A preparation of Polygonatum sibiricum vesicles for treating premature ovarian failure, its preparation method and application

Abstract

This invention discloses a Polygonatum sibiricum vesicle preparation for treating premature ovarian failure, its preparation method, and its application. The preparation method includes: cleaning Polygonatum sibiricum, juicing, filtering to remove residue, and collecting the juice; subjecting the juice to gradient centrifugation to remove residual plant tissue, plant cells, and plant cell debris; then using an extraction reagent for stepwise precipitation, collecting the precipitate, and resuspending it to obtain the final product. The Polygonatum sibiricum vesicle preparation prepared by this invention can be efficiently taken up by ovarian granulosa cells. It effectively treats premature ovarian failure by activating the Nrf2 signaling pathway, upregulating antioxidant enzyme expression, scavenging reactive oxygen species, restoring mitochondrial membrane potential, inhibiting apoptosis, and restoring the expression levels of estrogen, follicle-stimulating hormone, and anti-Müllerian hormone. The preparation method of this invention is standardized, biocompatible, and safe, and has broad clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a preparation of Polygonatum sibiricum vesicles for treating premature ovarian failure, its preparation method, and its application. Background Technology

[0002] Premature ovarian failure (POF) refers to the decline of ovarian function in women before the age of 40, manifested as amenorrhea, decreased estrogen levels, and increased gonadotropin levels, accompanied by a series of clinical symptoms such as infertility, hot flashes, night sweats, and osteoporosis, severely impacting women's reproductive health and quality of life. The pathogenesis of POF is not yet fully understood, but oxidative stress damage is considered one of its important pathophysiological mechanisms. Ovarian granulosa cells are important functional cells of the ovary, and their normal function directly affects ovarian endocrine function and follicle development. Oxidative stress factors such as hydrogen peroxide (H2O2) can induce oxidative damage in ovarian granulosa cells, leading to inhibited cell proliferation, increased apoptosis, and mitochondrial dysfunction. Simultaneously, it causes a decrease in the secretion levels of hormones such as estrogen (e.g., E2) and anti-Müllerian hormone (AMH), ultimately leading to ovarian dysfunction.

[0003] Current treatments for premature ovarian failure primarily rely on hormone replacement therapy. While this can alleviate symptoms related to low estrogen levels to some extent, it cannot fundamentally repair damaged ovarian granulosa cells. Furthermore, long-term hormone use carries the risk of complications such as thrombosis and breast disease. In addition, although some traditional Chinese medicine extracts have been shown to have certain antioxidant and ovarian function-protecting effects, their active ingredients are complex and have poor targeting. Moreover, traditional extraction methods cannot preserve the biological effects of their active ingredients, thus limiting their clinical application.

[0004] Plant exovesicles are nanoscale membranous vesicles secreted by plant cells, containing various bioactive substances such as nucleic acids, proteins, and lipids. They possess good biocompatibility, targeting, and bioactivity, and can be taken up by animal cells to regulate cellular physiological functions, providing a new research direction for the treatment of premature ovarian failure. Polygonatum, a traditional Chinese medicine and food, has the effects of tonifying qi and nourishing yin, strengthening the spleen and kidneys, and replenishing essence and marrow. Modern pharmacological studies have confirmed that Polygonatum has antioxidant, anti-inflammatory, and endocrine-regulating effects. However, there are currently no reports on the application of Polygonatum exovesicles in the treatment of premature ovarian failure, and there is also a lack of standardized methods for preparing Polygonatum exovesicles. Summary of the Invention

[0005] To address the shortcomings of existing treatments for premature ovarian failure (POF), such as treating symptoms but not the root cause, significant side effects, and poor targeting and insufficient retention of active ingredients in traditional Chinese medicine extracts, the primary objective of this invention is to provide a Polygonatum sibiricum vesicle preparation for treating PFO and its preparation method. This preparation method can efficiently extract the vesicles from Polygonatum sibiricum while preserving their biological activity. Another objective of this invention is to provide the application of this Polygonatum sibiricum vesicle preparation, demonstrating that it can improve the proliferation, apoptosis, and endocrine function of ovarian granulosa cells by repairing H2O2-induced oxidative damage, thereby providing a new effective ingredient for the preparation of drugs for treating PFO and solving the technical pain points of existing treatments.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a Polygonatum sibiricum vesicle preparation for treating premature ovarian failure.

[0008] A method for preparing a Polygonatum sibiricum vesicle preparation for treating premature ovarian failure includes the following steps:

[0009] (1) Preparation of Polygonatum juice: After cleaning Polygonatum, juice it, filter it to remove residue, and collect the Polygonatum juice;

[0010] (2) Extraction of Polygonatum sibiricum vesicles: The Polygonatum sibiricum juice was subjected to gradient centrifugation to remove residual plant tissue, plant cells and plant cell fragments in sequence. Then, different extraction reagents were used for stepwise precipitation. The precipitates were collected and resuspended to obtain the Polygonatum sibiricum vesicle preparation.

[0011] Preferably, in step (1), a juicer is used to juice the cleaned Polygonatum odoratum, and 150-mesh sterilized gauze is used for filtration, and the filtration is performed at least once.

[0012] Preferably, in step (2), the gradient centrifugation process involves centrifuging in stages under conditions of progressively increasing centrifugal force to remove residual plant tissue, plant cells, and plant cell fragments.

[0013] In some preferred embodiments of the present invention, the gradient centrifugation process includes:

[0014] (2.1.1) Take the Polygonatum juice and centrifuge it at 4℃ and 500~1000×g for 5~20min to remove residual plant tissue and collect the supernatant; more preferably, centrifuge it at 4℃ and 500×g for 10min.

[0015] (2.1.2) Centrifuge the supernatant from step (2.1.1) at 4°C and 2000~5000×g for 5~20 min to remove plant cells and collect the supernatant; more preferably, centrifuge at 4°C and 2000×g for 10 min.

[0016] (2.1.3) Centrifuge the supernatant from step (2.1.2) at 4°C and 8000~15000×g for 10~30 min to remove plant cell debris and collect the supernatant; more preferably, centrifuge at 4°C and 10000×g for 20 min.

[0017] Preferably, in step (2), the stepwise precipitation includes: sequentially using different extraction reagents for precipitation and collecting the precipitate.

[0018] In some preferred embodiments of the present invention, the stepwise precipitation includes:

[0019] (2.2.1) According to volume ratio V 样1 :V A =2:1 Add Isolation Reagent A, where V 样1 V is the volume of the supernatant after gradient centrifugation. A The volume of Isolation Reagent A is mixed, and the mixture is allowed to stand at 4°C for 5-20 min. Then, it is centrifuged at 4°C and 8000-15000×g for 5-20 min, and the supernatant is collected. More preferably, the mixture is allowed to stand at 4°C for 10 min, and then centrifuged at 4°C and 12000×g for 10 min.

[0020] (2.2.2) Take the supernatant from step (2.2.1) by volume ratio V 样2 :V B =3:1 Add Isolation Reagent B, where V 样2 V is the volume of the supernatant obtained in step (1). B The volume of Isolation Reagent B is mixed, and the mixture is allowed to stand at 4°C for 0.5-2 hours. Then, it is centrifuged at 4°C and 8000-15000×g for 20-40 minutes to collect the precipitate. More preferably, the mixture is allowed to stand at 4°C for 1 hour and then centrifuged at 4°C and 13500×g for 30 minutes.

[0021] Preferably, Isolation Reagent A and Isolation Reagent B are extraction reagents from the TW4001 plant tissue fluid exosome extraction kit.

[0022] Preferably, in step (2), the resuspension involves resuspending the precipitate in sterile saline and collecting the supernatant by centrifugation.

[0023] In some preferred embodiments of the present invention, the resuspension is performed by adding 5 mL of sterile physiological saline to every 500 mL centrifuge bottle, then centrifuging at 2000-5000×g for 5-20 min, and collecting the supernatant. More preferably, centrifuging at 2000×g for 10 min.

[0024] Secondly, the present invention provides a Polygonatum sibiricum vesicle preparation obtained by the aforementioned preparation method.

[0025] Preferably, the particle size distribution of the Polygonatum sibiricum vesicle formulation is 30-200 nm, the average particle size is 30-60 nm, and the particle concentration is 10. 6 ~1×10 10 More preferably, the average particle size is 45.2 nm and the particle concentration is 5.55 × 10⁻⁶ particles / mL. 8 Particles / mL.

[0026] Thirdly, the present invention provides the application of the aforementioned Polygonatum sibiricum vesicle preparation.

[0027] The application of the aforementioned Polygonatum sibiricum vesicle preparation in the preparation of drugs for treating premature ovarian failure.

[0028] The aforementioned Polygonatum sibiricum vesicle preparation is taken up by ovarian granulosa cells, activating the Nrf2 signaling pathway to upregulate the expression of antioxidant enzymes to clear intracellular reactive oxygen species (ROS), restore mitochondrial membrane potential, inhibit the apoptosis cascade triggered by oxidative stress, and simultaneously restore the expression levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) in ovarian granulosa cells, repairing ovarian granulosa cell damage caused by oxidative stress, thereby playing a role in treating premature ovarian failure.

[0029] The antioxidant enzyme includes superoxide dismutase SOD1. The Polygonatum sibiricum vesicle preparation can upregulate the mRNA expression level of superoxide dismutase SOD1, reduce the level of malondialdehyde (MDA), a lipid peroxidation product, and increase SOD enzyme activity.

[0030] The aforementioned Polygonatum sibiricum vesicle preparation can upregulate the mRNA expression levels of the anti-apoptotic gene Bcl-2 and the follicle-stimulating hormone receptor gene FSHR in ovarian granulosa cells, and downregulate the mRNA expression levels of the apoptosis genes Caspase3 and Bax, thereby reducing ovarian granulosa cell apoptosis and promoting cell proliferation.

[0031] Preferably, the concentration of the Polygonatum sibiricum vesicle preparation in the drug is 50-1000 μg / mL, used to repair an ovarian granulosa cell oxidative damage model induced by a 400 μM H2O2 solution. More preferably, the concentration of the Polygonatum sibiricum vesicle preparation in the drug is 100 μg / mL.

[0032] Preferably, the dosage form of the drug for treating premature ovarian failure is an oral liquid, prepared by diluting the outer vesicle preparation of Polygonatum sibiricum with physiological saline.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] (1) A standardized method for preparing Polygonatum exovesicles is provided: This invention uses Polygonatum as raw material and achieves efficient extraction of Polygonatum exovesicles by gradient centrifugation combined with a plant exosome extraction kit. Compared with traditional methods, this invention does not require the preparation of sucrose solutions of various concentrations, avoids cumbersome operation steps and ultracentrifugation equipment requirements (traditional methods require ultracentrifugation of more than 90,000g). Only conventional centrifugation equipment (maximum 15,000g) is needed to complete the extraction. The operation is simple, the conditions are controllable, and the reproducibility is good. The extracted Polygonatum exovesicles have uniform particle size (30~200nm) and high biological activity. It also avoids the problems of loss of active ingredients and contamination by impurities in traditional extraction methods, providing a more economical and convenient technical basis for the industrial production of Polygonatum exovesicles.

[0035] (2) First application of Polygonatum sibiricum extracellular vesicles to the treatment of premature ovarian failure: This invention confirms that Polygonatum sibiricum extracellular vesicles can be efficiently taken up by ovarian granulosa cells. As a single active ingredient, it has strong targeting and can directly act on damaged ovarian granulosa cells. Although existing studies have reported that Polygonatum sibiricum extract or Polygonatum sibiricum extracellular vesicles have in vitro antioxidant activity (such as DPPH and ABTS free radical scavenging ability), they all remain at the level of chemical activity. There are no studies on its ability to repair oxidative damage and restore cell function at the cellular level, let alone its application in the treatment of premature ovarian failure. Starting from the pathological mechanism of oxidative stress damage, this invention verifies the therapeutic effect of Polygonatum sibiricum extracellular vesicles at the cellular level for the first time. This includes activating the Nrf2 antioxidant signaling pathway, upregulating the expression of antioxidant enzymes such as SOD1, clearing excess ROS in cells, and reducing the level of lipid peroxidation product MDA, thereby alleviating oxidative stress damage from the root cause.

[0036] (3) Multi-target repair of ovarian granulosa cell function: Existing technologies (such as the traditional ultracentrifugation method for extracting extracellular vesicles of Polygonatum sibiricum) have only verified chemical antioxidant activity, but have not verified its effect on cell function. This invention is the first to demonstrate that Polygonatum sibiricum extracellular vesicles can not only alleviate oxidative stress damage, but also restore the mitochondrial membrane potential of damaged ovarian granulosa cells, increase intracellular ATP levels, and improve mitochondrial energy metabolism function; at the same time, it can inhibit the expression of apoptosis genes Caspase3 and Bax, upregulate the expression of anti-apoptotic gene Bcl-2, reduce ovarian granulosa cell apoptosis, and promote cell proliferation; in addition, it can significantly restore the hormone secretion levels of E2, FSH, and AMH in ovarian granulosa cells, improve ovarian endocrine function, and achieve a comprehensive treatment for premature ovarian failure. These multi-target functional repair effects have not been revealed in Polygonatum sibiricum extracellular vesicles extracted by traditional methods.

[0037] (4) Good biocompatibility and high safety: Polygonatum is a Chinese medicine that is both food and medicine. Its outer vesicles are plant-derived vesicles without artificial chemical synthesis components. They have good biocompatibility and are not prone to causing immune rejection. Compared with hormone replacement therapy, it avoids side effects such as thrombosis and breast disease. Compared with chemically synthesized drugs, it is safer and more suitable for long-term use.

[0038] (5) Provides a new direction for drug development for the treatment of premature ovarian failure: This invention is the first to confirm the anti-oxidative damage effect of the outer vesicles of Polygonatum sibiricum on ovarian granulosa cells, providing a new natural active ingredient for the treatment of premature ovarian failure. The preparation of the outer vesicles of Polygonatum sibiricum can be used as the core ingredient to prepare anti-premature ovarian failure drugs in various dosage forms such as oral liquid, capsules, and tablets, which have broad clinical application prospects. Attached Figure Description

[0039] Figure 1 The images show a transmission electron microscope (TEM) image (top) and a particle size distribution diagram (bottom) of the Polygonatum sibiricum vesicle preparation obtained in Example 1 of this invention.

[0040] Figure 2 This is a fluorescence microscope image of the DIO fluorescence labeling method used in Example 2 of this invention to detect the uptake effect of mouse ovarian granulosa cells (mGCs) on the outer vesicles of Polygonatum sibiricum.

[0041] Figure 3 This is a bar chart showing the recovery of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) levels in a mouse ovarian granulosa cell (mGCs) premature aging model induced by H2O2 in the outer vesicles of Polygonatum sibiricum in Example 3 of this invention. Detailed Implementation

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments. The embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0043] Example 1: Preparation of Polygonatum sibiricum vesicle formulation

[0044] 1. Experimental materials: Fresh Polygonatum sibiricum; Plant tissue fluid exosome extraction kit (TW4001, Shenzhen Shifangjie Technology Co., Ltd.); Sterile physiological saline; 150-mesh sterile gauze; Refrigerated high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); Juicer; Sterile centrifuge bottles, etc.

[0045] 2. Preparation steps:

[0046] (1) Take 500g of fresh Polygonatum, wash it with deionized water to remove surface impurities, drain the water and weigh it, put it into a juicer to extract the juice, filter the extracted Polygonatum juice twice with 150 mesh sterile gauze to remove the residue, and collect the filtrate for later use.

[0047] (2) Transfer the filtrate to a sterile centrifuge bottle and centrifuge at 4℃ and 500×g for 10 min. Discard the bottom precipitate and collect the supernatant.

[0048] (3) Take the above supernatant and centrifuge it at 4℃ and 2000×g for 10 min. Discard the bottom precipitate and collect the supernatant.

[0049] (4) Take the supernatant above, centrifuge at 4℃ and 10000×g for 20 min, discard the cell debris at the bottom, and collect the supernatant;

[0050] (5) Take the above supernatant and mix it according to the volume ratio V 样1 V A Add Isolation Reagent A from the kit at a ratio of 2:1, gently invert to mix, let stand in a 4°C refrigerator for 10 min, then centrifuge at 4°C and 12000×g for 10 min and collect the supernatant.

[0051] (6) Take the above supernatant and mix it according to the volume ratio V 样2 V B Add Isolation Reagent B from the kit at a ratio of 3:1, gently invert to mix, and let stand in a 4°C refrigerator for 1 hour. Then centrifuge at 4°C and 13500×g for 30 minutes, carefully discard the supernatant, and retain the pale white precipitate at the bottom.

[0052] (7) Add sterile physiological saline to the above precipitate at a ratio of 5 mL sterile physiological saline per 500 mL centrifuge bottle, gently blow and resuspend the precipitate, then centrifuge at 2000×g for 10 min, collect the supernatant, which is the preparation of Polygonatum sibiricum vesicles, and store it in a 4℃ refrigerator for short-term storage or a -80℃ refrigerator for long-term storage.

[0053] 3. Characterization and identification of Polygonatum sibiricum vesicle preparations

[0054] (1) Transmission electron microscopy (TEM) observation: 10 μL of the above-mentioned Polygonatum sibiricum vesicle preparation was added to a copper grid and allowed to precipitate for 1 min. The floating liquid was then absorbed by filter paper. 10 μL of uranium acetate was added to a copper grid and allowed to precipitate for 1 min. The floating liquid was then absorbed by filter paper. After drying at room temperature, electron microscopy was performed at 100 kV for imaging. The results showed that the Polygonatum sibiricum vesicles exhibited typical cup-shaped morphological characteristics, consistent with the morphological characteristics of vesicles.

[0055] (2) Nanoparticle tracking analysis (NTA): 200 μL of Polygonatum sibiricum vesicle preparation was diluted 5 times. After adjusting the instrument to its optimal state with diluent, the sample was injected for particle size and concentration analysis. The results showed that the particle size of Polygonatum sibiricum vesicles ranged from 30 to 200 nm, with an average particle size of 45.2 nm and a particle concentration of 5.55 × 10⁻⁶. 8 particles / mL, where the unit particles / mL means particles per mL. Results are as follows: Figure 1 As shown, the transmission electron microscopy (TEM) image reveals that the outer vesicles of Polygonatum sibiricum exhibit a typical cup-shaped morphology, with a scale bar of 100 nm; the nanoparticle tracking analysis image shows a particle size distribution ranging from 30 to 200 nm, with an average particle size of 45.2 nm. The ordinate represents the concentration (×10⁻⁶). 6 Particles / ml), with the x-axis representing particle size (nm).

[0056] Example 2: Uptake of Polygonatum sibiricum vesicle preparation by mouse ovarian granulosa cells (mGCs)

[0057] 1. Experimental materials: 7-8 week old SPF-grade female mice; Polygonatum sibiricum vesicle preparation prepared in Example 1; Dio cell membrane green fluorescent probe; DMEM / F12 culture medium; FBS; penicillin-streptomycin mixture; clean bench (Suzhou Antai Air Technology Co., Ltd.); CO2 incubator (Sanyo Corporation, Japan); fluorescence microscope (Guangzhou Mingmei Technology Co., Ltd.), etc.

[0058] 2. Experimental Methods:

[0059] (1) Isolation and culture of primary mGCs: After 7-8 week old female mice were acclimatized for 3 days, they were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin for superovulation. After 48 hours, they were euthanized by cervical dislocation. The abdomen was disinfected with 75% alcohol, and the bilateral ovaries were removed and placed in preheated DMEM / F12 medium. Under a stereomicroscope, the surface of the ovary was repeatedly punctured with a 1 mL syringe needle to release granulosa cells. The granulosa cells were transferred into T10 culture flasks containing DMEM / F12 high glucose medium with a finger volume fraction of 10% FBS and cultured in a 37℃, 5% CO2 incubator until the cell confluence reached about 90%.

[0060] (2) Dio-labeled Polygonatum vesicles: Centrifuge the Polygonatum vesicle preparation at 13500×g for 30min, discard the supernatant to obtain the precipitate; prepare the staining working solution according to the volume ratio of Dio probe: staining buffer = 1:250, add the staining working solution according to the ratio of outer vesicle: Dio staining working solution = 1μg: 0.9μL to resuspend the precipitate, incubate at 37℃ in the dark for 30min; centrifuge at 13500×g for 30min to remove excess dye, resuspend the precipitate with 1×PBS to obtain Dio-labeled Polygonatum vesicles.

[0061] (3) Cell uptake experiment: mGCs were seeded at 5000 cells / well in 96-well plates, with 5 replicates / groups, and divided into CK group (without Polygonatum sibiricum vesicles) and Polygonatum sibiricum vesicle group. After overnight culture, Polygonatum sibiricum vesicle group was added with Dio-labeled Polygonatum sibiricum vesicle preparation, and CK group was added with an equal volume of 1×PBS. They were cultured for 24h and 36h respectively. After culture, the culture medium was discarded, and the cells were washed once with 1×PBS. The cells were fixed with 4% paraformaldehyde fixative (i.e., 4g paraformaldehyde per 100mL of solution) for 10min, and the nuclei were stained with DAPI for 5min. The cells were observed and photographed under a fluorescence microscope. The relative fluorescence intensity was calculated as: fluorescence intensity of experimental group / fluorescence intensity of CK group.

[0062] 3. Experimental Results: Compared with the control group, the Polygonatum sibiricum vesicle group showed significant green fluorescence at both 24h and 36h, with a highly significant increase in relative fluorescence intensity (P<0.01), indicating that the Polygonatum sibiricum vesicles could be efficiently taken up by mGCs, and the take-up efficiency showed a certain time dependence with incubation time. The results are as follows... Figure 2 As shown, the three columns in the figure, from left to right, are: DIO-labeled Polygonatum vesicles (green fluorescence), DAPI-stained cell nuclei (blue fluorescence), and a combined image of the former two. The figure includes two time points (24 hours and 36 hours), with three rows under each time point, from top to bottom: control group (no Polygonatum vesicles added), 100 μg / mL Polygonatum vesicle group, and 200 μg / mL Polygonatum vesicle group. The results show that Polygonatum vesicles can be efficiently taken up by mouse ovarian granulosa cells, and the uptake effect is concentration-dependent and time-dependent.

[0063] Example 3: Repair effect of Polygonatum sibiricum exovesicle preparation on H2O2-induced oxidative damage model of mGCs

[0064] 1. Experimental Materials: Polygonatum sibiricum vesicle preparation prepared in Example 2; H2O2 standard (Shanghai Maclean Biochemical Technology Co., Ltd.); mGCs; CCK-8 kit; DCFH-DA probe; Hoechst 33258 staining solution; JC-1 mitochondrial membrane potential detection kit; MDA test kit; SOD typing kit; ATP detection kit; E2, FSH, and AMH ELISA kits; qPCR related reagents; multi-functional microplate reader (Thermo Fisher Scientific); qPCR instrument (Hangzhou Borui Technology Co., Ltd.).

[0065] 2. Experimental Methods:

[0066] (1) Establishment of H2O2-induced oxidative damage model of mGCs: mGCs were seeded in culture plates and cultured in DMEM / F12 medium containing 10% FBS and 1% penicillin-streptomycin. Cells were treated with 100, 200, 300, 320, 350, and 400 μM H2O2 for 2 h and 4 h, respectively. Cell proliferation was detected by CCK-8 assay to screen the optimal modeling concentration. The results showed that when mGCs were treated with 400 μM H2O2 for 4 h, cell proliferation was significantly inhibited (P<0.01), which was determined to be the optimal modeling condition.

[0067] (2) Experimental grouping: mGCs were divided into three groups, namely control group (CK group, normal culture, without H2O2 and Polygonatum sibiricum vesicles), H2O2 model group (400μM H2O2 treatment for 4h), and H2O2 + Polygonatum sibiricum vesicle group (400μM H2O2 treatment for 4h, followed by treatment with 100μg / mL (based on total protein of Polygonatum sibiricum vesicles) Polygonatum sibiricum vesicle preparation for 24h and 48h).

[0068] (3) Testing of various indicators:

[0069] ① Cell proliferation detection: The OD450nm value of cells in each group was detected by CCK-8 assay, and the relative proliferation rate was calculated;

[0070] ② ROS level detection: DCFH-DA probe staining, fluorescence microscopy observation and calculation of relative ROS levels;

[0071] ③ MDA and SOD level detection: MDA test kit and SOD typing kit were used respectively, and the OD532nm and OD550nm values ​​were detected by enzyme-linked immunosorbent assay (ELISA) according to the instructions. The MDA content and SOD activity were calculated.

[0072] ④ Apoptosis detection: Hoechst 33258 staining and JC-1 mitochondrial membrane potential detection, fluorescence microscopy observation and calculation of relative apoptosis level;

[0073] ⑤ ATP level detection: The ATP detection kit was used to detect the RLU value of cells in each group and to calculate the ATP content;

[0074] ⑥ Hormone level detection: The expression levels of E2, FSH, and AMH in cells of each group were detected by ELISA.

[0075] ⑦ Gene expression detection: The mRNA expression levels of Nrf2, SOD1, Caspase3, Bax, Bcl-2 and FSHR in each group of cells were detected by qPCR, with β-actin as the internal reference gene.

[0076] (4) Data statistics: Graphpad Prism 6.0 was used for data analysis. Two-way ANOVA was used for multiple group comparisons. P<0.05 was considered statistically significant and P<0.01 was considered extremely statistically significant.

[0077] 3. Experimental Results:

[0078] (1) Effect on cell proliferation: Compared with the CK group, cell proliferation in the H2O2 model group was significantly inhibited at 24h and 48h (P<0.01); compared with the H2O2 model group, cell proliferation in the H2O2+Polygonatum sibiricum vesicle group was significantly promoted at 24h (P<0.01), and had a slight promoting effect at 48h.

[0079] (2) Effects on oxidative stress indicators: Compared with the CK group, the 24h ROS and MDA levels in the H2O2 model group were significantly increased (P<0.01), and the SOD level was significantly decreased (P<0.05); compared with the H2O2 model group, the 24h ROS and MDA levels in the H2O2+Polygonatum sibiricum vesicle group were significantly decreased (P<0.05 / P<0.01), and the SOD level was significantly increased (P<0.05).

[0080] (3) Effects on apoptosis and mitochondrial function: Compared with the CK group, the mitochondrial membrane potential of the H2O2 model group was significantly reduced at 24h and 48h, and the ATP level was significantly decreased (P<0.01); compared with the H2O2 model group, the mitochondrial membrane potential of the H2O2+Polygonatum sibiricum vesicle group was significantly restored at 24h and 48h, and the ATP level was significantly increased (P<0.01). At the same time, it can regulate the expression of apoptosis genes and inhibit apoptosis.

[0081] (4) Effects on hormone levels: Compared with the CK group, the expression of E2, FSH and AMH in the H2O2 model group was significantly inhibited at 24h and 48h (P<0.01); compared with the H2O2 model group, the expression of the above hormones in the H2O2+Polygonatum sibiricum vesicle group was significantly increased at 24h and 48h (P<0.01).

[0082] (5) Effects on gene expression: Compared with the CK group, the mRNA expression of Nrf2, SOD1, Bcl-2, and FSHR in the H2O2 model group was significantly decreased at 24h and 48h (P<0.01), while the mRNA expression of Caspase3 and Bax was significantly increased (P<0.01); compared with the H2O2 model group, the mRNA expression of Nrf2, SOD1, Bcl-2, and FSHR in the H2O2 + Polygonatum sibiricum vesicle group was significantly increased at 24h and 48h (P<0.01), while the mRNA expression of Caspase3 and Bax was significantly decreased (P<0.01). Results are as follows. Figure 3 As shown.

[0083] Example 4: Dosage Form Application of Polygonatum sibiricum Vesicle Formulation in the Preparation of Drugs for Treating Premature Ovarian Failure

[0084] Using the Polygonatum sibiricum vesicle preparation of this invention as the core active ingredient, it is compounded with sterile physiological saline to prepare an oral liquid dosage form. This dosage form has a simple preparation process and is convenient to take. Moreover, according to component analysis, the preparation can still stably retain the biological activity of Polygonatum sibiricum vesicles after being stored at 4°C for 90 days, and can effectively play a role in treating premature ovarian failure. The specific preparation method is as follows:

[0085] The Polygonatum sibiricum vesicle preparation of this invention is diluted with sterile physiological saline to a final concentration of 100 μg / mL (based on total protein from Polygonatum sibiricum vesicles), thus obtaining a Polygonatum sibiricum vesicle oral solution. The oral solution is then dispensed into sterile pharmaceutical containers, sealed, and stored at 4°C.

[0086] This Polygonatum sibiricum vesicle oral solution is an oral dosage form that does not require complex reconstitution or auxiliary processing, resulting in high patient compliance. It is suitable for the clinical treatment and long-term management of patients with premature ovarian failure of varying degrees. Tests have shown that even after 90 days of storage at 4°C, the particle size, concentration, and bioactivity of the Polygonatum sibiricum vesicles do not significantly decrease. They can still be effectively taken up by ovarian granulosa cells, exerting antioxidant, mitochondrial function repair, cell apoptosis regulation, and ovarian endocrine function restoration effects.

[0087] Comparative Example 1: A comparative experiment between Polygonatum sibiricum vesicle preparations and Polygonatum sibiricum vesicles prepared by traditional methods.

[0088] 1. Preparation of Polygonatum sibiricum exovesicles by traditional sucrose density gradient centrifugation method

[0089] The extraction of Polygonatum sibiricum vesicles was performed using a combination of traditional ultracentrifugation and sucrose density gradient centrifugation. The specific steps are as follows:

[0090] (1) Preparation of Polygonatum extract: Same as step (1) in Example 1.

[0091] (2) Centrifugation extraction: Same as steps (2)-(4) in Example 1.

[0092] (3) Filtering: Same as step (5) in Example 1.

[0093] (4) Concentration: Centrifuge the filtered Polygonatum juice at 95000g for 120min, discard the supernatant and collect the precipitate.

[0094] (5) Sucrose density gradient centrifugation: Prepare a discontinuous sucrose gradient solution, with sucrose solutions of 8wt%, 30wt%, 45wt%, and 60wt% from top to bottom, in a volume ratio of 2:3:3:3. Resuspend the precipitate from step (4) in PBS, transfer it to the upper layer of the sucrose gradient solution, and centrifuge at 95000g for 60min. Take the sample layer between the 30wt% and 45wt% sucrose solutions, add 3 times the volume of PBS to resuspend it, centrifuge again at 95000g for 60min, and collect the precipitate, which is the outer vesicle of Polygonatum sibiricum.

[0095] 2. Comparative Experiment

[0096] The Polygonatum sibiricum vesicles prepared in Example 1 (hereinafter referred to as "the present invention group") were compared with the Polygonatum sibiricum vesicles prepared in Comparative Example 1 (hereinafter referred to as "the conventional group"), and the following indicators were tested:

[0097] (1) Comparison of extraction efficiency: The present invention group only requires conventional centrifugation equipment (up to 13500g), without the need for ultracentrifugation, and the operation steps are simple; the traditional group requires ultracentrifugation equipment (95000g), and the operation steps are complicated.

[0098] (2) Comparison of cell uptake efficiency: Following the method in Example 2, the uptake efficiency of vesicles by mouse ovarian granulosa cells in the two groups was detected. The results showed that the uptake efficiency of vesicles in the present invention group was comparable to or higher than that in the conventional group.

[0099] (3) Comparison of cell function repair effects: Following the method in Example 3, the repair effects of the two groups of vesicles on the 400 μM H2O2-induced ovarian granulosa cell oxidative damage model were detected. The results showed:

[0100] Cell proliferation (CCK8 assay): The cell proliferation promoting effect of the present invention group (relative absorbance, 1.46±0.17) was significantly better than that of the traditional group (1.15±0.25, n=6, P=0.031).

[0101] ROS scavenging (DCFH-DA fluorescent probe method): The scavenging effect of the present invention group on intracellular ROS (relative fluorescence intensity 0.57±0.12) was significantly better than that of the conventional group (0.84±0.17, n=6, P=0.009).

[0102] Apoptosis regulation (qPCR detection): The downregulation of apoptosis genes Caspase3 and Bax and the upregulation of anti-apoptotic gene Bcl-2 in the present invention group were significantly better than those in the conventional group (P<0.05). As shown in Table 1.

[0103] Table 1

[0104] Gene This invention group Traditional Group P Caspase 3 0.45±0.18 0.74±0.13 0.009 Bax 0.57±0.20 0.86±0.24 0.046 Bcl-2 1.56±0.09 1.35±0.12 0.006

[0105] Hormone recovery (ELISA test): The recovery effect of E2, FSH and AMH hormone levels in the present invention group was significantly better than that in the traditional group (P<0.05), as shown in Table 2.

[0106] Table 2

[0107] hormone This invention group Traditional Group P E2 (pg / mL) 20.45±3.64 14.78±4.23 0.032 FSH (ng / mL) 62.79±4.56 53.98±5.78 0.015 AMH (ng / mL) 38.49±5.10 25.14±6.28 0.002

[0108] 3. Conclusion

[0109] The preparation method of the present invention is not only simpler to operate and requires less equipment, but the resulting Polygonatum vesicles also exhibit significantly better biological activity than those prepared by the traditional sucrose density gradient centrifugation method in terms of cell uptake, anti-oxidation, anti-apoptosis, and hormone restoration.

[0110] The preparation method of the Polygonatum sibiricum vesicle preparation provided by this invention is standardized and the conditions are controllable, and it can be mass-produced using conventional biopharmaceutical equipment. The Polygonatum sibiricum vesicle preparation has a clear anti-oxidative damage effect on ovarian granulosa cells and can repair ovarian function at multiple targets. It can be used as the core active ingredient to prepare drugs for treating premature ovarian failure in various dosage forms such as oral liquid, capsules, and tablets, and has significant industrial application value and clinical application prospects.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a Polygonatum sibiricum vesicle preparation for treating premature ovarian failure, characterized in that, Includes the following steps: (1) Preparation of Polygonatum juice: After cleaning Polygonatum, juice it, filter it to remove residue, and collect the Polygonatum juice; (2) Extraction of Polygonatum sibiricum vesicles: Polygonatum sibiricum juice is subjected to gradient centrifugation, followed by stepwise precipitation. The precipitate is collected and resuspended to obtain Polygonatum sibiricum vesicle preparation. The gradient centrifugation process includes: (2.1.1) Take the Polygonatum juice and centrifuge it at 4℃, 500~1000×g for 5~20min to remove residual plant tissue and collect the supernatant; (2.1.2) Centrifuge the supernatant from step (2.1.1) at 4℃ and 2000~5000×g for 5~20min to remove plant cells and collect the supernatant; (2.1.3) Centrifuge the supernatant from step (2.1.2) at 4℃ and 8000~15000×g for 10~30min to remove plant cell debris and collect the supernatant; The stepwise precipitation includes: (2.2.1) According to volume ratio V 样1 :V A =2:1 Add Isolation Reagent A, where V 样1 V is the volume of the supernatant after gradient centrifugation. A The volume of Isolation Reagent A is used to mix the liquid. After mixing, the mixture is allowed to stand at 4°C for 5-20 min, then centrifuged at 4°C and 8000-15000×g for 5-20 min. The supernatant is then collected. (2.2.2) Take the supernatant from step (2.2.1) by volume ratio V 样2 :V B =3:1 Add Isolation Reagent B, where V 样2 V is the volume of the supernatant obtained in step (1). B The volume of Isolation Reagent B is used to mix the precipitate. After mixing, the precipitate is allowed to stand at 4°C for 0.5-2 hours, then centrifuged at 8000-15000×g for 20-40 minutes at 4°C. The precipitate is then collected. Isolation Reagent A and Isolation Reagent B are extraction reagents from the TW4001 Plant Tissue Fluid Exosome Extraction Kit.

2. The preparation method according to claim 1, characterized in that, In step (1), a juicer is used to juice the cleaned Polygonatum sibiricum. The filter is made with 150-mesh sterilized gauze and is filtered at least once.

3. The preparation method according to claim 1, characterized in that, In step (2), the resuspension is to resuspend the precipitate by adding 5 mL of sterile physiological saline to every 500 mL centrifuge bottle, then centrifuging at 2000~5000×g for 5~20 min, and collecting the supernatant.

4. A Polygonatum sibiricum vesicle preparation obtained by the preparation method according to any one of claims 1-3, characterized in that, The particle size distribution of the Polygonatum sibiricum vesicle preparation is 30-200 nm, the average particle size is 30-60 nm, and the particle concentration is 1×10⁻⁶. 6 ~1×10 10 Particles / mL.

5. The use of the Polygonatum sibiricum vesicle preparation according to claim 4 in the preparation of a drug for treating premature ovarian failure.

6. The application according to claim 5, characterized in that, The concentration of the Polygonatum sibiricum vesicle preparation used in the drug is 50~1000μg / mL, which repairs the oxidative damage model of ovarian granulosa cells induced by 400μM H2O2 solution.

7. The application according to claim 5, characterized in that, The drug for treating premature ovarian failure is in the form of an oral liquid, prepared by diluting a preparation of Polygonatum sibiricum vesicles with physiological saline.

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