Composition for resisting premature ovarian failure

Abstract

The invention discloses a composition for resisting premature ovarian failure. The composition comprises a ginsenoside monomer Rg1 and fucoidin. The composition can be applied to preparation of anti-premature ovarian failure drugs or preparation of drugs for reducing expression of neutrophile granulocyte extracellular traps. The invention also provides an anti-premature ovarian failure drug containing the ginsenoside monomer Rg1 and the fucoidin. The composition is based on the intestinal-ovarian axis, improves tissue microcirculation disturbance, inhibits overexpression of NETs, relieves adverse side effects caused by chemotherapeutic drugs in a multi-target mode, and fundamentally inhibits occurrence and development of premature ovarian failure, so that the safe and effective treatment purpose is achieved, and a new treatment strategy is provided for clinic.

Description

Technical Field

[0001] The present invention relates to a natural medicine composition, and particularly to a composition for resisting premature ovarian failure. Background Art

[0002] Chemotherapy is one of the most commonly used means for treating malignant tumors, and chemotherapy often brings serious side effects, such as vomiting, dizziness, hair loss and sexual function decline. The decline of female reproductive function caused by chemotherapy, especially chemotherapy-induced premature ovarian failure (CIPOF), refers to the phenomenon of increased gonadotropin in women caused by chemotherapy. Clinically, it is specifically manifested as symptoms of estrogen reduction, including amenorrhea, infertility, vasomotor instability (hot flashes, night sweats), sleep disorders, etc. Moreover, the reduction of estrogen will also induce an increase in the fragility of women's bones and increase the risk of suffering from cardiovascular system diseases and cognitive disorders.

[0003] Cytotoxic anti-tumor drugs, such as cyclophosphamide, doxorubicin, and cisplatin, are likely to induce premature ovarian failure. However, the induction mechanism is complex and mainly focuses on the loss of oogonia, the direct disappearance of primordial follicles, the overactivation of primordial follicles, and follicular atresia. Research shows that chemotherapeutic drugs can cause ovarian interstitial fibrosis in mice. After using doxorubicin (Dox), the ovarian toxicity of mice is the greatest and the degree of fibrosis is the most obvious. Doxorubicin will increase oxidative stress and induce ovarian fibrotic damage in mice.

[0004] Neutrophil extracellular traps (NETs) are large reticular structures with chromatin as the skeleton and composed of membrane proteins, granule proteins, etc. The formation of suicidal NETosis and vital NETs has established two main types of neutrophil release of nucleic acid skeletons. The characteristics of suicidal NETosis are the production of ROS and the rupture of neutrophils; the formation of vital NETs is initiated by stimuli, such as Staphylococcus aureus through TLR2 and complement receptors, or Gram-negative bacteria through TLR4 or indirectly through LPS produced by platelets activated by TLR4. NETs have always been closely related to the formation of thrombus. Existing research points out that NETs can promote vascular occlusion and thrombus formation; NETs can also trigger the coagulation cascade reaction, bind to tissue factor TF, and increase the stability of thrombus. After doxorubicin chemotherapy, the relevant indicators of NETs in the patient's blood increase, increasing the risk of thrombus formation.

[0005] The existing drugs for treating CIPOF mainly include hormone replacement therapy (HRT), oocyte donation, cryopreservation, and psychotherapy. The above therapies can only relieve the vasomotor and genitourinary symptoms of patients, cannot fundamentally improve infertility or reduce the incidence of systemic symptoms, and may also induce breast cancer or coronary heart disease. In recent years, studies have found that the etiology of premature ovarian failure is complex, highly heterogeneous, and lacks effective treatment. Therefore, it is urgent to explore the core pathophysiological mechanisms of the occurrence and development of premature ovarian failure in order to find new therapeutic drugs. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide a composition to solve the problem of how to treat premature ovarian failure. Another object of the present invention is to propose the application of a composition in the preparation of drugs for anti-premature ovarian failure to solve the problem of how to prepare drugs for anti-premature ovarian failure. The third object of the present invention is to provide an anti-premature ovarian failure drug containing ginsenoside monomer Rg1 and fucoidan. The fourth object of the present invention is to provide the application of a composition in the preparation of drugs for reducing the expression of neutrophil extracellular traps to solve the problem of how to reduce the expression of neutrophil extracellular traps.

[0007] Technical Solution: A composition according to the present invention is characterized by comprising ginsenoside monomer Rg1 and fucoidan.

[0008] Preferably, the mass ratio of the ginsenoside monomer Rg1 to the fucoidan is 1:1 - 5.

[0009] The second aspect of the present invention discloses the application of the above composition in the preparation of drugs for anti-premature ovarian failure.

[0010] Preferably, the premature ovarian failure is induced by chemotherapy drugs.

[0011] Preferably, the chemotherapy drugs include at least one of doxorubicin, paclitaxel, cyclophosphamide, and cisplatin.

[0012] More preferably, the chemotherapy drug is doxorubicin.

[0013] The third aspect of the present invention discloses an anti-premature ovarian failure drug containing ginsenoside monomer Rg1 and fucoidan.

[0014] Preferably, the anti-premature ovarian failure drug further contains pharmaceutically acceptable excipients.

[0015] The fourth aspect of the present invention discloses the application of the above composition in the preparation of drugs for reducing the expression of neutrophil extracellular traps.

[0016] Advantageous Effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0017] The composition of the present invention can inhibit the ovarian shrinkage caused by chemotherapy drugs, significantly improve the low hormone symptoms of premature ovarian failure, inhibit the expression of NETs (neutrophil extracellular traps), relieve the adverse side effects caused by chemotherapy drugs, and delay the development of CIPOF, so as to achieve a safe and effective treatment purpose and provide a new treatment strategy for clinical practice. The composition provided by the present invention can significantly increase the ovarian volume of CIPOF model mice induced by doxorubicin and improve the estrogen reduction symptoms induced by doxorubicin, and has a significant effect on the treatment of CIPOF. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Results of the determination of ovarian wet weight of mice in each experimental group;

[0019] Figure 2 Results of the determination of estrous cycle of mice in each experimental group;

[0020] Figure 3 Follicle count in the ovaries of mice in each experimental group;

[0021] Figure 4 Paraffin sections of HE of ovaries of mice in each experimental group;

[0022] Figure 5 Results of the determination of CIPOF-related hormone levels in peripheral blood of mice in each experimental group;

[0023] Figure 6 Results of the determination of cf-DNA content in plasma of mice in each experimental group. DETAILED DESCRIPTION OF THE INVENTION

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Example 1: A preparation method of a composition for anti-premature ovarian failure is as follows:

[0026] Mix ginsenoside Rg1 powder and fucoidan powder in a mass ratio of 2:5 as the drug for anti-premature ovarian failure. The purity of ginsenoside Rg1 is ≥95%, and the purity of fucoidan is ≥95%. There are no special requirements for the particle size and diameter.

[0027] Configuration of the administration liquid in animal experiments: Dissolve 80 mg of ginsenoside Rg1 powder and 200 mg of fucoidan powder in 10 mL of sterile water, shake and vortex to make it evenly mixed and completely dissolved.

[0028] Example 2: A preparation method of a composition for anti-premature ovarian failure is as follows:

[0029] Mix the ginsenoside Rg1 powder and the fucoidan powder in a mass ratio of 1:1 as an anti-ovarian premature aging drug. The purity of ginsenoside Rg1 is ≥95%, and the purity of fucoidan is ≥95%. There are no special requirements for the particles and diameter.

[0030] Preparation of the administration liquid in animal experiments: Dissolve 80 mg of ginsenoside Rg1 powder and 80 mg of fucoidan powder in 10 mL of sterile water, shake and vortex to make it uniformly mixed and completely dissolved.

[0031] Example 3: The preparation method of a composition for anti-ovarian premature aging is as follows:

[0032] Mix the ginsenoside Rg1 powder and the fucoidan powder in a mass ratio of 1:5 as an anti-ovarian premature aging drug. The purity of ginsenoside Rg1 is ≥95%, and the purity of fucoidan is ≥95%. There are no special requirements for the particles and diameter.

[0033] Preparation of the administration liquid in animal experiments: Dissolve 40 mg of ginsenoside Rg1 powder and 200 mg of fucoidan powder in 10 mL of sterile water, shake and vortex to make it uniformly mixed and completely dissolved.

[0034] Comparative Example 1: The rest are the same as in Example 1, except that:

[0035] Only use the ginsenoside Rg1 powder as the test drug sample.

[0036] Preparation of the administration liquid in animal experiments: Dissolve 80 mg of ginsenoside Rg1 powder in 10 mL of sterile water, shake and vortex to make it uniformly mixed and completely dissolved.

[0037] Comparative Example 2: The rest are the same as in Example 1, except that:

[0038] Only use the fucoidan powder as the test drug sample.

[0039] Preparation of the administration liquid in animal experiments: Dissolve 200 mg of fucoidan powder in 10 mL of sterile water, shake and vortex to make it uniformly mixed and completely dissolved.

[0040] Comparative Example 3: The rest are the same as in Example 1, except that:

[0041] Replace ginsenoside Rg1 with ginsenoside Rg5.

[0042] Comparative Example 4: The rest are the same as in Example 1, except that:

[0043] Replace ginsenoside Rg1 with ginsenoside Rg3.

[0044] Comparative Example 5: The rest are the same as in Example 1, except that:

[0045] Replace ginsenoside Rg1 with ginsenoside RH1.

[0046] Comparative Example 6: The rest are the same as in Example 1, except that:

[0047] Replace fucoidan with algal oligosaccharide.

[0048] Comparative Example 7: The rest are the same as in Example 1, except that:

[0049] Replace fucoidan with alginate.

[0050] Evaluate the effects of the administration solutions prepared in Example 1 and Comparative Examples 1-7 on the treatment of CIPOF as follows:

[0051] Experimental animals: Female C57BL / 6J mice, SPF grade, 6-8 weeks old, provided by Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. All experimental animals were housed in an independent environment with a 12h light-dark cycle, with free access to water and food. The breeding environment temperature was maintained at 22±2°C, and the experiment was carried out after 1 week of environmental adaptation.

[0052] Randomly divide the experimental mice into 5 groups (n = 11), namely the control group, doxorubicin group (model group), doxorubicin + Comparative Example 1 group, doxorubicin + Comparative Example 2 group, doxorubicin + Comparative Example 3 group, doxorubicin + Comparative Example 4 group, doxorubicin + Comparative Example 5 group, doxorubicin + Comparative Example 6 group, doxorubicin + Comparative Example 7 group, doxorubicin + Example 1 group. The specific administration plan is as follows: 7 days before modeling, intragastrically administer the solutions prepared in Comparative Examples 1-7 and Example 1 at 10 mL / kg twice a day, and continue administration until 14 days after modeling. The control group was intragastrically administered normal saline at 0.1 ml / 10 g twice a day 7 days before modeling and continued administration until 14 days after modeling. When using doxorubicin for modeling, intraperitoneally inject doxorubicin (10 mg / kg) once into the mice in the model group and the five treatment groups to prepare a CIPOF animal model. Behavioral assays were performed daily starting from the day of mouse modeling, and samples were taken 14 days after modeling.

[0053] Determination of mouse estrous cycle and ovarian wet weight: The behavioral assay before sampling of the present invention is to collect vaginal smears from mice to evaluate their estrous cycle changes. Reference: Qin X, Zhao Y, Zhang T, Yin C, Qiao J, Guo W, Lu B. TrkB agonist antibody ameliorates fertility deficits in aged and cycloph osphamide-induced premature ovarian failure model mice. Nat Commun. 2022 Feb 17; 13 (1): 914. doi: 10.1038 / s41467-022-28611-2. PMID: 35177657. For at least three consecutive estrous cycles (12 days), vaginal smears were performed at a fixed time every morning to detect the estrous cycle. During the experiment, 10 μL of physiological saline was drawn into the vagina using a pipette, and the liquid was transferred to a glass slide for microscopic analysis after repeated aspiration 2-3 times. The estrus cycle is determined by the cell morphology of the main cell types in the vagina: proestrus: round nucleated epithelial cells; estrus: cornified squamous epithelial cells; metestrus: epithelial cells and leukocytes; diestrus: nucleated epithelial cells and leukocytes predominate. After sampling, the mouse ovaries were extracted and their weights were measured. The test results are shown in Figure 1 and Figure 2 .

[0054] Mouse ovary HE paraffin sections and follicle counting: 14 days after mouse modeling, the mouse ovaries were fixed in 4% paraformaldehyde for 24 hours. A series of ethanol from low to high concentrations were used as dehydrating agents to gradually remove the water in the tissue; then the tissue was placed in a transparent agent xylene dewaxing liquid that is soluble in both ethanol and paraffin to make the tissue transparent. After that, the transparent tissue was placed in a mold containing melted paraffin, the tissue position was adjusted, and then placed on a cooling table to cool. Preheat the bleaching machine (water injection scale line) and the spreader to 42-45℃ in advance, fix the embedded wax block on the slicer, first adjust the slice thickness to 15 microns, trim the paraffin section to flatness, and then modify the slice thickness to 4 microns for slicing. The cut slices were placed in the bleaching machine for bleaching, and after the slices were stretched, they were fished out with a pre-marked anti-slip slide. After pasting, the slides were placed in the spreader for temporary drying, and after all the patches were completed, they were placed in an oven and dried overnight at 40℃. Use xylene dewaxing solution to remove the paraffin in the slices, then completely elute the paraffin through high to low concentrations of ethanol, and finally wash with ultrapure water before staining.

[0055] First, perform hematoxylin staining for 5 minutes. After rinsing off the floating stain with a gentle stream of tap water, place it in 0.1% hydrochloric acid and oscillate for 2 - 3 seconds, then rinse off the excess hydrochloric acid with a gentle stream of tap water. After that, perform eosin staining for 20 seconds. After dehydrating by oscillation with 70% and 90% ethanol, wash twice with 100% absolute ethanol. Finally, use xylene for clearing. Drop neutral resin onto the cleared section, cover it with a coverslip for mounting. After mounting, wipe off the excess resin, place it in an oven at 37°C overnight, and observe under a microscope. Images were taken by two researchers who were unaware of the grouping under a panoramic slide scanner, imaging while moving to scan and image all the tissue information on the tissue section to form a folder, which contains all the tissue information on the tissue section. After opening the folder with CaseViewer 2.2 software, it can be magnified arbitrarily from 1 - 400 times for observation. Count the primordial follicles, primary follicles, secondary follicles, and atretic follicle-like follicles in each section respectively. The HE result images and follicle count statistical graphs are shown in Figure 4 and Figure 3 。

[0056] ELISA was used to detect CIPOF-related hormones (E2: estrogen; FSH: follicle-stimulating hormone; AMH: anti-Müllerian hormone): 14 days after establishing the CIPOF model in mice, collect the mouse plasma. Take out the required strips from the aluminum foil bag that has been equilibrated at room temperature for 20 minutes, and seal the remaining strips with a self-sealing bag and return them to 4°C. Set up the standard wells and sample wells. Add 50 μL of standards with different concentrations to each standard well. First, add 10 μL of the sample to be tested to the sample well, and then add 40 μL of sample diluent; do not add anything to the blank well. Except for the blank well, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each standard well and sample well. Seal the reaction wells with a sealing film and incubate in an incubator at 37°C for 60 minutes. After 60 minutes, discard the liquid, pat it dry on absorbent paper. Fill each well with washing solution, let it stand for 1 minute, discard the washing solution, and pat it dry on absorbent paper. Repeat the washing process 5 times. Add 50 μL of substrate A and B to each well, and incubate in the dark at 37°C for 15 minutes. Add 50 μL of stop solution to each well, and within 15 minutes, measure the absorbance (OD) value of each well at a wavelength of 450 nm. Draw a standard curve based on the OD values obtained from the standards. Calculate the concentration values of each sample according to the curve equation. The results are shown in Figure 5 。 Figure 5 Among them, from left to right are the statistical graphs of estrogen level, anti-Müllerian hormone level, and follicle-stimulating hormone level in sequence.

[0057] NETs are composed of circulating free DNA (cf-DNA), histones, and neutrophil cytoplasmic-derived proteins such as proteases. Therefore, measuring the content of cf-DNA in plasma can reflect the content of NETs to a certain extent. The laboratory uses the NETosis As sayKit kit to detect the content of plasma cf-DNA. First, dilute the TE stock solution 20-fold to prepare the TE working solution, that is, mix 1 mL of the TE stock solution evenly with 19 μL of deionized water for later use. Dilute the dye 200-fold, that is, mix 50 μL of the dye evenly with 950 μL of the TE working solution to prepare the staining solution. The standard DNA concentration is 100 μg / mL. Mix 7 μL of the standard DNA with 313 μL of the TE working solution to make standard A with a concentration of 2 μg / mL; dilute 125 μL of standard A with 125 μL of the TE working solution to become standard B with a concentration of 1 μg / mL; dilute 25 μL of standard B with 225 μL of the TE working solution to become standard C with a concentration of 100 ng / mL, 1 ng / mL, and 0 ng / mL of standard D, E, and F. Secondly, place the blood sample in an EP tube containing EDTA at room temperature for 10 minutes, then set the centrifuge to centrifuge at 3000 rpm for 15 minutes at 4°C, and aspirate the supernatant to obtain plasma. Dilute the plasma with the TE working solution at a ratio of 1:4. Then, add 100 μL of the standard and 100 μL of the staining solution to the standard curve wells in sequence, add 100 μL of the diluted sample and 100 μL of the staining solution to the sample wells, and react at room temperature for 2 - 5 minutes. Detect the fluorescence intensity in the microplate reader under the conditions of 480 nm excitation light and 520 nm absorption light. Finally, make a standard curve based on the standard products, and thus convert the concentration of cf-DNA in the sample. It is recommended to use plastic products instead of glass products during the experimental process because this dye is easily adsorbed on the surface of plastic products. The results are shown in Figure 6 。

[0058] In Figures 1-6 ,"**" represents a significant difference compared with the control group, "##" represents a significant difference compared with the DOX model group, and the unlabeled experimental groups represent no difference compared with the DOX model group. According to the above experimental results, it can be seen that when ginsenoside Rg1 or fucoidan is administered alone, it cannot relieve doxorubicin-induced CIPOF. Only when ginsenoside Rg1 and fucoidan are used in combination can it effectively relieve doxorubicin-induced CIPOF, indicating that there is a synergistic effect in enhancing the drug efficacy between ginsenoside Rg1 and fucoidan. When ginsenoside Rg1 is replaced with other types of ginsenosides or fucoidan is replaced with polysaccharide substances extracted from other algae, the anti-CIPOF drug efficacy will be lost, indicating that ginsenoside Rg1 and fucoidan are indispensable and interdependent when exerting the drug efficacy of relieving CIPOF. The combination of ginsenoside Rg1 and fucoidan may inhibit the production of NETs by enhancing the intestinal barrier, thereby reducing the possibility of thrombosis and achieving the therapeutic purpose of protecting ovarian function.

Claims

1. A composition, characterized in that It includes ginsenoside monomer Rg1 and fucoidan.

2. The composition according to claim 1, characterized in that The mass ratio of the ginsenoside monomer Rg1 to fucoidan is 1:1-5.

3. Use of the composition according to claim 1 or 2 in the preparation of an anti-premature ovarian failure drug.

4. The use according to claim 3, characterized in that: The premature ovarian failure is induced by chemotherapy drugs.

5. The use according to claim 4, characterized in that: The chemotherapy drugs include at least one of doxorubicin, paclitaxel, cyclophosphamide and cisplatin.

6. The use according to claim 4, characterized in that: The chemotherapy drug is doxorubicin.

7. An anti-premature ovarian failure drug comprising ginsenoside monomer Rg1 and fucoidan.

8. The anti-premature ovarian failure drug according to claim 4, characterized in that: Also contains pharmaceutically acceptable excipients.

9. Use of the composition according to claim 1 or 2 in the preparation of a drug for reducing the expression of neutrophil extracellular traps.

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