain the phylloxetine-myricetin-epicatechin composite hydrogel; Step 3: preparing oxidized indica rice starch with deionized water into an oxidized indica rice starch solution with a concentration of 7.5 mg / mL, transferring the oxidized indica rice starch solution to a reactor, heating at 80-85° C. for 25-35 min, then adding the phyllotoxin-myricetin-epicatechin composite hydrogel, stirring at 450-480 r / min for 4.5-5.5 h, centrifuging at 3000 r / min for 10 min, collecting the supernatant, and uniformly mixing 1.2-1.5% of the mass of the dispersant into the supernatant to obtain a composite hydrogel co-encapsulating myricetin and epicatechin; In step 3, oxidized indica rice starch is prepared by TEMPO oxidation method: Add indica rice starch and deionized water into a reactor, gelatinize at 95° C. for 15 min, cool naturally, add TEMPO, sodium bromide, sodium hypochlorite and a sodium hydroxide solution with a molar concentration of 0.5 mol / L into the reactor, adjust the oxidation degree of the oxidized indica rice starch by controlling the amount of sodium hydroxide solution added, stir and react at 200-300 r / min for 4 h, add a saturated sodium sulfate solution to terminate the reaction, then add sodium borohydride under stirring, let stand for 2 h, transfer the reaction product into anhydrous ethanol for precipitation, filter, wash the filter cake with anhydrous ethanol for 3-5 times, and vacuum dry to obtain oxidized indica rice starch; the oxidation degree of the oxidized indica rice starch is 25-100%; The dispersant is prepared by the following steps: Add 15wt% sodium hydroxide aqueous solution and hyaluronic acid into a reactor, stir at 200-300r / min for 1-1.5h, then adjust the pH value to 6.5-7 with L-lactic acid, continue stirring for 1-1.5h, and finally transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 12000Da, dialyze with ultrapure water for 24h, and freeze-dry the dialyzed product to obtain a pretreated hyaluronic acid powder; The sodium hydroxide aqueous solution and hyaluronic acid powder are used in a ratio of 20-25 mL: 1 g; The pretreated hyaluronic acid powder, L-lactide and dimethyl sulfoxide were added into a reaction kettle and stirred and mixed, vacuum dehydrated at 110-120° C., and then stannous isooctanoate was added, and the reaction was kept warm for 6-8 hours. 31wt% hydrochloric acid was added dropwise to terminate the reaction, and the filter cake was filtered, and the filter cake was washed with deionized water until the last washing liquid was neutral, vacuum dried, ground, and passed through a 180-mesh sieve to obtain a dispersant; The dosage ratio of the pretreated hyaluronic acid powder, dimethyl sulfoxide, L-lactide, stannous isooctanoate and 31wt% hydrochloric acid is 3g:30mL:1g:0.015-0.02g:0.15-0.2mL.

2. The method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin according to claim 1, characterized in that: The volume ratio of the phylloprotein solution to the myricetin solution in step 1 is 1:

20.

3. The method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin according to claim 1, characterized in that: The volume ratio of the epicatechin solution to the phytoplanin-myricetin hydrogel in step 2 is 1:

20.

4. The method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin according to claim 1, characterized in that: The volume ratio of the oxidized indica rice starch solution and the phylloxera protein-myricetin-epicatechin composite hydrogel in step three is 2:

1.

5. The method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin according to claim 1, characterized in that: The usage ratio of the indica rice starch, deionized water, TEMPO, sodium bromide, sodium hypochlorite and sodium borohydride is 5g:200mL:0.1g:1g:1.75g:0.1g.

6. A composite hydrogel co-encapsulating myricetin and epicatechin, characterized in that: The composite hydrogel is prepared by the method for preparing a composite hydrogel for co-encapsulating myricetin and epicatechin as described in any one of claims 1 to 5.

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