Use of vitamin-magnesium complex in preparation of medicine for preventing and treating miscarriage during pregnancy and early pregnancy period and preparation thereof

Abstract

The application provides application of a vitamin-magnesium compound in preparation of a medicine for preventing and treating miscarriage in a pregnant period and an early pregnant period and a preparation, relates to the technical field of biological medicines, and the vitamin-magnesium compound is a magnesium-based coordination chelate with a repairing effect on endometrial tissue. The vitamin-magnesium compound is an organic ligand of the magnesium-based coordination chelate, and the organic matter is any one of vitamin B13, vitamin B3 and vitamin C. The magnesium-based coordination chelate acts on endometrial stromal cells, promotes cell proliferation, and promotes normal implantation of an embryo. The magnesium-based coordination chelate acts on endometrial tissue vascular endothelial cells, promotes cell proliferation, and promotes angiogenesis to meet the nutritional supply of the embryo. The magnesium-based coordination chelate acts on endometrial tissue macrophages, M1 type macrophages are polarized into M2 type macrophages, immune tolerance to the embryo is promoted, and the normal growth and development of the embryo are prevented from being affected by the attack of maternal immune cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application and formulation of vitamin-magnesium complex in the preparation of drugs for preventing miscarriage during the preconception period and early pregnancy. Background Technology

[0002] Miscarriage is one of the most common and challenging adverse pregnancy outcomes for women, causing harm to both their physical and mental health. Physically, it can lead to bleeding, infection, thinning of the uterine lining, and intrauterine adhesions, and may also affect fertility, increasing the risk of infertility and ectopic pregnancy. Psychologically, it can trigger negative emotions such as sadness, guilt, anxiety, and depression, impacting daily life and mental well-being.

[0003] Clinically, the main treatments for miscarriage are still hormones, immunosuppressants, and magnesium sulfate. However, high-dose or long-term use of hormones can lead to metabolic disorders and osteoporosis. Although immunotherapy has potential benefits, it is accompanied by adverse reactions such as bleeding, nausea, and infection, and there is a lack of sufficient clinical trial data to support its safety and efficacy. Magnesium sulfate antagonizes the effect of calcium ions on uterine contractions, inhibiting uterine contractions to prevent embryonic abortion. It is often used for threatened miscarriage, preeclampsia, and eclampsia, but in clinical obstetrics and gynecology treatment, blood magnesium levels must be strictly monitored to avoid side effects such as maternal pain, palpitations, dizziness, and nausea. Therefore, in addition to the significant challenges faced in the clinical diagnosis and treatment of miscarriage during pregnancy, prevention of miscarriage during the preconception period and early pregnancy presents even greater difficulties: ① Target gap—Existing drugs all target "inhibition of uterine contractions" as their endpoint, acting on uterine smooth muscle (magnesium sulfate) or the systemic immune-endocrine axis (hormones, immunosuppressants), but neglecting the fact that the "endometrial microenvironment" is the "first gateway" that determines whether the embryo can implant and continue to develop. Therefore, there is a lack of clearly defined target tissues for intervention during the preconception period and the implantation window; ② Dosage form gap—Magnesium sulfate must be administered intravenously and monitored in real time. Blood magnesium concentration and the instantaneous release of magnesium ions cannot reach the endometrial tissue locally and are prone to causing systemic toxicity; hormones and immunosuppressants have dose-limiting toxicity such as metabolic disorders and infection risks due to systemic exposure, and there are currently no endometrial microenvironment repair agents that can achieve "local-slow-release-multi-cell synergy"; ③ Mechanism gap - existing technologies have not been able to solve the three major pathological links of "endometrial stromal cell damage, immune tolerance imbalance, and angiogenesis defects" at the same time, and there is a lack of a comprehensive solution that can be intervened in the preconception period to reduce the incidence of miscarriage from the source. Summary of the Invention

[0004] The present invention provides the application and formulation of vitamin-magnesium complex in the preparation of drugs for preventing miscarriage during the preconception period and early pregnancy, so as to at least partially solve the problems existing in the prior art.

[0005] The first aspect of this invention provides the application of a vitamin-magnesium complex in the preparation of a drug for preventing miscarriage during the preconception and early pregnancy periods. The vitamin-magnesium complex is a magnesium-based coordination chelate with a repairing effect on endometrial tissue. The vitamin-magnesium complex uses an organic ligand, such as vitamin B13, vitamin B3, or vitamin C, as the ligand. The magnesium-based coordination chelate acts on endometrial stromal cells, promoting cell proliferation to facilitate normal embryo implantation. It also acts on vascular endothelial cells of the endometrial tissue, promoting cell proliferation to promote angiogenesis and meet the nutritional needs of the embryo. Furthermore, the magnesium-based coordination chelate acts on macrophages of the endometrial tissue, polarizing M1-type macrophages to M2-type macrophages, promoting immune tolerance to the embryo, and preventing the embryo from being attacked by maternal immune cells, thus affecting normal growth and development.

[0006] Optionally, the magnesium-based coordination chelate decomposes and releases divalent magnesium ions in the endometrial tissue microenvironment. The magnesium ions promote the antioxidant stress of endometrial stromal cells, reduce the level of reactive oxygen species in cells, promote mitochondrial metabolism, and promote cell proliferation.

[0007] Optionally, the vitamin-magnesium complex is a vitamin B13-magnesium chelate, wherein the magnesium-based coordination chelate decomposes in the endometrial tissue microenvironment to release free vitamin B13 monomers, wherein the vitamin B13 monomers are biocompatible and used to promote the proliferation of endometrial stromal cells.

[0008] Optionally, the concentration range of the vitamin-magnesium complex is greater than 1 μg / mL. -1 Less than 100 μg / mL -1 .

[0009] Optionally, the release rate of the magnesium-based coordination chelate can be controlled by pH response or temperature change, or the release rate of the magnesium-based coordination chelate in the vitamin-magnesium complex formulation can be controlled by controlling the degradation rate of the vitamin-magnesium complex itself.

[0010] A second aspect of this invention provides a vitamin-magnesium complex formulation, which is used to prepare a drug for preventing miscarriage during the preconception period and early pregnancy. The vitamin-magnesium complex formulation is composed of a magnesium-based coordination chelate and an organic carrier. The magnesium-based coordination chelate is synthesized by chelating a magnesium salt compound and an organic ligand. The carrier is any one or more of agarose, chitosan, gelatin, thermosensitive hydrogel, sodium alginate, polyethylene glycol, polylactic acid, liposomes, and acrylic acid.

[0011] Optionally, the magnesium compound includes any one of magnesium chloride, magnesium nitrate, magnesium acetate, magnesium sulfate, and their hydrates.

[0012] Optionally, the organic ligand includes any one of vitamin B13, vitamin B3, and vitamin C.

[0013] Optionally, the synthesis methods of the magnesium-based coordination chelate include solvothermal, liquid-phase synthesis, solid-phase synthesis, ultrasonic-assisted, high-temperature-assisted, surfactant-assisted, and microwave oven-assisted methods.

[0014] Optionally, the vitamin-magnesium complex preparation is administered after the formation of endometrial tissue, or in early pregnancy after the embryo invades and implants in the endometrium; the administration methods of the vitamin-magnesium complex preparation include minimally invasive abdominal intrauterine administration, minimally invasive back intrauterine administration, intravenous injection, and intrauterine perfusion administration.

[0015] In this embodiment of the invention, the vitamin-magnesium complex is used in the preparation of drugs for preventing miscarriage during the preconception period and early pregnancy. The vitamin-magnesium complex promotes the proliferation of endometrial stromal cells, promotes angiogenesis and immune regulation, ensures normal fetal growth and development, and prevents miscarriage.

[0016] In this embodiment of the invention, the magnesium element in the magnesium-based coordination chelate is an essential element for human life and is also the effective component of MgSO4, a clinical drug for the treatment of threatened miscarriage. In addition to playing a role in preventing and treating miscarriage, it can play a vital protective role in the development of the fetal nervous system.

[0017] The vitamin-magnesium complex used in this embodiment of the invention employs vitamin B13, an organic ligand of a magnesium-based coordination chelate. Vitamin B13 is a human-friendly organic compound with good tissue compatibility, plays a vital role in normal life functions, and can enhance the effect of magnesium-based chelates. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram illustrating the action of a vitamin-magnesium complex on endometrial tissue to prevent miscarriage, as provided in the embodiments of this application.

[0020] Figure 2 This is a schematic diagram and result diagram of the synthesis of a vitamin-magnesium complex provided in the embodiments of this application;

[0021] Figure 3This is a diagram showing the cell compatibility results of a vitamin-magnesium complex provided in the embodiments of this application;

[0022] Figure 4 This is a graph showing the magnesium ion release results of a vitamin-magnesium complex provided in the embodiments of this application;

[0023] Figure 5 This is a diagram showing the results of a vitamin-magnesium complex in resisting oxidative stress and repairing mitochondrial damage, as provided in the embodiments of this application.

[0024] Figure 6 This is a schematic diagram illustrating how a vitamin-magnesium complex promotes endometrial cell proliferation, as provided in an embodiment of this application.

[0025] Figure 7 This is a schematic diagram illustrating the therapeutic effect of a vitamin-magnesium complex provided in this application embodiment on an animal. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0028] The endometrial tissue is a key regulatory hub for pregnancy outcomes, playing a crucial role in supporting embryo implantation, establishing the maternal-fetal interface, and maintaining pregnancy. After fertilization, the endometrial microenvironment transforms into endometrial tissue under the influence of progesterone. Damage to endometrial stromal cells, immune imbalance, and angiogenesis defects can all lead to an imbalance in the endometrial microenvironment, which is an important mechanism for adverse pregnancy outcomes such as implantation failure, expulsion, and even recurrent miscarriage. For example, oxidative stress damages endometrial stromal cells, reducing the receptivity of the endometrial tissue to the embryo and causing expulsion. Damage to vascular endothelial cells leads to impaired and defective angiogenesis in the endometrial tissue, resulting in insufficient nutrient supply and inducing miscarriage. An increase in M1 macrophages and Th1 cells and enhanced toxicity of dNK cells in the microenvironment leads to an imbalance in immune tolerance, causing the embryo to be attacked by the maternal immune system and resulting in developmental arrest. However, current research still needs to explore the application of synergistic repair of endometrial tissue from multiple aspects, including stromal cells, immunity, and blood vessels, in the prevention of miscarriage during the preconception and early pregnancy periods, as well as vitamin-magnesium complex preparations and methods.

[0029] Therefore, this invention proposes a vitamin-magnesium complex for synergistic repair of endometrial tissue, including promoting the proliferation of stromal cells during the preconception period to improve uterine receptivity to the embryo, regulating early pregnancy immune tolerance to prevent fetal attack by the maternal immune system, and promoting early pregnancy angiogenesis to ensure adequate nutrient supply to the fetus. It is a key regulatory hub for improving pregnancy outcomes. The vitamin-magnesium complex differs from currently used magnesium sulfate injections in terms of drug composition, mechanism of action, and duration of action, and neither has been publicly reported domestically or internationally. The complex is self-assembled from vitamins and magnesium ions, offering the advantage of sustained-release magnesium ions, avoiding the side effects of hypermagnesemia associated with magnesium sulfate in clinical practice. The sustained-release free vitamins further reduce this effect. In this invention, the vitamin-magnesium complex targets uterine stromal cells, immune cells, and vascular cells, thereby preventing miscarriage during the preconception and early pregnancy periods, unlike magnesium sulfate injections which act on uterine smooth muscle cells to treat threatened miscarriage, preeclampsia, and eclampsia. Furthermore, this invention, for the first time, moves the window for miscarriage prevention forward to the "preconception period - implantation period," fundamentally overcoming the limitation of existing technologies that can only passively handle "threatened miscarriage" or "eclampsia."

[0030] The following specific embodiments illustrate the research and exploration process of the embodiments of the present invention:

[0031] Specifically, such as Figure 1 As shown, embodiments of the present invention propose that the vitamin-magnesium complex (VB13-Mg) releases Mg 2+ It activates cell signaling pathways, promotes mitochondrial metabolism and anti-oxidative stress to regulate uterine decidual stromal cells to maintain the receptivity of decidual tissue to the embryo, promotes M1 macrophage polarization to M2 type to mediate embryonic immune tolerance, promotes angiogenesis to increase embryonic nutrient supply, and synergistically remodels the microenvironment of uterine decidual tissue to ensure normal fetal development and improve pregnancy outcomes of recurrent miscarriage.

[0032] An imbalance in the uterine microenvironment, including stromal cell damage, immune cell imbalance, and impaired angiogenesis and remodeling, collectively leads to miscarriage. In this invention, based on the biological effects of Mg, including promoting angiogenesis, cell proliferation, and polarization of M1 macrophages to M2 macrophages, and the natural cell compatibility of VB13, a VB13-Mg coordination chelate was synthesized. VB13-Mg releases Mg... 2+ By activating the Notch signaling pathway within vascular endothelial cells, cell proliferation is promoted, thereby regulating angiogenesis and remodeling, thus ensuring the nutrient supply to the embryo. In this embodiment of the invention, VB13-Mg releases Mg... 2+ It inhibits the death of endometrial stromal cells due to oxidative stress by activating the Nrf2 antioxidant pathway.

[0033] In addition, Mg 2+After being transported into the cell via the MagT1 protein on the stromal cell membrane, it can enter the mitochondria via the MrS2 protein on the mitochondrial membrane to regulate energy metabolism, thereby providing sufficient energy. At the same time, it activates the MAPK proliferation signaling pathway, synergistically promoting stromal cell proliferation and improving the uterus's receptivity to the embryo.

[0034] In addition, Mg 2+ It can promote the polarization of M1 macrophages into M2 macrophages. M2 macrophages regulate CD4+ T cell differentiation and mediate Treg / Th2 cell homeostasis by secreting IL4I1. M2 macrophages inhibit the toxicity of dNK cells by secreting IL-10, reduce the attack of the maternal immune system on the embryo, reshape the immune tolerance homeostasis at the maternal-fetal interface, promote the healthy growth and development of the embryo in the uterus, and prevent miscarriage.

[0035] Example 1: Synthesis of VB13-Mg coordination chelate

[0036] In this embodiment of the invention, raw materials for purchasing or synthesizing VB13-Mg coordination chelates are discussed, and different solvent systems, reaction temperatures, reaction times, reaction concentrations, and reaction ratios in the synthesis reaction are explored.

[0037] The magnesium salts and organic central ligands used in the synthesis of VB13-Mg coordination chelates can be purchased from companies such as Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai McLean Biochemical Technology Co., Ltd., Merck Co., Ltd., or other companies qualified to synthesize VB13-Mg coordination chelates.

[0038] The chemical methods for synthesizing VB13-Mg coordination chelates include: coprecipitation, microwave oven-assisted synthesis, liquid-phase synthesis, solvothermal synthesis, ultrasonic-assisted synthesis, solid-phase synthesis, surfactant synthesis, and high-temperature assisted synthesis. In this invention, the solvothermal method is preferred for synthesizing VB13-Mg coordination chelates, and the parameter ranges used are as follows:

[0039] The temperature range is 30℃ to 120℃; the solvent system includes water reaction system, DMF (N,N-dimethylformamide) reaction system, organic alcohol reaction system, DMSO (dimethyl sulfoxide) reaction system, organic ether reaction system, etc., and their mixtures; the reaction time is 0.5h to 48h; the purification system includes water reaction system, DMF (N,N-dimethylformamide) reaction system, organic alcohol reaction system, DMSO (dimethyl sulfoxide) reaction system, organic ether reaction system, etc., and their mixtures; the freeze-drying method includes freeze-drying, natural evaporation, and baking. The magnesium salt raw materials of the VB13-Mg coordination chelate include magnesium chloride, magnesium sulfate, magnesium acetate, etc.; the VB13-Mg coordination chelate includes mono- or multi-metal magnesium-based chelates, single-component or multi-component metal compound magnesium-based chelates; the central ligand of the magnesium-based chelate includes natural organic materials and artificially synthesized organic materials, such as vitamin B3, vitamin B13, tannic acid, etc.; the constituent elements of the magnesium-based complex are all essential elements for human life.

[0040] In this invention, VB13-Mg coordination chelates are prepared by a co-precipitation method, specifically as follows: Figure 2 As shown in Part A, 0.33 g of vitamin B13 and 0.41 g of magnesium nitrate were placed in separate reaction glass flasks, and solvent systems (a solvent reaction system prepared by mixing DMF and water in a 1:1 volume ratio, and a solvent reaction system prepared by mixing anhydrous ethanol and water in a 1:1 volume ratio) were added. The mixture was heated to 90°C until completely dissolved. The two solutions were then mixed and reacted at 90°C for 30 min to obtain a brown precipitate. Figure 2 (Part B). Then, after centrifugation at 5000 rpm for 10 minutes, a brown precipitate was obtained. The precipitate was washed with water, DMF, and a mixed solution of water and DMF, followed by centrifugation, repeating the washing process three times. The precipitate was then placed in a clean freeze-drying solution to obtain the sample. The VB13-Mg coordination chelate was dispersed in pure water at a concentration of 200 μg / mL. -1 After mixing, 3 µL was dropped onto a 200-mesh copper grid used for transmission electron microscopy (TEM). The copper grid was placed between two pieces of polydimethylsiloxane (PDMS), with the bottom center of the grid suspended to allow solvent evaporation. The grid was then placed in a plastic petri dish and allowed to dry naturally overnight. Finally, the particle morphology and size of the VB13-Mg coordination chelate were observed under a TEM. Figure 2 As shown in section C, the VB13-Mg coordination chelate is a rhombic polyhedral single crystal with a size of 1-3 μm.

[0041] In summary, the co-precipitation method of this invention yields VB13-Mg coordination chelates with good dispersibility and uniform size.

[0042] Example 2: Cell compatibility of VB13-Mg coordination chelate

[0043] CCK8 reagent and a double staining reagent for dead and live cells were used to stain different concentrations of VB13-Mg coordination chelates (0.31 μg / mL). -1 0.63 μg mL -1 The cytocompatibility of human endometrial stromal cells (HESCs) was assessed at 0h, 4h, 8h, 12h, and 24h. HESCs are the main fibroblast-like cells in the lamina propria of the endometrium, playing a crucial role in the menstrual cycle and pregnancy. They respond to hormonal stimulation (such as estrogen and progesterone), undergoing decidualization and differentiating into secretory decidual cells, preparing for embryo implantation and placental development. HESCs can mimic the physiological changes of the endometrial stroma under hormonal influence, making them suitable for assessing the effects of VB13-Mg coordination chelates on endometrial cell function. Due to their responsiveness to hormones and ability to mimic endometrial function, HESC cell lines have become an important tool for assessing the biocompatibility of VB13-Mg coordination chelates. Therefore, as... Figure 3 As shown in Part A, 0.31 μg mL - 1 The VB13-Mg coordination chelate showed the following cell viability increases in HESCs at 0h, 4h, 8h, 12h, and 24h: 101.74±2.47%, 105.10±6.76%, 111.91±14.89%, 123.21±21.66%, and 123.25±19.84%, respectively. This indicates that 0.31 μg / mL... - 1 VB13-Mg coordination chelates can promote the proliferation of endometrial stromal cells and increase the receptivity of the endometrium to the embryo during pregnancy, thus preventing miscarriage. Figure 3 As shown in Part B, exposure to 0.63 μg mL -1 In the VB13-Mg coordination chelate, HESC cells showed green fluorescence at 0h, 4h, 8h, 12h, and 24h. In the double staining of dead and live cells, green fluorescence represents live cells, and red fluorescence represents dead cells. Therefore, 0.63 μg mL -1 The VB13-Mg coordination chelate still exhibits good biocompatibility with HESC cells.

[0044] In summary, VB13-Mg coordination chelates have good biocompatibility with HESC cells and can promote the proliferation of endometrial stromal cells. During pregnancy, they can increase the receptivity of the endometrium to the embryo and prevent miscarriage.

[0045] Example 3: Release of magnesium ions and free VB13 from VB13-Mg coordination chelates

[0046] Gene sequencing-RNA transcriptome analysis, gas chromatography-mass spectrometry (GCMS), liquid chromatography-mass spectrometry (LCMS), gas chromatography-liquid chromatography-liquid chromatography (GCLC), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), secondary ion mass spectrometry (SIMS)-cell metabolomics analysis, high performance liquid chromatography (HPLC-UV), qPCR analysis of the relative expression levels of relevant genes, and Western blotting analysis of copper ion release and the expression of related genes and proteins of copper ion transporters are provided. For the results of this example, please refer to [link to relevant documentation]. Figure 4 , Figure 4 This image shows the release results of magnesium ions and free VB13 in simulated uterine cavity fluid, as provided in an embodiment of the present invention. Simulated uterine cavity fluid is an artificially prepared in vitro solution designed to accurately replicate the chemical and physical microenvironment within the human uterine cavity. It uses a basic salt solution as its core, containing key ions and energy substrates such as sodium, potassium, calcium, phosphate, bicarbonate, and glucose. A buffer system stabilizes the pH at 7.2–7.4, controls the osmotic pressure at 280–300 mOsm / kg, and maintains a constant temperature of 37 °C, thus closely mimicking the composition of uterine cavity fluid under physiological conditions. To more closely approximate the uterine cavity environment during the actual secretory period or early pregnancy, researchers can supplement the basic formula with human serum albumin, urea, lactic acid, hormones, or antimicrobial peptides to simulate fluctuations in proteins, hormones, and metabolites that may occur at different stages of the menstrual cycle or under pathological conditions. Therefore, as... Figure 4 As shown in Part A, 0.31 μg mL -1 The magnesium ion concentration released by the VB13-Mg coordination chelate in simulated intrauterine fluid at 0h, 4h, 8h, 12h, and 24h was 0.0034 ± 0.000005 μg / L. -1 0.59±0.096 μg L -1 0.94±0.067 μg L -1 1.20±0.13 μg mL -1 1.39±0.11 μg L -1 This indicates that magnesium ions are released rapidly at first and then steadily. These concentrations of magnesium ions can promote the proliferation of HESC cells and increase the receptivity of the endometrium to the embryo, thus preventing miscarriage. Figure 4 As shown in Part B, 0.31 μg mL -1The concentrations of free VB13 released by the VB13-Mg coordination chelate in simulated intrauterine fluid at 0h, 4h, 8h, 12h, and 24h were 0.026±0.0060%, 0.040±0.0082%, 0.074±0.011%, 0.11±0.025%, and 0.098±0.025%, respectively. This indicates that the release of free VB13 initially increases and then decreases. The decrease may be due to excessive magnesium ions re-aggregating with VB13 to form a complex. VB13 has natural biocompatibility with the body, tissues, and cells, and is fundamental to maintaining life activities. VB13 is a key intermediate in the de novo synthesis pathway of pyrimidine nucleotides. For cells, it is both the "brick and tile transport vehicle" of nucleic acid metabolism and the "signal light" for energy metabolism and cell fate determination. In mammalian cells, orotic acid is condensed with 5-phosphoribose-1-pyrophosphate (PRPP) by orotic acid phosphoribosyltransferase (OPRT) to form orotic nucleotide (OMP), which is then decarboxylated to obtain uridine monophosphate (UMP). UMP can be further phosphorylated to generate UTP and CTP, providing substrates for the synthesis of RNA, DNA, glycogen and phospholipids.

[0047] Example 4: VB13-Mg coordination chelate's antioxidant effect on oxidative stress and repair of mitochondrial damage

[0048] Studies have shown that excessive reactive oxygen species (ROS) can lead to oxidative stress in endometrial stromal cells, resulting in cell damage and even death, and impairing endometrial function. Therefore, by eliminating the dose-response curve of ROS as a signaling molecule, and precisely regulating the redox network, mitochondrial homeostasis, and immune metabolism, spatiotemporally specific regulators targeting Nrf2 or mitochondrial ROS may become a novel strategy for restoring tissue cell function and preventing chronic diseases. Firstly, the antioxidant stress function of VB13-Mg coordination chelates was explored. ROS inducer Rosup and 0.01 μg / mL VB13 Mg-MOF were added to the cell culture medium to treat cells. Green fluorescence in cells represents the level of ROS. DCHF-DA (2',7'-Dichlorodihydrofluorescein diacetate) is a classic fluorescent probe for detecting reactive oxygen species (ROS) in cell biology. Its ingenious design combines lipid solubility, esterase hydrolysis, and an oxidation-sensitive green fluorescence generation mechanism, allowing for real-time, in-situ, and semi-quantitative reflection of ROS levels in living cells. DCHF-DA utilizes a cascade mechanism of "membrane permeation-esterase hydrolysis-ROS oxidation" to convert invisible free radicals into quantifiable green fluorescence, providing a direct tool for studying oxidative stress, drug toxicology, signal transduction, and aging. Its advantages lie in its simplicity and compatibility with live-cell dynamic monitoring. Therefore, such as Figure 5As shown in Part A, treatment with the endometrial stromal cell ROS inducer Rosup resulted in an increasing trend of ROS in the cells at 4 h, followed by a decreasing trend from 8 to 24 h, indicating that the VB13-Mg coordination chelate can reduce the intracellular ROS level in endometrial cells. Under the detection of the JC-1 probe, red fluorescence represents normal mitochondrial membrane potential, while green fluorescence represents decreased mitochondrial membrane potential and mitochondrial damage. The JC-1 probe is considered the gold standard for detecting mitochondrial membrane potential (ΔΨm) because it transforms this abstract electrochemical quantity into a visible and proportional color change. The entire process requires no external calibration voltage, relying solely on the spontaneous aggregation and deaggregation of molecules. The chemical backbone of JC-1 is a positively charged cyanine dye with a small molecular weight and high lipid solubility, allowing it to freely penetrate the lipid bilayer along the negative potential of the outer side of the inner mitochondrial membrane. ΔΨm in normal cells is approximately -180 mV; such a strong electrochemical gradient is sufficient to drive a large amount of JC-1 into the mitochondrial matrix in monomeric form. Due to the narrow matrix space and the extremely high energy provided by the membrane potential, the incoming JC-1 monomers rapidly accumulate. Once the concentration exceeds the threshold, J-type π-π accumulation occurs, forming a red fluorescent complex called a J-mer. The emission spectrum of the mer shows a significant redshift to 590 nm, and the color changes from the green (529 nm) of the monomeric state to orange-red. This color change is discernible to the naked eye, allowing differentiation between normal and abnormal mitochondrial membrane potentials under a fluorescence microscope. Therefore, as shown in section B of Figure 5, the mitochondrial membrane potential is significantly reduced at 4 h due to the influence of ROS, and gradually returns to normal between 8 and 24 h, indicating that the VB13-Mg coordination chelate can promote mitochondrial metabolism to enhance cell viability.

[0049] In summary, VB13-Mg coordination chelates can reduce the abnormally high ROS levels in endometrial stromal cells, restore abnormal mitochondrial membrane potential, reduce the risk of mitochondrial damage, and synergistically promote and enhance the survival of endometrial cells.

[0050] Example 5: VB13-Mg coordination chelate promotes endometrial cell proliferation and migration

[0051] Endometrial cell proliferation is a highly coordinated physiological process finely regulated by hormones and the microenvironment, playing a central role in menstrual regeneration, embryo implantation, maintenance of tissue homeostasis, and damage repair. During menstruation, the functional layer of the endometrium sheds, leaving only the basal layer. Under estrogen stimulation, the epithelial and stromal cells in the basal layer rapidly enter the proliferative phase, restoring the endometrial thickness from less than 1 mm to 5–6 mm within 7–10 days, preparing for the next pregnancy. This process relies on the upregulation of cyclin D1 and CDK4 / 6, driving the G1 / S phase transition and achieving a 4–5 fold increase in cell number, preventing uterine cavity exposure, adhesions, or infertility. During the proliferative phase, cells not only increase in number but also secrete matrix molecules such as laminin, fibronectin, and type IV collagen, forming a loose, vascularized framework that provides space for embryonic invasion. If proliferation is insufficient, the embryo implantation rate is <10% when the endometrial thickness is <7 mm; however, with normal proliferation and an endometrial thickness ≥8 mm, the implantation rate recovers to 30–40%. Furthermore, proliferating cells highly express homeobox genes HOXA10 and VEGF, promoting glandular proliferation and angiogenesis, and enhancing endometrial receptivity. During the proliferative phase, endometrial cells recruit endothelial progenitor cells via the HIF-1α / VEGF axis, forming spiral arteries. Newly formed microvessels dilate during the secretory phase, establishing early maternal-fetal circulation and ensuring nutrient supply to the embryo. Animal experiments show that inhibiting VEGFR2 can reduce vascular density by 60%, with embryo resorption rates as high as 85%, suggesting that angiogenesis is a key benefit of proliferation. Therefore, endometrial cell proliferation is not merely an increase in number, but rather a process driven by the cell cycle, angiogenesis, cell renewal, and microenvironment remodeling, constructing a dynamic, nutrient- and immune-friendly microenvironment that provides "golden soil" for embryo implantation and maintains long-term uterine health and fertility. Therefore, as... Figure 6 As shown, compared with the control group, the VB13-Mg coordination chelate significantly accelerated the migration rate of endometrial cells in the same time period, indicating that the VB13-Mg coordination chelate has a good cell migration promotion function.

[0052] Example 6: Efficacy of VB13-Mg coordination chelate in the treatment of recurrent miscarriage

[0053] In this embodiment of the invention, based on existing research, an animal model of recurrent miscarriage caused by endometrial damage was established using SD rats. In this embodiment, magnesium-based coordination chelates were used as the carrier material for magnesium ions, primary endometrial stromal cells, vascular endothelial cells, and macrophages were used as model cells, and a rat pregnancy model was used as the animal model. The animal model exhibited characteristics of endometrial tissue damage, including endometrial stromal cell damage, impaired angiogenesis, and immune regulation.

[0054] Animal model illustration, such as Figure 7As shown in Section A, on the first day of pregnancy in SD rats, 95% alcohol was injected into the upper left uterus to induce pregnancy. The image shows the actual animal receiving the medication. The upper uterus was chosen as the injection site to avoid alcohol affecting the fertilized egg in the upper part of the uterus, as alcohol can cause endometrial tissue damage after remaining in the uterine cavity for 3 minutes. Figure 7 As shown in Part B, the fur on the dorsal and ventral areas of the female rat was shaved clean with a razor to facilitate the surgery. Any remaining hair was wiped away with alcohol and iodine, and the surgical area was disinfected. Using surgical scissors, a 2 cm incision was made in the surgical area to the left of the midline of the rat's dorsal and ventral region. Subcutaneous tissue, fat, and muscle were then dissected until the abdominal cavity was exposed. The rat's uterus is typically buried under a thick layer of fat; by manipulating the fat with forceps, the tubular uterus was exposed. 25 μL of the appropriate solution was injected into the uterus, approximately one-third of the way from the ovary, using an insulin syringe. The small orifice of the insulin syringe prevented extensive or severe damage to the rat's uterus and also prevented solution leakage. This was performed during the first pregnancy, inducing damage to the decidual tissue of the uterus in early pregnancy as follows... Figure 7 As shown in section C, compared to the decidual tissue on the right side that provides normal embryo implantation, the left uterus experienced abnormal decidualization and swelling after alcohol treatment, leading to miscarriage. In a second pregnancy, induced damage to the uterine decidual tissue in early pregnancy was observed... Figure 7 As shown in section D, compared to the right side which provides normal embryo implantation with uterine decidual tissue, the left uterus, after alcohol treatment, exhibited abnormal decidualization and swelling, leading to miscarriage. Two miscarriages satisfy the clinical diagnostic criteria for recurrent miscarriage, and the abnormal decidualization and swelling of the left uterus in rats satisfies the study of microenvironmental imbalance in uterine decidual tissue remodeling. In a third pregnancy, after 95% alcohol was left in the uterus for 2 minutes to induce decidualization and damage to the left uterine tissue, treatment was administered with an injection of a VB13-Mg coordination chelate. Figure 7 As shown in section E, the embryos in the left uterus of rats were able to develop normally after treatment with the VB13-Mg coordination chelate, indicating that the VB13-Mg coordination chelate can improve the pregnancy outcome of rats with decidual tissue damage.

[0055] Based on the research and exploration of the above embodiments, this invention proposes the application of a vitamin-magnesium complex in the preparation of drugs for preventing miscarriage during the preconception period and early pregnancy. The vitamin-magnesium complex is a magnesium-based coordination chelate with a repairing effect on endometrial tissue. The vitamin-magnesium complex uses an organic ligand, such as vitamin B13, vitamin B3, or vitamin C, as the magnesium-based coordination chelate. The magnesium-based coordination chelate acts on endometrial stromal cells, promoting cell proliferation to facilitate normal embryo implantation. It also acts on vascular endothelial cells of the endometrial tissue, promoting cell proliferation to promote angiogenesis and meet the nutritional needs of the embryo. Furthermore, the magnesium-based coordination chelate acts on macrophages of the endometrial tissue, polarizing M1-type macrophages to M2-type macrophages, promoting immune tolerance to the embryo, and preventing the embryo from being attacked by maternal immune cells, thus affecting normal growth and development.

[0056] In this embodiment of the invention, the magnesium-based coordination chelate releases magnesium ions to promote the proliferation of endometrial stromal cells, enhance antioxidant stress and mitochondrial metabolism, and meet the conditions for embryo implantation. The magnesium ions released by the magnesium-based coordination chelate promote the proliferation of vascular endothelial cells in endometrial tissue, thereby promoting angiogenesis and providing more sufficient nutrition for embryonic growth. The magnesium ions released by the magnesium-based coordination chelate also polarize M1 macrophages to M2 macrophages, promoting immune tolerance to the embryo and preventing the embryo from being attacked by maternal immune cells, thus affecting normal growth and development.

[0057] In this embodiment of the invention, the vitamin-magnesium complex decomposes and releases divalent magnesium ions in the endometrial tissue microenvironment. The magnesium ions promote the anti-oxidative stress of endometrial stromal cells, reduce the level of reactive oxygen species in cells, promote mitochondrial metabolism, and promote cell proliferation.

[0058] In this embodiment of the invention, the vitamin-magnesium complex is a vitamin B13-magnesium chelate. The magnesium-based coordination chelate decomposes in the endometrial tissue microenvironment to release free vitamin B13 monomers. The vitamin B13 monomers have good biocompatibility and are used to promote the proliferation of endometrial stromal cells.

[0059] In this embodiment of the invention, the concentration range of the vitamin-magnesium complex used is greater than 1 μg / mL. -1 Less than 100 μg / mL -1 It can be selected as 10 μg mL -1 35 μg mL -1 65 μg mL -1 95 μg mL -1 100 μg mL -1And so on. Those skilled in the art can configure it according to their needs, and this application does not limit it, but only to actual use.

[0060] In this embodiment of the invention, during application, the release rate of the magnesium-based coordination chelate is controlled by pH response or temperature change, or the release rate of the magnesium-based coordination chelate in the vitamin-magnesium complex preparation is controlled by controlling the degradation rate of the vitamin-magnesium complex preparation itself.

[0061] The mechanism of pH-responsive controlled release is as follows: the microenvironment of the uterus is acidic, and magnesium-based coordination chelates can decompose and release magnesium ions under acidic conditions; the decomposition rate of the magnesium-based coordination chelate vitamin-magnesium complex preparation can be adjusted according to the pH value, thereby achieving controlled release of magnesium ions from the magnesium-based coordination chelate.

[0062] The mechanism of temperature-controlled release is as follows: the microenvironment temperature of the uterus is a dynamic process and is regulated by the physiological cycle; according to the temperature change, the decomposition rate of magnesium-based coordination chelate vitamin-magnesium complex preparation can be adjusted. The increase of temperature can increase the decomposition rate of magnesium-based coordination chelate, thereby achieving controlled release of magnesium ions from magnesium-based coordination chelate.

[0063] The mechanism of self-degradation of vitamin-magnesium complex preparations is as follows: Uterine mucus is a hydrogel-like liquid containing various ions, which can promote the decomposition of magnesium ion carriers in the magnesium-based coordination chelate vitamin-magnesium complex preparations and release magnesium ions. The rate of decomposition of vitamin-magnesium complex preparations can be controlled by changing the physicochemical properties of the preparations, thereby controlling the rate of magnesium ion release.

[0064] In this embodiment of the invention, a vitamin-magnesium complex formulation is also proposed. The vitamin-magnesium complex is used to prepare a drug for preventing miscarriage during the preconception period and early pregnancy. The vitamin-magnesium complex formulation is composed of a magnesium-based coordination chelate and an organic carrier. The magnesium-based coordination chelate is synthesized by chelating a magnesium salt compound and an organic ligand. The carrier is any one or more of agarose, chitosan, gelatin, thermosensitive hydrogel, sodium alginate, polyethylene glycol, polylactic acid, liposomes, and acrylic acid.

[0065] In this embodiment of the invention, the magnesium compound includes any one of magnesium chloride, magnesium nitrate, magnesium acetate, magnesium sulfate, and their hydrates.

[0066] In this embodiment of the invention, the organic ligand includes any one of vitamin B13, vitamin B3, and vitamin C. The organic ligands used as raw materials for synthesizing the above-mentioned magnesium-based coordination chelates may include organic compounds such as nicotinic acid, orotic acid, and ascorbic acid.

[0067] In this embodiment of the invention, the magnesium-based coordination chelate promotes the proliferation of endometrial stromal cells and endometrial vascular endothelial cells by releasing magnesium ions, and mediates the polarization of M1 macrophages to M2 type; the carrier is used to maintain the slow release of magnesium ions from the magnesium-based chelate.

[0068] In this embodiment of the invention, the magnesium group in the magnesium-based coordination chelate is derived from inorganic metal materials, including mono- or multi-metal-doped magnesium-based coordination chelates, and single-component or multi-component metal compound-doped magnesium-based coordination chelates.

[0069] In this embodiment of the invention, the central ligand constituting the magnesium-based coordination chelate is an organic compound that is biocompatible and friendly to the organism; the magnesium element constituting the magnesium-based coordination chelate is a vital element.

[0070] In this embodiment of the invention, the synthesis methods of the magnesium-based coordination chelate include solvothermal synthesis, liquid-phase synthesis, solid-phase synthesis, ultrasonic-assisted synthesis, high-temperature-assisted synthesis, surfactant synthesis, and microwave oven-assisted synthesis.

[0071] In this embodiment of the invention, the vitamin-magnesium complex preparation is administered after the formation of endometrial tissue, or in early pregnancy after the embryo invades and implants in the endometrium; the administration methods of the vitamin-magnesium complex preparation include minimally invasive intrauterine administration via the abdomen, minimally invasive intrauterine administration via the back, and intravenous injection; the administration method of the vitamin-magnesium complex preparation also includes intrauterine instillation. The vitamin-magnesium complex preparation is administered after the formation of the trophoblast and before invasion; the administration method of the vitamin-magnesium complex preparation is intrauterine instillation.

[0072] Under the influence of progesterone, the endometrial tissue transforms into decidual tissue, preparing for embryo implantation. Endometrial stromal cells are relatively large and rounded, with abundant nutrient-rich cytoplasm, large nuclei, prominent nucleoli, and loosely distributed chromatin, indicating an active metabolic state. In early pregnancy, endometrial stromal cells primarily play a role in immune regulation and nutritional support; while in late pregnancy, their function may shift more towards maintaining placental stability and fetal development. Furthermore, the proliferation of vascular endothelial cells in the endometrial tissue promotes angiogenesis, providing more ample nutrition for embryonic growth. The polarization of M1 macrophages into M2 macrophages promotes immune tolerance to the embryo, preventing attacks by maternal immune cells that could affect normal growth and development.

[0073] The aforementioned vitamin-magnesium complex preparation can be administered to animals via minimally invasive dorsal administration, minimally invasive abdominal administration, and intrauterine instillation. Due to the special environment of the uterine cavity, intrauterine instillation is preferred in this embodiment to directly administer the vitamin-magnesium complex preparation into the uterine cavity. Similarly, in future clinical applications, intrauterine instillation is also preferred for directly administering the vitamin-magnesium complex preparation into the uterine cavity.

[0074] In this embodiment of the invention, a method for preventing miscarriage during the preconception period and early pregnancy is also provided. Specifically, the vitamin-magnesium complex preparation described above can be placed in the uterine cavity via intrauterine administration or injected intravenously, so that the magnesium-based coordination chelate can promote the proliferation of endometrial stromal cells, vascular endothelial cells and immune regulation based on the sustained release of magnesium ions; the vitamin-magnesium complex is a vitamin B13-magnesium chelate, which simultaneously releases magnesium ions and free vitamin B13 monomers.

[0075] Because the embodiments of this application can be administered after the formation of endometrial tissue, or in early pregnancy after the embryo invades and implants in the endometrium, acting on endometrial stromal cells to promote cell proliferation, enhance uterine receptivity to the embryo, and promote successful implantation; acting on vascular endothelial cells of uterine tissue to promote angiogenesis, providing sufficient nutrition for fetal growth and development; and acting on immune macrophages to promote the polarization of M1 macrophages to M2 macrophages, promoting fetal immune tolerance and preventing the fetus from being attacked by the maternal immune system. Therefore, the magnesium-based coordination chelate in the embodiments of this application has a promoting effect on endometrial stromal cells, vascular endothelial cells, and immune macrophages; and the mechanism of controlled release of magnesium ions from the magnesium-based coordination chelate in the vitamin-magnesium complex preparation is based on the above-mentioned pH response and temperature response controlled release or controlled degradation rate of the vitamin-magnesium complex preparation itself.

[0076] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0077] The application and formulation of a vitamin-magnesium complex provided by this invention in the preparation of drugs for preventing miscarriage during the preconception period and early pregnancy have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. The application of vitamin-magnesium complex in the preparation of drugs for preventing miscarriage during the preconception period and early pregnancy, characterized in that, The vitamin-magnesium complex is a magnesium-based coordination chelate that has a repairing effect on endometrial tissue. The magnesium-based coordination chelate is synthesized by chelation of a magnesium salt compound and an organic ligand. The organic ligand is vitamin B13, and the magnesium salt compound includes any one of magnesium chloride, magnesium nitrate, magnesium acetate, and magnesium sulfate.

2. The application of the vitamin-magnesium complex according to claim 1 in the preparation of a drug for preventing miscarriage during the preconception period and early pregnancy, characterized in that, The concentration range of the vitamin-magnesium complex is greater than 1 μg / mL. -1 Less than 100 μg / mL -1 .

3. A vitamin-magnesium complex formulation, characterized in that, The vitamin-magnesium complex is used to prepare a drug for preventing miscarriage during the preconception period and early pregnancy; the vitamin-magnesium complex formulation is composed of a magnesium-based coordination chelate and an organic carrier, wherein the magnesium-based coordination chelate is synthesized by chelation of a magnesium salt compound and an organic ligand, and the organic carrier includes any one or more of agarose, chitosan, gelatin, thermosensitive hydrogel, sodium alginate, polyethylene glycol, polylactic acid, liposomes, and acrylic acid; The concentration range of the vitamin-magnesium complex is greater than 1 μg / mL. -1 Less than 100 μg • mL -1 ; The magnesium salt compound in the vitamin-magnesium complex includes any one of magnesium chloride, magnesium nitrate, magnesium acetate, and magnesium sulfate; The organic ligand is vitamin B13; The methods for synthesizing the magnesium-based coordination chelates include solvothermal synthesis, liquid-phase synthesis, solid-phase synthesis, ultrasonic-assisted synthesis, high-temperature-assisted synthesis, surfactant synthesis, and microwave oven-assisted synthesis.

4. The vitamin-magnesium complex formulation according to claim 3, characterized in that, The vitamin-magnesium complex preparation is administered after the formation of endometrial tissue, or in early pregnancy after the embryo invades and implants in the endometrium; the administration methods of the vitamin-magnesium complex preparation include minimally invasive abdominal intrauterine administration, minimally invasive back intrauterine administration, intravenous injection, and intrauterine perfusion administration.

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