Procyanidine C1 nano-particles as well as preparation method and application thereof

Abstract

The invention provides procyanidine C1 nanoparticles as well as a preparation method and application thereof, and relates to the technical field of food processing. The nanoparticles are prepared by using lysine as a coupling agent, polysaccharide (such as polygonatum polysaccharide, dendrobium polysaccharide and lycium barbarum polysaccharide) as an embedding wall material and procyanidine C1 as a core material through a self-assembly method. The nanoparticles can significantly improve the embedding rate and stability of the proanthocyanidins C1, enhance the anti-oxidation and anti-aging effects of the proanthocyanidins C1, can be used for constructing a nano delivery system or preparing anti-aging and anti-oxidation medicines and foods, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a proanthocyanidin C1 nanoparticle and a preparation method and application thereof. Background Art

[0002] Aging is the main risk factor for all age-related diseases. With the progressive decline of the body's physiological functions, the continuous accumulation of cell damage and the systematic decline of repair capacity lead to molecular changes such as abnormal DNA methylation, telomere shortening, imbalance of protein homeostasis, and mitochondrial dysfunction. These biological changes not only directly cause degenerative lesions 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 (ie, "inflammatory aging"). Therefore, more and more researchers and institutions have begun to pay attention to 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 senolytic drug development aim to achieve cross-disease therapeutic breakthroughs by targeting the senescence-associated secretory phenotype (SASP).

[0003] However, compared with anti-aging drugs, natural active substances in fruits, vegetables and edible crops have the advantages of high safety, multi-target synergy, easy access and dual nutritional and functional properties. It is well known that grape seeds and their extracts are important components of a variety of anti-aging products. The latest research has found that proanthocyanidin C1 is the key substance that mediates the anti-aging effect of grape seed extract. However, during oral administration, proanthocyanidin C1 is easily degraded in the oral and gastric environments, and the content reaching the intestinal target organs is low. Therefore, increasing the concentration of proanthocyanidin C1 entering the intestine is of great significance to improving its oral utilization rate. Summary of the invention

[0004] In view of this, the present invention provides a proanthocyanidin C1 nanoparticle and a preparation method thereof. The system is constructed with polysaccharide as a carrier, which can effectively improve the oral absorption rate of active small molecule substances and enhance the antioxidant, anti-aging and other effects of the product.

[0005] The first aspect of the present invention provides a proanthocyanidin C1 nanoparticle, which is prepared by a self-assembly method using a coupling agent, an embedding wall material and a core material as raw materials.

[0006] Preferably, the coupling agent is lysine, the embedding wall material is polysaccharide, and the core material is proanthocyanidin C1. More preferably, the polysaccharide is at least one of polygonatum polysaccharide, dendrobium polysaccharide, and wolfberry polysaccharide.

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

[0008] The proanthocyanidin C1 ethanol aqueous solution and the lysine ethanol aqueous solution are mixed in proportion and stirred for a period of time to obtain a proanthocyanidin C1-lysine mixed solution, and then the proanthocyanidin C1-lysine mixed solution is quickly injected into the 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), 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 1.5-2.5h, and the stirring speed is 500-700rpm, and the stirring time after mixing the proanthocyanidin C1-lysine mixed solution with the polysaccharide aqueous solution is 1-3h, and the stirring speed is 500-700rpm.

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

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

[0012] The fourth aspect of the present invention is to provide an application of proanthocyanidin C1 nanoparticles in the preparation of medicines 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] The present invention uses lysine as a coupling agent, which significantly improves the embedding rate of proanthocyanidin C1, and makes it easier for polysaccharides to form lysine-polysaccharide complexes through electrostatic action, thereby improving the stability of nanoparticles. The nanoparticles constructed with polysaccharides as carriers of the present invention can effectively improve the oral absorption rate of active small molecules, and use synergistic effects to improve the antioxidant and anti-aging effects of nanoparticles. The nanoparticles of the present invention are not only highly stable, but also have precise targeted release, can remain stable in heat, light and simulated in vitro digestion environments, and can significantly extend the maximum lifespan and average lifespan of fruit flies. At the same time, the nanoparticles of the present invention have significantly improved the motility and intestinal permeability of fruit flies, and can be used to construct a proanthocyanidin C1 nano delivery system or to prepare drugs and foods with anti-aging and antioxidant functions, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 1 is the result of determination of embedding efficiency and drug loading, where: Figure 1 A is the effect of polysaccharide concentration on the embedding rate, Figure 1 B is the effect of polysaccharide concentration on drug loading. Figure 1 C is 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 with polygonatum polysaccharide; Figure 1 E shows the Zeta potential of the nanoparticles prepared when proanthocyanidin C1, polygonatum polysaccharide, and lysine solution were added simultaneously;

[0016] Figure 2 is the embedding condition characterization result, where: Figure 2 A and Figure 2 B is a scanning electron microscope image. Figure 2 C and Figure 2 D transmission electron microscopy image; Figure 2 E is the infrared spectrum; Figure 2 F is the XRD pattern;

[0017] Figure 3 is the stability test result, where: Figure 3 A is the photostability test result, Figure 3 B is the result of thermal stability test. Figure 3 C is the release rate of nanoparticles incubated in simulated oral, gastric, and intestinal fluids in vitro;

[0018] Figure 4 Results of animal experiments, including: Figure 4 A is the longest life span of female fruit flies, Figure 4 B is the longest life span of male fruit flies, Figure 4C is the climbing ability of female fruit flies, Figure 4 D is the climbing ability of male fruit flies, Figure 4 E is the intestinal permeability results of female fruit flies. Figure 4 F is the intestinal permeability results of male fruit flies. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The first aspect of the present invention provides a proanthocyanidin C1 nanoparticle, which is prepared by a self-assembly method using a cross-linking agent, an embedding wall material, and a core material as raw materials.

[0021] The coupling agent of the present invention is lysine, the embedding wall material is a polysaccharide, and the core material is proanthocyanidin C1. In some specific embodiments of the present invention, the polysaccharide is at least one of polygonatum polysaccharide, dendrobium polysaccharide, and wolfberry polysaccharide. The polysaccharide of the present invention can be a commercially available product, or it can be prepared by the following method:

[0022] The crude polysaccharide is extracted from the raw material by water-soluble alcohol precipitation method, and then the protein is removed by Sevag method, the color is decolored by resin, and the impurities are removed by dialysis to obtain polysaccharide (PSP).

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

[0024] The proanthocyanidin C1 ethanol aqueous solution and the lysine ethanol aqueous solution are mixed in proportion and stirred for a period of time to obtain a proanthocyanidin C1-lysine mixed solution, and then the proanthocyanidin C1-lysine mixed solution is quickly injected into the 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 the proanthocyanidin C1 ethanol aqueous solution and the lysine ethanol aqueous solution are mixed is 1.5-2.5 hours, and the stirring speed is 500-700 rpm, and the stirring time after the proanthocyanidin C1-lysine mixed solution is mixed with the polysaccharide aqueous solution is 1-3 hours, and the stirring speed is 500-700 rpm.

[0026] The drying method of the present invention is freeze drying, and the drying time is not less than 48 hours.

[0027] In order to further illustrate the present invention, the following examples are provided for detailed description. The raw materials used in the following examples of the present invention are all commercially available.

[0028] Unless otherwise specified, all experiments were repeated three times. SPSS 21.0 was used for analysis of variance (ANOVA) and Duncan's multiple comparison analysis. The results were expressed as mean ± standard deviation, and P < 0.05 indicated a significant difference.

[0029] The processing method of the macroporous resin in the embodiment of the present 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 there was no white turbidity, and then washed with distilled water until there was no ethanol smell. The macroporous resin was then soaked in 3 column volumes of 5% HCl solution for 3 hours, washed with deionized water to neutralize, and then soaked in 5% NaOH solution for 3 hours. Finally, it was washed with distilled water to neutralize to obtain the treated macroporous resin.

[0031] Example 1 A method for preparing nanoparticles of Polygonatum sibiricum polysaccharide encapsulating proanthocyanidin C1, comprising the following steps:

[0032] 0.25 mg of proanthocyanidin C1 (PCC1) was accurately weighed and fully dissolved in 5 mL of anhydrous ethanol. After stirring and ultrasonication until fully dissolved, a PCC1 solution with a concentration of 50 μg / mL was obtained. Different amounts of polygonatum sibiricum polysaccharide were dissolved in ultrapure water to prepare a series of polygonatum sibiricum 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 quickly injected into 3 mL of different concentrations of polygonatum polysaccharide solutions, and the mixed solution was continuously stirred at 600 rpm for 2 h by a magnetic stirrer, and then 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-encapsulated proanthocyanidin C1 nanoparticles (PCC1-PSPNPs);

[0036] Under room temperature conditions, 50 μg / mL of PCC1 solution and 50 μg / mL of lysine ethanol aqueous solution were first uniformly mixed to form a PCC1-Lys mixture, and then 1 mL of the PCC1-Lys mixture was taken and quickly injected into 3 mL of polygonatum polysaccharide solutions of different proportions, and 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] Take the dried rhizome of Polygonatum cyrtonema, grind it into powder, and pass it through a 60-mesh sieve to obtain Polygonatum cyrtonema powder; weigh 5g of Polygonatum cyrtonema powder, put it in a conical flask, add anhydrous ethanol at a solid-liquid ratio of 1:8, soak it for 48h, filter it with suction, and dry it to obtain a defatted Polygonatum cyrtonema sample; weigh 4g of the defatted Polygonatum cyrtonema sample, add deionized water at a solid-liquid ratio of 1:40, ultrasonicate it at 400W for 50min, take the supernatant, repeat it twice, combine the filtrates obtained twice, put it in a rotary evaporator to concentrate it to 20mL, add 4 times the volume of anhydrous ethanol, mix it quickly, precipitate it at 4℃ overnight, centrifuge it, and obtain crude polysaccharide;

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

[0040] Take 1g of the treated macroporous resin, put it into a 150mL conical flask, add 30mL of polysaccharide aqueous solution, place it on a 35℃ constant temperature shaker and oscillate for 3h for adsorption, filter the adsorbed macroporous resin to obtain the supernatant, and then add the polysaccharide liquid after resin adsorption to a 3500KD dialysis bag and dialyze it with distilled water for 3d. After changing the water once in the morning and evening, the dialysate is freeze-dried, and the protein removal is determined using the Biyuntian protein kit. After confirming the protein removal, Polygonatum sibiricum polysaccharide is obtained.

[0041] Example 2 A method for preparing nanoparticles of proanthocyanidin C1 embedded in dendrobium polysaccharide is the same as that in Example 1, except that the polysaccharide is dendrobium polysaccharide.

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

[0043] Place the Dendrobium officinale in a drying oven, dry to constant weight, grind into powder, and pass through a 60-mesh sieve to obtain Dendrobium powder; weigh 5 g of Dendrobium powder, add deionized water at a ratio of 1:50, add 0.1 g of pectinase, and perform ultrasonic treatment at 250W for 30 min, take the supernatant, repeat 2 times, combine the filtrates obtained from the two times, place in a rotary evaporator and concentrate to 20 mL, add 4 times the volume of ethanol, mix quickly, precipitate at 4°C overnight, and centrifuge to obtain crude polysaccharide;

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

[0045] Take 1g of the treated macroporous resin, put it into a 150mL conical flask, add 30mL of polysaccharide aqueous solution, place it on a 35℃ constant temperature shaker for 3h for adsorption, filter the adsorbed macroporous resin to obtain the supernatant, and then add the polysaccharide liquid after resin adsorption to a 3500KD dialysis bag and dialyze it with distilled water for 3d. After changing the water once in the morning and evening, the dialysate is freeze-dried, and the protein removal is determined using the Biyuntian protein kit. After confirming the protein removal, Dendrobium polysaccharide is obtained.

[0046] Example 3 A method for preparing nanoparticles of Lycium barbarum polysaccharide-embedded proanthocyanidin C1 is the same as Example 1, except that the polysaccharide is Lycium barbarum polysaccharide.

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

[0048] Place wolfberry in a drying oven, dry to constant weight, grind into powder, and pass through a 60-mesh sieve to obtain wolfberry powder; weigh 10g of wolfberry powder, add deionized water at a ratio of 1:50, extract with hot water at 62°C for 80min, centrifuge, collect the supernatant, rotary evaporate the supernatant, concentrate to 50mL, add 4 times the volume of ethanol, mix quickly, precipitate at 4°C for 12h, centrifuge, take the precipitate, and obtain crude polysaccharide;

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

[0050] Take 1g of the treated macroporous resin, put it into a 150mL conical flask, add 30mL of polysaccharide aqueous solution, place it on a 35℃ constant temperature shaker for 3h for adsorption, filter the adsorbed macroporous resin to take the supernatant, and then add the polysaccharide liquid after resin adsorption to a 3500KD dialysis bag and dialyze it with distilled water for 3d. After changing the water once in the morning and evening, the dialysate is freeze-dried, and the protein removal is determined using the Biyuntian protein kit. After confirming the protein removal, wolfberry polysaccharide is obtained.

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

[0052] (1) Experimental methods

[0053] After the self-assembly of Example 1 was completed, the supernatant of the nanoparticles was collected to calculate the embedding rate and drug loading. Among them, the nanoparticles embedded with a single Polygonatum sibiricum polysaccharide were recorded as PCC1-PSPNPs, and the cross-linked nanoparticles formed after adding lysine were recorded as PCC1-P / LNPs. The average particle size, PDI and Zeta potential were measured using a laser particle size analyzer.

[0054] The embedding rate calculation formula is as follows:

[0055] Wtotal-Wfree / Wtotal×100%;

[0056] Wherein, Wfree is the weight of polysaccharide in the supernatant, and Wtotal is the total weight of polysaccharide.

[0057] The drug loading calculation formula is as follows:

[0058] Wtotal-Wfree / Wnp×100%;

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

[0060] (2) Experimental results

[0061] By investigating the polysaccharide concentration and the addition of lysine coupling agent, experimental results were obtained, such as Figure 1 shown.

[0062] Depend on Figure 1 It can be seen that the embedding efficiency (EE) of PCC1-PSPNPs gradually increased with the increase of polysaccharide concentration, reaching only 79.6% at 16 mg / mL. Figure 1 A, B), the embedding amount and drug loading capacity of the nanosystem were significantly improved ( Figure 1B), when the polysaccharide concentration was 8 mg / mL, it had the highest embedding amount of 93.57%, which was 21.9% higher than that of the single polysaccharide-embedded nanoparticles (71.67%). With the increase of polysaccharide content, the particle size of the cross-linked PCC1-P / LNPs first decreased and then increased, from 1489nm to 313nm, and then increased to 424nm, and 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 the other groups, indicating that the nanoparticles prepared under this condition had better dispersibility and more uniform particle size distribution ( Figure 1 C).

[0063] From the Zeta potential results, it can be seen that the Zeta potential of all nanoparticles is recorded as a negative value, indicating that the nanoparticles have a charge in the suspended state, and the absolute value of the Zeta potential is greater than 20mV, indicating that the nanoparticles of the present invention have excellent physical and chemical stability ( Figure 1 D).

[0064] At the same time, it was found during the experiment that when lysine, proanthocyanidin C1 and polygonatum polysaccharide solution were mixed and assembled at the same time, the absolute value of the Zeta potential was significantly reduced, resulting in a negative impact on the stability of the nanoparticles ( Figure 1 E). Therefore, the preparation process of nanoparticles should follow the following addition order: first, the proanthocyanidin C1 and lysine are fully mixed to promote cross-linking, and then the mixture is injected into the polysaccharide solution to ensure their full combination.

[0065] Test Example 2 Characterization of Embedding Condition

[0066] (1) Experimental methods

[0067] The Polygonatum sibiricum polysaccharide-embedded proanthocyanidin C1 nanoparticles (PCC1-P / LNPs), 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] Among them, the preparation process of 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 in a volume ratio of 0.5:3, and stirred at 600 rpm in a magnetic stirrer for 2 hours, and then centrifuged at 12000 r / min for 15 minutes, 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 showed a porous mesh particle morphology, with a large number of subspherical small particles distributed on the surface and relatively smooth, without obvious aggregation phenomenon ( Figure 2 A); in contrast, the wall material nanoparticles are aggregated, showing loose packing and porous structure ( Figure 2 B). The transmission electron microscopy results show that the nanoparticles prepared by the present invention are spherical or sub-spherical, have a core-shell structure with alternating light and dark, and have high dispersibility ( Figure 2 C). However, the wall material nanoparticles without PCC1 showed a loose spherical structure ( Figure 2 D). The interaction between the proposed nanoparticle components was elucidated by Fourier transform infrared spectroscopy (FT-IR). The characteristic absorption band of proanthocyanidin C1 almost disappeared or overlapped with the characteristic absorption band of the encapsulating material. This may be due to the encapsulation effect of the nanocarrier limiting the expansion and contraction of the chemical groups on PCC1, resulting in a weakening of its characteristic signal ( Figure 2 E). X-ray diffraction results showed that both polygonatum polysaccharide and lysine showed a certain crystalline structure. The diffraction curve showed flat peaks at diffraction angles of 22.73° and 23.40°, respectively, and no sharp characteristic peaks were observed in the rest of the area. After the nanoparticles were formed, the intensity of the diffraction peak of lysine decreased significantly, probably because the shrinkage of the hydrophobic region during the anti-solvent precipitation method destroyed the crystal structure of lysine ( Figure 2 F).

[0071] Test Example 3 Stability Determination

[0072] (1) Experimental methods

[0073] Thermal and light environment stability:

[0074] 5 mL of the suspension of Polygonatum sibiricum polysaccharide embedded proanthocyanidin C1 prepared in Example 1 was placed in a boiling water bath at different temperatures (25, 45, 65 and 85°C) for 30 min, and then cooled to room temperature (25°C). 5 mL of the suspension of freshly prepared nanoparticles in a transparent glass vial was exposed to 36W of ultraviolet light at a distance of 15 cm, and the irradiation time was 15, 30, 45, 60, and 90 min, respectively. Subsequently, the average particle size was measured by a laser particle size analyzer, and the content of residual PCC1 in the sample was determined by UV-visible spectrophotometer analysis.

[0075] In vitro digestion:

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

[0077] ②Simulated gastric juice digestion: After oral digestion, the mixture was added to 100 mL of simulated gastric juice and incubated at 37°C for 2 h (wrapped in tin foil). 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 juice digestion.

[0078] ③Simulated small intestinal digestion: The simulated intestinal fluid is composed of 12 mg / mL bile salt and 2.25 mg / mL pancreatic enzymes, and the pH is adjusted to 7.0 with 1 m NaOH solution. The digestive fluid is mixed with the above 45 mL small intestinal fluid, and incubated for 2 hours under the same conditions, and finally heated at 95 ° C for 5 minutes to end the digestion process.

[0079] At each stage of gastrointestinal digestion, 2 mL of sample was collected from the digestion mixture every 5 min for the first 30 min, and the same volume of digestion solution was added. Then, 2 mL of sample was collected from the mixture every 10 min, and the same volume of digestion solution was added. The pH of the collected samples was quickly adjusted to 7.0, centrifuged at 12000 rpm for 15 min, and the polysaccharide content in the supernatant was determined. The release amount was calculated by dividing the polysaccharide content released in the supernatant by the total polysaccharide content loaded in the nanoparticles.

[0080] (2) Experimental results

[0081] Depend on Figure 3 As shown in Figures A and 3B, the nanoparticles prepared by the present invention did not undergo significant degradation after being treated under UV light for 90 min or placed in a water bath at 25, 45, 65, and 85°C for 30 min. Figure 3 As shown in Figure C, after incubation of free proanthocyanidin C1 in the oral cavity and simulated gastric fluid for 4 hours, the total release rate of free PCC1 was 39.73%, while the release amount of nanoparticles under simulated intestinal conditions was 50.38%, which was significantly higher than the release amount of 22.67% under simulated gastric environment conditions. This shows that the nanoparticles prepared by the present invention effectively increase the concentration of proanthocyanidin C1 reaching the target organs.

[0082] Test Example 4 Animal Experiment

[0083] (1) Experimental methods

[0084] Synergistic extension of fruit fly lifespan study:

[0085] The nanoparticles of proanthocyanidin C1 embedded in polygonatum polysaccharide prepared in Example 1 were used as samples, and wild-type W118 Drosophila melanogaster were used as model animals, and were raised with corn yeast medium under controlled conditions (25°C, 65% relative humidity, 12-hour light-dark cycle). The fruit flies hatched within 72 hours were randomly assigned to the control group and the experimental group, and each vial of each group contained 20 fruit flies.

[0086] The experimental groups included the Polygonatum sibiricum polysaccharide (PSP) group, the Proanthocyanidin C1 (PCC1) group, the high-dose nanoparticle group (NP-H) and the low-dose nanoparticle group (NP-L), of which the Polygonatum sibiricum polysaccharide group and the Proanthocyanidin C1 (PCC1) group were referred to as the single-dose groups. Four replicates were set for each group. The culture medium was changed every three days, and the number of dead fruit flies was recorded daily until all fruit flies died, and the data of each group was recorded at least four times independently to calculate the survival time.

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

[0088] 1. Basic culture medium: Weigh 20.87g of Drosophila culture medium and dissolve it in 100mL of distilled water. After it cools down slightly, add 0.65mL of propionic acid and mix thoroughly. While it is still hot, put the culture medium into a sterilized culture bottle for later use. The basic culture medium should be replaced approximately every three weeks.

[0089] 2. Polygonatum sibiricum polysaccharide (PSP) group: 16 mg of Polygonatum sibiricum polysaccharide was accurately weighed and dissolved in 2 mL of deionized water to prepare Polygonatum sibiricum polysaccharide stock solution. Then 2.25 mL of 8 mg / 100 mL PSP stock solution was added to the basal culture medium and stirred evenly to finally 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 finally obtain a fruit fly culture medium with a concentration of 0.042%.

[0091] 4. Nanoparticle group: Add 0.5 mL and 5.0 mL of a 1 mg / 100 mL nanoparticle solution 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] Improved motility and intestinal permeability in Drosophila:

[0093] The negative geotaxis climbing test was used to evaluate the motility of fruit flies fed for 5, 10, and 20 days, and the Smurf test was used to detect intestinal permeability.

[0094] (2) Experimental results

[0095] Figure 4 A and Figure 4B The results 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; 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. The lifespan experiment showed that the nanoparticles prepared by the present invention can extend the median lifespan and maximum lifespan of fruit flies, and the lifespan extension effect is greater than the effect of adding the components alone, indicating that the raw materials in the nanoparticles have a good synergistic effect on the lifespan of fruit flies.

[0096] Figure 4 C and Figure 4 D The results showed that as the age increased, the motor ability of fruit flies gradually decreased. On the 20th day, the effects of high and low nanoparticle dose groups on male and female fruit flies were significantly higher than those of the single drug group (p < 0.001). Compared with the single component, the high and low dose nanoparticle groups showed the most significant improvement in climbing ability. These effects were statistically significantly different from the observations of the single proanthocyanidin C1 and polygonatum polysaccharide treatment groups (p < 0.05), indicating that the synergistic effect of the two enhanced motor behavior.

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

[0098] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A proanthocyanidin C1 nanoparticle, characterized in that: The nanoparticles are prepared by a self-assembly method using a coupling agent, an embedding wall material, and a core material as raw materials; The coupling agent is lysine, the embedding wall material is polysaccharide, and the core material is proanthocyanidin C1.

2. The proanthocyanidin C1 nanoparticle according to claim 1, characterized in that: The polysaccharide is at least one of polygonatum polysaccharide, dendrobium polysaccharide and wolfberry polysaccharide.

3. The method for preparing the proanthocyanidin C1 nanoparticles according to any one of claims 1 to 2, characterized in that: The following steps are involved: The proanthocyanidin C1 ethanol aqueous solution and the lysine ethanol aqueous solution are mixed in proportion and stirred for a period of time to obtain a proanthocyanidin C1-lysine mixed solution, and then the proanthocyanidin C1-lysine mixed solution is quickly injected into the polysaccharide aqueous solution, mixed in proportion and stirred for a period of time, washed and dried to obtain proanthocyanidin C1 nanoparticles.

4. The preparation method according to claim 3, characterized in that: 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; and the concentration of polysaccharide in the polysaccharide aqueous solution is 8-16 mg / mL.

5. The preparation method according to claim 3, characterized in that: 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 to the polysaccharide is 1:(2-4).

6. The preparation method according to claim 3, characterized in that: The drying method is freeze drying, and the drying time is not less than 48 hours.

7. Use of proanthocyanidin C1 nanoparticles in constructing a proanthocyanidin C1 nano delivery system, characterized in that: The proanthocyanidin C1 nanoparticles are the proanthocyanidin C1 nanoparticles according to any one of claims 1 to 2 or the proanthocyanidin C1 nanoparticles prepared by the method according to any one of claims 3 to 6.

8. Use of proanthocyanidin C1 nanoparticles in the preparation of medicines and foods with anti-aging and antioxidant functions, characterized in that: The proanthocyanidin C1 nanoparticles are the proanthocyanidin C1 nanoparticles according to any one of claims 1 to 2 or the proanthocyanidin C1 nanoparticles prepared by the method according to any one of claims 3 to 6.

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