Cornu cervi pantotrichum polypeptide-diosgenin self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses pilose antler polypeptide-diosgenin self-assembled nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of natural medicine composite nanoparticles. The cornua cervi pantotrichum polypeptide-diosgenin self-assembled nano-particles are an amorphous state sphere-like compound formed by self-assembly of cornua cervi pantotrichum polypeptide and diosgenin. The self-assembled nano-particles are applied to the aspect of improving blood testis barrier injury caused by aging, solve the problems of low utilization efficiency of natural components and insufficient stability of the existing self-assembled nano-particles based on natural active components, and have the characteristics of good stability and high drug loading capacity.

Description

Technical Field

[0001] This invention belongs to the field of natural drug composite nanoparticle technology, and particularly relates to a deer antler polypeptide-diosgenin self-assembled nanoparticle, its preparation method and application. Background Technology

[0002] The deer antler-yam medicinal pair was first recorded in Volume 219 of *Puji Fang*. Deer antler wine, used to tonify the kidneys and strengthen yang, invigorate the spleen and replenish essence, is primarily used to treat weakness, impotence, pale complexion, frequent urination, and loss of appetite. Among its components, deer antler polypeptide (PAP) is one of the important physiologically active substances in deer antler. It can improve sexual function by increasing serum and testicular testosterone levels and enhance the antioxidant capacity of mice by reducing TNF-α production, thus delaying aging. Diosgenin (DGN), a natural active ingredient extracted from yam, possesses various activities including anti-inflammatory and anti-aging properties. Studies have shown that diosgenin can improve sperm count and density in mice with oligospermia and asthenospermia, enhance the antioxidant capacity of the testes, and protect the reproductive system.

[0003] Self-assembly refers to the process by which molecules spontaneously combine under equilibrium conditions to form well-defined and stable aggregates linked by non-covalent bonds, such as van der Waals forces, hydrogen bonds, π-π stacking, and ionic bonds. Studies have shown that peptides can self-assemble under the drive of weak bonds, forming supramolecular complexes with specific structures and targeting functions. Diosgenin, however, is highly lipid-soluble and poorly water-soluble, with an oral bioavailability of only about 7%. Therefore, it is hoped that self-assembled diosgenin can effectively improve bioavailability. Research has shown that diosgenin can inhibit tumor cell metastasis, and doxorubicin can effectively induce apoptosis. Based on this, a self-assembled nanopolymer of diosgenin and doxorubicin was prepared. This polymer is uniformly spherical with a high drug loading capacity. Compared with free drugs, this polymer can improve drug penetration within cells and its accumulation efficiency at tumor sites, reduce the rate of clearance in vivo, and does not affect the normal functioning of tissues, exhibiting good anti-metastasis and anti-proliferation effects. Meanwhile, studies have used high-pressure homogenization and single-factor experiments to optimize the formulation and prepare diosgenin albumin nanoparticles.

[0004] Although nanomedicine carriers have attracted much attention in the biomedical field in recent years due to their drug loading and targeted delivery capabilities, and self-assembled nanoparticles based on natural active ingredients have become a research hotspot due to their good biocompatibility, biodegradability, and multifunctionality, the development and utilization of deer antler peptide-diosgenin self-assembled nanoparticles have not yet been reported. Furthermore, existing self-assembled nanoparticles based on natural active ingredients currently face the following challenges: 1. Low utilization efficiency of natural ingredients: Nanoparticles with a single active ingredient often have problems such as limited drug loading capacity and single function; 2. Complex preparation process: Existing technologies mostly rely on chemical cross-linking or emulsification methods, which require the introduction of additional reagents and may lead to biosafety risks; 3. Insufficient stability: Natural ingredients are easily affected by environmental factors (such as pH and temperature) during self-assembly, leading to particle aggregation or structural damage; 4. Lack of synergistic effect: Traditional composite carriers are difficult to achieve efficient synergy among multiple components, which limits the improvement of their therapeutic effect.

[0005] Based on the above, the technical problem to be solved by this invention is to efficiently obtain deer antler polypeptide-diosgenin self-assembled nanoparticles with good stability and high drug loading capacity, and to explore their application in the biomedical field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is to overcome the problems of low utilization efficiency and insufficient stability of existing self-assembled nanoparticles based on natural active ingredients. This invention provides a self-assembled nanoparticle of deer antler polypeptide-diosgenin with good stability and high drug loading capacity, its preparation method and application.

[0007] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: In one aspect, this invention provides a deer antler polypeptide-diosgenin self-assembled nanoparticle, which is an amorphous spherical complex formed by the self-assembly of deer antler polypeptide and diosgenin.

[0008] In some embodiments, the deer antler polypeptide includes peptides as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0009] In some embodiments, diosgenin is encapsulated in the core of deer antler polypeptide-diosgenin self-assembled nanoparticles.

[0010] Another aspect of the present invention provides a method for preparing self-assembled nanoparticles of deer antler polypeptide-diosgenin according to any of the above technical solutions, comprising: dropping an ethanol organic phase containing diosgenin into an aqueous phase containing deer antler polypeptide at a set rate, adding the mixture until the mass ratio of deer antler polypeptide to diosgenin reaches a set mass ratio and the concentration of ethanol in the mixture reaches a set concentration, then ultrasonically treating to remove ethanol, and ultrafiltration to obtain self-assembled nanoparticles of deer antler polypeptide-diosgenin.

[0011] In some embodiments, the mass ratio is set to 5.5:1.

[0012] In some embodiments, the concentration is set to 64%.

[0013] In some embodiments, the ultrasonic treatment time is 40 minutes.

[0014] In some embodiments, under magnetic stirring at 400-600 rpm, the organic phase is added dropwise to the aqueous phase at a rate of 0.5-1.0 mL / min. After ultrasonic treatment using a probe sonicator, the liquid obtained by ultrasonication is subjected to reduced pressure rotary evaporation to remove ethanol. Subsequently, it is pre-filtered through a microporous membrane, the filtrate is collected, and ultrapure water is added to obtain a coarse nanoparticle suspension. The coarse nanoparticle suspension is loaded into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuged and ultrafiltered, and ultrapure water is added to the original volume. After pre-freezing, it is placed in a freeze dryer to obtain deer antler polypeptide-diosgenin self-assembled nanoparticles.

[0015] The present invention also provides the application of the above-mentioned deer antler polypeptide-diosgenin self-assembled nanoparticles in the preparation of drugs to improve blood-testis barrier damage caused by aging.

[0016] In some embodiments, deer antler polypeptide-diosgenin self-assembled nanoparticles improve age-related blood-testis barrier damage by regulating the TNF-α / p38MAPK signaling pathway.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a deer antler polypeptide-diosgenin self-assembled nanoparticle, which forms stable nanoparticles through molecular self-assembly, achieving efficient co-loading of natural active ingredients; This invention provides a method for preparing self-assembled nanoparticles of deer antler polypeptide-diosgenin. By controlling a specific concentration of ethanol and a precise mass ratio of deer antler polypeptide to diosgenin, stable nanoparticles are formed through molecular self-assembly. Utilizing the principle of directional arrangement and hydrophobic interaction of amphiphilic molecules in a controllable polar environment, combined with ultrasonic and low-speed dropwise processes, uniform nucleation and structural stabilization are promoted. This achieves the technical effect of constructing deer antler polypeptide-diosgenin self-assembled nanoparticles (PDN) with well-defined components and stable structure, successfully solving the technical problems of traditional methods that are difficult to achieve efficient co-loading of natural active ingredients, poor system stability, and low bioavailability. This invention also provides the application of deer antler polypeptide-diosgenin self-assembled nanoparticles in the preparation of drugs to improve blood-testis barrier damage caused by aging. The nanoparticles can effectively improve blood-testis barrier damage caused by aging by regulating the TNF-α / p38MAPK signaling pathway, providing a novel nano-formulation and treatment strategy for male reproductive diseases caused by aging. Attached Figure Description

[0018] Figure 1 This is the mass spectrum baseline of PAP; Figure 2 The x-axis represents the molecular weight of the protein, where the x-axis represents the relative molecular weight of the identified protein; the main y-axis corresponds to the bar chart in the figure, representing the number of proteins identified with the corresponding relative molecular mass; and the secondary y-axis corresponds to the cumulative curve in the figure, representing the cumulative percentage of proteins with a molecular mass not exceeding the corresponding relative molecular mass. Figure 3 Functional annotations (GO) for PAP; Figure 4 Annotation explaining the function of PAP in the KEGG pathway; Figure 5 The effect of PAP and DGN ratio on encapsulation efficiency; Figure 6 The effect of ethanol concentration on encapsulation efficiency; Figure 7 The effect of ultrasonic time on encapsulation efficiency; Figure 8 A three-dimensional response surface plot showing the effect of the interaction between ethanol concentration and time on chromogen content; Figure 9 Three-dimensional response surface plot of the effect of time and PAP-DGN ratio on chromogen content; Figure 10 Three-dimensional response surface plot showing the effect of ethanol concentration and PAP-DGN ratio on chromogen content; Figure 11 These are transmission electron microscopy results for PDN; Figure 12 This is a schematic diagram of PDN particle size. Figure 13 The results are for the Zeta potential of the PDN; Figure 14 The results are Fourier transform infrared spectra. Figure 15 Ultraviolet spectroscopy results; Figure 16 X-ray diffraction pattern; Figure 17 The results are from the thermogravimetric analysis of PAP; Figure 18 The results are from the DGN thermogravimetric analysis. Figure 19 The results are from the thermogravimetric analysis of PDN; Figure 20 The variation of PDN particle size at different pH values; Figure 21 The change in potential of PDN at different pH values; Figure 22This represents the change in PDN particle size over different time periods. Figure 23 This represents the change in potential of the PDN at different times; Figure 24 The variation of PDN particle size at different temperatures; Figure 25 The change in potential of PDN at different temperatures; Figure 26 The release rate of DGN in gastric juice; Figure 27 The release rate of DGN in intestinal fluid; Figure 28 Morphological appearance of mouse testes and epididymis induced by PDN-induced D-gal; Figure 29 For the determination of the testicular index in mice induced by PDN and D-gal; Figure 30 For the determination of the epididymal index in mice induced by PDN and D-gal; Figure 31 PDN staining for sperm morphology in D-gal-induced mice in various groups; Figure 32 For the determination of D-gal-induced sperm density in mice by PDN; Figure 33 For the determination of D-gal-induced sperm motility in mice by PDN; Figure 34 To determine the sperm abnormality rate induced by PDN in mice with D-gal; Figure 35 The effect of PDN on the β-galactosidase content in D-gal-induced mouse testicular tissue; Figure 36 HE staining of mouse testicular tissue; Figure 37 HE staining of mouse epididymal tissue; Figure 38 The percentage of ZO-1 positive cells according to immunohistochemistry; Figure 39 Immunohistochemical staining for ZO-1; Figure 40 The effect of PDN on Occludin, a tight junction protein in D-gal-induced mouse testicular tissue; Figure 41 The effect of PDN on D-gal-induced blood-testis barrier in mice; Figure 42 The effect of PDN on luteinizing hormone levels in D-gal-induced mouse serum; Figure 43 The effect of PDN on follicle-stimulating hormone in D-gal-induced mouse serum; Figure 44 The effect of PDN on D-gal-induced serum testosterone in mice; Figure 45 The effect of PDN on D-gal-induced TNF-α in mouse serum; Figure 46 The effect of PDN on malondialdehyde in D-gal-induced mouse testicular tissue; Figure 47 The effect of PDN on D-gal-induced glutathione peroxidase in mouse testicular tissue; Figure 48 The effect of PDN on catalase in D-gal-induced mouse testicular tissue; Figure 49 The effect of PDN on D-gal-induced superoxide dismutase in mouse testicular tissue; Figure 50 Results of Western blot analysis of testicular tissue; Figure 51 Gray-scale analysis of protein blots of N-cadherin in testicular tissue; Figure 52 Gray-scale analysis of protein blots of β-catenin in testicular tissue; Figure 53 Western blot grayscale analysis of p38 MAPK kinase in testicular tissue; Figure 54 Gray-scale analysis of phosphorylated p38 protein blots in testicular tissue; Figure 55 Gray-scale analysis of Western blot for c-Jun protein in testicular tissue; Figure 56 This study presents a grayscale analysis of the protein blot of tumor necrosis factor-α in testicular tissue. Detailed Implementation

[0019] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0020] In one aspect, this invention provides a deer antler polypeptide-diosgenin self-assembled nanoparticle, which is an amorphous spherical complex formed by the self-assembly of deer antler polypeptide and diosgenin.

[0021] Transmission electron microscopy revealed that the above-mentioned deer antler polypeptide-diosgenin self-assembled nanoparticles (PDN) showed good dispersion in aqueous solution with no obvious aggregation. The overall morphology of the particles was relatively uniform, exhibiting a near-spherical structure with a diameter of 300-400 nm, which is consistent with the morphological characteristics of self-assembled nanoparticles.

[0022] XRD patterns clearly characterized the differences in crystal structure between antler polypeptide (PAP), diosgenin (DGN), and their complex. Pure DGN exhibited typical sharp diffraction peaks (e.g., ~15°, ~17°, ~23°) in the 2θ = 5°–30° range, indicating a highly ordered crystalline structure. In contrast, PAP only showed a broad diffuse band at 2θ ≈ 20°, consistent with the flexible chain characteristics of amorphous polypeptides. Notably, the XRD pattern of the self-assembled nanoparticles (PDN) showed a significant change: the characteristic crystalline peaks of DGN completely disappeared, leaving only a broad diffuse band at 2θ ≈ 20°. This phenomenon indicates that during self-assembly, DGN molecules detached from the ordered lattice and recombinated with PAP molecules through hydrophobic interactions and hydrogen bonds, forming a uniform amorphous complex. This molecular arrangement confirms the successful construction of PAP-DGN self-assembled nanoparticles.

[0023] The above-mentioned self-assembled deer antler polypeptide-diosgenin nanoparticles, constructed through self-assembly, have the characteristics of structural stability and efficient co-loading of natural active ingredients.

[0024] In some embodiments, the deer antler polypeptide includes peptides as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0025] A total of 3210 peptides and 854 proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS), with proteins in the molecular weight range of 10-80 kDa accounting for 60% of the total. The 10 peptides in the above sequences were the 10 most abundant peptides detected by LC-MS, and their signal intensity values ​​were calculated based on LC-MS data. The deer antler peptides containing these 10 peptides can self-assemble with diosgenin to construct structurally stable nanoparticles with high bioactivity loading, effectively ensuring the efficacy of the nanoparticles in improving age-related blood-testis barrier damage.

[0026] In some embodiments, diosgenin is encapsulated in the core of deer antler polypeptide-diosgenin self-assembled nanoparticles.

[0027] Encapsulating diosgenin in the core of deer antler polypeptide-diosgenin self-assembled nanoparticles can improve the bioavailability of this hydrophobic component, while also providing sustained release and synergistic effects.

[0028] Another aspect of the present invention provides a method for preparing self-assembled nanoparticles of deer antler polypeptide-diosgenin according to any of the above technical solutions, comprising: dropping an ethanol organic phase containing diosgenin into an aqueous phase containing deer antler polypeptide at a set rate, adding the mixture until the mass ratio of deer antler polypeptide to diosgenin reaches a set mass ratio and the concentration of ethanol in the mixture reaches a set concentration, then ultrasonically treating to remove ethanol, and ultrafiltration to obtain self-assembled nanoparticles of deer antler polypeptide-diosgenin.

[0029] The above method utilizes molecular self-assembly to form stable nanoparticles, leveraging the directional alignment and hydrophobic interactions of amphiphilic molecules in a controllable polar environment, combined with ultrasound and dropwise addition processes to promote uniform nucleation and structural stabilization. In some embodiments, the mass ratio is set to 5.5:1. In some embodiments, the concentration is set to 64%. In some embodiments, the ultrasound treatment time is 40 min.

[0030] It should be noted that this invention utilizes diosgenin and deer antler polypeptides under specific ethanol concentration (64%) and precise mass ratio (5.5:1) conditions to form stable nanoparticles through molecular self-assembly. By leveraging the principle of directional arrangement and hydrophobic interaction of amphiphilic molecules in a controllable polar environment, combined with probe ultrasound and low-speed drop addition process to promote uniform nucleation and structural stabilization, the invention achieves the technical effect of constructing deer antler polypeptide-diosgenin self-assembled nanoparticles (PDN) with well-defined composition and stable structure. This successfully solves the technical problems of traditional methods, such as difficulty in achieving efficient co-loading of natural active ingredients, poor system stability, and low bioavailability.

[0031] The above-mentioned technical solution of the present invention uses deer antler polypeptide and hydrophobic diosgenin as raw materials, 64% ethanol as solvent and adds an ultrafiltration step, which makes the purified deer antler polypeptide-diosgenin self-assembled nanoparticles more stable and simpler to operate, and explores the mechanism of action in improving blood-testis barrier damage caused by aging.

[0032] In some embodiments, under magnetic stirring at 400-600 rpm, the organic phase is added dropwise to the aqueous phase at a rate of 0.5-1.0 mL / min. After ultrasonic treatment using a probe sonicator, the liquid obtained by ultrasonication is subjected to reduced pressure rotary evaporation to remove ethanol. Subsequently, it is pre-filtered through a microporous membrane, the filtrate is collected, and ultrapure water is added to obtain a coarse nanoparticle suspension. The coarse nanoparticle suspension is loaded into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuged and ultrafiltered, and ultrapure water is added to the original volume. After pre-freezing, it is placed in a freeze dryer to obtain deer antler polypeptide-diosgenin self-assembled nanoparticles.

[0033] This invention also provides the application of the above-described deer antler polypeptide-diosgenin self-assembled nanoparticles in the preparation of drugs to improve age-related blood-testis barrier damage. In some embodiments, the deer antler polypeptide-diosgenin self-assembled nanoparticles improve age-related blood-testis barrier damage by regulating the TNF-α / p38MAPK signaling pathway.

[0034] To more clearly and in detail introduce the deer antler polypeptide-diosgenin self-assembled nanoparticles, their preparation method, and applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0035] The following describes the materials, reagents, and instruments involved in the embodiments of the present invention.

[0036] (1) Materials The fresh sika deer antlers were purchased from Shuangyang Deer Farm in Changchun, Jilin Province, and identified by Professor Wei Jixiang of Jilin Agricultural University as sika deer antlers.

[0037] Male KM mice were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd., and the experimental procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University (Ethics No.: 20211011003). One hundred male KM mice (18-22g) were used, with 10 mice per cage. After one week of acclimatization, the mice were put into the experiment. Each group of mice had free access to food, and the environment was maintained with alternating light and dark conditions at 12h / 12h.

[0038] (2) Reagents D-galactose was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; diosgenin (DGN) was purchased from Yuan Ye Company; TNF-α inhibitor (Pomalidomide) was purchased from MCE Company; Vitamin E was purchased from Meilun Biotechnology Co., Ltd.; CCK-8 reagent, BCA protein concentration assay kit, reactive oxygen species, SOD, MDA, CAT, and GSH-Px detection kits were all purchased from Beyotime Biotechnology Co., Ltd.; ready-to-use dialysis bags and β-galactosidase staining kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; TNF-α, FSH, LH, and sex hormone T detection kits were all purchased from Shanghai Youxuan Biotechnology Co., Ltd.; hematoxylin and eosin were purchased from Zhuhai Beso Biotechnology Co., Ltd.; LC-MS grade methanol was purchased from Fisher Scientific; 4% paraformaldehyde was purchased from Biosharp; total protein extraction kit was purchased from Bebo Biotechnology Co., Ltd.; Vimentin / FITC rabbit polyclonal antibody was purchased from Beijing Biosen Biotechnology Co., Ltd.; N Cadherin and P38 were also purchased. The primary antibodies for MAPK and Occludin were purchased from Chengdu Zhengneng Biotechnology Co., Ltd.; the primary antibodies for ZO-1 and Phospho-p38 MAPK (Tyr182) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; the primary antibody for c-Jun was purchased from Wanlei Biotechnology Co., Ltd.; the primary antibodies for beta Catenin and JAM1 were purchased from Affnity Biotechnology Co., Ltd.; and the antibody and secondary antibody for β-actin were purchased from Wuhan Yilairuit Biotechnology Co., Ltd.

[0039] (3) Instruments Hitachi transmission electron microscope (Hitachi HT7700 TE); Setline TGA thermogravimetric analyzer; Rigaku XRD polycrystalline X-ray diffractometer (SmartLab); Brookhaven Zeta potential analyzer (NanoBrook Omni); Nicolet iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific); V3000 ultraviolet spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.); Changsha Gaoke Xiangyi Centrifuge Co., Ltd. low-speed benchtop centrifuge (TDZ4K); Beckman Coulter paraffin embedding machine (Leica EG1140) and paraffin sectioning machine (Leica RM2255), Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.; Electronic balance (FA1004N) Shanghai Precision Scientific Instrument Co., Ltd.; Optical microscope (Olympus BX51); Nikon image analysis system (NIS-ELEMNT BR); Q Exactive Plus liquid chromatography-mass spectrometry system (Thermo Fisher Scientific); TurboIonSpray ion source (AB). SCIEX Corporation); UPLC (Ultra-High Performance Liquid Chromatography) system (Thermo Fisher Scientific, USA); H1850-R Refrigerated Centrifuge (Xiangyi); MB-96 Tissue Grinder (Meibi); KQ-800DE Ultrasonic Cleaner (Shumei); FW-100 High-Speed ​​Grinder (Beijing Zhongxing Weiye Instrument Co., Ltd.); DHG-9140A Constant Temperature Drying Oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); Mili-Q Advantage A10 Ultrapure Water System (Millibert, Inc., USA).

[0040] (4) Extraction of deer antler polypeptides After thawing frozen fresh sika deer antlers, they are cut into thin slices approximately 1-2 mm thick. The slices are rinsed with pre-cooled distilled water at 4°C to remove surface blood and impurities. They are then placed in a vacuum freeze dryer at -50°C and 0.05 mBar for 12 hours to dehydrate. The thoroughly dried antler slices are removed, the surface hairs are removed, and the powder is pulverized using a high-speed grinder and passed through a 100-mesh sieve to obtain coarse antler powder. Soxhlet extraction is then performed using anhydrous ethanol as the solvent for 6 hours to remove fats and pigments, yielding defatted antler powder. This powder is then sealed and stored at -20°C for later use.

[0041] Accurately weigh 0.5g of defatted deer antler powder and add it to 30% ethanol solution at a liquid-to-solid ratio of 16mL / g. Sonicate the solution at 9℃ for 30min, then centrifuge at 4℃ and 4000r / min for 15min. Collect the supernatant as the deer antler protein extract. Take this supernatant and add enzyme at a ratio of deer antler protein to pepsin of 100:3. Incubate the solution in a 50℃ water bath for 4h. After hydrolysis, heat in a 100℃ water bath for 10min to inactivate the enzyme. After cooling, centrifuge at 4℃ and 3800r / min for 20min. Filter the supernatant and dialyze the resulting filtrate in ultrapure water at 4℃ for 12h (changing the water every 4h). Finally, freeze-dry the dialysate to obtain deer antler polypeptide (PAP), which should be stored in a sealed container at 4℃.

[0042] Example 1 An appropriate amount of diosgenin was completely dissolved in a certain amount of 100% ethanol and sonicated until clear and transparent to form the organic phase. An appropriate amount of deer antler polypeptide was dissolved in a certain amount of water and gently vortexed to ensure complete dissolution to form the aqueous phase. Under magnetic stirring (500 rpm), the organic phase was slowly added dropwise to the aqueous phase at a constant rate (1 mL / min). The mixture was ultrasonically treated for a certain time using a probe sonicator (250 W power, pulse mode, 2 s on / 1 s off). The resulting liquid was then subjected to reduced pressure rotary evaporation below 40℃ to slowly remove the ethanol. Subsequently, the liquid was pre-filtered using a 0.45 μm microporous membrane, and the filtrate was collected. Ultrapure water was added to obtain a coarse nanoparticle suspension. The coarse nanoparticle suspension was loaded into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4℃ and 4000 rpm for 15 min. Ultrapure water was then added to the original volume. After pre-freezing at -20℃ for 2 days, the mixture was placed in a freeze dryer at -20℃ for 1 day to obtain PDN.

[0043] This invention provides a method for preparing self-assembled nanoparticles of deer antler polypeptide-diosgenin, and investigates its synergistic mechanism in improving age-related blood-testis barrier damage. Simultaneously, it explores methods for preparing self-assembled nanoparticles and uses response surface methodology to evaluate the state of the deer antler polypeptide-diosgenin self-assembled nanoparticles based on indicators such as drug loading, stability, and in vitro dissolution. The optimal method for preparing deer antler polypeptide-diosgenin self-assembled nanoparticles is thus selected, providing a paradigmatic reference for the in-depth application of self-assembly of traditional Chinese medicine components in the modernization research of traditional Chinese medicine.

[0044] The following analysis examines the deer antler polypeptide used in Example 1 and the PDN prepared from it. Single-factor experiments were conducted to investigate the key parameters (PAP-DGN ratio, ethanol concentration, and ultrasonic time) in the PDN preparation process of Example 1. The application effect of PDN was also investigated. The specific methods used are as follows.

[0045] 1. Analysis of deer antler polypeptides (1) Determination of deer antler polypeptide composition by label-free proteomics A 30 µg protein sample was treated with 10 mM dithiothreitol (DTT) at 37 °C for 1 hour. Subsequently, it was placed in a dark environment and alkylated with 50 mM ethyl isothiocyanate (IAA) at room temperature for another hour. After enzymatic digestion with 5 mM DTT for 10 minutes, the protein was ultrafiltered using an Amicon Ultra-0.5 centrifuge filter. It was then washed twice with 100 μL of 8 M urea solution and three times with 100 μL of 50 mM ammonium bicarbonate solution. The protein was then mixed with trypsin at a 50:1 ratio and enzymatically digested at 37 °C for 18 hours. The resulting peptides were purified, dried, and dissolved in 40 μL of a solution containing 0.1% formic acid (FA). The peptide concentration was determined by measuring the absorbance (OD280) at 280 nm.

[0046] (2) Liquid chromatography-tandem mass spectrometry analysis DBHE samples were separated using a high-performance liquid chromatography (HPLC) system. The mobile phase consisted of A (0.1% formic acid aqueous solution) and B (80% acetonitrile, containing 0.08% formic acid). Before injection, the column was equilibrated with 100% mobile phase A. The elution gradient was set as follows: at 0 min, 95% A and 5% B, with a constant flow rate of 600 nL / min; at 8 min, it was adjusted to 90% A and 10% B; at 58 min, it became 76% A and 24% B; at 70 min, it was 68% A and 32% B; and from 71 to 78 min, it switched to 5% A and 95% B. The samples were then analyzed automatically using a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer. The mass spectrometry parameters were set as follows: ion source voltage 4 kV, ion source temperature 350 °C, desolvation gas flow rate 10 L / min, cone gas flow rate 50 L / hr, collision energy 30 eV, and scan time 0.2 s. The mass spectrometer was operated in positive ion mode with a mass scan range of m / z 100–1500. Specific parameters also included: analysis time 78 min, positive ion detection mode, scan range m / z 300–1400, primary mass spectrometry resolution 120,000, and AGC target value of 5 × 10⁻⁶. 5 Maximum injection time is 50ms, and dynamic exclusion time is 20s. MS 2 The HCD fragmentation mode was used, with an isolation window of 1.6 m / z, a single micro-scan, a maximum secondary injection time of 35 ms, and a normalized collision energy of 33 eV.

[0047] (3) Database retrieval and bioinformatics analysis Protein and peptide identification was performed using a searchable database, specifically the Canadian red deer protein database [Cervus_canadensis_GCF_019320065.1_protein.faa] downloaded from NCBI. The search software used was MaxQuant (version 2.1.4.0). Gene ontology and KEGG pathway enrichment analyses were performed using the EBI database and InterProScan software (versions 5.31-70.0).

[0048] 2. Determination of encapsulation efficiency by high performance liquid chromatography Encapsulation efficiency and drug loading are two important indicators of drug content in nanoparticles, and also crucial metrics for evaluating nanoparticle preparation processes. Encapsulation efficiency refers to the proportion of drug encapsulated within the nanoparticles relative to the total added drug. Drug loading refers to the proportion of drug within the nanoparticles in the entire drug delivery system, calculated using the following formula: Encapsulation rate % = [(Total drug mass - Free drug mass) / Total drug mass added] × 100% Drug loading % = [(Total drug - Free drug) / Total mass of the drug delivery system] × 100% (1) Preparation of the test solution The coarse nanoparticle suspension from Example 1 was loaded into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4°C and 4000 rpm for 15 min. The lower layer of filtrate was then collected for later use.

[0049] (2) Preparation of reference solution Accurately weigh 5 mg of diosgenin and pour it into a 5 mL volumetric flask. Add methanol to the mark and sonicate for 15 min. This solution is used as a reference solution (1 mg / mL). -1 ).

[0050] (3) Selection of chromatographic conditions Chromatographic column: Agilent TC-C18 silica gel column (250 mm × 4.6 mm, 5 μm); mobile phase: water (A) – methanol (B), gradient elution (0–20 min, 5% → 5% A; 95% → 95% B); flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 10 μL; wavelength: 205 nm.

[0051] 3. Single-factor experiment (1) Investigation on the ratio of PAP-DGN Seven 0.01g portions of diosgenin powder were completely dissolved in 6mL of 100% ethanol. Then, deer antler polypeptides were dissolved in 4mL of water at ratios of 10:1, 7:1, 3:1, 1:1, 1:3, 1:7, and 1:10 (total mass of diosgenin). The organic phase was added dropwise to the aqueous phase at a constant rate until the solvent became 60% ethanol. The mixture was then analyzed in groups and sonicated for 30min using a probe sonicator. The resulting liquid was then subjected to rotary evaporation under reduced pressure below 40℃ to slowly remove the ethanol. Subsequently, the liquid was initially filtered using a 0.45μm microporous membrane. The filtrate was collected and replenished with ultrapure water to obtain a coarse nanoparticle suspension. This suspension was then placed in ultrafiltration centrifuge tubes with a molecular weight cutoff of 10kDa and centrifuged at 4℃ and 4000rpm for 15min. The lower filtrate was used for analysis as the free drug concentration. The encapsulation efficiency and drug loading were calculated using the formula described above.

[0052] (2) Investigation of ethanol concentration Seven 0.01g portions of diosgenin powder were placed in a certain volume of 100% ethanol, and seven 0.07g portions of deer antler polypeptide were placed in a certain volume of water. The organic phase was added dropwise to the aqueous phase at a constant rate, so that the solvent was changed to ethanol with concentrations of 100%, 80%, 60%, 50%, 40%, 20%, and 0%, respectively. The samples were then analyzed in groups, and ultrasonic treatment was performed for 30 min using a probe sonicator. The resulting liquid was then subjected to rotary evaporation under reduced pressure below 40℃ to slowly remove the ethanol. Subsequently, the liquid was initially filtered using a 0.45μm microporous membrane, and the filtrate was collected and replenished with ultrapure water to obtain a coarse nanoparticle suspension. The coarse nanoparticle suspension was loaded into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa and centrifuged at 4℃ and 4000rpm for 15 min. The lower filtrate was collected for analysis and used as the free drug component. The encapsulation efficiency and drug loading were calculated using the above formula. (3) Ultrasonic time study Seven 0.01g portions of diosgenin powder were completely dissolved in 6mL of 100% ethanol, and seven 0.07g portions of deer antler polypeptide were completely dissolved in 4mL of water. The organic phase was added dropwise to the aqueous phase at a constant rate until the solvent became 60% ethanol. The samples were ultrasonically treated with a probe sonicator for 5, 10, 20, 30, 60, 120, and 180 minutes for measurement. The resulting liquid was then subjected to rotary evaporation under reduced pressure below 40℃ to slowly remove the ethanol. Subsequently, the liquid was initially filtered using a 0.45μm microporous membrane, and the filtrate was collected and replenished with ultrapure water to obtain a coarse nanoparticle suspension. The coarse nanoparticle suspension was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa and centrifuged at 4℃ and 4000rpm for 15 minutes. The lower filtrate was measured as the free drug concentration, and the encapsulation efficiency and drug loading were calculated using the above formula.

[0053] (4) Box-Behnken response surface methodology for process optimization Design-Expert 13 statistical software was used to design experiments based on the results of single-factor experiments, with PAP-DGN ratio (A), ethanol concentration (B), and ultrasonic time (C) as the subjects of study, and encapsulation rate (Y) as the index. The Box-Behnken method was used for the experimental design.

[0054] 4. Characterization of PDN (1) Transmission electron microscopy (TEM) detection Place a copper mesh on a self-adhesive backing paper, add 30 μL of PDN to the copper mesh, and let it stand for 10 min. Remove excess sample from the copper mesh using filter paper, add 30 μL of phosphotungstic acid staining solution to the copper mesh, and stain for 2 min. Remove excess staining solution from the copper mesh using filter paper, place the copper mesh under an infrared lamp to dry for 10 min, and observe under a microscope.

[0055] (2) Particle size (DLS) and potential (ELS) measurement The particle size (nm) and zeta potential (mV) of the samples were determined using a Brookhaven Zeta potential analyzer. Before measurement, the samples were equilibrated at 25°C for 120 seconds, and all samples were measured three times.

[0056] (3) Fourier transform infrared spectroscopy detection (FTIR) Three groups of samples were prepared: PAP (deer antler polypeptide), DGN (diosgenin), and PDN (self-assembled nanoparticles). Fourier transform infrared spectroscopy was used for analysis. 2 mg of sample powder was mixed with 100 mg of dry potassium bromide, thoroughly ground, compressed into tablets, and placed in a sample chamber at 400-4000 cm⁻¹. -1 The sample is scanned within the wavenumber range.

[0057] (4) Ultraviolet spectroscopy analysis Weigh a certain amount of sample powder and prepare a 1 mg / mL sample solution with deionized water. Scan the sample in the near-ultraviolet region of 200-800 nm using a UV-Vis spectrophotometer.

[0058] (5) X-ray diffraction analysis (XRD) Start the circulating water system. After the water temperature and pressure reach the standard values, turn on the XRD analyzer. Set the accelerating voltage to 40kV, the current to 20mA, 20 = 2-80°, and the scanning speed to 2° / min to determine the X-ray diffraction intensity of the sample.

[0059] (6) Thermogravimetric analysis (TG) The mass change of the sample was measured using a Setline TGA thermogravimetric analyzer in the range of 30-600℃, and observed at a heating rate of 10℃ / min.

[0060] (7) Stability test 1) pH stability evaluation Sample solutions with a concentration of 0.5 mg / mL and pH values ​​of 4, 5, 6, 7, 8 and 9 were prepared and incubated at 4°C for 24 h. The particle size and zeta potential were then measured to evaluate their pH stability.

[0061] 2) Time stability evaluation Seven sample solutions with a concentration of 0.5 mg / mL were prepared and placed at 4°C for 0, 3, 7, 14, 21, and 28 days, respectively. The particle size and zeta potential were then measured to evaluate their pH stability.

[0062] 3) Temperature stability evaluation A 0.5 mg / mL sample solution was prepared and incubated in a water bath at 30, 40, 50, 60, 70, 80, and 90 °C for 1 h. After the samples were cooled to room temperature, the particle size and zeta potential were measured to evaluate their temperature stability.

[0063] 5. Stability analysis of simulated gastrointestinal digestion Preparation of artificial gastric juice: Accurately weigh 10g of pepsin, measure 16.4mL of hydrochloric acid (0.1mol / L), add water and stir the two evenly, filter and make up to 1000mL in a volumetric flask, adjust the pH to 1.2, filter sterilize with 0.22μm filter and set aside for use.

[0064] Preparation of artificial intestinal fluid: Accurately weigh 6.5g of potassium dihydrogen phosphate, add 50mL of water to dissolve it, adjust the pH to 7.4 using sodium hydroxide solution (0.1mol / L), then dissolve 10g of trypsin in water, mix the two evenly, filter, and make up to 1000mL in a volumetric flask, adjust the pH to 1.2, filter sterilize with 0.22μm filter and set aside for use.

[0065] Take diosgenin and an appropriate amount of PDN (both containing 10 mg of diosgenin), add 5 mL of simulated gastric fluid, then place it in a dialysis bag (molecular weight cutoff 8000-14000 Da), tie both ends tightly, and place it in 1000 mL of distilled water containing 0.5% SDS at 37℃. Take samples at 0, 3, 6, 9, and 12 h, filter through a 0.45 μm microporous membrane, and inject the samples under the above chromatographic conditions to determine the cumulative release rate. At the same time, investigate the drug release from simulated intestinal fluid.

[0066] 6. Study on the effect of PDN in improving D-galactose-induced blood-testis barrier damage in aging mice (1) Animal model establishment and grouping One hundred Kunming mice were randomly divided into 10 groups: a blank control group (Control), a model group (D-gal), a positive drug group (VE), an inhibitor group (TNF-α inhibitor), a high-dose and low-dose group of deer antler polypeptide (high dose: PAPH, low dose: PAPL), a high-dose and low-dose group of diosgenin (high dose: DGNH, low dose: DGNL), and a high-dose and low-dose group of deer antler polypeptide-diosgenin self-assembled nanoparticles (high dose: PDNH, low dose: PDNL), with 10 mice in each group. Injection dosage: D-gal 200 mg / kg; intraperitoneal injection dosage: 0.1 mL / 10 g; high dose of deer antler polypeptide: 400 mg / kg, low dose: 200 mg / kg; high dose of diosgenin: 100 mg / kg, low dose: 50 mg / kg; high and low dose groups of deer antler polypeptide-diosgenin self-assembled nanoparticles: 100 mg / kg, low dose: 50 mg / kg (calculated based on drug loading); TNF-α inhibitor group: 0.5 mg / kg intraperitoneal injection, 3 times a week. Vitamin E was 400 mg / kg, gavage dosage: 0.1 mL / 10 g. Except for the blank control group, mice in the other 8 groups were intraperitoneally injected with D-gal to establish the model for 8 weeks. Each treatment group was gavage with the corresponding drug, while the model group and the blank control group were gavage with an equal volume of physiological saline.

[0067] After the last administration, the mice were weighed, euthanized, and the adipose tissue surrounding the epididymis and testis was extracted under aseptic conditions using forceps. The testis and epididymis were carefully removed, weighed, and then fixed and frozen separately. Blood was collected in sterile centrifuge tubes, labeled, and centrifuged at 3000g for 20 minutes at room temperature. Serum was collected and stored at -80℃.

[0068] (2) Detection of mouse sperm parameters When collecting specimens from mice anesthetized with isoflurane, the epididymis on both sides are quickly dissected, washed in room temperature saline, and then cut into small pieces. The specimens are then incubated in a 37°C water bath for 5 minutes to allow the sperm to swim out of the epididymis, thus preparing a mouse sperm suspension.

[0069] 1) Sperm density measurement Add 30 μL of sperm suspension to a hemocytometer and observe under a high-power microscope. Record the number of sperm in the five squares within the large central square. Add the sperm counts in the five squares together. The sperm count per milliliter = total number of squares × 5 × 10 × 10 3 .

[0070] 2) Sperm motility assay Take 10 μL of sperm suspension and drop it into a hemocytometer. Record the video for 10 seconds. Evaluate the sperm in the middle squares of the four corner squares of the hemocytometer in the video. Randomly select a clear field of view from each middle square (generally, the total number of sperm in each field of view should not be less than 50, and the total number of sperm in all four fields of view should not be less than 200). Count the number of motile and dead sperm in each field of view and calculate sperm motility.

[0071] 3) Sperm deformity rate measurement 10 μL of sperm suspension was dropped onto a clean glass slide. A second slide was then used to push the suspension forward at a 45° angle to the surface of the first slide, creating a smear of appropriate thickness. Sperm morphology staining was applied, and the results were observed and recorded under a microscope. At least 1000 intact sperm were counted from each mouse sperm suspension. Sperm abnormalities were categorized as hookless, banana-shaped, swollen-headed, curled-tailed, double-headed, and double-tailed, and the abnormality rate was recorded.

[0072] (3) Histopathological staining Testicular sections embedded in paraffin were dewaxed with hot water and stained with hematoxylin and eosin. Frozen sections were stained with β-galactosidase. The sections were blocked with 3% bovine serum albumin (BSA) at room temperature for 30 minutes, incubated with ZO-1 primary antibody overnight at 4°C, washed three times with PBS, and then incubated with secondary antibody at room temperature for 50 minutes. After further washing with PBS, the sections were developed with 3,3'-diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, mounted, and observed under a microscope (Nikon). Digital pathological image analysis was performed using Aipathwell software to quantify the percentage of ZO-1 positive cells.

[0073] (4) Immunofluorescence experiment Fresh mouse testicular tissue was immediately fixed with 4% paraformaldehyde solution for 24 hours at room temperature to ensure adequate fixation. After fixation, the tissue was dehydrated using a series of ethanol solutions with increasing concentrations, followed by clearing and embedding in paraffin. After the paraffin block solidified, it was sliced ​​into 46-micrometer-thick sections using a microtome and transferred to glass slides. The paraffin sections were dewaxed and hydrated, and antigen retrieval was performed by microwave heating in a sodium citrate buffer solution at pH 6.0. After blocking, the sections were incubated with primary and secondary antibodies against Occludin. The sections were washed three times with 1xPBS solution for 5 minutes each time. DAPI solution (diluted to an appropriate concentration) was added to the sections and incubated at room temperature in the dark for 5-10 minutes for nuclear staining. The immunofluorescence staining results were observed and recorded under a fluorescence microscope to analyze the expression and localization of target proteins in the testicular tissue.

[0074] (5) Blood-testis barrier (BTB) biotin tracer assay Under anesthesia, mice underwent a subtunic injection of biotin into one testis and a PBS solution injection into the other. Sixty minutes later, the testis was removed using the same method and quickly fixed in 12% neutral formaldehyde solution for paraffin sectioning.

[0075] (6) Detection of hormone, inflammatory factors and antioxidant levels in serum and testes of aged mice Serum and testicular tissue were collected separately, and the levels of LH, FSH, T, TNF-α, MDA, SOD, CAT and GSH-Px in the cells were measured according to the instructions of the detection kit.

[0076] (7) Western blot experiment to detect the effect of PDN on related proteins in testicular tissue of aging mice Testicular tissue was frozen and ground to extract total cellular protein, and the protein was quantified using the BCA method. All samples were diluted to the same concentration and then subjected to Western blotting experiments.

[0077] 7. Data Processing Experimental data are reported as mean ± standard deviation (SD). Statistically significant differences were assessed using one-way ANOVA (p < 0.05). The analyses in this study were performed using Origin 8.5 software. Performance Test Results and Analysis 1. Results of liquid chromatography-tandem mass spectrometry of deer antler polypeptides (1) Protein identification and quantification results Detection was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Figure 1 A total of 3210 peptides and 854 proteins were identified, of which proteins with a molecular weight range of 10-80 kDa accounted for 60% of the total. Figure 2 Table 1 lists the 10 most abundant peptides detected by LC-MS, and their signal intensity values ​​are calculated based on LC-MS data.

[0078] (2) Functional annotation and enrichment pathway of deer antler peptides Functional annotation and pathway enrichment analysis of PAPs revealed that their protein composition primarily involves multiple important entries from the three major GO categories, specifically including biological processes such as biological regulation, cellular processes, developmental processes, localization, metabolic processes, multicellular biological processes, stimulus-response, and signal transduction; cellular components such as cellular anatomy; and molecular functions such as binding, catalytic activity, molecular functional regulatory activity, molecular transduction activity, and structural molecular activity. Among these, the number of proteins corresponding to binding, cellular anatomy, and cellular processes was the most significant. Figure 3Further analysis using KEGG pathway annotation revealed that these proteins are primarily enriched in several key pathways, including carbon metabolism, neurodegenerative disease pathways, cancer pathways, the PI3K-Akt signaling pathway, ribosomes, the MAPK signaling pathway, the TGF-β signaling pathway, and the TNF signaling pathway. Figure 4 Next, in vitro and in vivo experiments will be conducted to further validate the MAPK and TNF signaling pathways.

[0079] Table 1 shows the 10 most abundant peptides identified by PAP using LC-MS.

[0080] In Table 1, the structural formula of peptide 1 is as follows:

[0081] The structural formula of peptide 2 is as follows:

[0082] The structural formula of peptide 3 is as follows:

[0083] The structural formula of peptide 4 is as follows:

[0084] The structural formula of peptide 5 is as follows:

[0085] The structural formula of peptide 6 is as follows:

[0086] The structural formula of peptide 7 is as follows:

[0087] The structural formula of peptide 8 is as follows:

[0088] The structural formula of peptide 9 is as follows:

[0089] The structural formula of peptide 10 is as follows:

[0090] 2. Single-factor experiment (1) Effect of PAP-DGN ratio on encapsulation efficiency By systematically investigating the encapsulation efficiency of self-assembled nanoparticles of deer antler polypeptide (PAP) and diosgenin (DGN) at different mass ratios, the results showed that the ratio of the two components had a significant impact on the nanoparticle formation efficiency. Figure 5-10 As shown, the encapsulation efficiency reached its maximum when the mass ratio of PAP to DGN was 7:1, suggesting that the peptides might provide more self-assembly sites and spatial structures. Ultimately, the optimal PAP-DGN ratio was determined to be 5.524:1 using single-factor experiments and response surface methodology. Figure 5 ).

[0091] (2) Effect of ethanol concentration on encapsulation efficiency Ethanol, as an organic solvent, plays a crucial role in regulating intermolecular interactions and nanoparticle morphology during self-assembly. Appropriate amounts of ethanol facilitate molecular diffusion and assembly. Single-factor experiments show that the encapsulation efficiency is highest when the ethanol concentration is 60%. Figure 6 Finally, the optimal ethanol concentration was determined to be 63.895% using response surface methodology.

[0092] (3) Effect of ultrasound time on encapsulation efficiency Ultrasonic treatment, as a key step in promoting the uniformity and stability of self-assembled systems, has a significant impact on encapsulation efficiency due to its duration. For example... Figure 7 As shown, the encapsulation efficiency gradually increased from 5 min to 30 min, reaching a peak at 30 min, indicating that moderate sonication can promote molecular dispersion and self-assembly structure optimization. Further extending the sonication time (60-180 min) led to a decrease in encapsulation efficiency, possibly related to peptide structure destruction or nanoparticle disassembly caused by prolonged sonication. The optimal sonication time was finally determined to be 39.430 min using response surface methodology.

[0093] (4) Fitting of a quadratic polynomial model Table 2 shows the star-shaped design and level. The Box-Behnken response surface methodology was analyzed using Design-Expert 13.0 software (Table 3). After regression fitting of each factor, the response value equations are as follows: Y (%) = 82.42-5.76A-0.8160B-0.0064C+0.1173AB-0.2410AC+1.59BC-10.36A 2 -6.11B 2 -1.02C 2 .

[0094] The results showed that R 2 =0.9768, CV%=2.40%, indicating that this model can explain 97.68% of the data variability. The lack-of-fit term P=0.6749>0.05, indicating that the lack-of-fit term is not significant, indicating that the model fits well. The model P<0.0001 indicates that the regression model is highly significant, and the equation can reflect the relationship between each factor and the response surface well, as detailed in Table 4.

[0095] Table 2 Star Design and Horizontal

[0096] Table 3. Box-Behnken Experimental Design and Response Values ​​for Extraction Processes

[0097] Table 4. Analysis of variance results for the Box-Behnken experimental design fitted by binary multiple regression.

[0098] (5) Response surface optimization and prediction Using Design-Expert 13.0 software, based on quadratic polynomial equations, a 3D response surface plot can be comprehensively evaluated to assess the content of chromone. The optimal process parameter range can be visually observed, with the highest vertex representing the optimal conditions. Response surface model optimization and prediction were performed using Design Expert 13.0 software to obtain the response surface plots of the interaction between PAP-DGN ratio, ethanol concentration, and ultrasonic time. Figure 8-10 ).

[0099] (6) Optimal process verification The optimal process parameters can be obtained by solving a quadratic polynomial equation, and the mathematical model predicts the encapsulation efficiency. The model predicts the optimal extraction process parameters as follows: PAP-DGN ratio 5.524:1, ethanol concentration 63.895%, and ultrasonic time 39.430 min. Based on actual operation, the verified optimal process is: PAP-DGN ratio 5.5:1, ethanol concentration 64%, and ultrasonic time 40 min. Under these conditions, the encapsulation efficiency is 83.246%, and the drug loading is 22.416%. Three experiments were conducted according to the optimal process parameters, and the encapsulation efficiency was calculated. The average encapsulation efficiency was 84.0122%, and the RSD (n=3) was 0.23%, which is very close to the model prediction, indicating that the model prediction is accurate and reliable, and the optimal extraction process is acceptable.

[0100] 3. Characterization of PDN (1) Morphological characteristics of PDN Transmission electron microscopy revealed that PDN exhibited good dispersion in aqueous solution, with no obvious aggregation. The overall morphology of the particles was relatively uniform, exhibiting a near-spherical structure. Figure 11 (The diameter is 300-400nm, which conforms to the morphological characteristics of self-assembled nanoparticles.)

[0101] (2) Results of particle size distribution and surface potential measurement PDN was prepared according to the optimized formulation process, diluted with distilled water, and the particle size was measured using a laser dynamic scattering instrument. Figure 12) and Zeta potential ( Figure 13 The results showed that the particle size, polydispersity index and zeta potential of PDN were (366.29±2.5) nm, (0.211±0.03) mV and (-9.41±0.4) mV, respectively, indicating that the nanoparticles were uniform in size and the whole system was relatively stable.

[0102] (3) Fourier transform infrared spectroscopy analysis Figure 14 There are 4 samples at 4000-400cm -1 The infrared spectrum within the range is shown. In the PAP spectrum, a typical characteristic absorption band of peptide amide can be observed: located at approximately 3280 cm⁻¹. -1 The broad absorption peak is attributed to the amide A band (NH stretching vibration with OH), 1645 cm⁻¹ -1 The vicinity is the amide I band (C=O stretching vibration), 1540 cm. -1 The vicinity is the amide II band (NH bending and CN stretching coupled vibration). As a saponin, DGN's characteristic absorption mainly occurs at 2920 cm⁻¹. -1 With 2850cm -1 (CH stretching vibration) and 1050cm -1 Nearby (CO stretching vibration).

[0103] Comparative analysis revealed significant shifts in both amide I and II bands in the PAP-DGN sample, with some characteristic peaks showing reduced intensity or broadening. This indicates that the interaction between PAP and DGN is not a simple physical mixture, but rather a structural reorganization and assembly through intermolecular interactions such as hydrogen bonding and hydrophobic interactions, forming a nanocomposite with a novel microstructure. Furthermore, PAP-DGN exhibits enhanced performance in the low wavenumber region (e.g., 1000-1200 cm⁻¹). -1 The absorption profile also showed changes, suggesting that COC and C-OH groups participated in the interaction, further supporting the alteration of molecular orientation and local environment during self-assembly. Changes in the position, intensity, and shape of characteristic absorption peaks in the infrared spectrum indicate that PAP and DGN undergo molecular self-assembly under specific preparation conditions to form PAP-DGN nanoparticles.

[0104] (4) Ultraviolet spectroscopy analysis Ultraviolet absorption spectrum such as Figure 15As shown, diosgenin exhibits characteristic absorption peaks at 212 nm and 235 nm, attributed to the π-π* transitions of its conjugated structure in its steroidal skeleton and the electronic transitions of its aromatic ring system, respectively. Notably, the absorption peak of the PAP-DGN complex at 235 nm shows a significant shift, and the absorbance decreases, indicating a change in the microscopic polar environment of diosgenin and verifying the occurrence of molecular self-assembly. However, at the maximum absorption wavelength of diosgenin, 212 nm, the complex does not exhibit a corresponding characteristic absorption peak. This phenomenon can be attributed to the following mechanism: this wavelength region simultaneously encompasses the n-π* transitions (200-220 nm) of the antler polypeptide peptide bond and the absorption of the conjugated structure of diosgenin, with their absorption bands overlapping to form a spectral masking effect; simultaneously, after self-assembly, diosgenin is encapsulated within the nanoparticle core, and its local concentration is significantly reduced, resulting in its apparent absorbance at 212 nm being covered by the strong absorption of PAP. This synergistic change confirms that PAP and DGN form a self-assembly through interaction and hydrogen bonding.

[0105] (5) X-ray diffraction analysis XRD patterns clearly characterized the differences in crystal structure between the polypeptide (PAP), diosgenin (DGN), and their complexes. Figure 16 Pure DGN exhibits typical sharp diffraction peaks (e.g., ~15°, ~17°, ~23°) in the 2θ = 5°–30° range, indicating its highly ordered crystalline structure. PAP, on the other hand, only shows a broad, diffuse envelope at 2θ ≈ 20°, consistent with the flexible chain characteristics of amorphous peptides. Notably, the XRD spectrum of the self-assembled nanoparticles (PDN) shows a significant change: the characteristic crystalline peaks of DGN completely disappear, leaving only a broad, diffuse envelope at 2θ ≈ 20°. This phenomenon indicates that during self-assembly, DGN molecules detach from the ordered lattice and recombine with PAP molecules through hydrophobic interactions and hydrogen bonds, forming a uniform amorphous complex. This confirms the successful construction of PAP-DGN self-assembled nanoparticles at the molecular arrangement level.

[0106] (6) Thermogravimetric analysis Thermogravimetric analysis results show that PAP (deer antler polypeptide), as an organic biomolecule, generally has low thermal stability and is prone to chain breakage, dehydration, and decomposition when heated. Figure 17 DGN (diosgenin) remains stable at higher temperatures, then undergoes rapid and steep decomposition over a relatively narrow temperature range, a typical thermal decomposition behavior of many small-molecule crystalline compounds. Figure 18 The weight loss initiation point of PDN (self-assembled nanoparticles) shifts to the right, and the main decomposition steps shift towards higher temperatures. Figure 19 It demonstrates that PAP and DGN self-assemble through intermolecular interactions, and that breaking the structure requires more energy at high temperatures.

[0107] (7) Stability test Stability is a key indicator for evaluating the practical application of nanomedicine delivery systems. This study systematically investigated the stability of PDN under different pH environments, storage times, and temperatures, using particle size and dispersion index (PDI) as evaluation indicators.

[0108] 1) pH stability evaluation like Figure 20 , 21 As shown, PDN exhibits good stability in environments with different pH values ​​(3-11). Within a wide pH range of 3 to 9, the particle size of the nanoparticles remains around 200 nm, and the PDI value is consistently below 0.2, indicating good monodispersity of the particle system without significant aggregation or precipitation. However, under strongly alkaline conditions (pH=11), the particle size significantly increases to approximately 350 nm, while the PDI value rises above 0.25, suggesting that the structure of the nanoparticles is disrupted and stability decreases under this environment. These results indicate that PDN is structurally stable under acidic, neutral, and weakly alkaline conditions, while its instability under strongly alkaline conditions may be related to changes in interactions such as deprotonation of the polypeptide chains and intermolecular hydrogen bonds, leading to dissociation or aggregation of the nanostructure.

[0109] 2) Time stability evaluation like Figure 22 , 23 As shown, after 28 days of storage at 4°C, neither the particle size nor the PDI of PDN changed significantly. The particle size remained stable at around 200 nm, and the PDI value remained below 0.2 throughout the period. This result fully demonstrates the good storage stability of PDN. During the observation period of up to 4 weeks, no aggregation or sedimentation of the nanoparticles occurred, which is of great significance for its long-term storage as a pharmaceutical formulation.

[0110] 3) Temperature stability evaluation like Figure 24 , 25 As shown, PDN exhibits excellent thermal stability in the temperature range of 30℃ to 60℃, with its particle size and PDI remaining essentially unchanged. When the temperature rises to 70℃, the particle size begins to increase slightly. When the temperature further increases to 80℃ and 90℃, the particle size increases sharply to over 300 nm, and the PDI also increases significantly, indicating that the nanoparticle structure is destroyed at high temperatures, resulting in severe aggregation. This phenomenon is consistent with the results observed in thermogravimetric analysis that nanoparticles have higher thermal decomposition initiation temperatures, suggesting that the self-assembled structure raises the energy barrier of the system, but excessively high thermal energy is still sufficient to destroy the intermolecular forces maintaining the nanostructure, leading to its disintegration.

[0111] (8) Results of in vitro gastrointestinal simulation experiment Release behavior of free DGN and PDN in simulated gastrointestinal fluids, such as Figure 26 and Figure 27 As shown, free DGN is released at nearly 90% within 2 hours. In stark contrast, PDN exhibits significant sustained-release characteristics: an initial burst release of approximately 25% within the first 2 hours, followed by a gradual increase in release. This release profile reflects the characteristics of the nanomedicine delivery system; PDN effectively delays the release of DGN, contributing to a longer duration of drug action in vivo.

[0112] 4. Mechanism of action of PDN in improving D-galactose-induced blood-testis barrier damage in aging mice (1) Results of observation of organ index and sperm morphology in mice Observation of testicular size revealed that, compared with the Control group, the D-gal group mice showed significant atrophy of both the testes and epididymis. Compared with the D-gal group, all drug-treated groups showed varying degrees of recovery in both the testes and epididymis. Figure 28 ).

[0113] Analysis of testicular and epididymal organ indices ( Figure 29 and 30 The results showed that compared with the Control group (testicular organ index approximately 0.60%, epididymal organ index approximately 0.28%), the model group (D-gal group) mice had significantly reduced testicular organ index to approximately 0.40% and epididymal organ index to approximately 0.15% (P<0.01). Compared with the model group, the testicular and epididymal organ indices of each treatment group increased to varying degrees. Among them, the improvement effect of the high-dose PDN group was significantly better than that of the PAP group and the DGN group: Testicular organ index: The high-dose PDN group recovered to approximately 0.54%, which was 3.5 times that of the PAP group (recovered to 0.45%) and 2.33 times that of the DGN group (recovered to 0.43%); Epididymal organ index: The high-dose PDN group recovered to approximately 0.38%, which was 3.29 times that of the PAP group (recovered to 0.22%) and 2.3 times that of the DGN group (recovered to 0.25%).

[0114] Sperm morphology and motility were observed by staining of sperm from each group of mice. Overall, sperm density in the model group was lower than in the control group, and abnormal sperm morphologies such as curled tails, double tails, and amorphous sperm were observed in the model group. Compared with the model group, all treatment groups showed improvement, with the PDN group showing significantly higher levels than the PAP and DGN groups, demonstrating a good improvement effect. Figure 31 ).

[0115] Sperm quality results analysis ( Figure 32 , 33 34) It was found that compared with the control group, the sperm count in the model group was significantly higher (approximately 4.5 × 10⁻⁶ in the control group). 6 / mL, model group approximately 2.5×10 6 Sperm count and motility (approximately 80% in the control group and approximately 40% in the model group) were significantly reduced, while sperm abnormality rate (approximately 5% in the control group and approximately 35% in the model group) was significantly increased (P<0.0001). In mice treated with the drug, sperm count and motility improved in all dosage groups, and sperm abnormality rate decreased (P<0.01). The improvement effect in the PDN group was significantly better than that in the PAP and DGN groups: sperm count in the PDN group recovered to approximately 4 × 10⁹ / mL. 6 / mL (recovery volume 1.5×10) 6 / mL), the recovery rate was the same as that of the PAP group (recovered to 3×10⁹ / mL). 6 / mL, recovery rate 0.5×10 6 The concentration of the drug was 3 times that of the DGN group (which recovered to 2.8 × 10⁹ / mL). 6 / mL, recovery rate 0.3×10 6 The PDN group showed a 5-fold increase in sperm motility (5 / mL); in terms of sperm motility, the PDN group recovered to approximately 70% (a 30% increase), which was 3 times that of the PAP group (recovered to 50%, a 10% increase) and 2 times that of the DGN group (recovered to 55%, a 15% increase); while in terms of improvement in sperm abnormality rate, the PDN group decreased to approximately 15% (a 20% decrease), which was 2 times that of the PAP group (reduced to 25%, a 10% decrease) and 2.5 times that of the DGN group (reduced to 27%, an 8% decrease).

[0116] n=6, ##p<0.01, ####p<0.0001vs.Control; **p<0.01, ***p<0.001, ****p<0.0001vs.D-gal.

[0117] (2) Histopathological experimental results The results of in situ staining of mouse testicular tissue for aging-related β-galactosidase are as follows: Figure 35 As shown in the figure, compared with the control group, the D-gal-induced aging model mice showed a significantly enhanced positive signal for β-galactosidase staining in the testicular tissue, indicating the presence of significant senescent cell accumulation in the testicular tissue. Treatment with the positive-positive drugs VE and TNF-α inhibitors effectively reduced the staining intensity. All treatment groups showed varying degrees of improvement, with the high-dose PDN group (PDNH) showing the most significant positive regulation of aging signals, suggesting that PDN can effectively alleviate D-gal-induced testicular aging.

[0118] Histopathological analysis of testicular tissue showed that the seminiferous tubules in the Control group mice were intact, with neatly arranged and clearly layered spermatogenic epithelial cells, and a large number of mature sperm were visible in the lumen. In contrast, the testicular tissue in the model group showed typical spermatogenic dysfunction, including damage to the seminiferous tubule structure, a significant reduction in the number and disordered arrangement of spermatogenic cells, accompanied by obvious luminal vacuolation, and almost complete absence of mature sperm. After intervention in the drug-treated groups, the above-mentioned pathological damage was repaired to varying degrees, with the high-dose group of deer antler polypeptide-diosgenin self-assembled body showing the most significant effect, with a recovery in the number of spermatogenic cells and the reappearance of mature sperm in the lumen. Figure 36 The control group in the epididymal tail was filled with mature sperm, while the model group showed sperm loss and abnormal apoptosis. All drug-treated groups showed varying degrees of improvement in sperm storage in the epididymal tail, with mature sperm reappearing in the lumen. The PDN group showed the best effect, significantly superior to the PAP and DGN groups. Figure 37 ).

[0119] (3) Effects of PDN on ZO-1 and Occludin in testicular tissue of aging mice Tight junctions are primarily composed of various transmembrane proteins (Occludin) and ankyrin (Z0-1). These two proteins bind to each other to stably anchor tight junction structures to the cytoskeleton. In this part of the study, we used immunohistochemistry and immunofluorescence staining to analyze the expression and distribution of tight junction-related proteins.

[0120] Regarding ankyrin expression ( Figure 38 and 39 Immunohistochemical experiments showed that compared with the Control1 group, the expression of ankylosing spondylogenetic protein Z0-1 in the testicular tissue of mice in the model group was significantly decreased (P<0.0001); compared with the D-gal group, the expression of Z0-1 in each group was significantly increased after intervention with drug administration (P<0.0001). The Z0-1 expression recovery in the high-dose PDN group reached 35% (from 60% to 95%), which was 3.5 times that of the high-dose PAP group (10%) and 1.75 times that of the high-dose DGN group (20%). This result clearly indicates that PDN has a significantly better effect on enhancing the expression of ankylosing spondylogenetic protein Z0-1 in testicular Sertoli cells than the PAP and DGN groups, and can more effectively restore the tight junction function of Sertoli cells. These experimental results indicate that the model group can lead to a decrease in the expression of ankylosing spondylogenetic protein in Sertoli cells, and PDN intervention can increase the expression of ankylosing spondylogenetic protein in testes to a certain extent.

[0121] n=6, ####p<0.0001vs.Control; **p<0.01, ****p<0.0001vs.D-gal.

[0122] Regarding transmembrane protein expression ( Figure 40 Immunofluorescence assays showed that, compared with the control group, the expression of the tight junction transmembrane protein Occludin in the testicular tissue of the model group mice was significantly decreased; compared with the model group, the expression of Occludin was significantly increased after intervention in the high-dose PDN group. These results suggest that PDN can enhance the expression of tight junction transmembrane proteins in Supporting cells, and is significantly superior to the PAP and DGN groups.

[0123] (4) Effects of PDN on the blood-testis barrier (BTB) in aging mice Biotin tracing is a commonly used method for observing blood-tissue barrier permeability. In this study, biotin was injected into the testes of mice in various groups using biotin labeling technology. FITC combined with streptomycin was used for fluorescent labeling to track biotin distribution and assess BTB permeability. Results showed that in the Control group, biotin-positive reactions were mainly concentrated in the interstitial region of the testes. Compared with the Control1 group, biotin-positive signals were observed in the seminiferous tubules of the model group mice. Compared with the model group, after intervention with different doses of Wuzi Zong Wan (a traditional Chinese medicine), biotin-positive signals appeared only in the interstitial region of the testes, and no biotin-positive signals were detected inside the seminiferous tubules. These results indicate that D-gal can increase testicular BTB permeability, and PDN can repair D-gal-induced testicular BTB permeability damage, significantly better than the PAP and DGN groups. Figure 41 ).

[0124] (5) Detection of hormone, inflammatory factors and antioxidant levels in serum and testes of aged mice To assess the systemic effects of PDN on the overall condition of aging mice, we measured serum levels of sex hormones, gonadotropins, inflammatory factors, and oxidative stress indicators.

[0125] like Figures 42-43As shown, compared with the blank control group (LH: approximately 25 mIU / ml, FSH: approximately 75 mIU / ml, T: approximately 38 ng / ml), the serum luteinizing hormone (LH) level in the D-gal model group mice increased to approximately 50 mIU / ml, the follicle-stimulating hormone (FSH) level increased to approximately 130 mIU / ml, and the key testosterone (T) level decreased to approximately 18 ng / ml (all P < 0.0001). After intervention in each treatment group, the levels of the aforementioned hormones all showed a rebound. The regulatory effect of the PDN group was significantly better than that of the PAP and DGN groups: the high-dose PDN (PDNH) group reduced LH to approximately 28 mIU / ml, a decrease of 22 mIU / ml, which is 1.29 times that of the high-dose PAP group (LH reduced to approximately 33 mIU / ml, a decrease of 17 mIU / ml) and 1.47 times that of the high-dose DGN group (LH reduced to approximately 35 mIU / ml, a decrease of 15 mIU / ml). Simultaneously, the PDNH group reduced FSH to approximately 80 mIU / ml, a decrease of 50 mIU / ml, which is significantly better than that of the high-dose PAP group (FSH reduced to approximately 100 mIU / ml). The PDNH group showed a 1.67-fold increase in FSH (FSH decreased to approximately 115 mIU / ml, a decrease of 30 mIU / ml) and a 3.33-fold increase in FSH (FSH decreased to approximately 115 mIU / ml, a decrease of 15 mIU / ml) compared to the high-dose DGN group. Regarding the recovery of testosterone levels, the PDNH group increased T to approximately 30 ng / ml, an increase of 12 ng / ml, which is 1.71 times that of the high-dose PAP group (T increased to approximately 25 ng / ml, an increase of 7 ng / ml) and the high-dose DGN group (T increased to approximately 25 ng / ml, an increase of 7 ng / ml). This demonstrates that the PDNH group can more effectively approach the hormone levels of the control group and has a more significant regulatory effect on age-related endocrine imbalances.

[0126] Regarding inflammation levels ( Figure 44 In the control group, the TNF-α concentration was approximately 50 pg / ml, while in the model group it significantly increased to approximately 100 pg / ml (P<0.0001), indicating the presence of a systemic inflammatory state. All treatment groups reduced TNF-α levels to varying degrees, with the PDN group showing the most significant inhibitory effect: the PDNH group reduced TNF-α to approximately 60 pg / ml, a decrease of 40 pg / ml. This reduction was twice that of the high-dose PAP group (TNF-α reduced to approximately 80 pg / ml, a decrease of 20 pg / ml) and 1.6 times that of the high-dose DGN group (TNF-α reduced to approximately 75 pg / ml, a decrease of 25 pg / ml).

[0127] Regarding oxidative stress indicators in testicular tissue ( Figures 45-48Compared with the control group (MDA: approximately 8 nmol / mg prot, GSH-Px: approximately 80 U / mg prot, CAT: approximately 10 U / mg prot, SOD: approximately 22 U / mg prot), the model group showed an increase in MDA to approximately 15 nmol / mg prot, and a decrease in GSH-Px, CAT, and SOD to approximately 40 U / mg prot, 7 U / mg prot, and 11 U / mg prot, respectively (all P < 0.0001), suggesting that D-gal-induced aging is accompanied by the depletion of the endogenous antioxidant defense system in testicular tissue and the aggravation of oxidative damage. PDN treatment effectively reversed this trend in a dose-dependent manner, with the PDNH group showing significantly better recovery than the PAP and DGN groups: the PDNH group reduced MDA to approximately 9 nmol / mg protein, a reduction of 6 nmol / mg protein, which is twice that of the high-dose PAP group (MDA reduced to approximately 12 nmol / mg protein, a reduction of 3 nmol / mg protein) and the high-dose DGN group (MDA reduced to approximately 12 nmol / mg protein, a reduction of 3 nmol / mg protein). Regarding the recovery of antioxidant enzyme activity, the PDNH group restored GSH-Px to approximately 75 U / mg protein, an increase of 35 U / mg protein, which is 1.94 times that of the high-dose PAP group (GSH-Px restored to approximately 58 U / mg protein, an increase of 18 U / mg protein) and the high-dose DGN group (GSH-Px restored to approximately 55 U / mg protein, an increase of 15 U / mg protein). The PDNH group increased CAT to approximately 9.5 U / mg prot, an increase of 2.5 U / mg prot, which is 1.67 times that of the high-dose PAP group (CAT increased to approximately 8.5 U / mg prot, an increase of 1.5 U / mg prot) and 2.5 times that of the high-dose DGN group (CAT increased to approximately 8 U / mg prot, an increase of 1 U / mg prot). Regarding the increase in SOD, the PDNH group increased it to approximately 19 U / mg prot, an increase of 8 U / mg prot, which is 2 times that of the high-dose PAP group (SOD increased to approximately 15 U / mg prot, an increase of 4 U / mg prot) and 2.67 times that of the high-dose DGN group (SOD increased to approximately 14 U / mg prot, an increase of 3 U / mg prot).

[0128] (6) Effects of PDN on the expression of TNF-α / p38MAPK signaling pathway-related proteins in the testes of aging mice To further explore its molecular mechanisms, we analyzed the expression of key proteins related to blood-testis barrier function and specific signaling pathways in testicular tissue using Western blot. The results are as follows: Figures 49-56As shown, compared with the control group, the expression levels of key adhesion proteins β-catenin and N-cadherin in the testicular tissue of model mice were significantly downregulated (P<0.001), indicating that the structural integrity of the blood-testis barrier was disrupted. Simultaneously, the TNF-α / p38MAPK signaling pathway was abnormally activated, as evidenced by significant upregulation of TNF-α protein levels, downstream transcription factor c-Jun, and the phosphorylation level of p38 MAPK (p-p38), as well as their total protein levels (P<0.0001). After high-dose PDN intervention, these abnormal changes were improved: the expression of β-catenin and N-cadherin in testicular tissue was upregulated, while the overexpression of TNF-α, c-Jun, p38, and p-p38 was effectively inhibited (P<0.05). These results indicate that the protective effect of PDN in vivo is closely related to its ability to negatively influence the overactive TNF-α / p38MAPK signaling pathway in testicular tissue. Its mechanism of action may lie in inhibiting the phosphorylation activation process of p38 MAPK, thereby blocking the transmission of downstream pro-inflammatory and stress signals, ultimately alleviating inflammatory damage to testicular tissue and promoting the restoration of cell junction structures.

[0129] Figures 42-56 Medium, n=6, ####p<0.0001vs.Control; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001vs.D-gal.

Claims

1. A pilose antler polypeptide-diosgenin aglycone self-assembled nanoparticle, characterized in that, An amorphous spherical complex formed by the self-assembly of deer antler polypeptide and diosgenin.

2. The Cervus polypeptide-diosgenin aglycone self-assembled nanoparticle of claim 1, wherein, The deer antler polypeptide includes peptides as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10. 3.The Cervus polypeptide-diosgenin self-assembled nanoparticle of claim 1, wherein, The diosgenin is encapsulated in the core of the deer antler polypeptide-diosgenin self-assembled nanoparticles.

4. The method for preparing deer antler polypeptide-diosgenin self-assembled nanoparticles according to any one of claims 1-3, characterized in that, include: The ethanol organic phase containing the diosgenin was added dropwise at a set rate to the aqueous phase containing the antler polypeptide. After the mass ratio of the antler polypeptide to the diosgenin in the mixed system reached the set mass ratio and the concentration of ethanol in the mixed system reached the set concentration, the mixture was ultrasonically treated to remove the ethanol and ultrafiltered to obtain the antler polypeptide-diosgenin self-assembled nanoparticles.

5. The method for preparing deer antler polypeptide-diosgenin self-assembled nanoparticles according to claim 4, characterized in that, The set mass ratio is 5.5:

1.

6. The method for preparing deer antler polypeptide-diosgenin self-assembled nanoparticles according to claim 4, characterized in that, The set concentration is 64%.

7. The method for preparing deer antler polypeptide-diosgenin self-assembled nanoparticles according to claim 4, characterized in that, The ultrasonic treatment lasted for 40 minutes.

8. The method for preparing deer antler polypeptide-diosgenin self-assembled nanoparticles according to claim 4, characterized in that, Under magnetic stirring at 400-600 rpm, the organic phase was added dropwise to the aqueous phase at a rate of 0.5-1.0 mL / min. After ultrasonic treatment using a probe sonicator, the resulting liquid was subjected to reduced pressure rotary evaporation to remove ethanol. Subsequently, it was pre-filtered using a microporous membrane, and the filtrate was collected. Ultrapure water was added to obtain a coarse nanoparticle suspension. The coarse nanoparticle suspension was loaded into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuged, and ultrafiltered. Ultrapure water was added to the original volume, and the mixture was pre-frozen and placed in a freeze dryer to obtain the deer antler polypeptide-diosgenin self-assembled nanoparticles.

9. The use of the deer antler polypeptide-diosgenin self-assembled nanoparticles according to any one of claims 1-3 in the preparation of drugs to improve blood-testis barrier damage caused by aging.

10. The application according to claim 9, characterized in that, The self-assembled nanoparticles of deer antler polypeptide-diosgenin improve age-related blood-testis barrier damage by regulating the TNF-α / p38MAPK signaling pathway.

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