Application of morinda officinalis-derived nano-vesicles in preparation of medicine for preventing and / or treating premature ovarian failure
By extracting nanovesicles (MO-EVs) from Morinaceae, the problem that existing Morinaceae extract cannot effectively summarize its efficacy in the treatment of premature ovarian failure is solved. MO-EVs have shown the effect of protecting ovarian cells and reversing premature ovarian failure, providing a new theoretical basis for treatment.
Patent Information
- Application Number
- CN202510209142.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-05-13
AI Technical Summary
The existing Morinacea extract cannot effectively summarize its efficacy in the treatment of premature ovarian failure, and its application in premature ovarian failure has not been reported.
Nanovesicles (MO-EVs) were extracted from Morinaceae. Through in vivo and in vivo experiments, they have the effects of protecting ovarian cells, promoting ovarian cell proliferation and migration, and reversing premature ovarian failure caused by cyclophosphamide.
MO-EVs show strong function of protecting ovarian cells and can reverse premature ovarian failure caused by cyclophosphamide, providing a new theoretical basis for the application and prevention of premature ovarian failure.
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Figure CN119970856A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of Morinda officinalis-derived nanovesicles in the preparation of drugs for preventing and / or treating premature ovarian failure. Background Art
[0002] Ovarian failure refers to the phenomenon of amenorrhea before the age of 40, which is manifested by infrequent menstruation or amenorrhea for at least 4 months, 2 FSH>25mIU / ml (4 weeks apart) or 1 FSH>40mIU / mL; it includes the early stage of diminished ovarian reserve (DOR) and the late stage of premature ovarian failure (POF). With the in-depth study of etiology and the continuous accumulation of clinical cases, people gradually realize that ovarian dysfunction is a progressive disease. In the DOR stage, the patient's ovarian function is impaired, the quality and quantity of eggs produced are reduced, the estrogen level decreases, and the patient has symptoms such as menstrual disorders. This period can last for several years. If it is not recognized and intervened early, it will eventually develop to the terminal stage of ovarian dysfunction, the POF stage. When the patient reaches the POF stage, the ovarian function is irreversible. Therefore, the FSH diagnostic threshold for POF is 40mIU / mL, and for POI it is reduced to 25mIU / mL. The purpose is to detect early ovarian dysfunction, diagnose early and take measures early, improve the patient's clinical symptoms, preserve the patient's fertility, guide the patient to give birth, improve the patient's quality of life, protect their right to have offspring, and promote family harmony.
[0003] According to the clinical symptoms of patients, Chinese medicine classifies them into categories such as "blood deficiency", "absence of menstruation", "infertility", "amenorrhea", "absence of menstruation due to blood deficiency", "cessation of menstruation before old age", and "symptoms before and after menstruation". The main causes and pathogenesis can be summarized as kidney deficiency, blood stasis, liver depression, heart fire, spleen dysfunction, qi and blood deficiency, qi stagnation, phlegm and dampness obstruction, etc.
[0004] In terms of Chinese medicine treatment: such as Sicuzugu Yiluru Pill, its function is to nourish essence and blood, stop bleeding and remove blood stasis. For patients with kidney deficiency, it is necessary to start with nourishing kidney yin, warming and nourishing kidney yang, regulating yin and yang, nourishing kidney and replenishing qi, nourishing blood and regulating menstruation. Representative prescriptions include Jinkui Shenqi Pill, Zuogui Pill, Yougui Pill, etc. When the blood sea is insufficient and the uterine vessels are blocked, it is necessary to replenish the blood sea to fill it up, and then regulate Chongren and take advantage of the situation. The representative prescription is Siwu Decoction. For patients with spleen dysfunction, tonify the spleen and replenish qi, tonify the kidney and strengthen the spleen, and coordinate the prenatal and postnatal. The prescriptions are Wentu Yulin Decoction, Shenling Baizhu San plus or minus, etc. For patients with heart-kidney disharmony, it is necessary to calm the mind and calm the nerves, and connect the heart and kidney. The prescription is Huanglian Ejiao Decoction plus or minus, etc. When encountering patients with liver depression and qi stagnation, it is necessary to soothe the liver and soften the liver, soothe the liver and regulate qi, nourish blood and activate blood circulation, and soothe the liver to relieve depression and promote qi. The prescription is Xiaoyao San plus or minus, etc. For patients with qi stagnation and blood stasis, the treatment is to promote qi, activate blood circulation, and remove blood stasis, and the prescriptions are Shenqi Pills, Shixiao Powder, etc. For patients with qi deficiency, qi and blood should be replenished, and the prescription is Sijunzi Decoction, etc. For patients with phlegm and dampness obstruction and disharmony between the gallbladder and stomach, the treatment is to regulate qi and remove phlegm, clear the gallbladder and harmonize the stomach, nourish yin and suppress yang, and the prescription is Wendan Decoction plus or minus, etc. There are also Yijing Decoction for the treatment of the four internal organs of the heart, liver, spleen and kidney. In the process of traditional Chinese medicine treatment, Morinda officinalis is a frequently used drug in treatment, and its extracts are also used in treatment in Western medicine research. However, the current Morinda officinalis extracts cannot fully summarize the efficacy of Morinda officinalis.
[0005] In recent years, the research on plant-derived nanovesicles, especially nanovesicles derived from traditional Chinese medicine, has gradually attracted the attention of the scientific community. However, there are no reports on the application of Morinda officinalis nanovesicles in the treatment of premature ovarian failure. Summary of the invention
[0006] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide the use of Morinda officinalis derived extracellular vesicles (MO-EVs) in the preparation of drugs for preventing and / or treating premature ovarian failure.
[0007] The applicant of the present invention extracted nanovesicles from Morinda officinalis and found in in vivo and in vitro experiments that the nanovesicles have a strong effect of protecting ovarian cells and reversing POF caused by cyclophosphamide, providing a new theoretical basis for the application and prevention of Morinda officinalis in POF.
[0008] The purpose of the present invention is achieved through the following solutions:
[0009] In a first aspect, the present invention provides the use of Morinda officinalis derived Extracellular vesicles (MO-EVs) in the preparation of a drug for preventing and / or treating premature ovarian failure.
[0010] The present invention found through in vivo and in vitro experiments that MO-EVs have a strong effect of protecting ovarian cells, promoting ovarian cell proliferation, promoting ovarian cell migration, and reversing POF caused by cyclophosphamide.
[0011] In a second aspect, the present invention provides the use of nanovesicles derived from Morinda officinalis in the preparation of a drug for promoting ovarian cell proliferation.
[0012] In a third aspect, the present invention provides the use of nanovesicles derived from Morinda officinalis in the preparation of drugs that promote ovarian cell migration.
[0013] In the technical solution of the present invention, the drugs are the same or different and include therapeutically effective amounts of nanovesicles derived from Morinda officinalis.
[0014] In the technical scheme of the present invention, the drugs that are the same or different can be made into various pharmaceutical dosage forms by conventional methods, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, drops and other oral dosage forms and injections and other dosage forms other than oral administration, such as injections.
[0015] In the technical solution of the present invention, the drugs described above, whether the same or different, may also contain one or more pharmaceutically acceptable carriers or excipients.
[0016] Furthermore, the carrier or auxiliary material may include a diluent, a wetting agent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, a disintegrant, a preservative, and the like.
[0017] The Morinda officinalis-derived nanovesicles (MO-EVs) of the present invention can be obtained by a nanovesicle preparation method known to those skilled in the art, such as being separated from Morinda officinalis.
[0018] The Morinda officinalis-derived nanovesicles (MO-EVs) can be separated and obtained by conventional separation methods, such as by crushing or breaking the wall of Morinda officinalis and then adding an extractant for extraction and separation.
[0019] Furthermore, the separated precipitate is the nanovesicles derived from Morinda officinalis.
[0020] Furthermore, the separation method may be to separate the extracted extract by ultracentrifugation.
[0021] Furthermore, the speed of the ultracentrifugation may be 100000 g or above.
[0022] Furthermore, the ultracentrifugation time may be 30-180 min.
[0023] Furthermore, the ultracentrifugation time may be 60-160 min.
[0024] Furthermore, the precipitate obtained by ultracentrifugation can be resuspended in a buffer such as PBS to obtain a suspension of nanovesicles derived from Morinda officinalis.
[0025] Furthermore, ultracentrifugation can be repeated once or more to obtain further purified Morinda officinalis-derived nanovesicles.
[0026] Furthermore, a filter can be used to remove larger impurities before ultracentrifugation; the filter can be a 0.45 μm filter.
[0027] Furthermore, the extract can be pre-centrifuged before ultracentrifugation to collect the supernatant and remove impurity precipitation.
[0028] Furthermore, the speed of the pre-centrifugation may be 10000 g or less.
[0029] Furthermore, the pre-centrifugation time may be 5-60 min.
[0030] Furthermore, the pre-centrifugation may be repeated once or more. The pre-centrifugation may separate and remove non-target substances, thereby reducing the influence of impurities on subsequent ultracentrifugation separation.
[0031] Furthermore, the extractant may be conventionally used water or an aqueous solution to which a buffer such as phosphate is added.
[0032] Furthermore, the suspension can be filtered through a filter before use to obtain a sterile suspension. The filter can be a 0.22 μm filter.
[0033] The present invention obtains Morinda officinalis-derived nanovesicles by isolating them from Morinda officinalis. The nanovesicles have natural ingredients, no toxic side effects, good biocompatibility and safety, and are proven to have a strong effect of protecting ovarian cells and reversing POF caused by cyclophosphamide through in vivo and in vitro experiments, indicating that the nanovesicles can be used to prevent and / or treat premature ovarian failure, providing a new theoretical basis for the application and prevention of Morinda officinalis in POF, and therefore have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.
[0035] Figure 1This is a transmission electron microscopy image of Morinda officinalis-derived nanovesicles (MO-EVs).
[0036] Figure 2 This is the particle size diagram of Morinda officinalis-derived nanovesicles (MO-EVs).
[0037] Figure 3 This is a diagram of the uptake of MO-EVs by KGN cells.
[0038] Figure 4 This figure shows the effect of MO-EVs on the proliferation ability of KGN cells.
[0039] Figure 5 This figure shows the effect of MO-EVs on the migration ability of KGN cells.
[0040] Figure 6 This figure shows the effect of MO-EVs on hormone secretion by KGN cells.
[0041] Figure 7 The distribution of MO-EVs in mouse organs.
[0042] Figure 8 This is a diagram of the uptake of MO-EVs by ovarian tissue.
[0043] Fig. 9 The morphological diagrams of the ovaries of the animals in each group are shown in Figure 2 .
[0044] Fig.10 The following are the ovarian sections of animals in each group.
[0045] Fig.11 To investigate the effect of MO-EVs on the animal model induced by cyclophosphamide, ELISA was used to detect the changes in estradiol (E2), progesterone and inhibin (Inh) in the serum of each group of animals.
[0046] Among them, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION
[0047] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can be obtained from commercial channels unless otherwise specified. The methods described are conventional methods unless otherwise specified.
[0048] In one embodiment, Morinda officinalis derived extracellular vesicles (MO-EVs) are used in the preparation of a drug for preventing and / or treating premature ovarian failure.
[0049] In one embodiment, the Morinda officinalis-derived nanovesicles have a strong effect of protecting ovarian granulosa cells and restoring POF caused by cyclophosphamide.
[0050] In one embodiment, the drugs are the same or different and each comprises a therapeutically effective amount of nanovesicles derived from Morinda officinalis.
[0051] In one embodiment, the drugs that are the same or different can be made into various pharmaceutical dosage forms by conventional methods, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, drops and other oral dosage forms and injections and other dosage forms other than oral administration, such as injections.
[0052] In one embodiment, the drugs, whether the same or different, may further contain one or more pharmaceutically acceptable carriers or excipients.
[0053] In one embodiment, the carrier or auxiliary material may include a diluent, a wetting agent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, a disintegrant, a preservative, and the like.
[0054] In one embodiment, the Morinda officinalis-derived nanovesicles (MO-EVs) described in the present invention can be obtained by a nanovesicle preparation method known to those skilled in the art, such as isolating them from Morinda officinalis.
[0055] In one embodiment, the Morinda officinalis-derived nanovesicles (MO-EVs) can be separated and obtained by conventional separation methods, such as by crushing or breaking the wall of Morinda officinalis and then adding an extractant for extraction and separation.
[0056] In one embodiment, the separated precipitate is the Morinda officinalis-derived nanovesicles.
[0057] Furthermore, the separation method may be to separate the extracted extract by ultracentrifugation.
[0058] In one embodiment, the speed of the ultracentrifugation can be 100,000 g or above. In one embodiment, the speed of the ultracentrifugation is 100,000 g; in another embodiment, the speed of the ultracentrifugation is 130,000 g; in another embodiment, the speed of the ultracentrifugation is 120,000 g.
[0059] In one embodiment, the ultracentrifugation time may be 30-180 min.
[0060] In one embodiment, the ultracentrifugation time may be 60-160 min. In one embodiment, the ultracentrifugation time is 70 min; in another embodiment, the ultracentrifugation time is 100 min; in another embodiment, the ultracentrifugation time is 80 min.
[0061] In one embodiment, the precipitate obtained by ultracentrifugation can be resuspended in a buffer such as PBS to obtain a suspension of nanovesicles derived from Morinda officinalis.
[0062] In one embodiment, ultracentrifugation can be repeated once or more to obtain further purified Morinda officinalis-derived nanovesicles.
[0063] In one embodiment, a filter may be used to remove larger impurities before ultracentrifugation; the filter may be a 0.45 μm filter.
[0064] In one embodiment, the extract may be pre-centrifuged before ultracentrifugation to collect the supernatant and remove impurity precipitates.
[0065] In one embodiment, the speed of the pre-centrifugation may be 10000 g or less. In one embodiment, the speed of the pre-centrifugation is 10000 g; in another embodiment, the speed of the pre-centrifugation is 2000 g; in another embodiment, the speed of the pre-centrifugation is 300 g.
[0066] In one embodiment, the pre-centrifugation time can be 5-60 minutes. In one embodiment, the pre-centrifugation time is 10 minutes; in another embodiment, the pre-centrifugation time is 20 minutes; in another embodiment, the pre-centrifugation time is 30 minutes.
[0067] In one embodiment, the pre-centrifugation may be repeated once or more. The pre-centrifugation may separate and remove non-target substances, thereby reducing the influence of impurities on subsequent ultracentrifugation.
[0068] In one embodiment, the extractant can be conventionally used water or an aqueous solution with a buffer such as phosphate added. In one embodiment, the extractant is water; in another embodiment, the extractant is a phosphate buffer.
[0069] In one embodiment, the suspension can be filtered through a filter before use to obtain a sterile suspension. The filter can be a 0.22 μm filter.
[0070] In one embodiment, the Morinda officinalis-derived nanovesicles (MO-EVs) can be obtained by crushing or breaking the wall of Morinda officinalis and then adding an extractant for extraction and separation, which may specifically include the following steps:
[0071] Fresh Morinda officinalis was cleaned and dried, and double distilled water was added. It was crushed by a wall breaker to separate the supernatant. The supernatant was pre-centrifuged at 100-10000g for multiple times, such as centrifuged at 300g, 2000g, and 10000g for 10-30min, and the supernatant was taken and the precipitate was discarded to remove non-target substances; and the supernatant was passed through a 0.45μm filter to remove impurities exceeding the size; the supernatant after pre-centrifugation was ultracentrifuged at a centrifugal force of 100000g or more (such as 135000g), and the centrifugation time could be 30-180min. The obtained precipitate was the Morinda officinalis-derived nanovesicle; it was resuspended in PBS to obtain a suspension of Morinda officinalis-derived nanovesicles. The suspension was passed through a 0.22μm filter to obtain a sterile MO-EVs suspension.
[0072] In one embodiment, the morphological identification of Morinda officinalis-derived nanovesicles (MO-EVs) was performed using transmission electron microscopy. Observation under transmission electron microscopy showed that MO-EVs were round or oval vesicles, such as tray-shaped; under the electron microscope, 50nm-100nm vesicle structures with membrane structures were clearly observed. Figure 1 .
[0073] In one embodiment, the particle size of MO-EVs in the suspension was measured using a nanoparticle tracking analyzer. The results showed that the particle size of MO-EVs was mainly distributed between 50-200 nm, which was consistent with expectations, with fewer impurities and a lower polydispersity index. Figure 2 .
[0074] Example 1: Identification of MO-EVs uptake by human ovarian granulosa cells KGN
[0075] (1) DIL fluorescently labeled MO-EVs: 10 mg DIL dye (Fushen Biotech) and 10 10 The MO-EVs were co-incubated with 10000 MO-EV particles at 37°C in the dark for 30 min to allow MO-EVs to fully combine with DIL; the mixture was centrifuged at 135000 g for 70 min, the supernatant was discarded, the precipitate was removed, and the mixture was resuspended in 1 mL of PBS buffer to obtain DIL-MO-EVs;
[0076] (2) Human ovarian granulosa cells KGN were seeded in a 6-well plate. When the cell confluence reached 50-60%, 10 μL DIL-MO-EVs were added and incubated at 37°C in the dark for 6 h. PBS was washed 2-3 times. 2 mL of basal 1640 medium was added to each well, and 10 μL of 1 mg / mL DAPI was added. The cells were incubated in an incubator for 30-40 min. Fluorescence microscope (Sunny Optical) was used to take pictures at different magnifications, and PBS and DIL incubation were used as negative controls. The results are shown in Figure 3 .
[0077] As can be seen from the figure, DIL-MO-EVs can be taken up by KGN cells and emit red fluorescence of DIL dye inside the cells, while no fluorescence was observed in the PBS control group. This result confirms that MO-EVs can be taken up by KGN cells and enter the cells.
[0078] Example 2: Effect of MO-EVs on KGN cell proliferation
[0079] (1) Grouping: blank control group (NC group); Morinda officinalis-derived nanovesicles group (MO-EVs); Icariin group (Icariin);
[0080] (2) CA determination of protein concentration, and preparation of complete cell culture medium containing MO-EVs at a final concentration of 10 μg / mL;
[0081] (3) KGN cells were plated into 96-well plates at 1000 cells / well and grouped according to the experimental design with 5 replicate wells per group at each time point;
[0082] (4) After 12 h of normal culture, the complete culture medium was used to replace the medium as the blank control group; the culture medium containing 10 μg / mL MO-EVs was used to replace the medium as the MO-EVs group; the culture medium containing 10 μg / mL Icariin was used to replace the medium as the Icariin group;
[0083] (5) Add 10 μL of CCK8 detection solution to each well, incubate at 37°C in the dark for 2 h, and measure the absorbance at 450 nm using an ELISA reader. Record and analyze the cell proliferation curve. The results are shown in Figure 4 .
[0084] As can be seen from the figure, MO-EVs significantly promoted the proliferation of KGN cells, and the effect was more significant than that of the positive control Icariin.
[0085] Example 3: Effects of MO-EVs on KGN cell migration
[0086] (1) Grouping: blank control group (NC group); Morinda officinalis-derived nanovesicles group (MO-EVs); Icariin group (Icariin);
[0087] (2) KGN cells were seeded into the upper chamber of the transwell chamber at a density of 10,000 cells. Serum-free medium was used in the upper chamber and complete medium was used in the lower chamber.
[0088] (3) Add sufficient amount of MO-EVs prepared in serum-free medium to make the volume of the upper chamber reach 300 μL, and make the MO-EVs concentration 10 μg / mL, as the MO-EVs group; add sufficient amount of Icariin prepared in serum-free medium to make the volume of the upper chamber reach 300 μL, and make the Icariin concentration 10 μg / mL, as the Icariin group; use serum-free medium to make up the upper chamber to 300 μL, as the control group;
[0089] (4) After 24 hours of normal culture, the chamber was removed, the upper liquid was removed, 4% paraformaldehyde was added for fixation for 30 minutes, and the cells in the upper chamber were gently removed with cotton fibers. 1% crystal violet staining solution was added for staining for 30 minutes. After staining, PBS was used for decolorization. Microscope photography and statistics were performed; the results are shown in Figure 5 .
[0090] As can be seen from the figure, MO-EVs significantly promoted the migration of KGN cells, and the effect was more significant than that of the positive control Icariin.
[0091] Example 4: Effects of MO-EVs on hormone secretion by KGN cells
[0092] (1) Grouping: blank control group (NC group); Morinda officinalis-derived nanovesicles group (MO-EVs); Icariin group (Icariin);
[0093] (2) KGN cells were plated in 96-well plates at 10,000 cells / well and grouped according to the experimental design with 5 replicate wells per group at each time point;
[0094] (3) After 12 h of normal culture, the complete culture medium was used to replace the medium as the blank control group; the medium was replaced with the medium containing 10 μg / mL MO-EVs as the MO-EVs group; the medium was replaced with the medium containing 10 μg / mL Icariin as the Icariin group;
[0095] (4) After 48 h of continuous culture, the supernatant was collected and the hormone changes in the supernatant of each group were detected using ELISA kits for the corresponding hormones (all kits were from Clone Cloud); the differences between the groups were statistically analyzed; the results are shown in Figure 6 .
[0096] As can be seen from the figure, both MO-EVs and Icariin can promote the secretion of E2, Prog and Inh in KGN cells, and MO-EVs has a more significant promoting effect than the positive control Icariin.
[0097] Example 5: In vivo imaging technology to track the distribution of MO-EVs in animals after intragastric administration
[0098] (1) The incubation of DIL-MO-EVs was the same as in Example 1; PBS and DIL were incubated as negative controls; a solution containing 10 μg / mL DIL-MO-EVs was prepared using PBS;
[0099] (2) SD rats were gavaged with DIL-MO-EVs and PBS-DIL at a volume of 1 mL / kg, and 10% chloral hydrate was used to anesthetize the animals 2 hours later at a volume of 100 μL / kg;
[0100] (3) After the animal is completely anesthetized, place the experimental animal in a small animal live animal imaging device and test it. The results are shown in the table below. Figure 7 .
[0101] As can be seen from the figure, after oral administration, MO-EVs can reach other parts of the body through the digestive tract, and are significantly enriched in the liver and kidneys, and are also enriched in the ovaries.
[0102] Example 6: Ovarian tissue sections were sliced using cryosectioning technology and inverted fluorescence microscopy was used to determine the uptake of MO-EVs by ovarian tissue
[0103] (1) The incubation of DIL-MO-EVs was the same as in Example 1; PBS and DIL were incubated as negative controls; a solution containing 10 μg / mL DIL-MO-EVs was prepared using PBS;
[0104] (2) SD rats were gavaged with DIL-MO-EVs and PBS-DIL at a dose of 1 mL / kg, and the animals were fed normally for 5 consecutive days, and then the experimental animals were killed;
[0105] (3) Rapidly dissect the mouse and remove the target tissues such as ovaries and uterus; cut part of the tissue with tissue scissors, wash with PBS, freeze to -80°C for WB use, soak part of it in 10% neutral formalin for fixation, and soak part of it in 30% sucrose solution. The fixative should be sufficient, generally more than 10 times the volume of the tissue block. The tissue fixed in sucrose solution should be moved to a -80 refrigerator for freezing as soon as possible after fixation for 4 hours;
[0106] (4) Fixing the tissue, freezing it, and then slicing it on a freezing microtome;
[0107] (5) Spread the slices on a glass slide and place them under an upright fluorescence microscope for observation and photography. The results of ovarian slices are shown in Figure 8 .
[0108] As can be seen from the figure, the results of ovarian section clearly show that MO-EVs can reach the ovary after oral administration and be absorbed by the ovarian tissue into the ovarian tissue cells.
[0109] Example 7: Regulatory effect of MO-EVs on POF animal model induced by cyclophosphamide
[0110] (1) Cyclophosphamide modeling SD rats: The animals were intraperitoneally injected with 0.6 mg / mL cyclophosphamide (CTX) PBS solution on day 1, and 0.8 mg / mL cyclophosphamide PBS solution on days 2-15, with an injection volume of 10 mL / kg body weight. The normal control group was injected with an equal volume of sodium chloride injection, once a day for a total of 15 days.
[0111] (2) Intervention groups: ① Normal control group (NC): the control group was fed normally; ② Cyclophosphamide group (CTX): after successful modeling, the mice were fed normally; ③ Cyclophosphamide + MO-EVs experimental group (CTX + MO-EVs): after successful modeling, MO-EVs were intragastrically administered at a volume of 1 mL / kg, and a solution containing 10 μg / mL MO-EVs was prepared using PBS; ④ Cyclophosphamide + Icariin experimental group (CTX + Icariin): after successful modeling, Icariin was intragastrically administered at a volume of 1 mL / kg, and a solution containing 10 μg / mL Icariin was prepared using PBS;
[0112] (3) Drug administration: On the third day after modeling, animals were intraperitoneally injected according to the group gavage dose, once every 3 days, for a total of 7 injections;
[0113] (4) The experiment was terminated on the second day after the last administration, and all mice were weighed. Blood collection from mouse orbits: The mice to be tested were anesthetized with ether, and blood was collected from below the inner orbits of the mice. The centrifuge tubes containing the blood were left to stand at room temperature for half an hour until the blood showed obvious stratification, and then centrifuged at high speed for 30 minutes. After the centrifugation, the upper serum was aspirated and placed in a newly coded centrifuge tube and stored in a -80°C refrigerator. After blood collection, the animals were killed by cervical dislocation, and the bilateral ovaries and uterus were fixed in 4% paraformaldehyde solution overnight. The remaining half of each organ was frozen at -80°C for subsequent experimental use.
[0114] (5) ELISA was used to detect the concentrations of estradiol, inhibin, and progesterone in the blood of each group of animals;
[0115] (6) Compare the morphology of the ovaries and the differences in HE inflammation among the groups;
[0116] The results are as follows Figure 9-11 As shown. Fig. 9 It can be seen that the ovaries of the animals in the cyclophosphamide group became elongated and shrunken in overall shape; while the condition of the ovaries in the MO-EVs group of the present invention was significantly improved compared with that in the cyclophosphamide group. Fig.10It can be seen that CTX has a damaging effect on ovarian tissue, which also suggests that the construction of the premature ovarian failure model was successful, and this inhibitory effect can be reversed by MO-EVs and Icarlin, and the effect of MO-EVs is better than that of Icarlin. Fig.11 It can be seen that cyclophosphamide can inhibit the secretion of estradiol and inhibin in animals, which also suggests that the premature ovarian failure model was successfully constructed. This inhibitory effect can be reversed by MO-EVs and Icarlin, and MO-EVs has a significantly better effect than the positive control group Icarlin.
[0117] The present invention obtains Morinda officinalis-derived nanovesicles by isolating them from Morinda officinalis. The nanovesicles have natural ingredients, no toxic side effects, good biocompatibility and safety, and are proven to have a strong effect of protecting ovarian cells and reversing POF caused by cyclophosphamide through in vivo and in vitro experiments, indicating that the nanovesicles can be used to prevent and / or treat premature ovarian failure, providing a new theoretical basis for the application and prevention of Morinda officinalis in POF, and therefore have broad application prospects.
[0118] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. Application of nanovesicles derived from Morinda officinalis in the preparation of drugs for preventing and / or treating premature ovarian failure.
2. Application of nanovesicles derived from Morinda officinalis in the preparation of drugs that promote ovarian cell proliferation.
3. Application of nanovesicles derived from Morinda officinalis in the preparation of drugs that promote ovarian cell migration.
4. The use according to any one of claims 1 to 3, characterized in that: The medicine comprises a therapeutically effective amount of nanovesicles derived from Morinda officinalis.
5. The use according to any one of claims 1 to 3, characterized in that: The medicine is prepared into various pharmaceutical dosage forms by conventional methods, and these dosage forms include: tablets, capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, oral dosage forms of drops and injections.
6. The use according to any one of claims 1 to 3, characterized in that: The drug is prepared into various pharmaceutical dosage forms by conventional methods, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, pills, suspensions, wine preparations, tinctures, oral dosage forms of drops and injections.
7. The use according to any one of claims 1 to 3, characterized in that: The medicine also contains one or more pharmaceutically acceptable carriers or excipients.
8. The use according to claim 7, characterized in that: The carrier or auxiliary material includes at least one of a diluent, a wetting agent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, a disintegrant, and a preservative.
9. The use according to any one of claims 1 to 3, characterized in that: The Morinda officinalis-derived nanovesicles are separated from Morinda officinalis.
10. The use according to any one of claims 1 to 3, characterized in that: The Morinda officinalis-derived nanocapsule is obtained by crushing or breaking the wall of Morinda officinalis and then adding an extractant for extraction and separation.