Glycyrrhizic acid / oxymatrine / berberine co-assembled hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of hydrogel and fruit preservation, and relates to glycyrrhizic acid / oxymatrine / berberine co-assembled hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving glycyrrhizic acid in water, continuously stirring at 70-90 DEG C until the glycyrrhizic acid is completely dissolved to obtain a glycyrrhizic acid solution, adding oxymatrine, then adding berberine, uniformly stirring, and then cooling to room temperature to form glycyrrhizic acid / oxymatrine / berberine co-assembled hydrogel; the molar ratio of oxymatrine to glycyrrhizic acid is below 1: 2, and the molar ratio of glycyrrhizic acid to berberine is 12: (0.25-6.0). According to the invention, glycyrrhizic acid, oxymatrine and berberine are taken as raw materials, and are co-assembled to form the hydrogel; the fruit preservative is sprayed before picking, is easy to adhere to and accumulate on the surfaces of fruits, enhances the fresh-keeping and antibacterial capabilities, improves the fresh-keeping effect of the fruits, and reduces the risk of physical damage caused by post-picking treatment at the same time.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of hydrogel and fruit preservation, and relates to a glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Soft-skinned berries such as strawberries, raspberries, blueberries and blackberries are the most nutritious fruits and are increasingly favored by people. However, these soft-skinned berries are prone to rot, mainly because these fruits are delicate, have thin skin, high respiration rate, are easy to be mechanically damaged and infected by microorganisms, resulting in a short shelf life and economic losses; therefore, preservation treatment is required.

[0003] In the prior art, the preservation of soft-skinned berries mainly relies on postharvest treatment techniques such as chemical agent soaking and coating preservation. These methods have certain preservation effect and can inhibit microbial growth and extend the shelf life to a certain extent. However, the following problems exist: (1) the existing preservation methods are all postharvest preservation treatments, which are difficult to achieve efficient preservation from the root; and the postharvest preservation treatment process is easy to cause physical damage to the fruits, increasing the risk of fruit rot; (2) the chemical agents are easy to be left over, which not only affects human health but also causes environmental pollution, resulting in poor safety and environmental protection. Therefore, the existing methods face great challenges in preventing the rot of soft-skinned berries during planting and harvesting.

[0004] Natural bioactive small molecules are known for their high efficiency and low toxicity, and have great potential in food preservation and plant biological stimulation. In food preservation, they can delay oxidative deterioration and inhibit microbial growth; in plant biological stimulation, they can precisely regulate plant growth and development while improving stress resistance. Oxymatrine (OMT) is one of the key active ingredients of Sophora flavescens, which has antibacterial and antioxidant effects, can inhibit the growth of various bacteria, can scavenge free radicals, reduce the damage of oxidative stress to food, and can achieve fruit preservation. However, due to its high water solubility, it has weak adhesion and short action time, making it difficult to achieve efficient preservation. SUMMARY

[0005] In view of the technical problems in the background art that postharvest preservation treatment is easy to cause secondary physical damage to the fruits and has weak adhesion, short action time and difficulty in achieving efficient preservation, the present application provides a glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel as well as a preparation method and application thereof.

[0006] The present application uses glycyrrhizic acid, oxymatrine and baicalein as raw materials to form a hydrogel by co-assembly. Spraying before harvesting, it is easy to adhere and accumulate on the surface of the fruits, enhancing the preservation and antibacterial ability, improving the preservation effect of the fruits, and reducing the risk of physical damage caused by postharvest treatment.

[0007] In order to achieve the above object, the technical scheme adopted by the present application is:

[0008] A preparation method of a glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel, comprising the following steps:

[0009] Dissolve glycyrrhizic acid in water, continuously stir at 70-90 DEG C until completely dissolved to obtain a glycyrrhizic acid solution, then add oxymatrine and subsequently add baicalein, stir uniformly, and then cool to room temperature to form a glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel.

[0010] Further limit, the molar ratio of the oxymatrine and glycyrrhizic acid is below 1:2; the molar ratio of the glycyrrhizic acid and baicalein is 12:(0.25-6.0).

[0011] Further limit, the molar ratio of the glycyrrhizic acid, oxymatrine and baicalein is 2:1:1.

[0012] Further limit, the concentration of the glycyrrhizic acid solution is above 0.5 mmol / L.

[0013] Preferably, the concentration of the glycyrrhizic acid solution is above 1.0 mmol / L.

[0014] Further limit, the time required for uniform stirring is 5-10 min.

[0015] A glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel prepared by the preparation method of the glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel.

[0016] Application of the glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel in preservation of soft-skinned berry fruits before picking.

[0017] Application of the glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel in preservation of strawberries before picking.

[0018] Further limit, in the green-ripe stage of strawberries, dilute the glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel by 20 times, and spray 3 mL per plant on the surface of the strawberries; spray every 7 days for a total of 3 times.

[0019] Further limit, when the glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel is applied in preservation of strawberries before picking, the content of soluble sugar, vitamin C, flavonoids and phenolic substances in the strawberries is significantly increased, and the activity of superoxide dismutase and peroxidase in the strawberries is significantly improved.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The mechanism of action of the glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel (referred to as GOB-gel) of the present application: baicalein (referred to as BBR) destroys the strong ionic hydrogen bond between glycyrrhizic acid (referred to as GA) and oxymatrine (referred to as OMT) through electrostatic attraction and pi-pi stacking, so that the carboxyl group of GA is released, thereby forming a crosslinked network and enhancing the structural strength of the hydrogel. The GOB-gel produces ROS (reactive oxygen species) under white light irradiation, which can destroy the cell membrane of bacteria, has a light-enhanced antibacterial effect, and can achieve preservation of soft-skinned berries such as strawberries, raspberries, blueberries and blackberries. The specific technical effects are as follows:

[0022] 1. The glycyrrhizic acid / oxymatrine / baicalein co-assembled hydrogel prepared by the present application, BBR can promote the co-assembly between GA and OMT, reduce the critical concentration of the formation of the hydrogel, and enhance the adhesion of the diluted GOB-gel; BBR also enhances the intermolecular interaction. When preserved before picking, it can improve the quality of strawberries, enhance the antioxidant capacity and prolong the shelf life.

[0023] 2. The research results of the present application show that spraying GOB-gel before picking can significantly increase the content of soluble sugar, vitamin C, flavonoids and phenolic substances in strawberries, effectively improve the flavor of strawberries and improve their nutritional value.

[0024] 3. The research results of the present application show that spraying GOB-gel before picking can significantly improve the activity of superoxide dismutase (SOD) and peroxidase (POD) in strawberries, thereby enhancing the ability of strawberries to resist oxidative damage.

[0025] 4. Spraying GOB-gel on strawberries before picking, and storing them at 25℃ for 6 days after picking, the weight loss rate of the strawberries is 23%, and the rot rate is 7%; storing them at 4℃ for 14 days, the strawberries have no rot phenomenon, and the preservation effect is obviously better than that of other treatment groups. It shows that GOB-gel can prolong the shelf life of strawberries and achieve efficient preservation.

[0026] 5. The GOB-gel of the present application is composed of natural ingredients GA, OMT and BBR, and has no cytotoxicity and hemolytic effect, which meets the requirements of food safety and is more environmentally friendly and safe.

[0027] 6. The GOB-gel of the present application is dissolved in water, and the preservation is completed by spraying before picking. Not only is the operation simple and convenient, but also the physical damage to strawberries caused by post-harvest treatment and the risk of fruit rot are avoided, further improving the shelf life of strawberries. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the inverted bottle physical map of GA and OMT in different proportions;

[0029] Figure 2Figure 1. Inverted bottle experiment of GA and OMT in 2:1 molar ratio at different concentrations;

[0030] Figure 3 Figure 2. GOB-gel phase transition diagram of GA, OMT and BBR in different ratios;

[0031] Figure 4 Figure 3. FT-IR spectra of GA+OMT mixture and GOB-gel at different concentrations in D20; 1 H NMR spectra;

[0032] Figure 5 Figure 4. Interaction diagram between GA, OMT and BBR molecules;

[0033] Figure 6 Figure 5. Photographs of GOB-gel preparation process and its fluorescence properties under 365 nm UV light;

[0034] Figure 7 Figure 6. Photographs of GOB-gel dissolution in water at each step under white light and 365 nm UV light;

[0035] Figure 8 Figure 7. Microscopic SEM and TEM images of GOB-gel;

[0036] Figure 9 Figure 8. Time sweep of GOB-gel measured at 1% strain and 1 Hz frequency for 540 s;

[0037] Figure 10 Figure 9. Strain dependent oscillatory shear rheology of GOB-gel at fixed frequency of 1 Hz;

[0038] Figure 11 Figure 10. Angular frequency sweep of GOB-gel at 1% strain;

[0039] Figure 12 Figure 11. Step strain results of GOB-gel at 1 Hz frequency and different strains for four cycles;

[0040] Figure 13 Figure 12. Zeta potential value of GOB-gel;

[0041] Figure 14 and Figure 15 Figure 13. UV-Vis spectra of GA+OMT and GOB-gel;

[0042] Figure 16 and Figure 17 Figure 14. Fluorescence spectra of GA+OMT and GOB-gel;

[0043] Figure 18 Figure 15. FT-IR spectra of GOB-P(GA+OMT+BBR powder) and GOB-gel;

[0044] Figure 19 FT-IR spectra of OMT, BBR, GA and GOB-gel;

[0045] Figure 20 and Figure 21 XRD analysis of OMT, BBR, GA and GOB-gel;

[0046] Figure 22 Structural changes of GOB system from 0 to 100 ns during simulation;

[0047] Figure 23 Structural changes of GOB-gel system at initial and final states;

[0048] Figure 24 Molecular interactions of GOB-gel system;

[0049] Figures 25-28 Molecular simulation parameters of GOB-gel; Figure 25 RMSD; Figure 26 SASA; Figure 27 Number of hydrogen bonds; Figure 28 Time evolution of radius of gyration of GOB system during simulation;

[0050] Figure 29 Cell viability assay after treatment of RAW264.7 cell line with different concentrations of GA and GOB-gel at 37 °C for 24 h;

[0051] Figure 30 Hemolysis assay results of RBCs;

[0052] Figure 31 Photos of respective RBC solutions after centrifugation;

[0053] Figure 32 Photos of RBC solutions treated with 1% TX-100, 0.9% physiological saline, BBR and GOB-gel after equilibration;

[0054] Figure 33 Photos of strawberries before and after spraying GOB-gel under natural and UV light;

[0055] Figure 34 Adhesion rate of GOB-gel on strawberry fruits;

[0056] Figure 35 SOD activity of fruits; Figure 36 POD activity of fruits; Figure 37 Flavonoid content of fruits; Figure 38 Phenolic content of fruits;Figure 39 for the soluble sugar content of the fruit; Figure 40 for the titratable acid content of the fruit; Figure 41 for the vitamin C content of the fruit;

[0057] Figure 42 Evaluation of the preservation effect of GOB-gel on strawberries: (A) visual morphological changes of strawberries stored at 25°C; (B) visual morphological changes of strawberries stored at 4°C; (C) weight loss rate of strawberries stored at 25°C; (D) weight loss rate of strawberries stored at 4°C; (E) rot rate of strawberries stored at 25°C; (F) rot rate of strawberries stored at 4°C;

[0058] In the figure: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant. DETAILED DESCRIPTION

[0059] The present application will be further described in conjunction with the accompanying drawings and examples, but the embodiments of the present application are not limited thereto. Other methods for preparing the compounds of the present application are considered to be within the scope of the present application, with some routine modifications of the reaction conditions according to the present application.

[0060] Unless otherwise defined, technical or scientific terms used in the present application shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0061] It should also be understood that the above-described embodiments are merely intended to explain the present application, and the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent substitutions or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

[0062] The present application co-assembles glycyrrhizin, oxymatrine and berberine to form a hydrogel, which is then used for fruit preservation before harvesting (pre-harvest period).

[0063] The raw materials used in the application: glycyrrhizinic acid (GA) is a triterpenoid saponin derived from licorice plants, having an amphiphilic structure; berberine (BBR) is a natural isoquinoline alkaloid derived from coptis, having broad-spectrum antibacterial activity. Glycyrrhizinic acid and a high proportion of oxymatrine co-assemble to form an ionic liquid, which can enhance drug solubility and transmembrane absorption; however, when glycyrrhizinic acid and oxymatrine co-assemble, oxymatrine will destroy the gel-forming ability of glycyrrhizinic acid; at this time, the addition of berberine (BBR) can reduce the critical concentration of GA to form a hydrogel, realize light-induced reactive oxygen species (ROS) generation and enhance the antibacterial effect. Therefore, the application uses the above three raw materials to prepare a nanofiber co-assembly hydrogel, which is driven by intermolecular electrostatic attraction and hydrogen bonding to cooperatively assemble nanofibers; the addition of berberine enhances the hydrogel formation ability, and the hydrogel can maintain a suspended state in water, easily adhere and accumulate on the surface of the fruit; the hydrogel is sprayed on the fruit in the pre-harvest period before picking, enhancing the preservation and antibacterial ability, greatly improving the fruit quality and post-harvest storage performance, and improving the flavor; at the same time, the risk of physical damage caused by post-harvest processing is reduced.

[0064] The technical solutions of the application are described in detail in several groups of examples.

[0065] It should be noted that in the following examples, unless otherwise specified, the chemicals and chemical reagents used are commercially available products commonly used in the art.

[0066] It should be noted that in the following examples, unless otherwise specified, the operations used are conventional operations; for example, unless otherwise specified, the operation temperature is room temperature operation. Unless otherwise specified, the test method is the existing standard test method in the art.

[0067] It should be noted that in the following examples, mM and μM both represent concentration units, mM refers to mmol / L, and μM refers to μmol / L.

[0068] The reagents and instruments used in the following examples are as follows.

[0069] Oxymatrine (purity > 98%, C 15 H 24 N2O2), berberine (purity > 98%, C 20 H 18 ClNO4) and glycyrrhizinic acid (purity > 95%, C 42 H 62 O 16) were purchased from Shanghai Adamas Reagent Co., Ltd. C57BL / 6 mice (6 weeks old) used for extracting mouse red blood cells were purchased from Shanghai Bikaike Biotechnology Co., Ltd. and used in accordance with the regulations approved by Shanghai University of Traditional Chinese Medicine (Approval No. PZSHUTCM210903005). Strawberry (Fragaria × ananassa Duch. ‘Akihime’) seeds were purchased from Shenyang Qiangfeng Seed Industry Co., Ltd. Other chemical reagents were purchased from Shanghai Titan Science and Technology Co., Ltd. and were all of analytical purity without further purification.

[0070] Preferred examples, preferred ratio of raw materials for preparing GOB-gel

[0071] In the preparation of co-assembled hydrogel, it was found that OMT significantly weakened the hydrogel formation ability of GA in a concentration-dependent manner, thereby limiting the ability of GA to load OMT, which could affect the performance of GA / OMT hydrogel. Therefore, the ratio of GA and OMT was optimized in the present application.

[0072] (1) Preferred molar ratio of GA and OMT

[0073] GA was dissolved in 100 mL ultrapure water, continuously stirred at 80°C until completely dissolved to obtain a GA solution; then OMT was added, stirred for 5 min, and cooled to room temperature to form a glycyrrhizinic acid / oxymatrine hydrogel.

[0074] In the glycyrrhizinic acid / oxymatrine hydrogel, the concentration of GA was 12 mM, and the concentration of OMT was 4 mM, 6 mM, 9 mM and 12 mM, respectively. Four groups of glycyrrhizinic acid / oxymatrine hydrogels were finally prepared.

[0075] The GA solution and the four groups of glycyrrhizinic acid / oxymatrine hydrogels were respectively loaded into inverted bottle experiments, and observed at 10 seconds, 20 seconds and 30 seconds of inversion; the results are shown in Figure 1 It can be seen that when the molar ratio of OMT to GA is higher than 1 / 2, GA and OMT cannot form stable hydrogel.

[0076] (2) Preferred concentration of GA

[0077] The molar ratio of GA and OMT was set to 2:1, and different concentrations were set, i.e. the concentration of GA was 16 mM, 12 mM, 8 mM, 6 mM, 4 mM, 2 mM, 1 mM and 0.5 mM, respectively. Eight groups of glycyrrhizinic acid / oxymatrine hydrogels were prepared by referring to the above method, and were respectively loaded into inverted bottle experiments, and observed at 10 seconds, 20 seconds and 30 seconds of inversion; the results are shown in Figure 2 .

[0078] From Figure 1 and Figure 2It can be seen that: when the molar ratio of OMT to GA is not higher than 1 / 2, the concentration of GA is above 0.5 mmol / L, and GA and OMT can be co-assembled to form hydrogel.

[0079] (3) Effect of GA and OMT ratio on co-assembled hydrogel

[0080] Add BBR to different glycyrrhizinic acid / oxymatrine systems to investigate the effect of GA and OMT ratio on the performance of BBR co-assembled hydrogel.

[0081] Dissolve GA in 100 mL ultrapure water, continuously stir at 80°C until completely dissolved, prepare GA solution. Add OMT, then add BBR, stir for 5 min, then cool to room temperature to form glycyrrhizinic acid / oxymatrine / berberine co-assembled hydrogel.

[0082] The concentration of each raw material in the system is designed as follows:

[0083] The first group: the concentration of GA and OMT is 9: 12 mM+24 mM, 12 mM+12 mM, 12 mM+6 mM, 12 mM+4 mM, 3 mM+9 mM, 3 mM+6 mM, 3 mM+1.5 mM, 2 mM+1 mM, 1 mM+0.5 mM, respectively, recorded as: 12(1:2), 12(1:1), 12(2:1), 12(3:1), 3.0(1:3), 3.0(1:2), 3.0(2:1), 2.0(2:1), 1.0(2:1); the concentration of BBR added under the above ratio is 0 mM, 0.25 mM, 0.5 mM, 1.0 mM, 1.5 mM, 3.0 mM, 6.0 mM.

[0084] The second group: the concentration of GA and OMT is 9: 16 mM+8 mM, 12 mM+6 mM, 8 mM+4 mM, 6 mM+3 mM, 4 mM+2 mM, 3 mM+1.5 mM, 2 mM+1 mM, 1 mM+0.5 mM, 0.5 mM+0.25 mM, respectively, recorded as: 16(2:1), 12(2:1), 8.0(2:1), 6.0(2:1), 4.0(2:1), 3.0(2:1), 2.0(2:1), 1.0(2:1), 0.5(2:1); the concentration of BBR added under the above ratio is 0 mM, 0.25 mM, 0.5 mM, 1.0 mM, 1.5 mM, 3.0 mM, 6.0 mM.

[0085] The glycyrrhizinic acid / oxymatrine / berberine co-assembled hydrogel of each group above is obtained, and its phase diagram is shown in Figure 3 .

[0086] Glycyrrhizin / Oxymatrine hydrogels formed at GA and OMT concentrations of 12 mM + 12 mM (1:1), 12 mM + 6 mM (2:1); GA, OMT and BBR concentrations of 12 mM + 6 mM + 2 mM (6:3:1), 12 mM + 4 mM + 4 mM (6:3:3), 6 mM + 3 mM + 6 mM (6:3:6). The NMR spectra of GOB-gels at different concentrations were measured by using a nuclear magnetic resonance spectrometer (AVANCE III, Bruker, Germany), and the results are shown in Figure 4 .

[0087] From Figure 3 it can be seen that when the proportion of OMT is not higher than 1 / 2 of GA, even at low concentrations, a certain concentration of BBR can promote the transition of GA + OMT from a solution state to a gel state, and when the proportion of OMT is higher than 1 / 2 of GA, it is difficult to form a gel even if BBR is added Figure 4 , but the solubility of BBR in the GA solution can be increased; BBR (0.25 mM) can trigger the gelation of GA + OMT when the concentration of GA is 1 mM and the concentration of OMT is 0.5 mM.

[0088] Referring to Figure 5 , further shows the interaction mechanism between GA, OMT and BBR molecules. There are N + -O- coordination bonds and C=O bonds in OMT, which endow OMT with significant proton accepting ability; on the other hand, GA can easily provide protons due to the presence of three polar and acidic similar carboxyl groups. When the molar concentration of OMT is less than 1 / 2 of GA, BBR can bind to part of OMT, thereby releasing part of the carboxyl groups of GA, so that it has enough cross-linking to support the network structure Figure 5 (A); this explains the phenomenon that BBR can promote and improve the gel formation of the mixture of GA and OMT. When the molar concentration of OMT exceeds 1 / 2 of GA, the strong ionic hydrogen bond interaction between N + -O- and -COOH makes GA lack enough cross-linking to support the network structure, resulting in OMT and GA having the properties of ionic liquid Figure 5 (B), which explains the phenomenon that high proportion of OMT can promote the solubility of BBR in GA.

[0089] In summary, when preparing glycyrrhizin / oxymatrine / berberine co-assembled hydrogels, the molar concentration of OMT is less than 1 / 2 of GA, and the concentration of GA is above 0.5 mmol / L, preferably the concentration of GA is above 1.0 mmol / L.

[0090] Example 1

[0091] The embodiment provides a preparation method of glycyrrhizinic acid / oxymatrine / baicalein co-assembled hydrogel, and comprises the following steps.

[0092] 988.3 mg (1.2 mmol) of GA is dissolved in 100 mL of ultrapure water, and stirring is continuously carried out at 80 DEG C until complete dissolution, so that a GA solution with a concentration of 12.0 mM is obtained;

[0093] The GA solution is kept at 80 DEG C, 159.3 mg (0.6 mmol) of OMT is added, then 223.2 mg (0.6 mmol) of BBR is added, stirring is carried out for 5 min, and then cooling is carried out to room temperature, so that glycyrrhizinic acid / oxymatrine / baicalein co-assembled hydrogel is formed, and is recorded as GOB-gel.

[0094] During the preparation process, photos of each raw material and reaction product and fluorescence characteristics thereof under 365 nm ultraviolet light are taken, as shown in the left green dashed box in FIG. 1, Figure 6 At the same time, the prepared GOB-gel is injected into water, Figure 6 The rightmost view in FIG. 1. Further, the GOB-gel is diluted with 20 times water at 40 DEG C, heated at 80 DEG C for 5 min, and incubated at 25 DEG C for 4 h, and a digital photo is taken under white light and 365 nm ultraviolet light at each step (as shown in FIG. 1, Figure 7 FIG. 1F, Figure 7 FIG. 1G is a close-up of the GOB-gel diluted 20 times at 40 DEG C under 365 nm ultraviolet light; Figure 7 FIG. 1H is a close-up of the GOB-gel diluted 20 times, heated and placed at 25 DEG C for 4 h under 365 nm ultraviolet light. It can be seen that the GOB-gel prepared in the embodiment has excellent fluorescence and gel characteristics.

[0095] Embodiment 2

[0096] The embodiment provides an application of glycyrrhizinic acid / oxymatrine / baicalein co-assembled hydrogel in fruit preservation before picking.

[0097] In the embodiment, strawberries are taken as examples for illustration. In the preservation application, the GOB-gel obtained in Embodiment 1 is diluted 20 times at the green-ripe stage of strawberries, so that the concentration after dilution is 12 mM GA+6 mM OMT+6 mM BBR, and the use amount is 3 mL per plant, and spraying is carried out every 7 days, and a total of 3 times.

[0098] Further, the glycyrrhizinic acid / oxymatrine / baicalein co-assembled hydrogel (GOB-gel) prepared in Embodiment 1 is taken as a research object, and performance advantages of the GOB-gel of the embodiment are verified through the following performance analysis.

[0099] Verification 1, morphology of the hydrogel

[0100] To more intuitively understand the microstructure inside the hydrogel, SEM and TEM were used to characterize the constructed hydrogel.

[0101] Scanning electron microscope images: 10 μL of GOB-gel was placed on a clean silicon wafer and frozen at -20 °C. After the sample was freeze-dried, it was imaged using a Zeiss Gemini 300 field emission scanning electron microscope (Carl Zeiss AG, Germany). Transmission electron microscope images: 10 μL of the GOB-gel solution was dropped on a copper grid with an ultra-thin carbon film, stained with 2% phosphotungstic acid, and TEM analysis was performed using a Talos L120C transmission electron microscope (Thermo Fisher Scientific, USA). The results are as Figure 8 shown, where A is SEM and B is TEM.

[0102] Figure 8 shown, the GOB-gel scaffold is a 3D network composed of nanofibers. It has been confirmed that GA self-assembles into nanofibers through hydrogen bonds, which is consistent with the observations in SEM and TEM.

[0103] Verification 2. Rheology

[0104] Rotational rheology was performed to analyze the viscoelastic characteristics of the hydrogel.

[0105] The rheological properties of the hydrogel were measured using a Kinexus ultra+ rheometer (Malvern Panalytical Ltd., UK), and all tests were carried out at 25 °C. The rheometer fixture was a steel cone plate with a diameter of 15 mm and a gap of 1.0 mm. The test conditions in different modes were as follows: 1) Time sweep, strain 1%, frequency 1 Hz, duration 540 s; 2) Dynamic frequency sweep, strain 1%, angular frequency range 0.1 - 100 rad·s -1 ; 3) Strain step sweep test, low strain 1%, high strain 100%, interval time 6 min, frequency 1 Hz; 4) Steady-state viscosity sweep mode, shear rate 0.1 - 100 s -1 . To test the injectability of the GOB-gel, the hydrogel was injected into a 5 mL sample bottle filled with ultrapure water at 25 °C using a 1.0 mL syringe, and the visual observation images were recorded.

[0106] As Figure 9 shown in the time sweep results, the GA+OMT solution showed relatively low storage modulus (G') and loss modulus (G”), and the values of G' and G” were quite similar, indicating that the pure GA+OMT solution did not form a hydrogel. After adding BBR, both G' and G” increased significantly, and G' was always much higher than G” (gel state: G'>G”, solution state: G'<G”), indicating that BBR promoted the gelation of the GA+OMT solution. As Figure 10As shown, GOB-gel started to undergo gel-sol transition when the strain exceeded 2%, while GA hydrogel only started to undergo this transition when the strain exceeded 17%. This indicates that the addition of BBR increased the gel strength of GA hydrogel. Dynamic frequency sweep results showed that G' was 4 times of G" for GOB-gel, while G' was 5 times of G" for GA. Figure 11 ) GOB-gel and GA hydrogel both exhibited stronger elasticity at higher frequency (10 Hz). These collectively indicated that GOB-gel had good stability.

[0107] As shown in Figure 12 , the step strain test results of GOB-gel showed that G' exceeded G" at a low strain level of 1%, which indicated that the structure of crosslinked hydrogel was stable. When the strain increased to 100%, G' decreased and was lower than the value of G", this change in modulus value provided evidence that GOB-gel exhibited excellent injectability, sprayability and self-repairing ability.

[0108] Verification 3, Zeta potential results

[0109] The Zeta potentials of GA, BBR, OMT and GOB-gel were measured by using a Zeta potential and particle size analyzer (Malvern Zetasizer Nano ZS90, Malvern Panalytical, UK) to study the electrostatic interaction between GA, OMT and BBR.

[0110] Referring to Figure 13 , the negatively charged OMT increased the surface potential of negatively charged ammonium glycyrrhizinate salt (AGAS) from -27.53 mV to -6.62 mV, indicating that OMT destroyed the nanofiber structure of GA and weakened its hydrogel formation ability. After loading positively charged BBR, the surface potential of negatively charged GA+OMT shifted from -6.62 mV to -21.07 mV, indicating that BBR partially replaced OMT combined with GA. This substitution enhanced the gel formation ability of GA+OMT, and BBR molecules adhered to GA nanofibers through electrostatic attraction.

[0111] Verification 4, UV-visible spectrum

[0112] The aggregation of BBR in hydrogel was verified using UV-visible spectrum. The UV absorption spectra of GA, BBR, OMT and GOB-gel were analyzed using a UV-visible spectrophotometer (UV-1900i, Shimadzu, Japan).

[0113] GOB-gel b, e, f, g and h were prepared by adding 3.0 mM, 1.5 mM, 1.0 mM, 0.5 mM and 0.25 mM BBR to 6 mM GA+3 mM OMT, respectively.

[0114] See also Figure 14 and Figure 15 UV absorption analysis revealed that BBR dominated the absorption, particularly at 320-360 nm and 400-440 nm, corresponding to π-π and n-π transitions in the isoquinoline ring. Furthermore, GOB-gel a (12 mM GA + 6 mM OMT + 6 mM BBR) was diluted 2-, 6-, and 12-fold to yield GOB-gel b, GOB-gel c, and GOB-gel d. Comparative studies revealed that the aromatic chromophore absorption of BBR in the hydrogels was red-shifted, indicating its involvement in the co-assembly process.

[0115] Verification 5. Photoluminescence (PL) spectroscopy

[0116] The photoluminescence (PL) spectra were collected using a fluorescence spectrophotometer (RF-6000, Hitachi, Japan), and the emission spectra of the hydrogels were measured in the range of 450–700 nm at λex = 365 nm.

[0117] See also Figure 16 and Figure 17 , the fluorescence emission spectrum of GOB-gel shows a single peak at about 560 nm. In addition, in the GOB-gel system, increasing the BBR concentration leads to a blue shift of the emission peak and a significant increase in intensity ( Figure 16 ), which indicates a strong interaction between BBR and GA / OMT.

[0118] Verification 6, FTIR spectroscopy

[0119] Fourier transform infrared spectroscopy (FTIR, Thermo Fisher Nicolet iS10, Thermo Fisher Scientific, USA) was used to investigate the functional groups of GA, BBR, OMT, and GOB-gels.

[0120] The GOB-gel peak at 3234 cm in the FTIR spectrum -1 The hydroxyl absorption peak intensity at the GA / OMT / BBR mixture is significantly higher than that at the GA / OMT / BBR mixture ( Figure 18 ) and GA( Figure 19 These results further confirmed the strong hydrogen bonding interaction between GA and OMT and BBR. By comparing the infrared spectra of pure OMT and GOB-gel, the N + -O-2998cm -1 The peak at 37° decreased in intensity in the GOB-gel, indicating that during the formation of the GOB-gel, N + -O- participates in the interaction with GA and BBR. The GOB-gel spectrum corresponds to the characteristic quaternary ammonium imine ion (C=N + ) of 1538cm-1 Peak reduction at 2theta Figure 18 ), attributed to the electrostatic interaction between BBR and GA, consistent with the zeta potential results.

[0121] Verification 7, XRD spectra

[0122] X-ray diffraction (XRD) analysis was performed using a Bruker D8 Avance (Bruker, Germany).

[0123] XRD of OMT and BBR Figure 20 and Figure 21 showed multiple characteristic crystalline peaks in the 0-40° angle range. However, the GOB-gel diffraction pattern showed complete disappearance of the crystalline peaks, indicating the formation of amorphous particles. Loss of crystalline structure in colloidal particles due to spontaneous interactions is a widely documented phenomenon. The combination of GA, OMT and BBR led to molecular reorganization, resulting in the formation of new two-dimensional structures in the GOB-gel. This reorganization involved the formation of weak bonds, subsequently leading to the disappearance or reduction in intensity of the broad peaks originally observed in GA in the 10-25° angle range.

[0124] Verification 8, Molecular dynamics simulation of GOB-gel

[0125] The co-assembly mechanism of GA, OMT and BBR was investigated using molecular dynamics (MD) simulations.

[0126] Atomistic molecular dynamics simulations were performed using the GROMACS 2020.6 software package, employing the GAFF2 force field and TIP3P water model. GA, BBR and OMT molecules were randomly placed in a cubic box of 6 nm x 6 nm x 6 nm, solvated with 6000+ water molecules. After energy minimization, the system was equilibrated under NPT conditions, followed by a 100 ns production run under NVT ensemble. The temperature was set to 298 K by the Nose-Hoover method, and the pressure was maintained at 1 atm by the Parrinello-Rahman method. Non-bonded interactions used a 1.2 nm cutoff, and long-range electrostatic interactions were treated using the Particle Mesh Ewald method (0.1 nm Fourier spacing). All bonds containing hydrogen atoms were constrained using LINCS. Hydrogen bonds were identified when the donor-acceptor angle < 30° and the donor-acceptor distance < 0.35 nm.

[0127] The molecular structure of the GA-OMT-BBR system evolved over time Figure 22 ). During the 20 ns simulation, the molecules in the system continuously aggregated, eventually forming a stable nanofiber cluster structure. After 100 ns of simulation, the aggregation of GA, OMT and BBR (2:1:1) with water molecules was observed in the simulation box system Figure 23 andFigure 24 ).

[0128] The average root mean square deviation (RMSD) for assessing the range of molecular motion was 3.46 ± 0.31 nm Figure 25 , indicating that the system remained stable throughout the simulation. The average solvent accessible surface area (SASA) of the GOB system was 69.08 ± 10.75 nm 2 . From 0 to 10 ns, the SASA decreased significantly, indicating reduced solvent exposure and stabilization of a denser nanocluster Figure 26 . There were an average of 23.35 ± 3.23 hydrogen bonds Figure 27 in the system. OMT and GA formed strong intermolecular ionic hydrogen bonds between N + -O- and -COOH, leaving GA with insufficient cross-linking to support a network structure Figure 24-1 . BBR disrupted the strong intermolecular ionic hydrogen bonds formed by OMT and GA, with intermolecular interactions of BBR being π-π stacking Figure 24-2 . Changes in the radius of gyration (Rg) over time indicated a transition from a dispersed state to a tightly connected aggregate Figure 28 . From 0 to 15 ns, the decrease in Rg indicated an increase in interactions between GA, OMT, and BBR, likely due to non-covalent bonds such as hydrogen bonds and π-π stacking. From 15 to 100 ns, the Rg values were consistent, suggesting the formation of stable complexes between GA, OMT, and BBR. In summary, the MD simulation indicated that electrostatic interactions, hydrogen bonds, and π-π stacking were key driving forces for GOB-gel formation.

[0129] Verification 9, AIE behavior of GOB-gel

[0130] Organic luminophores with aggregation-induced emission (AIE) have negligible fluorescence as single molecules but enhanced emission upon aggregation. When exposed to 365 nm UV light, the GA / OMT hydrogel and solution were non-fluorescent, while BBR alone emitted weak fluorescence. For the GOB-gel composed of GA, OMT, and BBR, the luminescence increased significantly with BBR concentration at a fixed level of GA / OMT Figure 17 , indicating that BBR had typical AIE behavior. Dilution of the GOB-gel with ultrapure water resulted in a concentration-dependent decrease in fluorescence, which was rapidly quenched below a critical threshold Figure 16 . In the hydrogel network, the close packing of BBR molecules limited their translational motion and conformational flexibility, minimizing non-radiative energy dissipation. However, isolated BBR molecules exhibited predominant non-radiative decay, resulting in weak fluorescence emission. AIE materials typically enhance ROS production under light exposure. Increased ROS effectively kills bacteria, making these materials promising for non-thermal sterilization methods.

[0131] Verification 10, biocompatibility of GOB-gel

[0132] The present study evaluated the biocompatibility by cytotoxicity and hemolytic activity.

[0133] (1) In vitro blood compatibility assay

[0134] Red blood cells (RBCs) were isolated by centrifugation of fresh mouse blood at 2000 rpm for 10 min at 4 °C, and the isolated RBCs were diluted with 0.9% physiological saline to a 2% (v / v) RBC suspension. The positive control group and the negative control group used 1% Triton X-100 (TX-100) and 0.9% physiological saline, respectively. 20 μL of each sample was added to 1 mL of the RBC suspension, incubated at 37 °C for 60 min, and centrifuged at 4 °C. For hemolysis testing of GOB-gel, 0.5 mL of positive / negative control supernatant was mixed with 0.5 mL of 0.12 mM GOB-gel solution (BBR concentration equivalent to 0.12 mM according to the molar ratio in GOB-gel), while the test sample was diluted with ultrapure water to 1.0 mL. The same protocol was used for BBR hemolysis testing, and photographs of the supernatant were taken and the absorbance was measured at 540 nm.

[0135] (2) Cytotoxicity

[0136] Cytotoxicity of GOB-gel was evaluated by CCK-8 assay using RAW264.7 macrophages. RAW264.7 cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated in a cell incubator at 37 °C, 5% CO2 until adherent. Test solutions were prepared for the experiment. After 24 h of incubation in a 5% CO2 environment, the medium was replaced with a medium containing 10% CCK-8 reaction solution, and the plate was further incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader to determine cell viability. The control group was the medium without hydrogel, and each experimental group was repeated three times.

[0137] The results show that after exposure to 100 μM of GA or GOB-gel for 24 hours, the cell viability remained above 90%, indicating that GOB-gel has good biocompatibility Figure 29 Due to the inherent color of BBR, the supernatant of the RBC suspension after incubation turned light yellow Figure 31 When BBR or GOB-gel was added to the control group supernatant, the color matched that observed when ultrapure water was added to the BBR or GOB-gel group supernatant Figure 32 Compared with the 1% TX-100 positive control, the hemolysis rate of the GOB-gel group was <5% Figure 30), indicating that GOB-gel has good compatibility with blood.

[0138] Verification 11, Adhesion effect of GOB-gel

[0139] The adhesion of hydrogel to the fruit surface and the subsequent sustained drug release are the basis of its efficacy.

[0140] Thirty similar-sized strawberries were selected, weighed, and placed in suitable containers. The strawberries were evenly sprayed with a sprayer. After spraying for 5 min, when the droplets stopped dripping, the strawberries were weighed again. The adhesion rate of the sprayed agent on the strawberries was calculated according to the following formula:

[0141] Adhesion rate (%) = [(weight after spraying - weight before spraying) / weight before spraying] x 100

[0142] By the foregoing Figure 7 It can be seen that: the diluted GOB-gel is not a simple aqueous mixture of GA, OMT, and BBR, but a suspension of GOB-gel particles in water. Therefore, the adhesion amount of ultrapure water, GA+OMT aqueous solution, and diluted GOB-gel was measured and compared, and the adhesion amount of diluted GOB-gel on the fruit surface was higher than that of the other two solutions Figure 34 ) After washing the sprayed strawberries with water, no green fluorescence was detected Figure 33 , which confirms that the sprayed GOB-gel exhibits easy washability.

[0143] Both GA and OMT exhibit good water solubility. Notably, OMT inhibits the ability of GA to form hydrogels and hinders the formation of stable gel structures. Therefore, at low concentrations, the adhesion amount of GA+OMT mixed solution on the fruit surface is low. In contrast, the addition of BBR significantly enhances the ability of GA to form stable hydrogels at low concentrations. These gel particles can easily adhere and accumulate on the fruit surface. This unique property ensures high adhesion and achieves controlled and sustained release of drugs.

[0144] Verification 12, Evaluation of GOB-gel on fruit flavor and quality

[0145] All plants have evolved enzymatic and non-enzymatic antioxidant systems to cope with various stresses. This study aims to investigate the antioxidant defense response of strawberries to GOB-gel.

[0146] Strawberries were planted in plastic pots in a greenhouse in Pingan District, Haidong City, Qinghai Province. At the green-ripe stage, the uniform plants were randomly divided into 3 groups (150 pots per group) using a block design.

[0147] GOB-gel pre-harvest group: GOB-gel was sprayed at 20-fold dilution, at which the solution contained 12 mM GA + 6 mM OMT + 6 mM BBR after dilution. GA + OMT pre-harvest group: GA + OMT was sprayed at 20-fold dilution, at which the solution contained 12 mM GA + 6 mM OMT after dilution. The control group was sprayed with distilled water.

[0148] Treatment (3 mL / plant) was applied once every 7 days for 3 times. Fruit and leaf samples were collected 3 days after the last spraying. Thirty strawberries in each group were weighed for fresh weight (FW), and then homogenized with distilled water.

[0149] The following indicators of strawberries were determined for each group: soluble sugar was determined by anthrone-sulfuric acid colorimetry, titratable acid was determined by alkali titration, vitamin C was determined by 2,6-dichloroindophenol method, total phenol and flavonoids were determined by Folin-Ciocalteu method and AlCl3 complex spectrophotometry, respectively. Peroxidase (POD) activity was determined by guaiacol colorimetry, and superoxide dismutase (SOD) was determined by water-soluble tetrazolium salt-1 (WST-1) method.

[0150] See Figure 35 and Figure 36 , the key enzymes SOD and POD in the active oxygen scavenging system showed significantly higher activity in GOB-gel treated strawberries than in the blank control, indicating that GOB-gel induced oxidative stress and activated the antioxidant system. Important phenolic compounds in plants increased after GOB-gel treatment, including flavonoids ( Figure 37 ) and phenols ( Figure 38 ). These secondary metabolites further enhanced the antioxidant and antibacterial capacity of strawberries. Strawberry flavor mainly depends on the content of sugar and acid flavor substances. GOB-gel increased the content of soluble sugar ( Figure 39 ), while the titratable acid content did not change significantly ( Figure 40 ), thereby improving the flavor balance. At the same time, the content of vitamin C also increased ( Figure 41 ). This study showed that GOB-gel treatment can improve the quality of strawberries.

[0151] Verification 13, Evaluation of GOB-gel on fruit preservation

[0152] Strawberries are highly perishable fruits, prone to rapid decay, significantly shortening their shelf life, while postharvest preservation practices often pose the risk of physical damage. A comparative study evaluated the preservation effect of GOB-gel applied in pre-harvest (pre-H) and post-harvest (post-H) on strawberries.

[0153] Strawberries were planted in plastic pots in a greenhouse in Ping'an District, Haidong City, Qinghai Province, and were used in a block design during the green-ripe period and after picking.

[0154] Control (Control), GA+OMT pre-harvest (GA+OMT pre-H), and GOB-gel pre-harvest (GOB-gel pre-H); GA+OMT post-harvest (GA+OMT post-H), and GOB-gel post-harvest (GOB-gel post-H).

[0155] The GOB-gel was sprayed at the above-mentioned rates for fruit flavor and quality evaluation onto the surface of the Control, GA+OMT post-harvest, and GOB-gel post-harvest strawberries. The fruits were stored at 25 °C, 50% relative humidity (RH) and 4 °C, 60% RH, and morphological changes and weight loss were evaluated at different time points.

[0156] Samples were stored at 25 °C and 4 °C, and the study focused on decay rate and weight loss rate Figure 42 At 25 °C, the Control strawberries exhibited severe decay with grayish mold Figure 42 A) within six days, accompanied by a decay rate Figure 42 E) of 54% and a weight loss Figure 42 C) of 36%. The GA+OMT pre-harvest and post-harvest groups exhibited similar severe decay Figure 42 E) within six days, while the GOB-gel pre-harvest and post-harvest groups exhibited slight discoloration Figure 42 A) and minimal decay (decay rates of 7% and 17%, respectively, Figure 42 E) and weight loss rates of 23% and 30% respectively Figure 42 C) after six days. At 4 °C, the storage time was extended for all groups. The GOB-gel pre-harvest group maintained good fruit shape Figure 42 B) and no decay Figure 42 F) after 14 days, with a weight loss rate of 20% Figure 42 D). After 26 days, its weight loss rate remained significantly lower than the other groups Figure 42 Figure 42 D).

[0157] The results indicate that spraying diluted GOB-gel either pre-harvest or post-harvest effectively delays decay and extends shelf life, with pre-harvest applications being the most effective, possibly due to increased production of endogenous antimicrobial and antioxidant substances from repeated pre-harvest treatments.

[0158] Based on the above performance research, the GOB-gel prepared by the application uses plant source co-assembly, BBR promotes the co-assembly between GA and OMT, reduces the critical concentration of the formation of the hydrogel, and enhances the adhesion of the diluted GOB-gel; BBR strengthens the intermolecular interaction; the in vitro biocompatibility test (hemolysis and cytotoxicity determination) shows that the GOB-gel has good safety; the GOB-gel increases the soluble sugar, vitamin C, flavonoids and phenolic substances in the strawberries, and enhances the SOD and POD activities. During the storage, the strawberries treated in the pre-harvest stage show the smallest weight loss and significantly lower rot rate compared with the control group, which is better than the post-harvest treatment. Therefore, the GOB-gel integrates the endogenous biosynthesis of antibacterial / antioxidant substances and the exogenous antibacterial effect, and provides a new technical path for improving the fruit quality and prolonging the postharvest shelf life.

[0159] The above performance verification is based on the GOB-gel prepared in Example 1, and when the molar ratio of oxymatrine and glycyrrhizic acid is still selected to be below 1:2, the molar ratio of glycyrrhizic acid and berberine is selected to be within 12:(0.25-6.0), the concentration of the glycyrrhizic acid solution is selected to be above 0.5 mmol / L, the stirring time is selected to be within 5-10 min, and the temperature is selected to be within 70-90℃, the GOB-gel formed also has the same or similar performance, and realizes the preservation of soft-skinned berries such as strawberries, raspberries, blueberries and blackberries.

[0160] The above are several more preferred embodiments of the preparation method of the application, but cannot be regarded as the limitation of the technical solutions protected by the application, and all the alternative solutions obtained by the ordinary skilled in the art based on the technical ideas of the application without creative labor should fall within the protection scope of the application.

Claims

1. A preparation method of a glycyrrhizinic acid / oxymatrine / baicalein co-assembled hydrogel, characterized in that, The method comprises the following steps: Dissolving glycyrrhizin in water, continuously stirring at 70-90℃ until completely dissolved to obtain a glycyrrhizin solution, adding oxymatrine, then adding berberine, stirring uniformly, and then cooling to room temperature to form a glycyrrhizin / oxymatrine / berberine co-assembled hydrogel.

2. The preparation method of the glycyrrhizinic acid / oxymatrine / berberine co-assembled hydrogel according to claim 1, characterized in that, The molar ratio of the oxymatrine and glycyrrhizin is below 1:2; the molar ratio of the glycyrrhizin and berberine is 12:(0.25-6.0).

3. The preparation method of the glycyrrhizinic acid / oxymatrine / berberine co-assembled hydrogel according to claim 2, characterized in that, The molar ratio of the glycyrrhizin, oxymatrine and berberine is 2:1:

1.

4. The preparation method of the glycyrrhizinic acid / oxymatrine / berberine co-assembled hydrogel according to claim 2, characterized in that, The concentration of the glycyrrhizin solution is above 0.5mmol / L.

5. The preparation method of the glycyrrhizinic acid / oxymatrine / berberine co-assembled hydrogel according to claim 1, characterized in that, The time required for uniform stirring is 5-10min.

6. A glycyrrhizin / oxymatrine / berberine co-assembled hydrogel prepared by the method for preparing a glycyrrhizin / oxymatrine / berberine co-assembled hydrogel according to claim 1.

7. Use of the glycyrrhizin / oxymatrine / berberine co-assembled hydrogel according to claim 6 in preserving soft-skinned berry fruits before picking.

8. Use of the glycyrrhizin / oxymatrine / berberine co-assembled hydrogel according to claim 6 in preserving strawberries before picking.

9. Use according to claim 8, characterized in that, In the green-ripe stage of strawberries, the glycyrrhizin / oxymatrine / berberine co-assembled hydrogel is diluted 20 times, and sprayed on the surface of the strawberries at a dosage of 3mL per plant; spraying is performed every 7 days, for a total of 3 times.

10. Use according to claim 8, characterized in that, When the glycyrrhizin / oxymatrine / berberine co-assembled hydrogel is used in preserving strawberries before picking, the content of soluble sugar, vitamin C, flavonoids and phenolic substances in the strawberries is significantly increased, and the activities of superoxide dismutase and peroxidase in the strawberries are significantly improved.

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