Procyanidin c1 nanoparticle, and preparation method and application thereof

Abstract

The application provides procyanidin C1 nanoparticles and a preparation method and application thereof, and relates to the field of food processing technology. The nanoparticles are prepared by using lysine as a coupling agent, polysaccharides (such as rhizoma polygonati polysaccharide, dendrobium polysaccharide and lycium barbarum polysaccharide) as embedding materials, and procyanidin C1 as core materials through a self-assembly method. The nanoparticles can significantly improve the embedding rate and stability of procyanidin C1, enhance the antioxidant and anti-aging effects of procyanidin C1, and can be used for constructing a nano delivery system or preparing anti-aging and antioxidant drugs and foods, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a proanthocyanidin C1 nanoparticle, its preparation method, and its application. Background Technology

[0002] Aging is a major risk factor for all age-related diseases. With the gradual decline of bodily physiological functions, the continuous accumulation of cellular damage and the systemic decrease in repair capacity lead to molecular-level changes such as abnormal DNA methylation, telomere shortening, protein homeostasis imbalance, and mitochondrial dysfunction. These biological changes not only directly cause degenerative diseases such as osteoporosis and muscle atrophy, but also accelerate the development of complex diseases such as neurodegenerative diseases, cardiovascular diseases, type 2 diabetes, and malignant tumors through the formation of a chronic inflammatory microenvironment (i.e., "inflammatory aging"). Therefore, more and more researchers and institutions are beginning to focus on anti-aging research. Current anti-aging research focuses on the regulation of the mTOR signaling pathway and NAD+. + Intervention strategies such as precursor supplementation and senolytics drug development aim to achieve breakthroughs in cross-disease treatment by targeting the aging-associated secretory phenotype (SASP).

[0003] However, compared to anti-aging drugs, natural active substances in fruits, vegetables, and medicinal and edible crops have advantages such as high safety, synergistic effects on multiple targets, easy accessibility, and dual nutritional and functional properties. Grape seed and its extracts are well known to be important components of many anti-aging products. Recent research has found that proanthocyanidin C1 is a key substance mediating the anti-aging effects of grape seed extract. However, during oral administration, proanthocyanidin C1 is easily degraded in the oral and gastric environment, resulting in low levels reaching the intestinal target organs. Therefore, increasing the concentration of proanthocyanidin C1 entering the intestine is crucial for improving its oral bioavailability. Summary of the Invention

[0004] In view of this, the present invention provides proanthocyanidin C1 nanoparticles and their preparation method. The system is constructed using polysaccharides as carriers, which can effectively improve the oral absorption rate of active small molecules and enhance the antioxidant and anti-aging effects of the product.

[0005] The first aspect of the present invention is to provide proanthocyanidin C1 nanoparticles, which are prepared by a self-assembly method using coupling agents, encapsulating wall materials and core materials as raw materials.

[0006] Preferably, the coupling agent is lysine, the embedding wall material is a polysaccharide, and the core material is proanthocyanidin C1. More preferably, the polysaccharide is at least one selected from Polygonatum polysaccharide, Dendrobium polysaccharide, and Lycium barbarum polysaccharide.

[0007] A second aspect of the present invention is to provide a method for preparing proanthocyanidin C1 nanoparticles, comprising the following steps:

[0008] Proanthocyanidin C1 ethanol aqueous solution and lysine ethanol aqueous solution were mixed in proportion and stirred for a period of time to obtain proanthocyanidin C1-lysine mixed solution. Then, the proanthocyanidin C1-lysine mixed solution was quickly injected into polysaccharide aqueous solution, mixed in proportion and stirred for a period of time, washed and dried to obtain proanthocyanidin C1 nanoparticles.

[0009] Preferably, the concentration of proanthocyanidin C1 in the proanthocyanidin C1 ethanol aqueous solution is 40-60 μg / mL, the concentration of lysine in the lysine ethanol aqueous solution is 4-6 μg / mL, the concentration of polysaccharide in the polysaccharide aqueous solution is 8-16 mg / mL, the volume ratio of the proanthocyanidin C1 ethanol aqueous solution to the lysine ethanol aqueous solution is 1:(1-2), the volume ratio of the proanthocyanidin C1 ethanol aqueous solution to the polysaccharide aqueous solution is 1:(2-4), the stirring time after mixing the proanthocyanidin C1 ethanol aqueous solution and the lysine ethanol aqueous solution is 1.5-2.5 h, the stirring speed is 500-700 rpm, and the stirring time after mixing the proanthocyanidin C1-lysine mixed solution and the polysaccharide aqueous solution is 1-3 h, the stirring speed is 500-700 rpm.

[0010] Preferably, the drying method is freeze drying, and the drying time is not less than 48 hours.

[0011] A third aspect of the present invention is to provide the application of proanthocyanidin C1 nanoparticles in constructing a proanthocyanidin C1 nanodelivery system, wherein the proanthocyanidin C1 nanoparticles are the proanthocyanidin C1 nanoparticles described in the above technical solution.

[0012] A fourth aspect of the present invention is to provide the application of proanthocyanidin C1 nanoparticles in the preparation of drugs and foods with anti-aging and antioxidant functions, wherein the proanthocyanidin C1 nanoparticles are the proanthocyanidin C1 nanoparticles described in the above technical solution.

[0013] This invention uses lysine as a coupling agent to significantly improve the encapsulation rate of proanthocyanidin C1 and facilitates the formation of lysine-polysaccharide complexes through electrostatic interactions, thereby enhancing the stability of nanoparticles. The nanoparticles constructed using polysaccharides as carriers in this invention can effectively improve the oral absorption rate of active small molecules and enhance the antioxidant and anti-aging effects of the nanoparticles through synergistic effects. The nanoparticles of this invention not only exhibit strong stability but also precise targeted release, remaining stable under heat, light, and simulated in vitro digestive environments, significantly extending the maximum and average lifespan of fruit flies. Simultaneously, the nanoparticles of this invention significantly improve the locomotion and intestinal permeability of fruit flies, and can be used to construct proanthocyanidin C1 nanodelivery systems or in the preparation of drugs and foods with anti-aging and antioxidant functions, showing promising application prospects. Attached Figure Description

[0014] The present invention will be further described below with reference to the accompanying drawings.

[0015] Figure 1 The results are for encapsulation efficiency and drug loading, where: Figure 1 A represents the effect of polysaccharide concentration on encapsulation efficiency. Figure 1 B represents the effect of polysaccharide concentration on drug loading. Figure 1 C represents the effect of polysaccharide concentration on particle size. Figure 1 D shows the zeta potential of nanoparticles prepared by first cross-linking lysine with proanthocyanidin C1 and then combining it with Polygonatum polysaccharide; Figure 1 E shows the zeta potential of nanoparticles prepared by simultaneously adding proanthocyanidin C1, polygonatum polysaccharide and lysine solution;

[0016] Figure 2 The results characterize the embedding situation, including: Figure 2 A and Figure 2 B is a scanning electron microscope image. Figure 2 C and Figure 2 Transmission electron microscopy image (D); Figure 2 E represents the infrared spectrum; Figure 2 F represents the XRD pattern;

[0017] Figure 3 The results are for stability testing, where: Figure 3 A represents the results of the photostability measurement. Figure 3 B represents the result of the thermal stability test. Figure 3 C represents the release rate of nanoparticles after incubation in simulated oral, gastric, and intestinal fluids in vitro;

[0018] Figure 4 These are results from animal experiments, including: Figure 4 A represents the longest lifespan of a female fruit fly. Figure 4 B represents the longest lifespan of male fruit flies. Figure 4C represents the climbing ability of female fruit flies. Figure 4 D represents the climbing ability of male fruit flies. Figure 4 E represents the intestinal permeability result in female fruit flies. Figure 4 F represents the intestinal permeability result in male fruit flies. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] The first aspect of the present invention is to provide proanthocyanidin C1 nanoparticles, which are prepared by a self-assembly method using crosslinking agent, embedding wall material and core material as raw materials.

[0021] The coupling agent of this invention is lysine, the wall material is a polysaccharide, and the core material is proanthocyanidin C1. In some specific embodiments of this invention, the polysaccharide is at least one selected from Polygonatum sibiricum polysaccharide, Dendrobium nobile polysaccharide, and Lycium barbarum polysaccharide. The polysaccharide of this invention can be a commercially available product or can be prepared by the following methods:

[0022] Crude polysaccharide was extracted from the raw material by water-soluble alcohol precipitation, followed by protein removal by Sevag method, resin decolorization, and dialysis to remove impurities, thus obtaining polysaccharide (PSP).

[0023] A second aspect of the present invention is to provide a method for preparing proanthocyanidin C1 nanoparticles, comprising the following steps:

[0024] Proanthocyanidin C1 ethanol aqueous solution and lysine ethanol aqueous solution were mixed in proportion and stirred for a period of time to obtain proanthocyanidin C1-lysine mixed solution. Then, the proanthocyanidin C1-lysine mixed solution was quickly injected into polysaccharide aqueous solution, mixed in proportion and stirred for a period of time, washed and dried to obtain proanthocyanidin C1 nanoparticles.

[0025] The concentration of proanthocyanidin C1 in the proanthocyanidin C1 ethanol aqueous solution is 40-60 μg / mL, and the concentration of lysine in the lysine ethanol aqueous solution is 4-6 μg / mL; the concentration of polysaccharide in the polysaccharide aqueous solution is 8-16 mg / mL; the volume ratio of the proanthocyanidin C1 ethanol aqueous solution to the lysine ethanol aqueous solution is 1:(1-2), and the volume ratio of the proanthocyanidin C1 ethanol aqueous solution to the polysaccharide aqueous solution is 1:(2-4); the stirring time after mixing the proanthocyanidin C1 ethanol aqueous solution and the lysine ethanol aqueous solution is 1.5-2.5 h, and the stirring speed is 500-700 rpm; the stirring time after mixing the proanthocyanidin C1-lysine mixed solution and the polysaccharide aqueous solution is 1-3 h, and the stirring speed is 500-700 rpm.

[0026] The drying method described in this invention is freeze drying, and the drying time is not less than 48 hours.

[0027] To further illustrate the present invention, the following embodiments are provided for detailed description. All raw materials used in the following embodiments of the present invention are commercially available.

[0028] Unless otherwise specified, all experiments were repeated three times. Analysis of variance (ANOVA) and Duncan's multiple comparison analysis were performed using SPSS 21.0. Results are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0029] The method for treating the macroporous resin in this embodiment of the invention is as follows:

[0030] The macroporous resin was washed with distilled water, soaked in 3 column volumes of 95% ethanol for 24 hours, washed with anhydrous ethanol until no white turbidity was observed, and then washed with distilled water until no ethanol odor was observed. The macroporous resin was then soaked in 3 column volumes of 5% HCl solution for 3 hours, washed with deionized water until neutral, soaked in 5% NaOH solution for 3 hours, and finally washed with distilled water until neutral to obtain the treated macroporous resin.

[0031] Example 1: A method for preparing nanoparticles containing proanthocyanidin C1 encapsulated in Polygonatum polysaccharide, the steps of which are as follows:

[0032] Accurately weigh 0.25 mg of proanthocyanidin C1 (PCC1), dissolve it completely in 5 mL of anhydrous ethanol, stir and sonicate until fully dissolved to obtain a PCC1 solution with a concentration of 50 μg / mL; dissolve different amounts of Polygonatum polysaccharide in ultrapure water to prepare a series of Polygonatum polysaccharide solutions with different concentrations (1, 4, 8, 12 and 16 mg / mL);

[0033] Weigh a certain amount of lysine and dissolve it in 80% ethanol to prepare an 80% (v / v) 50 μg / mL lysine ethanol aqueous solution;

[0034] The specific steps are as follows:

[0035] At room temperature, 1 mL of PCC1 solution was rapidly injected into 3 mL of different concentrations of Polygonatum polysaccharide solution. The mixture was stirred continuously at 600 rpm for 2 h with a magnetic stirrer. Then, it was centrifuged at 12000 r / min for 15 min, the precipitate was collected, washed with deionized water to remove residual ethanol, and freeze-dried to obtain single Polygonatum polysaccharide-embedded proanthocyanidin C1 nanoparticles (PCC1-PSPNPs).

[0036] Under normal temperature conditions, a PCC1-Lys mixture was first uniformly mixed with a 50 μg / mL PCC1 solution and a 50 μg / mL lysine ethanol aqueous solution to form a PCC1-Lys mixture. Then, 1 mL of the PCC1-Lys mixture was taken and quickly injected into 3 mL of a Polygonatum polysaccharide solution with different proportions. The mixture was then stirred at 600 rpm for 2 hours, followed by centrifugation, washing, and freeze-drying to finally obtain nanoparticles (PCC1-P / LNPs) formed by cross-linking lysine with Polygonatum polysaccharide and proanthocyanidin C1.

[0037] The preparation method of the Polygonatum polysaccharide is as follows:

[0038] The dried rhizomes of Polygonatum multiflorum were ground into powder and passed through a 60-mesh sieve to obtain Polygonatum powder. 5g of Polygonatum powder was weighed and placed in an Erlenmeyer flask. Anhydrous ethanol was added at a ratio of 1:8, and the mixture was soaked for 48 hours. The mixture was then filtered and dried to obtain a defatted Polygonatum sample. 4g of the defatted Polygonatum sample was weighed and deionized water was added at a ratio of 1:40. The mixture was sonicated at 400W for 50 minutes. The supernatant was collected and the process was repeated twice. The filtrates obtained from the two processes were combined and concentrated to 20mL in a rotary evaporator. Four times the volume of anhydrous ethanol was added, and the mixture was quickly mixed. The mixture was precipitated overnight at 4℃ and centrifuged to obtain crude polysaccharide.

[0039] Prepare Sevag reagent according to the ratio of chloroform: n-butanol = 4:1 (V:V). Add the crude polysaccharide obtained from S1 to 5 mL of distilled water, add 1.25 mL of Sevag reagent, centrifuge, repeat three times, and combine the supernatants to obtain polysaccharide aqueous solution.

[0040] Take 1g of the processed macroporous resin, put it into a 150mL Erlenmeyer flask, add 30mL of polysaccharide aqueous solution, and place it on a 35℃ constant temperature shaker for 3h for adsorption. Filter the macroporous resin after adsorption and collect the supernatant. Then add the polysaccharide liquid after resin adsorption into a 3500KD dialysis bag and dialyze with distilled water for 3 days, changing the water once in the morning and once in the evening. After lyophilizing the dialysate, use the Beyotime protein kit to determine the protein removal status. After confirming that the protein has been removed, Polygonatum polysaccharide is obtained.

[0041] Example 2: A method for preparing nanoparticles containing proanthocyanidin C1 encapsulated in Dendrobium polysaccharide, the same as in Example 1, except that the polysaccharide is Dendrobium polysaccharide.

[0042] The preparation method of the Dendrobium polysaccharide is as follows:

[0043] Dendrobium officinale was placed in a drying oven and dried to constant weight. After being ground into powder, it was passed through a 60-mesh sieve to obtain Dendrobium officinale powder. 5g of Dendrobium officinale powder was weighed and added to deionized water at a ratio of 1:50. 0.1g of pectinase was added and the mixture was sonicated at 250W for 30min. The supernatant was collected and the process was repeated twice. The filtrates obtained from the two processes were combined and concentrated to 20mL in a rotary evaporator. Four times the volume of ethanol was added and the mixture was quickly mixed. The mixture was precipitated at 4℃ overnight and centrifuged to obtain crude polysaccharide.

[0044] Prepare Sevag reagent according to the ratio of chloroform: n-butanol = 4:1 (V:V). Add the crude polysaccharide obtained from S1 to 5 mL of distilled water, add 1.25 mL of Sevag reagent, centrifuge, repeat three times, and combine the supernatants to obtain polysaccharide aqueous solution.

[0045] Take 1g of the processed macroporous resin and put it into a 150mL Erlenmeyer flask. Add 30mL of polysaccharide aqueous solution and place it on a 35℃ constant temperature shaker for 3h for adsorption. Filter the macroporous resin after adsorption and collect the supernatant. Then add the polysaccharide liquid after resin adsorption into a 3500KD dialysis bag and dialyze with distilled water for 3 days, changing the water once in the morning and once in the evening. After lyophilizing the dialysate, use the Beyotime protein kit to determine the protein removal. After confirming that the protein has been removed, Dendrobium polysaccharide is obtained.

[0046] Example 3: A method for preparing nanoparticles containing proanthocyanidins C1 encapsulated in Lycium barbarum polysaccharide, the same as in Example 1, except that the polysaccharide is Lycium barbarum polysaccharide.

[0047] The preparation method of the Lycium barbarum polysaccharide is as follows:

[0048] Place the goji berries in a drying oven and dry them to constant weight. After grinding them into powder, pass them through a 60-mesh sieve to obtain goji berry powder. Weigh 10g of goji berry powder and add deionized water at a ratio of 1:50. Extract the powder with hot water at 62℃ for 80min. Centrifuge and collect the supernatant. Evaporate the supernatant by rotary evaporation and concentrate it to 50mL. Add 4 times the volume of ethanol, mix quickly, and precipitate the ethanol at 4℃ for 12h. Centrifuge and collect the precipitate to obtain crude polysaccharide.

[0049] Prepare Sevag reagent according to the ratio of chloroform: n-butanol = 4:1 (V:V). Add the crude polysaccharide obtained from S1 to 5 mL of distilled water, add 1.25 mL of Sevag reagent, centrifuge, repeat three times, and combine the supernatants to obtain polysaccharide aqueous solution.

[0050] Take 1g of the processed macroporous resin and put it into a 150mL Erlenmeyer flask. Add 30mL of polysaccharide aqueous solution and place it on a 35℃ constant temperature shaker for 3h for adsorption. Filter the macroporous resin after adsorption and collect the supernatant. Then add the polysaccharide liquid after resin adsorption into a 3500KD dialysis bag and dialyze with distilled water for 3 days, changing the water once in the morning and once in the evening. After lyophilizing the dialysate, use the Beyotime protein kit to determine the protein removal. After confirming that the protein has been removed, wolfberry polysaccharide is obtained.

[0051] Test Example 1: Determination of Encapsulation Efficiency and Drug Loading

[0052] (1) Experimental methods

[0053] In Example 1, after self-assembly, the supernatant of the nanoparticles was collected to calculate the encapsulation efficiency and drug loading. Nanoparticles encapsulated with a single Polygonatum polysaccharide were designated PCC1-PSPNPs, and cross-linked nanoparticles formed after adding lysine were designated PCC1-P / LNPs. The average particle size, PDI, and Zeta potential were measured using a laser particle size analyzer.

[0054] The formula for calculating the embedding rate is as follows:

[0055] Wtotal-Wfree / Wtotal×100%;

[0056] Where Wfree is the weight of polysaccharides in the supernatant, and Wtotal is the total weight of polysaccharides.

[0057] The formula for calculating drug loading is as follows:

[0058] Wtotal-Wfree / Wnp×100%;

[0059] Where Wfree is the weight of proanthocyanidin C1 in the supernatant; Wnp is the total weight of the nanoparticles.

[0060] (2) Experimental Results

[0061] The experimental results were obtained by investigating the polysaccharide concentration and the addition of lysine coupling agent, as follows: Figure 1 As shown.

[0062] Depend on Figure 1 It can be seen that the encapsulation efficiency (EE) of PCC1-PSPNPs gradually increases with increasing polysaccharide concentration, reaching only 79.6% at 16 mg / mL. However, the cross-linking effect is further enhanced by the introduction of lysine. Figure 1 After A and B), the encapsulation capacity and drug loading of the nanosystem were significantly improved. Figure 1B), among which, when the polysaccharide concentration was 8 mg / mL, the highest encapsulation amount of 93.57% was observed, which was 21.9% higher than that of nanoparticles encapsulated with single polysaccharide (71.67%). With increasing polysaccharide content, the particle size of the cross-linked PCC1-P / LNPs first decreased and then increased, from 1489 nm to 313 nm, and then to 424 nm, while the PDI decreased from 1 to 0.35. When the polysaccharide concentration was 8 mg / mL, the PDI value of the nanoparticles was significantly lower than that of other groups, indicating that the nanoparticles prepared under this condition had better dispersibility and more uniform particle size distribution. Figure 1 C).

[0063] The Zeta potential results show that all nanoparticles have negative Zeta potentials, indicating that they carry a charge in the suspended state. The absolute values ​​of the Zeta potentials are all greater than 20 mV, demonstrating the excellent physicochemical stability of the nanoparticles of this invention. Figure 1 D).

[0064] Meanwhile, during the experiment, it was found that when lysine, proanthocyanidin C1, and Polygonatum polysaccharide solutions were mixed simultaneously for assembly, the absolute value of the Zeta potential decreased significantly, negatively impacting the stability of the nanoparticles. Figure 1 E). Therefore, the preparation process of nanoparticles should follow the following order of addition: first, thoroughly mix proanthocyanidin C1 with lysine to promote cross-linking, and then inject the mixture into the polysaccharide solution to ensure its full binding.

[0065] Test Example 2 Embedding Characterization

[0066] (1) Experimental methods

[0067] The nanoparticles (PCC1-P / LNPs) containing proanthocyanidin C1, the wall material nanoparticles (PSP-Lys NPs), PCC1, and lysine (Lys) prepared in Example 1 were analyzed by scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, and XRD.

[0068] The preparation process of the wall material nanoparticles is as follows: At room temperature, a lysine ethanol aqueous solution with a concentration of 50 μg / mL and a Polygonatum polysaccharide solution with a concentration of 8 mg / mL are mixed at a volume ratio of 0.5:3 and stirred at 600 rpm for 2 h in a magnetic stirrer. Then, the mixture is centrifuged at 12000 r / min for 15 min, the precipitate is collected, washed with deionized water to remove residual ethanol, and freeze-dried to obtain wall material nanoparticles (PSP-Lys NPs).

[0069] (2) Experimental Results

[0070] The surface morphology and internal structure of the nanoparticles were analyzed by scanning electron microscopy. The proanthocyanidin C1 nanoparticles embedded in Polygonatum polysaccharide exhibited a multilayered porous network morphology, with a large number of subspherical small particles distributed on the surface, which were relatively smooth and showed no obvious aggregation. Figure 2 A); in contrast, the wall material nanoparticles exhibit an aggregated state, displaying loose stacking and a porous structure. Figure 2 B). Transmission electron microscopy results show that the nanoparticles prepared in this invention are spherical or subspherical, possess a core-shell structure with alternating light and dark areas, and exhibit high dispersibility. Figure 2 C). Meanwhile, wall nanoparticles lacking PCC1 exhibit a more loosely structured spherical shape. Figure 2 D). Fourier transform infrared spectroscopy (FT-IR) elucidated the interactions between the proposed nanoparticle components. The characteristic absorption band of proanthocyanidin C1 almost disappeared or overlapped with the characteristic absorption band of the encapsulation material. This may be because the encapsulation effect of the nanocarrier restricts the stretching and contraction of the chemical groups on PCC1, leading to a weakening of its characteristic signal. Figure 2 E). X-ray diffraction patterns showed that both Polygonatum polysaccharide and lysine exhibited certain crystalline structures. The diffraction curves showed flat peaks at diffraction angles of 22.73° and 23.40°, respectively, with no sharp characteristic strong peaks observed in other regions. After the formation of nanoparticles, the diffraction peak intensity of lysine decreased significantly, possibly because the shrinkage of the hydrophobic region during the antisolvent precipitation process disrupted the crystal structure of lysine. Figure 2 F).

[0071] Test Example 3 Stability Determination

[0072] (1) Experimental methods

[0073] Stability in thermal and light environments:

[0074] Five mL of the Polygonatum polysaccharide-embedded proanthocyanidin C1 suspension prepared in Example 1 was placed in boiling water baths at different temperatures (25, 45, 65, and 85°C) for 30 min, and then cooled to room temperature (25°C). Five mL of freshly prepared nanoparticle suspension in a transparent glass vial was exposed to 36W ultraviolet light at a distance of 15 cm for 15, 30, 45, 60, and 90 min. Subsequently, the average particle size was measured using a laser particle size analyzer, and the residual PCC1 content in the sample was determined by ultraviolet-visible spectrophotometry.

[0075] In vitro digestion:

[0076] ① Oral stage: Take 3mg of nanoparticles, mix the nanoparticle suspension with 4mL of fresh oral saliva, and centrifuge at 2000g for 30s to thoroughly mix the nanoparticles and saliva. Then, adjust the pH of the digestive solution to 2.0 to inactivate salivary amylase.

[0077] ② Simulated gastric juice digestion: After oral digestion, the mixture was added to 100 mL of simulated gastric juice and incubated at 37°C for 2 hours (wrapped in aluminum foil). The gastric juice was simulated by 0.9% sodium chloride solution and 1.4 mg / mL pepsin solution. After digestion, the pH of the digestive juice was adjusted to 7.0 for further intestinal digestion.

[0078] ③ Simulated small intestinal digestion: The simulated intestinal fluid consisted of 12 mg / mL bile salts and 2.25 mg / mL pancreatic enzymes, with the pH adjusted to 7.0 using 1 mL NaOH solution. The digestive fluid was mixed with 45 mL of the above-mentioned small intestinal fluid, and incubated for another 2 hours under the same conditions. Finally, the digestion process was terminated by heating at 95°C for 5 minutes.

[0079] At each stage of gastrointestinal digestion, 2 mL samples were collected from the digestion mixture every 5 minutes for the first 30 minutes, and the same volume of digestion mixture was added. Thereafter, 2 mL samples were collected every 10 minutes, and the same volume of digestion mixture was added. The pH of the collected samples was rapidly adjusted to 7.0, and the samples were centrifuged at 12,000 rpm for 15 minutes. The polysaccharide content in the supernatant was determined, and the release amount was calculated by dividing the total polysaccharide content loaded in the nanoparticles by the polysaccharide content released in the supernatant.

[0080] (2) Experimental Results

[0081] Depend on Figure 3 As shown in A and 3B, after treatment under ultraviolet light for 90 min or in water baths at 25, 45, 65, and 85°C for 30 min, the nanoparticles prepared in this invention did not undergo significant degradation. In vitro digestion results are as follows... Figure 3 As shown in Figure C, after incubation in the oral cavity and simulated gastric juice for 4 hours, the total release rate of free proanthocyanidin C1 was 39.73%, while the release rate of nanoparticles under simulated intestinal conditions was 50.38%, which was significantly higher than the release rate of 22.67% under simulated gastric environment conditions. This indicates that the nanoparticles prepared in this invention effectively increased the concentration of proanthocyanidin C1 reaching the target organ.

[0082] Test Example 4: Animal Experiment

[0083] (1) Experimental methods

[0084] Study on synergistic extension of fruit fly lifespan:

[0085] Nanoparticles containing proanthocyanidin C1 encapsulated in Polygonatum polysaccharide prepared in Example 1 were used as samples. Wild-type Drosophila melanogaster W118 was used as a model organism and the flies were reared in corn yeast culture medium under controlled conditions (25°C, 65% relative humidity, 12-hour light-dark cycle). The Drosophila that hatched within 72 hours were randomly assigned to the control group and the experimental group, with 20 flies per vial in each group.

[0086] The experimental groups included the Polygonatum polysaccharide (PSP) group, the proanthocyanidin C1 (PCC1) group, the high-dose nanoparticle group (NP-H), and the low-dose nanoparticle group (NP-L). The Polygonatum polysaccharide group and the proanthocyanidin C1 (PCC1) group were designated as the single-drug treatment groups. Each group was replicated four times. The culture medium was changed every three days, and the number of dead fruit flies was recorded daily until all fruit flies died. Data for each group was recorded independently at least four times, and survival time was calculated.

[0087] The preparation process of the culture medium is as follows:

[0088] 1. Basal culture medium: Weigh 20.87g of Drosophila culture medium, dissolve it in 100mL of distilled water, let it cool slightly, add 0.65mL of propionic acid, mix thoroughly, and pour the medium into sterilized culture flasks while still warm. The basal culture medium should be replaced approximately every three weeks.

[0089] 2. Polygonatum polysaccharide (PSP) group: 16 mg of Polygonatum polysaccharide was accurately weighed and dissolved in 2 mL of deionized water to prepare Polygonatum polysaccharide stock solution. Then, 2.25 mL of PSP stock solution with a concentration of 8 mg / 100 mL was added to the basal culture medium and stirred evenly to obtain a Drosophila culture medium with a concentration of 18%.

[0090] 3. Proanthocyanidin C1 (PCC1) group: PCC1 was dissolved in deionized water to prepare PCC1 stock solution. 1.0 mL of PCC1 stock solution with a concentration of 42 μg / 100 mL was added to the basal culture medium and stirred evenly to obtain a Drosophila culture medium with a final concentration of 0.042%.

[0091] 4. Nanoparticle group: Add 0.5 mL and 5.0 mL of nanoparticle solution with a concentration of 1 mg / 100 mL to the basal culture medium, respectively, and stir evenly to obtain Drosophila culture medium with concentrations of 0.5% (NP-L) and 5.0% (NP-H), respectively.

[0092] Studies on improving motility and intestinal permeability in fruit flies:

[0093] Fruit flies fed for 5, 10, and 20 days were assessed for locomotion using a negative geotaxis climbing experiment, while the Smurf experiment was used to detect intestinal permeability.

[0094] (2) Experimental Results

[0095] Figure 4 A and Figure 4Results B showed that, compared with the blank control group, the median lifespan of male and female fruit flies in the low-dose nanoparticle group was extended by 10.3% and 15.4%, respectively, and the maximum lifespan was extended by 19.2% and 11.1%, respectively; while the median lifespan of male and female fruit flies in the high-dose nanoparticle group was extended by 25.6% and 16.6%, respectively, and the maximum lifespan was extended by 30.8% and 18.5%, respectively. Lifespan experiments indicate that the nanoparticles prepared in this invention can extend the median and maximum lifespan of fruit flies, and the lifespan extension effect is greater than that of adding any single component, indicating that the various raw materials in the nanoparticles have a good synergistic effect on the lifespan of fruit flies.

[0096] Figure 4 C and Figure 4 Results showed that the locomotion ability of fruit flies gradually decreased with increasing age. On day 20, the effects of high and low nanoparticle dosage groups on both male and female fruit flies were significantly higher than those on single-drug groups (p < 0.001). Compared with single-component treatments, the high and low nanoparticle groups showed the most significant improvement in climbing ability. These effects were statistically significantly different from the observations of proanthocyanidin C1 and Polygonatum polysaccharide treatment groups alone (p < 0.05), indicating that the synergistic effect of the two enhanced locomotion behavior.

[0097] Figure 4 E and Figure 4 Results showed that on day 20, the nanoparticles significantly improved the intestinal barrier function of aged male and female fruit flies. Specifically, the proportion of female "blue elves" fruit flies decreased by 34% and 27% in the high-dose and low-dose combined groups, respectively, while the proportion of male fruit flies decreased by 30% and 23%, respectively. Therefore, the combined treatment group demonstrated a positive effect in improving the intestinal morphological integrity of aging fruit flies.

[0098] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A type of proanthocyanidin C1 nanoparticle, characterized in that, The nanoparticles are prepared by a self-assembly method using coupling agents, encapsulating wall materials, and core materials as raw materials. The coupling agent is lysine, the embedding wall material is polysaccharide, and the core material is proanthocyanidin C1; The polysaccharide is at least one of Polygonatum polysaccharide, Dendrobium polysaccharide, and Lycium barbarum polysaccharide; The preparation method of the proanthocyanidin C1 nanoparticles includes the following steps: Proanthocyanidin C1 ethanol aqueous solution and lysine ethanol aqueous solution were mixed in proportion and stirred for a period of time to obtain proanthocyanidin C1-lysine mixed solution. Then, the proanthocyanidin C1-lysine mixed solution was quickly injected into polysaccharide aqueous solution, mixed in proportion and stirred for a period of time, washed and dried to obtain proanthocyanidin C1 nanoparticles. The concentration of proanthocyanidin C1 in the ethanol aqueous solution is 40-60 μg / mL; the concentration of lysine in the lysine ethanol aqueous solution is 4-6 μg / mL; and the concentration of polysaccharide in the polysaccharide aqueous solution is 8-16 mg / mL. The volume ratio of the proanthocyanidin C1 ethanol aqueous solution to the lysine ethanol aqueous solution is 1:(1-2), and the volume ratio of the proanthocyanidin C1 ethanol aqueous solution to the polysaccharide aqueous solution is 1:(2-4). The drying method is freeze drying, and the drying time is not less than 48 hours.

2. The application of proanthocyanidin C1 nanoparticles in constructing a proanthocyanidin C1 nanodelivery system, characterized in that, The proanthocyanidin C1 nanoparticles are the proanthocyanidin C1 nanoparticles as described in claim 1.

3. The application of proanthocyanidin C1 nanoparticles in the preparation of drugs with anti-aging and antioxidant functions, characterized in that, The proanthocyanidin C1 nanoparticles are the proanthocyanidin C1 nanoparticles as described in claim 1.

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