Colla corii asini collagen peptide modified zein animal and plant double-protein nanoparticles as well as preparation method and application thereof in preparation of medicines for treating diabetes mellitus

Abstract

The invention discloses colla corii asini collagen peptide modified zein animal and plant double-protein nanoparticles as well as a preparation method and application thereof in preparation of medicines for treating diabetes mellitus. The preparation method comprises the following steps: pretreating a colla corii asini material, performing ultrasonic assistance, extracting colla corii asini collagen by utilizing pepsin, preparing colla corii asini collagen peptide by utilizing a compound enzyme preparation, and preparing the double-protein nanoparticle CCAP-Z-Myr from the hydrophilic colla corii asini collagen peptide, hydrophobic zein and myricetin. The CCAP-Z-Myr has very high oxidation resistance and alpha-glucosidase inhibition activity, the alpha-glucosidase inhibition rate reaches up to 78.5%, the alpha-glucosidase inhibition rate of the CCAP-Z-Myr is equivalent to that of a common meal oral hypoglycemic drug acarbose in the range of 40-100 micrograms / mL, and the alpha-glucosidase inhibition rate of the CCAP-Z-Myr is remarkably higher than that of the acarbose when the CCAP-Z-Myr is 20 micrograms / mL. Therefore, the prepared compound has the hypoglycemic performance superior to that of acarbose at low concentration.

Description

Technical Field

[0001] The invention belongs to the technical field of protein peptides, and specifically relates to a zein animal and plant dual protein nanoparticle modified with donkey-hide gelatin collagen peptide, a preparation method thereof, and an application of the nanoparticle in preparing a drug for treating diabetes. Background Art

[0002] In recent years, low molecular weight peptides consisting of 2 to 20 amino acid residues with a molecular weight range of 0.4 to 2 kDa have been recognized for their various bioactivities, including antidiabetic, antihypertensive, anti-inflammatory, antiobesity and readily absorbable effects. Compared with synthetic low molecular weight peptides with defined amino acid sequences, which have high synthesis costs, limited dispersibility and potential safety risks, low molecular weight peptides from natural sources are preferred due to their low cost, ease of preparation and higher safety. Previous studies have shown that low molecular weight peptides extracted from natural sources have rich nutritional properties and contain binding sites that can strongly interact with certain ingredients. For example, soybean peptides were constructed as nanoparticle interaction systems for curcumin to enhance its bioactivity. Egg white-derived peptides interacted with chitosan to deliver curcumin, formed more compact nanoparticles, and exerted synergistic antioxidant effects. The self-assembly of deamidated zein peptide nanoparticles protected curcumin and effectively improved its solubility. Therefore, combining low molecular weight peptides extracted from natural sources with natural biopolymers to construct stable delivery systems is considered to be an effective strategy to improve the stability, applicability and bioactivity of certain hydrophobic bioactive substances.

[0003] Gelatin is an important natural protein obtained by hydrolyzing collagen in certain animal tissues, such as bones, skin, and fish scales. Due to its excellent properties, such as water binding capacity and gel-forming ability, gelatin has attracted widespread attention in the food and pharmaceutical fields. Colla Corii Asini Donkey hide glue (CCA) is a traditional Chinese medicine and functional food made from processed donkey hide, which is widely used for its therapeutic effects in hematopoiesis, immune enhancement and anti-aging. Reports indicate that amino acids and peptides are the main components affecting the efficacy of CCA. Wu et al. identified collagen-derived peptides with hematopoietic activity from CCA. However, low molecular weight peptides extracted from animal sources tend to have high water solubility and low natural self-assembly ability due to the rich hydrophilic amino acid residues. These phenomena limit its application in the field of protein self-assembly nanodelivery of hydrophobic bioactive ingredients.

[0004] In contrast, more than half of the amino acids in zein are hydrophobic, enabling it to deliver hydrophobic active compounds, nutrients, and other substances, making it an ideal self-assembly carrier. During the self-assembly process of zein, hydrophobic amino acid residues tend to aggregate inside, while hydrophilic residues bind to adjacent water molecules. As a result, various hydrophobic bioactive ingredients are encapsulated in the hydrophobic cavity of zein. However, the low water solubility and instability of zein, especially near its isoelectric point, pose a major challenge to the delivery of hydrophobic bioactive compounds by zein. Therefore, physical modification is a promising method to alter the structure and function of zein proteins.

[0005] Diabetes mellitus is a common and serious disease that can lead to chronic complications, including hyperglycemia and multi-organ damage syndrome. α- Glucosidase is a key catalytic enzyme in carbohydrate digestion and an important therapeutic target for regulating postprandial hyperglycemia. Inhibiting the activity of carbohydrate digestive enzymes is an effective strategy for treating diabetes. Common hypoglycemic drugs on the market are acarbose and metformin. Metformin lowers blood sugar by reducing hepatic glucose output and improving peripheral insulin resistance, while acarbose is an α-glucosidase inhibitor, which lowers postprandial blood sugar by inhibiting the absorption of carbohydrates in the upper small intestine. Therefore, materials with α-glucosidase inhibitory properties have potential value in the treatment of diabetes in the future. Summary of the invention

[0006] The purpose of the present invention is to provide a corn zein animal and plant dual protein nanoparticle modified with donkey-hide gelatin collagen peptide and its preparation method and application in the preparation of drugs for treating diabetes. The dual protein nanoparticle has excellent α -Glucosidase inhibitory activity can play an important role in the treatment of diabetes.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides a method for preparing zein animal and plant dual protein nanoparticles modified with donkey-hide gelatin collagen peptide, which comprises the following steps: (1) Grinding donkey-hide gelatin and dissolving it in a Na2CO3 solution, mixing and centrifuging the obtained precipitate, and then freeze-drying it to obtain defatted powder; dissolving the defatted powder in a NaOH solution, mixing and centrifuging the obtained supernatant, and then dialyzing and freeze-drying it to obtain a crude collagen powder; (2) dissolving the crude collagen powder in acetic acid, then adding pepsin and performing ultrasonic treatment. After the ultrasonic treatment, the supernatant obtained by centrifuging the mixture is dialyzed and freeze-dried to obtain donkey-hide gelatin collagen; (3) dissolving the donkey-hide gelatin collagen in distilled water, adjusting the pH value to 7.5-8 with a NaOH solution, then adding a composite enzyme preparation and stirring the mixture, centrifuging the obtained mixture to obtain a supernatant, and freeze-drying the supernatant to obtain a hydrophilic donkey-hide gelatin collagen peptide; (4) Zein and myricetin are dissolved in ethanol and subjected to ultrasonic treatment. The hydrophilic donkey-hide gelatin collagen peptide solution is then added and mixed evenly. The resulting mixture is centrifuged to obtain a supernatant, and the supernatant is freeze-dried to obtain zein animal and plant dual protein nanoparticles.

[0008] Furthermore, in step (1), the mass volume ratio of donkey-hide gelatin to Na2CO3 solution is 1:20-30; the mass volume ratio of defatted powder to NaOH solution is 1:20-25.

[0009] In the mass-to-volume ratio w / v mentioned in the present invention, the unit of mass w is g, and the unit of volume is mL.

[0010] Furthermore, in the step (2), the mass volume ratio of the crude collagen powder to the acetic acid is 1:40-45; the mass ratio of the pepsin to the crude collagen powder is 1:40-80, and the enzyme activity is ≥2500 U / mg.

[0011] Furthermore, in step (3), the mass volume ratio of donkey-hide gelatin collagen to distilled water is 1:5-6, and the added amount of the complex enzyme preparation is 3.5%-4.5% of the mass of the donkey-hide gelatin collagen.

[0012] Furthermore, in step (4), the mass ratio of zein to myricetin is 10-15:1-3, the concentration of the hydrophilic donkey-hide gelatin collagen peptide solution is 15 mg / mL-20 mg / mL, and the volume mass ratio of the hydrophilic donkey-hide gelatin to zein is 3-5:1. In the volume mass ratio mentioned in the present invention, the unit of volume is mL, and the unit of mass w is g.

[0013] The present invention also provides zein animal and plant dual protein nanoparticles prepared by the preparation method, and the particle size of the nanoparticles is 95-100 nm.

[0014] The present invention also provides the application of the zein animal and plant dual protein nanoparticles in antioxidants.

[0015] The present invention also provides the zein animal and plant dual protein nanoparticles in the preparation α- Application of glucosidase inhibitors.

[0016] The present invention also provides the use of the zein animal and plant dual protein nanoparticles in preparing medicines for treating diabetes.

[0017] Furthermore, the drug contains 20 μg / mL-100 μg / mL of zein animal and plant dual protein nanoparticles.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: The nanoparticle CCAP-Z-Myr of the present invention has a smaller particle size (98.2 nm), which is significantly lower than the carboxymethyl chitosan-ovalbumin peptide-γ-cyclodextrin-curcumin nanoparticles (particle sizes are all 172-242 nm) prepared in other studies, and the core-shell nanoparticles coexisting with curcumin and piperine (particle size is 599 nm). The smaller particle size helps to improve the dispersibility of the nanoparticles in the body, thereby enhancing its biological functionality. The CCAP-Z-Myr nanoparticles of the present invention exhibit a higher embedding efficiency (embedding efficiency of 91.4%). Li et al. prepared nanoparticles by encapsulating curcumin with deamidated zein peptide, and the embedding efficiency was 87.1%; Zhang et al. prepared soybean peptide-curcumin nanoparticles by pH-driven insoluble aggregates, and the embedding efficiency was 88%. The high embedding efficiency of CCAP-Z-Myr shows its high efficiency in delivering myricetin. Liu et al. prepared zein / caseinate-sodium alginate nanoparticles to encapsulate curcumin, with a particle size of 286 nm and an encapsulation rate of 88.8%; Yan et al. successfully prepared curcumin-zein-caseinate (Cur-ZC) with a particle size of 100 nm - 300 nm, a Cur encapsulation rate of 77.9% - 87.1%, and a DPPH scavenging ability of about 30%. DSC showed that the endothermic peaks of curcumin-zein-chondroitin sulfate nanoparticles and curcumin-zein-chondroitin sulfate / sophorolipid nanoparticles successfully prepared by Yuan et al. were 110.6°C and 128.3°C, respectively, while the endothermic peak of CCAP-Z-Myr was 138.5°C, which was significantly higher than the former two, and thus had good thermal stability. Compared with the curcumin-casein-ovalbumin peptide nanoparticles (ABTS ·+ Compared with the scavenging capacity of 31.4%), CCAP-Z-Myr has good antioxidant capacity (ABTS ·+ The scavenging ability was 60.4%). In another study, Kanwal et al. prepared curcumin nanoparticles (Cur-NSM, Cur-SM, Cur-ASP) by nanosuspension method (NSM), ultrasound method (SM) and antisolvent method (ASP), respectively. Their DPPH free radical scavenging abilities were all between 45% and 50%. α The glucosidase inhibition abilities of Cur-ASP, Cur-NSM and Cur-SM were 58.3%, 54.0% and 55.4%, respectively, which were lower than the DPPH free radical scavenging rates of CCAP-Z-Myr (57.6%) andα -Glucosidase inhibition capacity 78.5%.

[0019] The present invention uses donkey-hide gelatin collagen peptide (CCAP) as a modifier, and Zein is selected as a model protein due to its high self-assembly, biocompatibility and biodegradability, and myricetin (Myr) is selected as a hydrophobic active ingredient due to its potential role in treating postprandial hyperglycemia. CCAP-Z-Myr, i.e., double-protein nanoparticles, are prepared, and the nanoparticles form a compact, highly ordered structure, will not crystallize, and have high antioxidant properties and α -glucosidase inhibitory activity, α - The glucosidase inhibition rate can be as high as 78.5%, which is comparable to acarbose, a common oral hypoglycemic drug, within the range of 40-100 μg / mL. α The -glucosidase inhibition rate was significantly higher than that of acarbose at 20 μg / mL (55.43% vs. 40.88%). This indicates that the prepared compound has a blood sugar-lowering effect that exceeds acarbose at low concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SEC-MALS spectrum of CCA-digested peptides.

[0021] Figure 2 TEM images of zein (Z) NPs, CCAP-zein (CCAP-Z) NPs, zein-myricetin (Z-Myr) NPs, and CCAP-zein-myricetin (CCAP-Z-Myr) NPs.

[0022] Figure 3 Spectra, graphs and mechanism diagrams of CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, Myr and Z; a is the FTIR spectrum, b is the XRD graph and c is the mechanism diagram of CCAP-Z-Myr.

[0023] Figure 4 The graphs, spectra, and graphic illustrations of CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, Myr, and Z; a is the DSC spectrum, b is the fluorescence spectrum, and c is a graphic illustration of Myr-loaded NPs using the antisolvent precipitation method.

[0024] Figure 5 Analysis of the antioxidant capacity of CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, and Myr; a is ABTS ·+a is the scavenging ability of DPPH, c is the scavenging ability of FRAP, d is the scavenging ability of hydroxyl radicals, and e is the scavenging ability of superoxide anions.

[0025] Figure 6 Acarbose, Myr, Z-Myr, CCAP, CCAP-Z and CCAP-Z-Myr α -Inhibition of glucosidase. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0027] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0028] The donkey-hide gelatin of the present invention was provided by Wuji Kangyuan Food Engineering Co., Ltd., Dong'e County (Liaocheng, China). Pepsin (≥2500 U / mg) was obtained from Aladdin Biotechnology Co., Ltd. The compound enzyme preparations AP-200A and FF 104 were purchased from Angel Yeast Co., Ltd. Myricetin (Myr) (≥98%) was derived from MacLean Biochemical Co., Ltd. Zein (Z) was purchased from Merck & Co., Ltd. (Missouri, USA). α -Glucosidase (50 U / mg) and p-nitrophenyl-α-D-glucopyranoside (pNPG, purity ≥98%) were obtained from MacLean Biochemical Co., Ltd.

[0029] Example 1: Preparation of donkey-hide gelatin collagen peptide 1. Material pretreatment The donkey-hide gelatin block (CCA, 10.00 g) was crushed and dissolved in a 5% Na2CO3 solution, the mass volume ratio of donkey-hide gelatin to the Na2CO3 solution was 1:20, the unit of mass w in the mass volume ratio w / v in the present invention was g, and the unit of volume was mL, and the mixture was stirred continuously at a speed of 600 r / min for 24 h. The mixture was centrifuged at 10000 r / min for 30 min, and the obtained precipitate was freeze-dried to prepare a defatted powder.

[0030] The defatted powder (10.00 g) was then dissolved in 200 mL of 0.5 mol / L NaOH solution (1:20, w / v) and magnetically stirred at 600 r / min for 6 h to remove non-collagen components. The mixture was then centrifuged at 15000 r / min for 30 min, and the supernatant was transferred to a 10 kDa dialysis bag and dialyzed with 3 L of deionized water for 3 days, with the water changed every 6 h. The whole process was carried out at 4 °C. Subsequently, the inner dialysis bag solution was freeze-dried to obtain crude collagen powder.

[0031] 2. Ultrasound-assisted extraction of collagen Crude collagen powder (2.00 g) was dissolved in 80 mL of 0.5 mol / L acetic acid at a sample / solution ratio of 1:40 (w / v). Subsequently, 0.03 g of pepsin (≥2500 U / mg) was added to the above solution. The mixture was then subjected to ultrasonic treatment at 20 kHz and 360 W for 30 min. To prevent overheating, ultrasound was applied in pulse mode (on for 5 s and then off for 5 s), and the mixture was kept in an ice bath (temperature maintained at 4 °C). After ultrasonic treatment, the mixture was centrifuged at 10000 r / min for 30 min at 4 °C to obtain the supernatant. The supernatant was then dialyzed against 100 mL of distilled water (molecular weight = 15 kDa) until the pH reached neutrality, and the dialysis solution was replaced every 4 h. The dialysis solution (inner dialysis bag) was then frozen at -80 °C and freeze-dried. The resulting donkey-hide gelatin collagen was then obtained and stored at 4 °C for future use.

[0032] 3. Preparation of donkey-hide gelatin collagen peptide (CCAP) CCA collagen (10.00 g) was dissolved in 60 mL of distilled water at a ratio of 1:6 (w / v), and the pH was adjusted to 7.5 with NaOH solution (0.5 mol / L). Subsequently, the compound enzyme preparation AP-200A accounting for 3.75% (w / w) of the mass of donkey-hide gelatin collagen and the compound enzyme preparation FF 104 accounting for 0.45% (w / w) of the mass of donkey-hide gelatin collagen were added to the solution simultaneously. The solution was stirred at a constant temperature of 55 °C for 48 h (600 r / min). The enzyme was inactivated by heating the solution to 85 °C for 20 min. The mixture was then centrifuged at 9000 r / min for 30 min to remove the precipitate. The supernatant was freeze-dried to obtain CCAP.

[0033] 4. Size Exclusion Chromatography Coupled Multi-Angle Light Scattering (SEC-MALS) Analysis SEC-MALS experiments were performed by Boruitang Biotechnology Co., Ltd. CCAP was prepared as a 5 mg / mL solution containing 0.05 mol / L NaCl and passed through a 0.22 µm millipore filter before measurement. SEC-MALS analysis was performed using a high-performance liquid chromatography (HPLC) system (LC-10A, Shimadzu, Japan) combined with a MALS detector (DynaPro NanoStar, Wyatt, USA) and a refractive index (RI) detector (RI-10A, Shimadzu, Japan). Chromatographic separation was performed on a BRT105-103-101 series tandem gel filtration column (8 × 300 mm) at 25 °C. The mobile phase consisted of a 0.05 mol / L NaCl solution filtered through a 0.45 μm membrane before use. The injection volume was set to 20 μL and the flow rate was 1.0 mL / min. The laser wavelength of the MALS detector was 658.1 nm, and the RI increment (dn / dc) was set to 0.1850 mL / g, which is designed for peptides. Data acquisition and analysis were performed using Astra8 software (Wyatt Technology, Santa Barbara, USA).

[0034] The components separated by SEC are introduced into the MALS detector, where light scattering occurs when the laser illuminates the analyte. The intensity of the scattered light is measured from multiple angles, and the intensity is proportional to the molar mass, concentration, and the square of the refractive index increment, thus facilitating the direct analysis of the protein Mw range. MALS is a well-established technique for determining the molecular weight of a protein based on a direct correlation between its molecular weight and the intensity of scattered light. This method is highly accurate because it does not require standard proteins and is independent of elution retention time, enabling direct and accurate determination of protein molecular weight distribution. The results of SEC-MALS analysis of CCAP samples prepared with the compound enzyme AP-200A and FF104 (55°C, 48 h) are shown in Figure 2. Figure 1 , as shown in Table 1 and Table 2.

[0035] The results showed that the elution peaks of CCAP samples were mainly composed of three main peaks. Peak 1 ( Figure 1 , 7.316-9.138 min) showed an average molecular weight (Mw) of 1175.0 Da, peak 2 ( Figure 1 , 9.243-9.949 min) has an Mw of 295.5 Da, peak 3 ( Figure 1, 10.073-12.620 min) with a molecular weight of 526.1 Da. The relatively high PDI values ​​(Table 1) indicate significant heterogeneity within the sample, which is typical after extensive hydrolysis, resulting in a mixture of short and long peptide chains. The differences in PDI between the peaks (Table 1) indicate that the mixture of peptide fragments obtained after enzymatic hydrolysis contains a variety of small and medium fragments. The Mw distribution of the CCAP samples was further obtained by automatic calculation using the Zimm model in the Wyatt-Astra software (Table 2), with range 1 being 83.0-100.5 Da, range 2 being 100.5-785.6 Da, and range 3 being 785.6-2819.7 Da, with relative contents of 10.00%, 80.00%, and 10.00%, respectively.

[0036] Table 1 Peak analysis of CCA peptide by SEC-MALS

[0037] Table 2 Molecular weight distribution of CCA peptides analyzed by SEC-MALS

[0038] Example 2: Preparation of CCAP-Z-Myr Nanoparticles 1. Experimental steps 1. Preparation of Nanoparticles CCAP-Z-Myr, Z-Myr and CCAP-Z nanoparticles were prepared by antisolvent precipitation method. Since CCAP has high hydrophilicity, it cannot self-assemble into nanoparticles in aqueous solution. The details are as follows: (i) Z (10.00 mg) and Myr (1.00 mg, W total ) was dissolved in 10 mL of ethanol (80%, v / v), stirred at 500 r / min for 1 h to ensure complete dissolution and dispersion, and sonicated for 20 min. The solution was then injected into 30 mL of CCAP solution (15 mg / mL) and stirred at 600 r / min for 30 min, followed by evaporation at 40 °C for 15 min to remove ethanol. The resulting solution was then centrifuged at 3000 r / min for 10 min, and the supernatant was freeze-dried to obtain CCAP-Z-Myr nanoparticle powder.

[0039] (ii) Z (10.00 mg) and Myr (1.00 mg, W total) was dissolved in 10 mL of ethanol (80%, v / v) and sonicated for 20 min. The solution was injected into 30 mL of distilled water and stirred at 600 r / min for 30 min to prepare Z-Myr nanoparticles. The suspension was then centrifuged at 3,000 r / min for 10 min and freeze-dried to obtain Z-Myr.

[0040] (iii) Z (10.00 mg) was dissolved in 10 mL of ethanol (80%, v / v), and the solution was then injected into 30 mL of CCAP solution (15 mg / mL) and stirred at 600 r / min for 30 min. The solution was then centrifuged at 3,000 r / min for 10 min and freeze-dried to obtain CCAP-Z powder.

[0041] 2. Encapsulation efficiency (EE) and loading efficiency (LE) The encapsulation and loading efficiencies of Myr in CCAP-Z-Myr and Z-Myr were slightly modified. That is, 10.00 mg of freshly synthesized nanoparticles (W Nanoparticle ) was dissolved in 1 mL of distilled water. The mixture was then centrifuged at 4000 r / min for 10 min to remove insoluble or unencapsulated free Myr. The supernatant was collected, appropriately diluted with ethanol-water solution (75%, v / v), and then centrifuged at 10000 r / min for 20 min. The concentration of Myr in the supernatant (W) was quantified using a UV-visible spectrophotometer (TU-1810, Persee, Beijing, China). encapsulated ), the standard curve was established at 385 nm, R 2 ≥0.999. The EE and LE of Myr were calculated using the following equations:

[0042] Among them, W encapsulated , W total and W Nanoparticles represent the encapsulation value of Myr, the total value of input Myr, and the total mass of nanoparticles, respectively.

[0043] 3. Physical and structural characteristics of nanoparticles (1) Characteristics of nanoparticles The particle size, polydispersity index (PDI), and zeta potential of the Myr-loaded nanoparticles were measured at 25 °C using dynamic light scattering (Zetasizer Nano ZS90, Malvern Instruments Ltd, Malvern).

[0044] (2) Morphological characteristics The morphology of the nanoparticles was examined using a transmission electron microscope (TEM, Hitachi H-7650, Tokyo, Japan). The freshly prepared dispersion was diluted with water and deposited on a copper grid. Images of the nanoparticles were captured at 100 kV and 60,000× magnification.

[0045] (3) Fourier transform infrared spectroscopy (FTIR) The lyophilized samples (CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, Z, Myr) were mixed with potassium bromide (1:100, w / w) and compressed into tablets. -1 The sample was scanned at a resolution of 500 to 4000 cm -1 FTIR (Spectrum 100, Perkin Elmer, Warrington, UK) was repeated over a wavelength range of 1.5 Å. The scan was repeated 64 times.

[0046] (4) X-ray diffraction (XRD) The freeze-dried samples (CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, Z, Myr) were evaluated using XRD (Bruker D8, ADVANCE, Germany) with an angle of 2θ ranging from 5° to 55° and a scan speed of 5 s / 0.02°.

[0047] (5) Intrinsic fluorescence spectroscopy Intrinsic fluorescence measurements were performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). The excitation wavelength was set at 280 nm, and the emission spectra were collected between 300 and 400 nm at a scan speed of 100 nm / min. The intrinsic fluorescence of the lyophilized samples (CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, Z, Myr) was measured at a constant concentration of 0.2 mg / mL. The slit widths of 5.0 and 5.0 nm were fixed for excitation and emission, respectively.

[0048] (6) Differential Scanning Calorimetry (DSC) Lyophilized samples (CCAP-Z-Myr, Z-Myr, CCAP-Z, CCAP, Z, Myr) were analyzed using a differential scanning calorimeter (DSC, METTLER TOLEDO, Switzerland). Each sample (5 mg) was sealed in a standard aluminum pan and heated from 30 °C to 230 °C at a heating rate of 5 °C / min under a nitrogen flow rate of 10 mL / min.

[0049] 4. Functional properties of nanoparticles (1) Antioxidant activity assay The samples were tested for 1,1-diphenyl-2-picrylhydrazyl (DPPH), ABTS and ·+ , FRAP, superoxide anion and hydroxyl radical scavenging ability.

[0050] (2) Enzyme inhibitory activity A mixture of 80.0 μL phosphate-buffered saline (PBS, pH 6.8), 20.0 μL α-glucosidase (10 U / mL in PBS), and 20.0 μL peptide (in PBS) was incubated at 37 °C for 15 min. Subsequently, 20.0 μL pNPG (5 mM in PBS) was added to the mixture and incubated at 37 °C for another 15 min. To stop the reaction, 60 μL Na2CO3 (0.2 mol / L in PBS) was added, and the absorbance was measured at 405 nm using a SpectraMax m5 microplate reader (Molecular Devices, Sunnyvale, Canada). The percentage of inhibitory activity was calculated using the following equation:

[0051] Among them, A0 is the absorbance of the sample replaced by an equal amount of PBS buffer; A1 is the absorbance of the added sample; A2 is the absorbance of the sample replaced by an equal amount of PBS buffer. α -Absorbance of glucosidase and pNPG solutions.

[0052] 2. Experimental Results 1. Physical characteristics As shown in Table 3, the particle size of CCAP-Z (133.3 nm) was significantly larger than that of Z nanoparticles (104.0 nm) (p < 0.05). This increase in size was due to the deposition of CCAP on the surface of Z via electrostatic interactions. After the addition of Myr, the particle sizes of CCAP-Z-Myr and Z-Myr nanoparticles decreased to 98.2 nm and 82.7 nm, respectively. The decrease in size after the addition of Myr was attributed to the encapsulation of hydrophobic Myr into the internal hydrophobic sites of Z during the self-assembly process, forming a more compact nanoparticle structure. During the physical self-assembly process, the hydrophobic polyphenol molecules interact with the hydrophobic cavity of Z via non-covalent interactions. The addition of Myr may have affected the nanoparticle formation process, resulting in a decrease in particle size.

[0053] As shown in Table 3, the Zeta potentials of CCAP-Z-Myr and CCAP-Z were negatively charged at −24.2 and −19.9 mV, respectively, while Z and Z-Myr exhibited positive charges of 22.8 and 16.2 mV, respectively. These findings indicate that CCAP is negatively charged, suggesting that electrostatic interactions may be the main binding force between CCAP and Z. The PDI value of CCAP-Z nanoparticles was less than 0.14, indicating that the nanoparticles were relatively uniformly dispersed in the solution. As shown in Table 3, the encapsulation efficiency of CCAP-Z-Myr nanoparticles (91.4%) was higher than that of Z-Myr nanoparticles (87.1%). The presence of CCAP enhanced the encapsulation capacity of conventional Z nanoparticles, allowing more Myr to be successfully encapsulated. However, the LE of CCAP-Z-Myr (3.50%) was lower than that of Z-Myr (7.74%), which may be due to the increase in the total mass of CCAP-Z in the presence of CCAP-Z-Myr, which reduced the relative proportion of Myr within the prepared nanoparticles and led to a decrease in LE.

[0054] These results suggest that a compact, well-defined, and highly ordered structure and EE capacity are formed due to the noncovalent hydrophobic interactions of Myr with the hydrophobic cavity of Z and the appropriate electrostatic interactions between CCAP and Z.

[0055] Table 3 Particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE) and loading rate (LC) of different samples

[0056] Note: CCAP: donkey-hide gelatin peptide; Z: zein; Myr: myricetin; EE and LE: encapsulation efficiency and loading efficiency. Values ​​are expressed as mean ± standard error (n = 3). Different superscript letters indicate significant differences (p < 0.05).

[0057] 2. Morphological characteristics The microstructural characteristics of the newly prepared nanoparticles were observed by transmission electron microscopy (TEM). Figure 2 As shown, Z nanoparticles present a roughly spherical structure, while Z-Myr nanoparticles appear more regular, with smooth edges and smaller particle size than Z, which is consistent with the results of DLS (Table 3).

[0058] Compared with Z nanoparticles, CCAP-Z presents a virtual ring, which may represent the hydrated CCAP ring in the outermost layer of Z. The average particle size of CCAP-Z is also slightly larger than that of Z nanoparticles, which is consistent with the DLS results in Table 3. In addition, a virtual ring is observed outside the CCAP-Z-Myr nanoparticles, which may also represent the hydrated CCAP ring in the outermost layer of Z-Myr. The presence of Myr makes the CCAP-Z-Myr nanoparticles more compact and smaller than those of CCAP-Z, which is consistent with the DLS results in Table 3. These phenomena further indicate that a compact and highly ordered structure is formed during the self-assembly process of CCAP-Z-Myr; the hydrophobic Myr may be located in the Z core, while the CCAP may be located on the surface of the Z core.

[0059] 3. Structural characteristics (1) FTIR analysis In FTIR spectroscopy ( Figure 3 a) Z at 3,312 cm -1 ,2,958 cm -1 ,1,657 cm -1 and 1,542 cm -1 The characteristic peaks at 1657 cm-1 and 1667 cm-2 correspond to the strong stretching vibrations of hydroxyl (-OH), hydrophobic CH, C=O, and NH bending mode coupled with CN stretching (amide II), respectively. -1 (Z) Move to 1633cm -1 (CCAP-Z), while amide II from 1542 cm -1 (Z) Move to 1536cm -1 (CCAP-Z). These changes suggest that electrostatic interactions play a major role in the formation of nanoparticles, as shown by DLS (Table 3) and TEM analysis ( Figure 2 ) In addition, the CH vibration peak from 2,958 cm -1 (Z, Figure 3 a) Move to 2,963 cm -1 (CCAP-Z, Figure 3 a). TEM analysis results show that the hydrophobicity of CCAP-Z nanoparticles is significantly reduced, which may be due to the hydrophilic adsorption of CCAP on the Z surface.

[0060] For Myr, at 1,029cm -1 ,1,328cm -1 ,1,521cm -1 ,1,661cm -1 and 3407cm -1Characteristic peaks were observed at 3,312 cm-1, corresponding to the stretching vibrations of the C=C group in the trans-olefin, the CO group in the enol, the CC and CO groups, the benzene ring, and the phenolic hydroxyl group. When Myr is combined with Z (Z-Myr), the peak of the -OH stretching vibration in Z-Myr increases from 3,312 cm-1 to 3,312 cm-1. -1 (Z, Figure 3 a) Move to 3,376 cm -1 (Z-Myr, Figure 3 a), the characteristic peak of amide II is from 1,542 cm -1 (Z, Figure 3 a) Move to 1,559cm -1 (Z-Myr, Figure 3 a). Meanwhile, the corresponding peak in Z-Myr nanoparticles shifts from 2958 cm -1 (Z, Figure 3 a) Move to 2980cm -1 (Z-Myr, Figure 3 a) These results indicate that hydrogen bonding and hydrophobic interactions are the main driving forces for the successful capture of Myr by Z. In CCAP-Z-Myr nanoparticles, the absorption peak of -OH increases from 3,312 cm -1 (Z, Figure 3 a) Move to 3,314 cm -1 (CCAP-Z-Myr, Figure 3 ), and this change may be due to the hydrogen bonding between Z and CCAP. In contrast, the characteristic peak of natural Myr (1,029 cm -1 ,1,328 cm -1 ,1,521 cm -1 ,1661 cm -1 and 3,407 cm -1 ) is weakened (Z-Myr, Figure 3 a) or disappeared (CCAP-Z-Myr, Figure 3 a), which is attributed to the molecular structure of the protein encapsulating the chemical groups of Myr, effectively blocking its signal in the FTIR spectrum, thus confirming the successful encapsulation of Myr within the nanoparticles, which is supported by Table 3 and the TEM results.

[0061] (2) XRD analysis The physical state of the different types of nanoparticles was investigated using XRD. Thus, the XRD spectra of native Myr and nanoparticles with or without Myr were measured ( Figure 3 b). Two broad diffraction peaks appear at 9.0° and 19.9° ( Figure 3 b), a moderately broad diffraction peak appears near 20° ( Figure 3 b), indicating that CCAP and Z are amorphous in nature, and these amorphous properties are related to the length of the main chain of the α-helical structure. Figure 3 As shown in (b), in the XRD spectrum of Myr, strong and sharp diffraction peaks were observed at 5.8°, 11.4°, 14.1°, 16.6°, 26.3°, and 28.4°, indicating that it is in a highly crystalline state. However, when Myr is embedded in nanoparticles, the characteristic peaks of Myr almost disappear in Z-Myr and CCAP-Z-Myr nanoparticles, indicating that Myr transforms from a crystalline state to an amorphous state. These phenomena further confirm that encapsulating Myr in the hydrophobic cavity of nanoparticles can effectively prevent the crystallization of nanoparticles, thereby improving the physicochemical properties and biological activity of nanoparticles.

[0062] (3) DSC analysis The thermal stability of the nanoparticles (Z, Myr, CCAP, CCAP-Z, Z-Myr, CCAP-Z-Myr) was evaluated by differential scanning calorimetry (DSC). The observed peak represents the endothermic peak of the sample, and the change in this peak qualitatively reflects the change in the physical state and stability of the nanoparticles during the thermal treatment ( Figure 4 a). Highly crystalline natural Myr crystals show a distinct endothermic peak at approximately 199.5 °C ( Figure 4 a), which is attributed to the melting of Myr crystals. In contrast, Z and CCAP lack sharp melting peaks, indicating that they are mainly amorphous, which makes them suitable as wall materials for nanoparticles. Compared with native Myr, the sharp endothermic peaks disappear in CCAP-Z-Myr and Z-Myr ( Figure 4 a), indicating that Myr is completely encapsulated within the nanoparticle cavity, resulting in appropriate thermal stability due to its amorphous state, as confirmed by XRD. Characteristic endothermic peaks at 74.67 °C and 101.5 °C were observed in the DSC thermograms of Z and CCAP, respectively ( Figure 4 a). These peaks are due to the absorption of energy by water evaporation. Compared with Z (74.67 °C), the characteristic endothermic peak of Z-Myr ( Figure 4 a) Shift to a lower temperature (71.83 °C), resulting in a smoother curve. This indicates that the presence of Myr reduces the thermal stability of the nanoparticles and may promote the formation of an amorphous structure.

[0063] (4) Fluorescence spectrum Intrinsic fluorescence is an effective method to study the molecular interactions between proteins and other compounds, which may lead to changes in the protein microenvironment. Aromatic amino acids in protein molecules, including phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), can trigger intrinsic fluorescence at specific excitation wavelengths.

[0064] Z exhibits a strong affinity for hydrophobic or amphiphilic compounds, providing a basis for its role as a multifunctional carrier for hydrophobic substances in delivery applications. Z has a fluorescence emission peak around 305 nm ( Figure 4 b), which is attributed to its high ratio of Tyr residues. Compared with Z, the fluorescence intensity of Z-Myr nanoparticles is reduced ( Figure 4 b), λ max It exhibits a blue shift (from 305 nm to 304 nm), which may be due to the quenching of the fluorescence of the Tyr residue in Z by Myr and the microenvironment of the hydrophobic residues becomes polarized due to complexation with Myr.

[0065] CCAP, CCAP-Z NPs and CCAP-Z-Myr NPs showed strong emission peaks between 325 and 350 nm ( Figure 4 b), which is attributed to the presence of Trp residues in CCAP. In addition, the fluorescence intensity of CCAP-Z and CCAP-Z-Myr NP is stronger than that of CCAP ( Figure 4 b), which may be related to the presence of CCAP molecules. Due to the interaction between CCAP and Z, the Trp residues in the nanoparticles may be located in a more hydrophilic environment, resulting in increased exposure of the Trp residues.

[0066] Compared with CCAP, the maximum fluorescence emission peaks of CCAP-Z and CCAP-Z-Myr NPs ( Figure 4 b) shows a slight red shift from 330 nm (CCAP-Z) to 335 nm (CCAP-Z) and 332 nm (CCAP-Z-Myr), respectively. These results suggest that the polarity of the local environment increases during nanoparticle formation, with hydrophobic interactions and hydrogen bonding acting as the main driving forces for the change in microenvironment polarity in the formation of CCAP-Z-Myr or CCAP-Z NPs.

[0067] 4. Antioxidant analysis Choose different antioxidant measurement methods (ABTS ·+ , DPPH, FRAP, hydroxyl radicals and superoxide anions) were used to evaluate the antioxidant potential of Myr, CCAP, Z-Myr NPs, CCAP-Z NPs and CCAP-Z-Myr NPs. Figure 5 As shown, the ABTS of CCAP-Z-Myr ·+, DPPH, FRAP, hydroxyl radical and superoxide anion detection values ​​were higher than those of Z-Myr (60.40% vs. 25.28%, 57.57% vs. 32.96%, 0.65% vs. 0.42%, 50.37% vs. 30.15%, 50.88% vs. 29.65%) and Myr (60.40% vs. 11.50%, 57.57% vs. 12.83%, 0.65% vs. 0.24%, 50.37% vs. 13.86%, 50.88% vs. 8.87%, respectively), among which CCAP-Z-Myr was the highest among Myr, CCAP, Z-Myr, CCAP-Z and CCAP-Z-Myr. These phenomena indicate that CCAP can effectively improve the antioxidant properties of Z-encapsulated Myr nanoparticles.

[0068] In short, the antioxidant capacity of Myr is attributed to its functional groups, including phenolic hydroxyl groups and conjugated double bond systems, which are able to transfer electrons or donate hydrogen atoms. ·+ , FRAP, hydroxyl radical and superoxide anion assays are associated with electron and hydrogen atom transfer.

[0069] exist Figure 5 In the experiment, Myr showed relatively low free radical scavenging ability (ABTS: 11.50%, DPPH: 12.83%, FRAP: 0.24, hydroxyl radical: 13.86%, superoxide anion: 8.82%) due to its poor solubility, which significantly limited the effective interaction between dissolved samples and free radicals. ·+ The scavenging activities of Z-Myr NPs were significantly higher than those of free Myr (25.28%, 32.96%, 0.42%, 30.15%, and 29.65%, respectively). This enhancement may be due to the improved dispersibility of Z-Myr NPs after successful encapsulation. After the addition of CCAP (CCAP-Z-Myr), the ABTS ·+ , DPPH, FRAP, hydroxyl radicals and superoxide anion scavenging rates of Z-Myr were significantly higher than those of Z-Myr (60.40% vs. 25.28%, 57.57% vs. 32.96%, 0.65% vs. 0.42, 50.37% vs. 30.15%, 50.88% vs. 29.65%). This indicates that the hydrophilic CCAP is tightly adsorbed on the surface of NPs (CCAP-Z-Myr), which effectively improves the solubility of NPs (CCAP-Z-Myr) and greatly enhances the bioactivity of Myr in aqueous solution. CCAP is adsorbed on the surface of Z, forming a larger hydrophobic area, which enables Myr to fully interact with free radicals.

[0070] 5. α-Glucosidase inhibitory activity The results are as follows Figure 6 The results showed that within the dose range of 20.0-100.0 μg / mL, the activity of Myr, CCAP, Z-Myr NPs, CCAP-Z NPs, and CCAP-Z-Myr NPs was significantly higher than that of CCAP-Z-Myr NPs. α The inhibitory effects of -glucosidase increased gradually in a dose-dependent manner, with the inhibitory effects of CCAP-Z-Myr increasing from 55.4% to 78.5%, Z-Myr from 35.4% to 64.9%, CCAP-Z from 25.3% to 56.0%, CCAP from 24.0% to 49.4%, and Myr from 30.6% to 60.5%; all samples showed the highest inhibitory effects at the maximum concentration (100.0 μg / mL). α -glucosidase inhibitory activity, CCAP-Z-Myr NPs showed the greatest α -glucosidase inhibition potential (78.5%, 100 μg / mL). It is worth noting that as a control, the inhibitory ability of CCAP-Z-Myr is comparable to that of acarbose in the range of 40-100 μg / mL, while at 20 μg / mL, it significantly exceeds acarbose (40.88%). This indicates that the prepared compound has excellent oral hypoglycemic performance with meals that exceeds acarbose at low concentrations (55.43% vs. 40.88%).

[0071] Compared with free Myr (the inhibition rate increased from 30.6% to 60.5%), Z-Myr NPs α -glucosidase inhibition rate increased from 35.4% to 64.9%. This improvement was attributed to the enhanced dispersibility of Myr encapsulated in Z. CCAP-Z-Myr NPs. Compared with Z-Myr (64.8%, 100 μg / mL), Myr NPs showed the highest α -glucosidase inhibition potential (78.5%, 100 μg / mL). TEM, FTIR and XRD analysis showed that CCAP was adsorbed on the Z surface through hydrogen bonding and electrostatic effects, which increased the solubility of nanoparticles and thus enhanced the inhibitory effect of nanoparticles.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing zein animal and plant dual protein nanoparticles modified with donkey-hide gelatin collagen peptide, characterized in that: The following steps are involved: (1) Grinding donkey-hide gelatin and dissolving it in a Na2CO3 solution, mixing and centrifuging the obtained precipitate, and then freeze-drying it to obtain defatted powder; dissolving the defatted powder in a NaOH solution, mixing and centrifuging the obtained supernatant, and then dialyzing and freeze-drying it to obtain a crude collagen powder; (2) dissolving the crude collagen powder in acetic acid, then adding pepsin and performing ultrasonic treatment. After the ultrasonic treatment, the supernatant obtained by centrifuging the mixture is dialyzed and freeze-dried to obtain donkey-hide gelatin collagen; (3) dissolving the donkey-hide gelatin collagen in distilled water, adjusting the pH value to 7.5-8 with a NaOH solution, then adding a composite enzyme preparation and stirring the mixture, centrifuging the obtained mixture to obtain a supernatant, and freeze-drying the supernatant to obtain a hydrophilic donkey-hide gelatin collagen peptide; (4) Zein and myricetin are dissolved in ethanol and subjected to ultrasonic treatment. The hydrophilic donkey-hide gelatin collagen peptide solution is then added and mixed evenly. The resulting mixture is centrifuged to obtain a supernatant, and the supernatant is freeze-dried to obtain zein animal and plant dual protein nanoparticles.

2. The method for preparing zein double-protein nanoparticles according to claim 1, characterized in that: In the step (1), the mass volume ratio of donkey-hide gelatin to Na2CO3 solution is 1:20-30; the mass volume ratio of defatted powder to NaOH solution is 1:20-25.

3. The method for preparing zein double-protein nanoparticles according to claim 1, characterized in that: In the step (2), the mass volume ratio of the crude collagen powder to the acetic acid is 1:40-45; the mass ratio of the pepsin to the crude collagen powder is 1:40-80, and the enzyme activity is ≥2500 U / mg.

4. The method for preparing zein double-protein nanoparticles according to claim 1, characterized in that: In the step (3), the mass volume ratio of donkey-hide gelatin collagen to distilled water is 1:5-6, and the added amount of the complex enzyme preparation is 3.5%-4.5% of the mass of the donkey-hide gelatin collagen.

5. The method for preparing zein double-protein nanoparticles according to claim 1, characterized in that: In the step (4), the mass ratio of zein to myricetin is 10-15:1-3, the concentration of the hydrophilic donkey-hide gelatin collagen peptide solution is 15 mg / mL-20 mg / mL, and the volume mass ratio of the hydrophilic donkey-hide gelatin collagen peptide to zein is 3-5:

1.

6. Zein animal and plant dual protein nanoparticles prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The particle size of the zein animal and plant dual protein nanoparticles is 95-100 nm.

7. Use of the zein animal and plant dual protein nanoparticles according to claim 6 in antioxidants.

8. The zein animal and plant dual protein nanoparticles according to claim 6 are prepared α- Application of glucosidase inhibitors.

9. Use of the zein animal and plant dual protein nanoparticles according to claim 6 in the preparation of a drug for treating diabetes.

10. The use according to claim 9, characterized in that: The medicine contains 20 μg / mL-100 μg / mL of zein animal and plant dual protein nanoparticles.

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