Method for improving stability of chlorogenic acid by using zein and lycium barbarum polysaccharide and application of chlorogenic acid in preparation of liver protection medicine

Abstract

The invention discloses a method for improving the stability of chlorogenic acid by using zein and lycium barbarum polysaccharide and application of chlorogenic acid in preparation of a liver protection medicine. The method comprises the following steps: (1) dissolving raw materials; (2) mixing the raw materials; and (3) centrifuging and freeze-drying. The encapsulation efficiency of the zein / lycium barbarum polysaccharide / chlorogenic acid ternary complex prepared by the invention on the chlorogenic acid can reach 85.66%, and the bioavailability and the liver protection effect of the chlorogenic acid can be respectively improved by 12.99% and 19.48%. The product has the advantages of easily available raw materials, convenient process and easy large-scale production, has significant antioxidant and liver protection activities, and can be used in the fields of functional foods, antioxidants, alcoholic liver injury protection drugs and the like.

Description

Technical Field

[0001] The present invention belongs to the field of improving the performance of food functional components, and particularly relates to a method for encapsulating chlorogenic acid using zein and wolfberry polysaccharide, and its applications in functional foods, antioxidants, and liver protection. Background Art

[0002] Hepatocyte damage caused by long-term heavy drinking can further lead to alcoholic liver disease (ALD). In recent years, plant natural products have been proven to play a liver-protecting role by improving hepatocyte damage. Chlorogenic acid (CGA) is a natural polyphenolic compound widely present in plants, with significant antioxidant activity and liver-protecting function, but its application is limited by problems such as low stability and rapid metabolism (Trends in Food Science & Technology, 2022, 123, 172 - 186).

[0003] Currently, delivery systems such as nanoparticles are usually used to improve the stability of natural products. However, traditional nano-delivery systems (such as simple protein or polysaccharide carriers) have limited effects on improving the stability of chlorogenic acid and lack functional synergy. For example, zein can form nanoparticles through the anti-solvent precipitation method, but its stability in the physiological environment is insufficient (Food Hydrocolloids, 2022, 124, 107251); while wolfberry polysaccharide (LBP) is difficult to form stable nanoparticles with chlorogenic acid due to steric hindrance effects (Food Chemistry, 2025, 471, 142779). Existing studies have shown that constructing composite carriers through multi-component co-assembly may break through the limitations of single materials (Nanomaterials, 2024, 14(2), 197). For example, protein-polysaccharide complexes can enhance the stability of the carrier through electrostatic layer-by-layer self-assembly, while endowing the delivery system with functional diversity. However, the research on CGA delivery is still limited to single carriers or simple physical mixtures, without fully utilizing the molecular interactions between components to optimize the structure, nor exploring the synergistic potential of food and medicine homologous components.

[0004] Aiming at the deficiencies of the existing technology, the present invention uses zein and LBP to prepare a ternary nanoparticle (ZLC NPs) delivery system with CGA, and evaluates its antioxidant and liver-protecting activities, etc., to provide support for its application in the fields of functional foods, etc. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the stability of chlorogenic acid using zein and wolfberry polysaccharide and its application in the preparation of liver-protecting drugs.

[0006] To achieve the above object and other related objects, the technical solution provided by the present invention is: A method for improving the stability of chlorogenic acid using zein and wolfberry polysaccharide, comprising the following steps:

[0007] Step 1: Disperse zein and chlorogenic acid in ethanol to obtain a zein-chlorogenic acid solution, and disperse wolfberry polysaccharide in pure water to obtain a wolfberry polysaccharide solution;

[0008] Step 2: Under stirring conditions, add the zein-chlorogenic acid solution to the wolfberry polysaccharide solution and continue stirring to obtain a mixed solution;

[0009] Step 3: Adjust the pH value of the mixed solution, and then after centrifugation and freeze-drying, obtain zein / wolfberry polysaccharide / chlorogenic acid nanoparticles.

[0010] The preferred technical solution is: In Step 1, the mass ratio of zein to chlorogenic acid is 4:1 - 20:1, the concentration of zein is 1 - 3 mg / mL; the concentration of wolfberry polysaccharide is 1 - 3 mg / mL.

[0011] The preferred technical solution is: In Step 2, the volume ratio of the wolfberry polysaccharide solution to the zein-chlorogenic acid solution is 1:1 - 3:1, the temperature at which stirring occurs is 20 - 50 °C, the rotation speed is 600 - 100 r / min, the stirring time is 30 - 60 min, and the addition speed of the zein-chlorogenic acid solution is 5 - 15 mL / min.

[0012] The preferred technical solution is: In Step 3, the pH value is 4 - 6, and the centrifugation speed is 3000 - 5000 r / min.

[0013] To achieve the above object and other related objects, the technical solution provided by the present invention is: The application of the product obtained by the above method in the preparation of liver-protecting drugs.

[0014] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are:

[0015] 1. The encapsulation efficiency of the drug-loaded nanoparticles ZLC NPs prepared by the present invention reaches up to 85.66%, and the bioavailability of chlorogenic acid during the digestion process is increased by 12.99%.

[0016] 2. The drug-loaded nanoparticles ZLC NPs of the present invention can extend the safe and effective dose range of the administration concentration of ZLC NPs to 25 - 50 μg / mL, and the upper limit of the safe concentration is increased by 100% compared with both ZC NPs (the complex formed by Zein and CGA, 6.25 - 25 μg / mL) and free CGA (6.25 - 25 μg / mL). At a pretreatment concentration of 30 μg / mL, ZLC NPs increase the survival rate of alcohol-damaged cells to 69.56%, which is 19.48% higher than that of free CGA (50.08%).

[0017] 3. The raw materials of the present invention are easily available, the operation is simple, the requirements for equipment are low, there is no need to invest in expensive production facilities, the production cost is low, and it is suitable for industrial production. Brief Description of the Drawings

[0018] Figure 1 It is a synthesis schematic diagram and apparent morphology diagram of zein / Lycium barbarum polysaccharide / chlorogenic acid nanoparticles prepared in Example 1.

[0019] Figure 2 It is the characterization result of zein / Lycium barbarum polysaccharide / chlorogenic acid nanoparticles prepared in Example 1.

[0020] Figure 3 It is the research results of the antioxidant and bioavailability and other functions of zein / Lycium barbarum polysaccharide-chlorogenic acid nanoparticles prepared in Example 1.

[0021] Figure 4 It is the non-toxic concentration of zein / Lycium barbarum polysaccharide-chlorogenic acid nanoparticles prepared in Example 1 and the survival rate of AML-12 hepatocytes after alcohol treatment.

[0022] Figure 5 It is the liver protection effect result of zein / Lycium barbarum polysaccharide-chlorogenic acid nanoparticles prepared in Example 1.

[0023] Figure 6 It is the SEM image of zein / Lycium barbarum polysaccharide-chlorogenic acid nanoparticles prepared in Example 1. Detailed Embodiments

[0024] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this embodiment.

[0025] Please refer to Figure 1-6It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to match the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the conditions under which the present invention can be implemented. Therefore, they do not have any substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size. The following examples are provided to better understand the present invention, rather than to limit the present invention. Unless otherwise specified, the experimental materials used in the following examples are all obtained from conventional consumables and biochemical reagent stores.

[0026] Example 1: A method for improving the encapsulation efficiency of chlorogenic acid using zein and lycium barbarum polysaccharide composite nanoparticles I. Preparation method of zein / lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles

[0027] The specific operation steps are as follows:

[0028] (1) Dissolution of raw materials

[0029] Dissolve 0.3 g of Zein powder in 150 mL of 85% (v / v) ethanol aqueous solution, and stir the mixture with a magnetic stirrer for 4 hours to obtain a zein solution (2 mg / mL). Dissolve lycium barbarum polysaccharide powder (0.9 g) in 600 mL of distilled water, stir for 4 hours to obtain a 1.5 mg / mL LBP solution, and let it stand for hydration at 4 °C for 12 hours. CGA is mixed with Zein at a mass ratio of 0.094:1 to form a CGA-Zein organic phase solution.

[0030] (2) Assembly by anti-solvent precipitation method of raw materials: Dropwise add the CGA-Zein organic phase into the LBP aqueous phase (volume ratio 3:1, total volume 40 mL), and stir in the dark at 600 rpm for 1 hour. Remove ethanol by rotary evaporation, and supplement the lost volume with distilled water. Adjust the pH to 4.56, and use a refrigerated centrifuge to centrifuge (3000 rpm, 15 minutes) to collect the supernatant suspension of nanoparticles. The prepared nanoparticle suspension is stored in the dark at 4 °C. In addition, the sample is freeze-dried and stored at -20 °C for further testing.

[0031] (3) Centrifugal extraction and freeze-drying

[0032] (3.1) Put the zein / lycium barbarum polysaccharide-chlorogenic acid nano-suspension obtained in step 2 into a refrigerated centrifuge, and the temperature of freeze-centrifugation is 4 °C, and the centrifugation speed is 3000 r / min.

[0033] (3.2) Aspirate the upper layer of the nano-suspension and freeze-dry it to obtain zein / lycium barbarum polysaccharide-chlorogenic acid (ZLCNPs) powder.

[0034] II. Preparation of comparative samples

[0035] (1) Preparation of zein, wolfberry polysaccharide, and chlorogenic acid mixture

[0036] Dissolve 0.3 g of Zein powder and 28.2 mg of chlorogenic acid powder in 150 mL of 85% (v / v) ethanol aqueous solution. Use a magnetic stirrer to stir the mixture for 4 hours to obtain a mixed solution of zein and chlorogenic acid. Subsequently, measure 10 mL of the mixed solution and directly mix it with 30 mL of 1.5 mg / mL LBP solution. Stir overnight by exposure to air until the ethanol evaporates, and perform the whole process in the dark. Then adjust the pH to 4.56, and use a refrigerated centrifuge to centrifuge (3000 rpm, 15 minutes) to remove undissolved large particles, obtaining a suspended mixture, which is the zein, wolfberry polysaccharide, and chlorogenic acid mixture.

[0037] (2) Preparation of zein and chlorogenic acid binary complex

[0038] Dissolve 0.3 g of Zein powder and 28.2 mg of chlorogenic acid powder in 150 mL of 85% (v / v) ethanol aqueous solution. Use a magnetic stirrer to stir the mixture for 4 hours to obtain a mixed solution of zein and chlorogenic acid. Dropwise add the CGA-Zein organic phase into distilled water (volume ratio 3:1, total volume 40 mL), and stir at 600 rpm in the dark for 1 hour. Remove ethanol by rotary evaporation, and supplement the lost volume with distilled water. Adjust the pH to 4.56, and use a refrigerated centrifuge to centrifuge (3000 rpm, 15 minutes) to collect the supernatant suspended nanoparticle solution, namely ZC NPs.

[0039] (3) Preparation of zein and chlorogenic acid mixture

[0040] Dissolve 0.3 g of Zein powder and 28.2 mg of chlorogenic acid powder in 150 mL of 85% (v / v) ethanol aqueous solution. Use a magnetic stirrer to stir the mixture for 4 hours to obtain a mixed solution of zein and chlorogenic acid. Stir overnight by exposure to air until the ethanol evaporates, and perform the whole process in the dark. Then adjust the pH to 4.56, and use a refrigerated centrifuge to centrifuge (3000 rpm, 15 minutes) to remove large particles, obtaining a suspended mixture, which is the zein and chlorogenic acid mixture. III. Optimization of the preparation process of zein / wolfberry polysaccharide-chlorogenic acid nanoparticles

[0041] Adopt Box-Behnken design for optimization, with particle size and encapsulation efficiency (EE) as response variables, and the independent variables being the CGA / Zein mass ratio (1:5 - 1:20), water phase / organic phase volume ratio (1:1 - 3:1), and pH value (4.0 - 6.0).

[0042] Table 1: Observed values of experimental runs and response variables in the Box-Behnken design.

[0043]

[0044] The optimal formulation conditions were determined as the mass ratio of CGA to zein being 0.094, the volume ratio of the aqueous phase to the organic phase being 3, and the pH value being 4.56. The predicted particle size after experimental verification of these conditions was 114.90 nm, and the EE was 83.44%. The observed particle size was 122.73 ± 0.25 nm, and the EE was 85.66 ± 0.95%, with error rates of 6.40% and 2.60% respectively. The PDI was 0.070, and the zeta potential was -27.83 mV, indicating that the ternary composite nanoparticles could form a tight structure while improving the encapsulation efficiency of CGA.

[0045] IV. Structure Identification of the Zein / Lycium barbarum Polysaccharide-Chlorogenic Acid Composite Nanoparticles Prepared in Step 1

[0046] 1. Determination of the Encapsulation Efficiency of Zein / Lycium barbarum Polysaccharide-Chlorogenic Acid Composite Nanoparticles

[0047] The freshly prepared nanoparticle suspension was centrifuged at 8000×g for 15 minutes at 4 °C. Then, 200 μL of the supernatant was collected and diluted with 1.8 mL of 85% (v / v) ethanol aqueous solution. The CGA content was quantitatively detected using a microplate reader at a wavelength of 330 nm. The CGA concentration was determined according to the standard calibration curve. CGA was diluted in 85% (v / v) ethanol aqueous solution to prepare a series of CGA concentrations (0, 12.5, 25.0, 37.5, 50.0, and 62.5 μg / mL), and their absorbances were measured at a wavelength of 330 nm. The linear regression equation of the standard curve was: Y = 0.0258X + 0.0048 (R 2 = 0.9999), where X and Y represent the CGA concentration and absorbance, respectively. The encapsulation efficiency of different nanoparticles was calculated using the following formula.

[0048]

[0049] After calculation, under the above conditions, the encapsulation efficiency of the zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles was 85.66%, which was greater than 61.03% of the zein-chlorogenic acid composite nanoparticles.

[0050] 2. Determination of the Average Particle Size, Polydispersity Index, and Zeta Potential

[0051] At room temperature, the average particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were measured using a Malvern particle size analyzer (ZEN3700, PerkinElmer, USA). Distilled water was used as the solvent for measuring the samples, and after homogenization, the instrument was directly used for measurement.

[0052] After detection, the average particle size of ZLC NPs under this condition was 122.73 nm, the PDI was 0.070, and the Zeta potential was -27.83 mV.

[0053] 3. The shape, size, and surface morphology of the nanoparticles were observed by transmission electron microscopy (JEM-2100F, Japan) and scanning electron microscopy (SU8600, Japan). After diluting the nanoparticle solution by a certain multiple, two drops of the diluted solution were dropped on a copper grid, stained for about 3 min and then removed, and then taken off with forceps and placed in a fixed position on the transmission electron microscope. The size and morphology of the nanoparticles were observed on the display screen.

[0054] After detection, the morphology of ZLC NPs was round particles with relatively uniform texture ( Figure 1 of G);

[0055] 4. The functional groups in the sample were analyzed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) using a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), which is mainly used for molecular fingerprint recognition, identification, and quantification. The spectral acquisition range was from 650 to 4000 cm -1 , and the data were analyzed.

[0056] After detection, in ZLC NPs, the O-H stretching peak shifted to 3298.55 cm -1 , and the stretching vibration intensity of C=O in CGA (1685.73 cm -1 ) decreased, indicating that hydrogen bonds were formed between CGA and zein. The characteristic C=O peak of lycium barbarum polysaccharide at 1701.53 cm -1 decreased, and a broad band near 2934.29 cm -1 was observed. The spectral changes confirmed that CGA was encapsulated in ZLC NPs and stabilized by hydrogen bonds and hydrophobic interactions ( Figure 2 of A).

[0057] 5. X-ray diffraction (XRD) analysis was performed using a SmartLab SE X-ray diffractometer (Rigaku Corporation, Tokyo, Japan). The X-ray diffraction patterns of different samples were recorded. The data were collected in continuous mode in the 2θ range of 5° - 40°.

[0058] After detection, in ZLC NPs, the sharp peaks of CGA almost completely disappeared, and broad amorphous peaks occupied the entire spectrum. This indicates that CGA was incorporated into ZLC NPs and transformed into an amorphous state ( Figure 2B).

[0059] 6. Fluorescence spectroscopy (FS) of the nanoparticle samples was performed using a Horiba Scientific FluoroMax-4 (Horiba Scientific, USA) with an excitation wavelength of 280 nm and an emission wavelength ranging from 280 to 400 nm.

[0060] Upon detection, after the formation of nanoparticles with CGA, the fluorescence intensity of zein significantly decreased, indicating that the interaction between CGA and zein led to static quenching. The addition of LBP caused further fluorescence quenching, suggesting that LBP enhanced the interaction between zein and CGA through a synergistic effect ( Figure 2 C).

[0061] 7. The ultraviolet-visible absorption spectra of the nanoparticle samples were recorded using a Jasco V-730 ultraviolet-visible spectrophotometer (Jasco, Japan). The measurement wavelength range of absorbance was from 200 to 800 nm. To ensure accuracy, the spectra were collected in triplicate and baseline corrected using the solvent as a blank.

[0062] Upon detection, the absorption intensity of ZLC NPs was the highest at a wavelength of 330 nm, indicating a strong and stable binding force between zein and CGA, thereby enhancing electron precipitation. The results showed that ZLC NPs had strong interactions and stability ( Figure 2 D).

[0063] 8. The thermal properties of the dried samples were evaluated using a STA449F3 thermogravimetric analyzer (TGA-DSC, Netzsch, Germany). Briefly, in a nitrogen environment, at a heating rate of 10 °C / min, 5 mg of each sample was used in the temperature range from 25 °C to 800 °C. Curves of weight loss and heat flow as a function of temperature (percentage) were plotted, and the thermogravimetric (TGA) curves of the samples were obtained.

[0064] Upon detection, ZLC NPs showed a broad and flat thermal curve without an obvious melting peak ( Figure 2 E), indicating that CGA had been completely amorphous (compared with the melting peak of CGA at 326.62 °C). This transformation indicated that there was a strong synergistic effect among CGA, zein, and wolfberry polysaccharide, thus forming a more stable and uniform complex. The TGA results ( Figure 2F) Further shows that during the entire heating process, the weight loss of ZLC NPs is 63.81%, lower than 66.01% of CGA. The initial temperature of thermal decomposition of ZLC NPs is increased to 312 °C (287 °C for CGA). This indicates that ZLC NPs can withstand higher temperatures without significant degradation.

[0065] V. Evaluation of the functional results of the zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles prepared in Step 1

[0066] 1. Evaluation of the antioxidant activity of zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles

[0067] In a 96-well microplate, 100 μL of the sample was mixed with an equal volume of 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazine). The mixture was kept in the dark for 30 minutes, and the absorbance was recorded at a wavelength of 517 nm using a spectrophotometer. The blank control included only the solution without the sample. An equal volume of 7.4 mmol / L ABTS (AzBTS-(NH4)2) and 2.6 mmol / L potassium persulfate solution were mixed and stored in the dark for 16 hours. Then, the ABTS + radical solution was diluted with ethanol to make its absorbance reach 0.70 ± 0.02 at a wavelength of 734 nm. The ABTS + radical solution was mixed with various samples at a ratio of 1:20, and the mixture was reacted in the dark for 6 minutes. The absorbance was measured at a wavelength of 734 nm, and the DPPH and ABTS + radical scavenging ability was determined using the following formula:

[0068]

[0069] After detection, the DPPH scavenging rate: ZLC NPs (73.68%) > free CGA (51.44%) > ZC NPs (44.47%). The ABTS+ scavenging rate: ZLC NPs (67.95%) > free CGA (36.53%) > ZC NPs (28.80%). The results show that ZLCNPs not only significantly enhance the ability to scavenge free radicals but also effectively overcome the problem of the decrease in antioxidant activity caused by the encapsulation of chlorogenic acid by single zein ( Figure 3 of A).

[0070] 2. In vitro digestion evaluation of zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles

[0071] The ZLC NPs were placed in simulated gastric fluid (SGF) with a pH of 2 and stirred at 37 °C for 2 hours with an oscillation frequency of 2 Hz. After gastric digestion, the samples were transferred to simulated intestinal fluid (SIF) with a pH of 7 and cultured at 37 °C for another 4 hours. During the simulated digestion process, 1 mL of the sample was taken every 0.5 hours to determine the release rate and replenished with an equal volume of fresh gastric or intestinal fluid. The digested ZLC NPs were freeze-dried and then the microscopic morphology of the digested nanoparticles was observed using a scanning electron microscope.

[0072] After detection, the cumulative release rate of CGA from ZLC NPs was 41.3% (20.23% for ZC NPs and 28.4% for free CGA). This structure enhanced the resistance of ZLC NPs to digestive enzymes in the gastrointestinal tract, facilitating the slowdown of CGA degradation and controlled release ( Figure 3 of B). SEM showed that the structure of ZLC NPs was intact after digestion ( Figure 6 ), with no rupture on the surface (compared with the fragmented morphology of ZC NPs), which also indicated that ZLC NPs ensured the protective effect and effective controlled release of CGA under digestion conditions.

[0073] 3. Bioavailability evaluation of zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles

[0074] After digestion, the CGA content in the samples was analyzed using a microplate reader. The CGA concentration in simulated intestinal fluid (SIF) was measured to determine its potential absorption rate. In addition, the release curve of CGA over time was monitored to evaluate its bioavailability and release rate at different digestion stages. The in vitro bioavailability of CGA was calculated according to the following formula: where M1 is the CGA content in the supernatant and M2 is the total amount of CGA.

[0075]

[0076] After detection, the bioavailability of free CGA was the lowest (28.17%), followed by ZC NPs at 39.27%; due to the synergistic effect of Lycium barbarum polysaccharide, the bioavailability of ZLC NPs was the highest (41.16%). The addition of Lycium barbarum polysaccharide enhanced the stability of the nanoparticles, improved the anti-enzymatic digestion ability, and promoted the sustained release of CGA ( Figure 3 of D).

[0077] 4. Thermal stability evaluation of zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles

[0078] The ZLC NPs were incubated in the dark in a water bath at 70 °C for 90 minutes, and samples were taken every 15 minutes. The retention rate of CGA was calculated as a percentage of its initial content (100%). After the incubation, the CGA content was measured to evaluate the stability of CGA in the nanoparticles at different temperatures.

[0079] After detection, after acting at 50 °C, 70 °C and 90 °C for 3 h, the retention rates of CGA in ZLC NPs were 96.30%, 95.75% and 95.31% respectively, which were higher than those of ZC NPs (90.19% (50 °C), 89.87% (70 °C), 81.31% (90 °C))( Figure 3 E). After heating at 70 °C for 90 minutes, the CGA retention rate of ZLC NPs was 95.75%, which was higher than 89.87% of ZC NPs( Figure 3 F).

[0080] VI. Cytotoxicity test of the zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles prepared in Step 1 and evaluation of the protection results against alcoholic liver injury

[0081] 1. Screening of the non-toxic concentration of zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles

[0082] AML-12 mouse hepatocytes were passaged in DMEM / F12 medium containing 10% fetal bovine serum (37 °C, 5% CO). The growing AML-12 cells were seeded into a 96-well plate at a density of 0.8×10 5 cells / mL, and 100 μL of cell suspension per well was propagated for 24 hours. The alcohol treatment group was cultured in an incubator with 100 μL of alcohol (0 - 1600 mM, DMEM diluent) for 24 hours; 100 μL of CGA, ZC NPs and ZLC NPs (6.25 - 800 μg / mL, DMEM diluent) were added to each well in the incubator, and 120 μL of MTT (1 mg / mL) was added to each well and incubated for 4 h. The reaction solution was discarded, 150 μL of DMSO was added, the reaction solution was discarded, 150 μL of DMSO was added, and the OD value at a wavelength of 490 nm was measured after shaking in the dark for 10 minutes.

[0083] After detection, in the CGA group: the cell survival rate was >100% within 6.25 - 25 μg / mL. In the ZC NPs group: the cell survival rate was >100% within 6.25 - 25 μg / mL. In the ZLC NPs group: the cell survival rate was >100% within 25 - 50 μg / mL, and the safety window was significantly broadened.

[0084] 2. Evaluation of the protective effect of zein / Lycium barbarum polysaccharide-chlorogenic acid composite nanoparticles against alcoholic liver injury

[0085] Collect logarithmically growing AML-12 cells for digestion and inoculate them into 96-well plates at a density of 0.8×10 5 cells / mL. After the cells have proliferated for 24 hours, the control group is cultured with 100 μL of medium for 48 hours; after culturing for 24 hours in different groups, the original medium is discarded and 100 μL of medium containing 270 mM alcohol-diluted medium is added for culturing for 24 hours. The cell survival rate is calculated according to the following formula:

[0086]

[0087] After detection, in the model group: after treatment with 270 mM ethanol for 24 hours, the cell survival rate decreased to 50.1%. In the ZLC NPs pretreatment group: at a concentration of 30 μg / mL, the cell survival rate increased to 69.56%, which was 10.34% higher than that of ZC NPs (59.22%) and 19.48% higher than that of free CGA (50.08%) ( Figure 4 ). It shows that ZLC NPs show higher potential than CGA and ZC NPs to improve alcohol oxidative damage in AML-12 cells.

[0088] The above are only preferred embodiments for explaining the present invention and are not intended to limit the present invention in any form. Therefore, any modification or change to the present invention made in the same inventive spirit should still be included within the scope intended to be protected by the present invention.

Claims

1. A method for improving the stability of chlorogenic acid using zein and wolfberry polysaccharide, characterized in that: It includes the following steps: Step 1: Dissolve zein and chlorogenic acid in ethanol to obtain a zein-chlorogenic acid solution, and dissolve wolfberry polysaccharide in pure water to obtain a wolfberry polysaccharide solution; Step 2: Under stirring conditions, add the zein-chlorogenic acid solution to the wolfberry polysaccharide solution and continue stirring to obtain a mixed solution; Step 3: Adjust the pH value of the mixed solution, and then obtain zein / wolfberry polysaccharide / chlorogenic acid nanoparticles after centrifugation and freeze-drying.

2. The method for improving the stability of chlorogenic acid by using zein and wolfberry polysaccharide according to claim 1, wherein: In Step 1, the mass ratio of zein to chlorogenic acid is 4:1 - 20:1, the concentration of zein is 1 - 3 mg / mL; the concentration of wolfberry polysaccharide is 1 - 3 mg / mL.

3. The method for improving the stability of chlorogenic acid by using zein and wolfberry polysaccharide according to claim 1, wherein: In Step 2, the volume ratio of the wolfberry polysaccharide solution to the zein-chlorogenic acid solution is 1:1 - 3:1, the temperature at which stirring occurs is 20 - 50 °C, the rotation speed is 600 - 100 r / min, the stirring time is 30 - 60 min, and the addition rate of the zein-chlorogenic acid solution is 5 - 15 mL / min.

4. The method for improving the stability of chlorogenic acid by using zein and wolfberry polysaccharide according to claim 1, characterized in that: In Step 3, the pH value is 4 - 6, and the centrifugation speed is 3000 - 5000 r / min.

5. Use of the product obtained by the method according to any one of claims 1 - 4 in the preparation of liver-protecting drugs.

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