A complex for improving diminished ovarian reserve and use thereof

Abstract

The application discloses a kind of compound for improving diminished ovarian reserve and application thereof, belong to biological medicine field.The compound is composed of pueraria extract, cistanche extract, spina date seed extract and pyrroloquinoline quinone.The application carries out animal experiment by using cyclophosphamide to induce diminished ovarian reserve model of mouse, and the results show that the compound can improve estrous cycle disorder of model mouse, improve serum anti-mullerian hormone level, improve oocyte germinal vesicle breakdown rate and first polar body discharge rate, regulate oocyte reactive oxygen species level, mitochondrial function phenotype and filamentous actin homeostasis, can also improve oocyte fertilization capacity and preimplantation embryo development efficiency, and can relieve model animal liver and kidney function related biochemical index abnormality.The components of the compound are clear, and oral product can be prepared, which provides a new technical scheme for intervention of diminished ovarian reserve caused by chemotherapy and impaired oocyte development capacity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a complex for improving ovarian reserve and its application. Background Technology

[0002] Decreased ovarian reserve is a significant pathological condition leading to reduced female fertility. This condition not only involves a reduction in the number of primordial and developing follicles, but also includes defects in oocyte nuclear and cytoplasmic maturation, impaired mitochondrial function, elevated intracellular oxidative stress levels, and cytoskeletal structural disorder, ultimately resulting in fertilization failure and embryonic developmental arrest. Cyclophosphamide, a chemotherapy drug, is an alkylating agent commonly used in cancer treatment. After entering the body, it causes DNA damage, induces a large accumulation of reactive oxygen species, disrupts the balance between follicle recruitment and resting, significantly depletes ovarian follicle reserve, and directly damages oocyte quality, leading to a decline in female fertility after chemotherapy.

[0003] Current interventions for ovarian reserve damage fall into several categories, including clinical assisted reproductive technologies and single-substance interventions. Fertility preservation methods include ovarian tissue freezing and oocyte freezing, which focus on the in vitro preservation of reproductive cells and cannot improve the developmental quality of oocytes remaining after chemotherapy damage. Existing studies report that single substances such as kudzu root extract, cistanche extract, and pyrroloquinoline quinone can improve ovarian-related indicators in different animal models. However, single substances can only target certain pathways and mostly evaluate only a few indicators such as oxidative stress and hormone levels, making it difficult to simultaneously address ovarian endocrine status, oocyte meiotic maturation, cytoplasmic quality, and the entire process of continuous embryonic development after fertilization.

[0004] Current intervention protocols rely solely on ovarian weight, follicle count, and serum hormone levels to evaluate their effectiveness. These indicators only reflect the overall ovarian condition and cannot directly demonstrate the actual developmental capacity of oocytes to complete meiosis, fertilization, and blastocyst development. Furthermore, some intervention strategies depend on pathway inhibitors and cell therapy, which have stringent implementation conditions and high costs, making them difficult to develop into oral products. Currently, there is no publicly available comprehensive regimen that combines kudzu root extract, cistanche extract, jujube seed extract, and pyrroloquinoline quinone in a specific ratio to simultaneously improve chemotherapy-induced ovarian reserve decline, oocyte maturation disorders, and preimplantation genetic defects. Developing a controllable, orally administered compound intervention would be of practical significance for the intervention of chemotherapy-related ovarian damage. Summary of the Invention

[0005] The purpose of this invention is to provide a complex that improves ovarian reserve function, thereby solving the technical problem that existing intervention methods mostly use a single active substance, which cannot simultaneously improve ovarian reserve endocrine indicators, oocyte maturation capacity, cytoplasmic function, and subsequent embryonic development capacity.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A complex for improving diminished ovarian reserve, comprising kudzu root extract, cistanche extract, jujube seed extract, and pyrroloquinoline quinone; Pueraria lobata extract uses puerarin as the quality control component, with a labeled puerarin content of 95%~99%. The Cistanche deserticola extract uses echinacoside as the quality control component, with a labeled content of 70%~80%; The jujube seed extract uses jujube seed saponins as the quality control component, with a labeled content of 8%~10%.

[0007] Preferably, the mass ratio of kudzu root extract to cistanche extract is 7.5~8.5:9.0~11.0, the mass ratio of kudzu root extract to jujube seed extract is 7.5~8.5:75.0~85.0, and the mass ratio of kudzu root extract to pyrroloquinoline quinone is 7.5~8.5:1.5~2.0.

[0008] This complex can improve estrous cycle disorders in cyclophosphamide-induced ovarian reserve decline model mice, increase serum anti-Müllerian hormone levels, reduce ovarian tissue atrophy, downregulate intracellular reactive oxygen species levels in oocytes, improve oocyte mitochondrial function, alleviate abnormal remodeling of oocyte filament actin, enhance oocyte meiotic maturation capacity, improve oocyte fertilization capacity and the continuous developmental potential of preimplantation embryos, and alleviate abnormalities in liver and kidney function-related biochemical indicators in model animals. It can exert a comprehensive protective effect against chemotherapy-induced ovarian and oocyte damage through multiple targets. The intervention effect of this complex can be further translated into the restoration of the continuous developmental potential of 2-cell, 4-cell, morula to blastocyst stages after oocyte fertilization. The optimal response dose of the complex varies for different biological endpoints. High doses show significant improvement in oocyte maturation and embryonic development endpoints, while low- to medium doses show significant advantages in oocyte reactive oxygen species scavenging.

[0009] A complex formulation comprising a complex, wherein the formulation is one or more of a suspension, tablet, or granule.

[0010] Preferably, the preparation method of the suspension is as follows: the complex is added to physiological saline, stirred and mixed, vortexed for 2-4 minutes, and then ultrasonically treated: ultrasonic power 180-220W, single ultrasonic working time 25-35s, interval 15-25s, cycle 2-4 times.

[0011] Preferably, the mass-to-volume ratio of the complex to physiological saline is 50-400 mg: 10 mL.

[0012] Preferably, the preparation method of the granules is as follows: the complex is mixed with lactose, dextrin and steviol glycosides to obtain a mixed powder; purified water is added to the mixed powder to prepare a soft material; the soft material is fed into a swing granulator for wet granulation to obtain wet granules; the wet granules are granulated and then placed in a hot air circulating oven for drying; after drying, they are granulated again and sieved to obtain granules.

[0013] Preferably, the mass ratio of the mixed powder to purified water is 45~55:8~10.

[0014] Preferably, the mass ratio of the complex to lactose is 4~6:5~7, the mass ratio of the complex to dextrin is 3~5:0.8~1.2, and the mass ratio of the complex to steviol glycosides is 110~140:1.

[0015] Preferably, the mixed powder comprises 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one.

[0016] Preferably, the mass ratio of the complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one is 100:3~10.

[0017] Add 3,8 dihydroxy 6H Dibenzo[B,D]pyran 6 Ketones can further reduce serum lipid peroxidation product levels in model animals, alleviate oxidative stress damage in oocytes, increase mitochondrial membrane potential in oocytes, and enhance oocyte maturation and subsequent embryonic development. The underlying mechanism of action is speculated to be: this substance can induce mitophagy in oocytes, selectively clear damaged and dysfunctional mitochondria, reduce the continuous release of reactive oxygen species from damaged mitochondria, alleviate the persistent damage of oxidative stress to oocytes, and create a synergistic effect with the base composition.

[0018] More preferably, the mixed powder includes valerian root extract, and the mass ratio of the complex to the valerian root extract is 100:2~4.

[0019] The combined use of valerian root extract and its complex can further reduce lipid peroxidation damage levels in the body and improve the overall protective effect under chemotherapy-induced damage models. The underlying mechanism of action is speculated to be that active components such as valeric acid in valerian root extract can regulate stress-related neural pathways in the body. It regulates endocrine status, alleviates the amplified peroxidation effect of excessive stress, reduces the further deterioration of oocyte quality caused by stress-induced chemotherapy, and forms a multi-layered complementary protective effect with the complex, thereby further enhancing the overall protective ability of the composition for ovarian reserve and oocytes.

[0020] The present invention also discloses the use of complexes or formulations in the preparation of drugs for improving chemotherapy-induced ovarian reserve decline.

[0021] This invention also discloses the use of the complex or formulation in the preparation of products that simultaneously improve ovarian reserve endocrine indicators and / or oocyte maturation capacity and / or cytoplasmic function, and / or subsequent embryonic development capacity.

[0022] The present invention also provides a method for preparing the complex, as follows: Pueraria lobata extract, Cistanche deserticola extract, Ziziphus jujuba var. spinosa extract and pyrroloquinoline quinone were mixed evenly to obtain a complex.

[0023] Preferably, the kudzu root extract uses puerarin as the quality control component, and the puerarin content is labeled as 95%~99%.

[0024] Preferably, the Cistanche deserticola extract uses echinacoside as the quality control component, and the echinacoside content is labeled as 70%~80%.

[0025] Preferably, the jujube seed extract uses jujube seed saponins as the quality control component, and the jujube seed saponin content is labeled as 8%~10%.

[0026] Preferably, the mass ratio of kudzu root extract to cistanche extract is 7.5~8.5:9.0~11.0.

[0027] Preferably, the mass ratio of kudzu root extract to jujube seed extract is 7.5~8.5:75.0~85.0.

[0028] Preferably, the mass ratio of kudzu root extract to pyrroloquinoline quinone is 7.5~8.5:1.5~2.0.

[0029] This invention also provides a method for preparing a complex suspension, as detailed below: Add the complex to physiological saline, stir and mix thoroughly, and vortex for 2-4 minutes; then sonicate, with the sonic power set to 180-220W, a single sonication time of 25-35s, an interval of 15-25s, and repeat the process 2-4 times to obtain the complex suspension.

[0030] Preferably, the mass-to-volume ratio of the complex to physiological saline is 50-400 mg: 10 mL.

[0031] This invention also provides a method for preparing a complex tablet, as detailed below: The complex was thoroughly sieved and mixed with microcrystalline cellulose and croscarmellose sodium, and then magnesium stearate was added for total mixing to obtain a mixed powder. The powder was then compressed using a rotary tablet press to control the tablet hardness to 4-6 kgf to obtain tablets.

[0032] Preferably, the mass ratio of the complex to microcrystalline cellulose is 4~6:3~5; Preferably, the mass ratio of the complex to cross-linked sodium carboxymethyl cellulose is 20~30:2~4; Preferably, the mass ratio of the complex to magnesium stearate is 40~60:0.8~1.2.

[0033] This invention also provides a method for preparing granules, as detailed below: The complex was mixed with lactose, dextrin, and steviol glycosides to obtain a mixed powder. Purified water was added to the mixed powder to prepare a soft material. The soft material was fed into a swing granulator for wet granulation to obtain wet granules. After granulation, the wet granules were placed in a hot air circulating oven for drying at a temperature of 45-55°C. After drying, the granules were granulated again and sieved to obtain 26-34 mesh granules to obtain granules.

[0034] Preferably, the mass ratio of the complex to lactose is 4~6:5~7.

[0035] Preferably, the mass ratio of the complex to dextrin is 3~5:0.8~1.2.

[0036] Preferably, the mass ratio of the complex to steviol glycoside is 110-140:1.

[0037] Preferably, the mass ratio of the mixed powder to purified water is 45~55:8~10.

[0038] Preferably, the mixed powder comprises 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one.

[0039] Preferably, the mass ratio of the complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one is 100:3~10.

[0040] More preferably, the mixed powder includes valerian root extract.

[0041] More preferably, the valerian root extract uses valeric acid as a quality control component, with a labeled content of 0.6% to 0.8%.

[0042] More preferably, the mass ratio of the complex to the valerian root extract is 100:2~4.

[0043] This invention utilizes a specific ratio of kudzu root extract, cistanche deserticola extract, jujube seed extract, and pyrroloquinoline quinone to create a complex, resulting in the following beneficial effects: The components of the complex prepared in this invention exhibit synergistic effects, rather than being a simple additive effect, enabling multi-target intervention against cyclophosphamide-induced ovarian reserve decline; it can improve estrous cycle disorders in model mice, increase serum anti-Müllerian hormone levels, and alleviate chemotherapy-induced ovarian tissue atrophy; it can downregulate intracellular reactive oxygen species levels in oocytes, improve oocyte mitochondrial function, reduce abnormal remodeling of oocyte filament actin, enhance oocyte meiotic maturation capacity, and improve oocyte fertilization capacity and preimplantation genetic development potential. Simultaneously, it can alleviate lipid peroxidation damage and abnormal serum biochemical indicators related to liver and kidney function in model animals. Furthermore, 3,8- dihydroxy 6H Dibenzo[B,D]pyran 6 Ketones can further reduce lipid peroxidation damage in the body and improve mitochondrial function in oocytes and embryonic development; further combined with valerian root extract, it can regulate stress-related nerves in the body. The endocrine status is improved, further enhancing the overall protective effect of the composition. The complex of this invention can be processed into various oral formulations such as suspensions, tablets, and granules. The formulation process is simple, the components are controllable, and the raw materials are readily available, facilitating large-scale preparation. Therefore, this invention is a complex and its formulation that can improve chemotherapy-induced ovarian reserve decline and enhance oocyte development quality, possessing promising application prospects. Attached Figure Description

[0044] Figure 1 Microscopic images of vaginal smears at different stages of the estrous cycle in mice.

[0045] Figure 2 A stacked bar chart showing the percentage of each stage of the estrous cycle in each group of mice.

[0046] Figure 3 Line graphs showing the changes in body weight of mice in each group during the experimental period.

[0047] Figure 4 A bar chart showing the germinal follicle rupture rate and first polar body expulsion rate of oocytes in each group.

[0048] Figure 5 Bright field micrographs of oocytes in the germinal follicle rupture stage and oocytes in the MII stage from each group.

[0049] Figure 6 Bar chart showing the results of serum anti-Müllerian hormone and alanine aminotransferase in mice of each group.

[0050] Figure 7 Bar chart showing the results of serum aspartate aminotransferase and creatinine detection in mice of each group.

[0051] Figure 8 Fluorescence imaging images and magnified local images of reactive oxygen species, mitochondrial tracer probes, and nuclear fluorescent dyes in oocytes of each group.

[0052] Figure 9 Violin plots showing the relative fluorescence intensity of reactive oxygen species and the relative fluorescence intensity of mitochondrial tracer probes in oocytes of each group.

[0053] Figure 10 Fluorescence imaging images and magnified local images of microtubules, phalloidin, and nuclear fluorescent dyes in MII stage oocytes of each group.

[0054] Figure 11 The graph shows the statistical results of spindle abnormality rate and relative fluorescence intensity of phalloidin in oocytes of each group.

[0055] Figure 12 Bright-field micrographs of 2-cell stage embryos, 4-cell stage embryos, morula, blastocysts, and localized embryonic areas in each group.

[0056] Figure 13 A bar chart showing the fertilization rate of oocytes and the 4-cell embryo rate for each group.

[0057] Figure 14 A bar chart showing the morula rate and blastocyst rate of oocytes in each group. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0060] Example 1: This example provides a method for preparing a CPJP complex, as detailed below: Pueraria lobata extract, Cistanche deserticola extract, Ziziphus jujuba var. spinosa extract, and pyrroloquinoline quinone were uniformly mixed to obtain the CPJP complex. Pueraria lobata extract used puerarin as the quality control component, with a labeled content of 99%; Cistanche deserticola extract used echinacoside as the quality control component, with a labeled content of 80%; Ziziphus jujuba var. spinosa extract used jujuboside as the quality control component, with a labeled content of 10%. In the CPJP complex, the mass ratio of pueraria lobata extract to Cistanche deserticola extract was 8.09:10.01, the mass ratio of pueraria lobata extract to Ziziphus jujuba var. spinosa extract was 8.09:80.11, and the mass ratio of pueraria lobata extract to pyrroloquinoline quinone was 8.09:1.78.

[0061] Example 2: This example provides a method for preparing CPJP suspension, as detailed below: The CPJP complex prepared in Example 1 was added to physiological saline, initially stirred and mixed evenly, and then vortexed for 3 minutes; followed by sonication, with the sonication power set to 200W, a single sonication session duration of 30 seconds, and an interval of 20 seconds, and the process was repeated 3 times to obtain a low-dose suspension, namely the CPJP suspension. The mass-to-volume ratio of the CPJP complex to physiological saline was 85 mg: 10 mL.

[0062] Example 3: This example provides a method for preparing CPJP suspension, as detailed below: The CPJP complex prepared in Example 1 was added to physiological saline, initially stirred and mixed evenly, and then vortexed for 3 minutes; followed by sonication, with the sonication power set to 200W, a single sonication session duration of 30s, an interval of 20s, and repeated 3 times to obtain a medium-dose suspension, namely CPJP suspension. The mass-to-volume ratio of CPJP complex to physiological saline was 107mg:10mL.

[0063] Example 4: This example provides a method for preparing CPJP suspension, as detailed below: The CPJP complex prepared in Example 1 was added to physiological saline, initially stirred and mixed evenly, and then vortexed for 3 minutes; followed by sonication, with the sonication power set to 200W, a single sonication session duration of 30 seconds, and an interval of 20 seconds, and the process was repeated 3 times to obtain a high-dose suspension, namely CPJP suspension. The mass-to-volume ratio of CPJP complex to physiological saline was 340 mg: 10 mL.

[0064] Example 5: This example provides a method for preparing CPJP tablets, as detailed below: The CPJP complex prepared in Example 1 was thoroughly sieved and mixed with microcrystalline cellulose and croscarmellose sodium, and then magnesium stearate was added for further mixing to obtain a mixed powder. The powder was then compressed using a rotary tablet press to control the tablet hardness at 5 kgf, yielding CPJP tablets. The mass ratio of CPJP complex to microcrystalline cellulose was 5:4, the mass ratio of CPJP complex to croscarmellose sodium was 25:3, and the mass ratio of CPJP complex to magnesium stearate was 50:1.

[0065] Example 6: This example provides a method for preparing CPJP granules, as detailed below: The CPJP complex prepared in Example 1 was mixed with lactose, dextrin, and steviol glycosides to obtain a mixed powder. Purified water was added to the mixed powder to prepare a soft mass, which was then fed into a vibrating granulator for wet granulation to obtain wet granules. After granulation, the wet granules were placed in a hot air circulating oven for drying at 50°C. After drying, the granules were granulated again and sieved to obtain 30-mesh particles, thus obtaining CPJP granules. The mass ratio of CPJP complex to lactose was 5:6, the mass ratio of CPJP complex to dextrin was 4:1, and the mass ratio of CPJP complex to steviol glycosides was 125:1; the mass ratio of the mixed powder to purified water was 50:9.

[0066] Example 7: This example provides a method for preparing CPJP granules, as detailed below: The CPJP complex prepared in Example 1, 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one, lactose, dextrin, and steviol glycosides were thoroughly mixed to obtain a mixed powder. Purified water was added to the mixed powder to prepare a soft mass. The soft mass was then fed into a vibrating granulator for wet granulation to obtain wet granules. After granulation, the wet granules were placed in a hot air circulating oven for drying at 50°C. After drying, the granules were granulated again and sieved to obtain 30-mesh granules, thus obtaining CPJP granules. The mass ratio of CPJP complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one was 100:7; the mass ratio of CPJP complex to lactose was 5:6; the mass ratio of CPJP complex to dextrin was 4:1; the mass ratio of CPJP complex to steviol glycosides was 125:1; and the mass ratio of the mixed powder to purified water was 50:9.

[0067] Example 8: The only difference between this example and Example 7 is that the mass ratio of the CPJP complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one in Example 8 is adjusted from 100:7 to 10:1.

[0068] Example 9: This example provides a method for preparing CPJP granules, as detailed below: The CPJP complex prepared in Example 1, 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one, valerian root extract, lactose, dextrin, and steviol glycosides were thoroughly mixed to obtain a mixed powder. Purified water was added to the mixed powder to prepare a soft mass. The soft mass was fed into a swing granulator for wet granulation to obtain wet granules. After granulation, the wet granules were placed in a hot air circulating oven for drying at a temperature of 50°C. After drying, the granules were granulated again and sieved to obtain 30-mesh granules, thus obtaining CPJP granules. The valerian root extract uses valeric acid as the quality control component, with a labeled content of 0.8%; the mass ratio of CPJP complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one is 100:10, the mass ratio of CPJP complex to valerian root extract is 100:3; the mass ratio of CPJP complex to lactose is 5:6, the mass ratio of CPJP complex to dextrin is 4:1, the mass ratio of CPJP complex to steviol glycosides is 125:1; and the mass ratio of mixed powder to purified water is 50:9.

[0069] Comparative Example 1: The only difference between this comparative example and Example 3 is that the CPJP complex in Comparative Example 1 does not contain pyrroloquinoline quinone.

[0070] Comparative Example 2: The only difference between this comparative example and Example 3 is that the CPJP complex in Comparative Example 2 does not contain jujube seed extract.

[0071] Comparative Example 3: The only difference between this comparative example and Example 3 is that the CPJP complex in Comparative Example 3 does not contain Cistanche deserticola extract.

[0072] Comparative Example 4: The only difference between this comparative example and Example 3 is that the CPJP complex in Comparative Example 4 does not contain kudzu root extract.

[0073] Comparative Example 5: This comparative example differs from Example 7 only in that the mass ratio of the CPJP complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one in Comparative Example 5 is adjusted to 100:3.

[0074] Comparative Example 6: This comparative example differs from Example 7 only in that the 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one in Example 7 is adjusted to L Ascorbic acid, CPJP complex and L The mass ratio of ascorbic acid is 100:7.

[0075] Comparative Example 7: This comparative example differs from Example 9 only in that in this Comparative Example 7, the valerian root extract in Example 9 is adjusted to a licorice extract, with glycyrrhizic acid as the quality control component of the licorice extract, the labeled content of glycyrrhizic acid is 2.5%, and the mass ratio of the CPJP complex to the licorice extract is 100:3.

[0076] Test Example 1: Test of the effects of the CPJP complex on the general state, estrous cycle and serum biochemical indicators of cyclophosphamide-injured mice.

[0077] Test samples: CPJP suspensions prepared in Examples 2 to 4.

[0078] Test method: 50 specific pathogen free (SPF) female ICR mice aged 6 to 8 weeks, weighing 18 to 22 g, were purchased from Shanghai Slac Laboratory Animal Co., Ltd., with the experimental animal production license number SCXK (Hu) 2017-0005. The animals were housed in the barrier environment of the Laboratory Animal Center of Zhejiang Chinese Medical University. The housing conditions were set as room temperature (22±2) °C, relative humidity (50±10)%, 12h light / 12h dark photoperiod, and the animals had free access to food and water. Adaptive feeding was carried out for 7 days before the formal experiment. This animal experiment was reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang Chinese Medical University, with the ethical approval number IACUC-20220926-10, and all experimental operations followed the relevant specifications of laboratory animal welfare and ethics.

[0079] After the adaptive feeding, vaginal smear screening was carried out continuously, and mice with normal estrous cycle rhythm were selected for the experiment. The qualified mice were randomly divided into 5 groups: normal control group, model group, CPJP low-dose group, CPJP medium-dose group, and CPJP high-dose group, with 10 mice in each group. Except for the normal control group, the other groups were established with a cyclophosphamide-induced ovarian reserve injury model: on the first day, cyclophosphamide was intraperitoneally injected at 100 mg / kg; from day 2 to day 14, cyclophosphamide was intraperitoneally injected at 20 mg / kg daily. After the modeling was completed, the cell composition was observed through vaginal smears, and the modeling was judged to be successful when the mice showed estrous cycle disorder. The CPJP low-dose group used the CPJP low-dose suspension prepared in Example 2, the CPJP medium-dose group used the CPJP medium-dose suspension prepared in Example 3, and the CPJP high-dose group used the CPJP high-dose suspension prepared in Example 4. The normal control group and the model group were intragastrically administered with an equal volume of normal saline daily; each CPJP dose group was intragastrically administered with the CPJP suspension of the corresponding concentration, the intragastric administration volume was uniformly 10 mL / kg, administered once a day for 21 consecutive days; during the administration process, the suspension needs to be shaken and mixed intermittently to avoid sedimentation of solid components.

[0080] Mice body weight was continuously monitored throughout the experimental period. Vaginal smears were examined daily from 8:00 AM to 9:00 AM: a sterile cotton swab dipped in a small amount of physiological saline was gently inserted 3-5 mm into the mouse vaginal opening and rotated to collect the sample. Exfoliated cells were evenly spread on a glass slide, stained with Giemsa stain, and observed under a light microscope to determine cell morphology. The estrous stage was determined based on the proportion of nucleated epithelial cells, keratinized epithelial cells, and leukocytes in the smear: proestrus was dominated by nucleated epithelial cells; estrus was dominated by a large number of anucleated keratinized epithelial cells; mesestrus showed a mixture of keratinized epithelial cells and leukocytes; and mitrus was dominated by leukocytes, accompanied by a small number of epithelial cells. The proportion of each estrous stage was statistically analyzed for each group of mice.

[0081] After the last administration, five mice were randomly selected from each group, and whole blood was collected using the ocular blood sampling method. Blood samples were allowed to stand at room temperature for 30 minutes to fully coagulate, then centrifuged at 4°C and 3000 rpm for 10 minutes. The supernatant serum was separated, aliquoted, and stored at -80°C to avoid repeated freeze-thaw cycles. Commercially available kits were used to detect serum anti-Müllerian hormone (AMH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Cr). AMH was detected using a mouse AMH enzyme-linked immunosorbent assay kit (Elabscience, E-EL-M3015); ALT and AST were detected using an ALT / AST combined detection kit (Nanjing Jiancheng Bioengineering Institute, Z002-1-1); and Cr was detected using a creatinine detection kit (Nanjing Jiancheng Bioengineering Institute, C011-2-1). All samples were added, incubated, and subjected to color development and absorbance measurement strictly according to the kit instructions. The results of each serum indicator were obtained by referring to the kit's standard curve or calculation rules. Body weight data are expressed as mean ± standard deviation (SD); serum biochemical indicators are expressed as mean ± standard error (SEM); one-way ANOVA was used for multiple normally distributed data, and Tukey's test was used for multiple comparisons between groups; p < 0.05 was considered statistically significant.

[0082] Test results: Microscopic images of vaginal smears from different stages of the estrous cycle in mice are shown below. Figure 1 As shown; the statistical results of the proportion of different stages of the estrous cycle in each group of mice are as follows. Figure 2 As shown in the figure, the estrous cycle rhythm of the model group mice was significantly disrupted; after intervention with the CPJP complex, the distribution of the estrous cycle in each dose group was improved, with the high-dose group showing a significant increase in the proportion of the estrous phase. The body weight changes of each group of mice throughout the entire experimental period are shown in the figure. Figure 3As shown, the body weight gain of the model group mice was inhibited, while the body weight gain of the mice in each CPJP intervention group was restored to varying degrees. The serum AMH and ALT levels of the mice in each group are shown in the figures below. Figure 6 As shown; serum AST and Cr test results are as follows Figure 7 As shown in the figure, compared with the normal control group, the serum AMH levels of the model group mice were significantly reduced, while the ALT, AST, and Cr levels were significantly increased, suggesting that cyclophosphamide modeling not only caused a decrease in ovarian reserve but also induced abnormalities in liver and kidney function. AMH is mainly secreted by granulosa cells of growing follicles, and the recovery of this index suggests that CPJP complex intervention can improve the follicle-related endocrine status of model mice. The serum AMH level in the high-dose CPJP group was significantly higher than that in the model group; the ALT levels in all intervention groups were lower than those in the model group; the AST and Cr levels in the medium- and high-dose groups were significantly reduced, while the Cr level in the low-dose group was not statistically different from that in the model group, suggesting that the improvement of liver and kidney biochemical indicators is dose-dependent, and the improvement effect of medium- and high-dose intervention is more prominent. The results of this experiment show that the CPJP complex can improve the estrous cycle disorder in cyclophosphamide-induced model mice, increase serum AMH levels, and reduce the abnormally elevated levels of serum ALT, AST, and Cr in model mice.

[0083] Experimental Example 2: Effect of CPJP complex on in vitro nuclear maturation capacity of GV stage oocytes.

[0084] Test samples: CPJP suspensions prepared in Examples 2-4, and test animals were the same as in Example 1.

[0085] Test Method: Approximately 16 hours after the last administration, mice used for in vitro oocyte maturation experiments were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) at a dose of 5 IU / mouse. 44-48 hours after injection, the mice were sacrificed, and the ovaries were aseptically removed and placed in preheated M2 culture medium at 37°C. The ovarian cortex was punctured using a 29G syringe needle to release the cumulus-oocyte complex (COC). The COC was then treated with 80 IU / mL hyaluronidase at 37°C for 2-3 minutes, followed by gentle pipetting to remove cumulus cells. Under a stereomicroscope, GV-stage oocytes with homogeneous cytoplasm, intact membranes, and clearly defined germinal vesicles were selected for subsequent culture. The GV-stage oocytes were transferred to in vitro maturation culture medium (α-MEM basal medium supplemented with 10% fetal bovine serum and 10 mIU / mL follicle-stimulating hormone (FSH)) and cultured in a 37°C, 5% CO2 saturated humidity incubator. After culturing for 3-4 hours, the number of germinal follicle rupture (GVBD) oocytes was counted, and the germinal follicle rupture rate was calculated. After 16 hours of in vitro maturation culture, the number of MII stage oocytes that extruded the first polar body (PBE) was counted, and the first polar body extrusion rate was calculated. GVBD rate (%) = number of germinal follicle rupture oocytes / total number of GV stage cultured oocytes × 100%; PBE rate (%) = number of MII stage oocytes that extruded the first polar body / total number of GV stage cultured oocytes × 100%. The number of effective oocyte samples in each group of this experiment were: normal control group 52 oocytes, model group 88 oocytes, low-dose CPJP group 88 oocytes, medium-dose CPJP group 62 oocytes, and high-dose CPJP group 41 oocytes. The number of oocytes obtained was influenced by individual differences in ovarian reserve in mice, the loss during puncture and sampling, and the oocyte morphology screening criteria. In the model group, mice experienced abnormal follicle depletion and inconsistent oocyte quality after cyclophosphamide modeling, resulting in a larger pool of qualified samples. The high-dose intervention group showed more significant ovarian damage repair, leading to more stringent qualified oocyte screening criteria. Furthermore, a small number of oocytes with slight morphological abnormalities were excluded after high-dose administration, ultimately resulting in a gradient in sample size among the groups. All sample screening criteria were consistent throughout the process. GVBD and PBE rates were categorical data; contingency tables were constructed, and two-sided Fisher's exact test was used for inter-group comparisons. A p-value < 0.05 was considered statistically significant.

[0086] Test results: The statistical results of oocyte germinal follicle rupture rate and first polar body expulsion rate in each group are as follows: Figure 4 As shown; bright-field micrograph of oocytes as follows Figure 5As shown in the figure, compared with the normal control group, the germinal follicle rupture rate and first polar body expulsion rate of oocytes in the model group were significantly decreased. After intervention with CPJP complex, both indicators in each dose group rebounded compared with the model group, and the germinal follicle rupture rate in the high-dose group was close to the level of the normal control group. This result indicates that CPJP complex can promote the recovery of meiosis in oocytes damaged by cyclophosphamide and enhance the oocyte nuclear maturation capacity.

[0087] Experimental Example 3: Effects of CPJP complex on reactive oxygen species and mitochondrial functional phenotype in oocytes.

[0088] Test samples: CPJP suspensions prepared in Examples 2-4, with test animals and oocytes obtained in the same manner as in Example 2.

[0089] Test method: The intracellular reactive oxygen species (ROS) level of oocytes was detected using the DCFH-DA probe with a working solution concentration of 10 μmol / L. Oocytes were placed in the probe working solution and incubated at 37°C in the dark for 20 min. The preheated working solution was used to thoroughly wash away any residual probes outside the cells. The intensity of DCF green fluorescence represents the intracellular ROS level.

[0090] Functional mitochondria were labeled with Mito-Tracker Red CMXRos, and the nuclei were counterstained with Hoechst 33342. Oocytes were first incubated in mitochondrial probe working solution at 37°C in the dark for 20 min, followed by incubation with nuclear dye for 10 min in the dark. After washing three times, the cells were immediately subjected to fluorescence imaging. The fluorescence signal of Mito-Tracker Red CMXRos depends on the mitochondrial membrane potential, and the fluorescence intensity can reflect the level of functionally active mitochondria. All samples were acquired using identical microscope objectives, exposure time, gain, and other image acquisition parameters. Individual oocytes were selected as regions of interest (ROIs) using ImageJ / Fiji software, and the corrected cell fluorescence intensity was obtained after background noise subtraction. A uniform image analysis threshold was used for the same detection index. Oocyte fluorescence quantitative data were presented using violin plots, with each oocyte representing a statistical sample. Multiple comparisons were performed using one-way ANOVA combined with Tukey multiple comparisons; p < 0.05 was considered statistically significant. It should be noted that the Mito-Tracker Red CMXRos signal is regulated by mitochondrial membrane potential and reflects functional mitochondrial signaling, but is not equivalent to the total number of mitochondria.

[0091] Test results: Oocyte reactive oxygen species, mitochondrial tracer probes, nuclear fluorescent dye fluorescence imaging and magnified local images are shown below. Figure 8 As shown; the statistical results of the relative fluorescence intensity of reactive oxygen species and mitochondrial probes in oocytes are as follows: Figure 9As shown in the figure, compared with the normal control group, the fluorescence intensity of ROS in oocytes of the model group was increased, while the fluorescence intensity of mitochondrial probes was decreased. This indicates that cyclophosphamide damage caused an increase in oxidative stress in oocytes and a decrease in the number of functional mitochondria with membrane potential. Oxidative stress and mitochondrial dysfunction can mutually exacerbate each other. After intervention with the CPJP complex, the ROS level in oocytes of the low and medium dose groups was significantly downregulated, demonstrating the effect of clearing excess reactive oxygen species in cells. The fluorescence intensity of mitochondrial probes in the high dose group was significantly increased, suggesting that high doses are more conducive to restoring the functional phenotype related to mitochondrial membrane potential in oocytes. It should be noted that the Mito-Tracker Red CMXRos signal is regulated by mitochondrial membrane potential. This result represents an improvement in functional mitochondrial signaling, but is not equivalent to an increase in the total number of mitochondria. The results of this experiment show that the two indicators, ROS and mitochondrial function, do not show a simple linear dose-response relationship, indicating that the appropriate intervention dose varies for different biological endpoints. The above results suggest that the CPJP complex can regulate the oxidative stress state of oocytes, and high-dose intervention can significantly improve mitochondrial function in oocytes.

[0092] Experiment 4: Effects of CPJP complex on spindle and F-actin structure in MII oocytes.

[0093] Test samples: CPJP suspension prepared in Examples 2-4, and oocyte samples were taken from MII stage oocytes after in vitro maturation in Example 2.

[0094] Test Method: From fully matured oocytes, MII stage oocytes containing intact first polar bodies were selected; after washing with pre-warmed PBS, they were fixed in 4% paraformaldehyde fixative at 37°C for 30 min; washed with PBS-T, and permeabilized with 0.5% Triton X-100 at room temperature for 20 min; blocked with blocking buffer containing 5% BSA and 0.1% Tween-20 at room temperature for 1 h. Oocytes were then incubated overnight at 4°C with α-tubulin primary antibody (Beyotime, dilution 1:1000); after thorough washing, Alexa Fluor 488 secondary antibody (dilution 1:500) corresponding to the species was added, and incubated at room temperature in the dark for 1 h; then labeled with TRITC-Phalloidin (dilution 1:200) in the dark for 30 min; chromosomes were counterstained with DAPI dye. After staining and washing, anti-fluorescence quenching mounting buffer was added to complete the mounting. Images were acquired using a fluorescence microscope, with uniform imaging parameters maintained across all groups. The criteria for determining spindle morphology were as follows: A symmetrical bipolar spindle shape with continuous microtubule structure was considered normal; unipolar / multipolar spindles, broken and scattered microtubules, asymmetrical structures, and ectopic aggregation were considered abnormal. The abnormal spindle rate (%) was calculated as: (Number of oocytes with abnormal spindle morphology / Total number of oocytes) × 100%. The F-actin fluorescence signal region was selected using ImageJ / Fiji software, and the relative fluorescence intensity of phalloidin was calculated after background subtraction. The abnormal spindle rate was used for count data, employing a two-sided Fisher's exact test; phalloidin fluorescence intensity was assessed using one-way ANOVA with Tukey multiple comparisons.

[0095] Test results: Microtubules, phalloidin, and nuclear fluorescent dyes in MII stage oocytes were analyzed using fluorescence imaging and magnified local images, as shown below. Figure 10 As shown; the statistical results of oocyte spindle abnormality rate and relative fluorescence intensity of phalloidin are as follows. Figure 11As shown, F-actin is involved in oocyte spindle migration, cell polarity establishment, and first polar body expulsion. Increased fluorescence signal can represent abnormal F-actin accumulation or compensatory remodeling. The Phalloidin fluorescence intensity in oocytes of the model group was significantly higher than that of the normal control group, suggesting abnormal F-actin accumulation in damaged oocytes of the model. After intervention with the CPJP complex, the Phalloidin fluorescence intensity in the low, medium, and high dose groups was significantly lower than that in the model group, with the medium dose group showing the most significant decrease. This indicates that CPJP can alleviate abnormal F-actin remodeling in damaged oocytes, a phenotype that facilitates first polar body expulsion. Statistical results showed a statistically significant difference in the overall abnormal spindle rate among the five groups, but no statistically significant differences were found between the model group and each intervention group. This phenomenon is speculated to be related to the limited sample size of oocytes in each group and the insufficient power of count-based tests in this experiment. The results indicate that the CPJP complex can improve abnormal F-actin remodeling in chemotherapy-damaged oocytes. There were statistically significant differences in the overall spindle rate distribution among the five groups, but there were no statistically significant differences between the model group and each intervention group. This is likely due to the limited sample size of oocytes in this group, resulting in insufficient power of the counting indicators. The outstanding protective effect of this complex on oocyte structure lies in improving abnormal F-actin remodeling. The protective effect on spindle morphology needs to be further verified by expanding the sample size.

[0096] Experimental Example 5: Effects of CPJP complex on oocyte in vitro fertilization and preimplantation embryonic development.

[0097] Test samples: CPJP suspensions prepared in Examples 2-4, and test animals were the same as in Example 1.

[0098] Test Methods: Adult male ICR mice of the same strain and with normal fertility were selected and euthanized by cervical dislocation. The epididymal tail was quickly separated and placed in pre-equilibrated HTF fertilization solution. The epididymal tail was cut open to release sperm, which were incubated at 37°C and 5% CO2 for 30-60 minutes to complete sperm capacitation. The upper layer of progressively motile sperm was aspirated, counted, and their motility was assessed. The sperm concentration was adjusted for later use. For female mice used in in vitro fertilization, PMSG 7.5 IU / mouse was injected intraperitoneally. 46-48 hours later, human chorionic gonadotropin (hCG) 7.5 IU / mouse was injected to induce ovulation. Mice were euthanized 15 hours after hCG injection, and the oviduct was quickly removed and placed in pre-warmed HTF fertilization solution droplets. Under a stereomicroscope, the ampulla of the oviduct was torn open to release the cumulus-oocyte complex (COC), which was then transferred to pre-equilibrated fertilization droplets. Capacitated sperm were added to the COC-containing fertilization droplets to adjust the final sperm concentration to 1 × 10⁻⁶. 6Cells / mL; incubated at 37℃ and 5% CO2 for 4-6 hours. After fertilization and incubation, cumulus cells and unconjugated sperm were gently removed by pipetting; zygotes with two pronuclei (2PN) were identified as normal fertilized eggs. 2PN zygotes were washed and transferred to paraffin oil-coated, pre-equilibrated KSOM embryo culture medium droplets for continued in vitro culture. Embryo development was observed at regular intervals using hCG injection time as the benchmark: 2-cell embryos were counted 42-44 hours after hCG injection; 4-cell embryos were counted 54-56 hours after injection, observing blastomeres size and cleavage regularity; morulae were counted 80-90 hours after injection, observing the degree of compaction; and blastocysts were counted 96-100 hours after injection, observing blastocoel expansion, inner cell mass, and trophectoderm differentiation morphology. Fertilization rate (%) = number of 2PN zygotes / total number of oocytes participating in in vitro fertilization (MII) × 100%; 2-cell cleavage rate (%) = number of 2-cell embryos / number of 2PN zygotes × 100%; 4-cell embryo rate (%) = number of embryos developing to the 4-cell stage / number of 2PN zygotes × 100%; Morula rate (%) = number of morula-forming embryos / number of 2PN zygotes × 100%; Blastocyst formation rate (%) = number of blastocyst-forming embryos / number of 2PN zygotes × 100%. Embryo development rates at each post-fertilization stage were calculated using 2PN normal fertilized oocytes as the denominator. Embryo development-related indicators were expressed as mean ± standard error using SEM; one-way ANOVA combined with Tukey multiple comparisons were used; P < 0.05 was considered statistically significant.

[0099] Test results: Bright-field micrographs of 2-cell stage embryos, 4-cell stage embryos, morula, blastocysts, and localized areas of embryos in each group are shown below. Figure 12 As shown; the statistical results of oocyte fertilization rate and 4-cell embryo rate are as follows. Figure 13 As shown; the statistical results of morula rate and blastocyst rate are as follows. Figure 14 As shown in the figure, the oocyte fertilization capacity and subsequent embryonic development in the model group were significantly impaired, with a fertilization rate of only 9.93% and a blastocyst formation rate as low as 0.74%. This indicates that the oocyte damage caused by cyclophosphamide is not limited to the oocyte maturation stage but also continuously impairs the continuous development of the embryo before implantation. With intervention from the CPJP complex, oocyte fertilization and embryonic development indicators at all stages were improved, with the high-dose group showing the most significant improvement; the fertilization rate in the high-dose group increased to 30.26%, and the blastocyst formation rate increased to 16.82%. This result demonstrates that the improvement effect of the CPJP complex on oocyte maturation and cytoplasmic quality can be further translated into the restoration of oocyte fertilization capacity and the continuous development of the embryo before implantation. Combining the aforementioned multiple indicators, it is evident that the optimal response dose for different CPJP endpoints is not entirely consistent; the dose effect is prominent in some indicators, and the improvement effect of high doses is more significant at reproductive development endpoints.

[0100] Experimental Example 6: Effect of CPJP complex on ovarian organ index in mice with cyclophosphamide injury.

[0101] Test Sample: Example 2 4. The suspensions prepared in comparison examples 1-4.

[0102] Test method: The animal modeling, grouping, and administration regimen were the same as in Experiment 1. After the last administration, both ovaries were dissected and removed. The adipose tissue attached to the ovaries was carefully removed, and the wet weight of the ovaries was measured using a precision electronic balance. The ovarian organ index was calculated as follows: Ovarian organ index = Total wet weight of both ovaries (mg) / Mouse body weight (g) × 100%.

[0103] The statistical results of ovarian organ indices in each group of mice are shown in Table 1.

[0104] Table 1. Statistical results of ovarian organ index in mice of each group

[0105] Test Results: Compared with the normal control group, the ovarian organ index of the model group mice was significantly decreased. Combined with the follicle and oocyte-related phenotypes observed in the aforementioned experiments of this invention, it indicates that cyclophosphamide chemotherapy damage not only causes massive follicle depletion but also leads to overall ovarian tissue atrophy. The ovarian organ index can directly reflect the overall degree of ovarian damage. Example 2 was given. 4. After intervention with different doses of the complete four-component CPJP complex, the ovarian organ index showed a dose-response relationship of gradual recovery with increasing dosage; among them, the high-dose group in Example 4 showed the largest recovery of the ovarian organ index, which was closest to the level of the normal control group, indicating that the complete CPJP complex can effectively antagonize ovarian tissue atrophy and damage induced by cyclophosphamide. Comparison with the default three-component comparative example 1 4. It can be observed that after removing any one of the active components—pyrroloquinolinequinone, jujube seed extract, cistanche extract, and kudzu root extract—the effect of the composition in increasing the ovarian organ index was significantly reduced. The ovarian organ index of all comparative examples was significantly lower than that of the high-dose group of Example 4 with the complete four components. Each of the four raw materials plays an irreplaceable role in the protection of ovarian tissue. The absence of any one component reduces the overall protective effect, and the degree of efficacy reduction due to the absence of different components varies. Based on the above trends, it can be concluded that only the combination of kudzu root extract, cistanche extract, jujube seed extract, and pyrroloquinolinequinone can achieve more comprehensive protection of chemotherapy-damaged ovarian tissue. Retaining only any three of these components cannot achieve the protective effect achievable by the complete composition.

[0106] Experiment 7: Effect of CPJP complex on serum malondialdehyde levels in mice Test samples: Granules prepared in Examples 6-9 and granules prepared in Comparative Examples 5-7.

[0107] Test methods: All granules were resuspended in physiological saline to prepare the corresponding concentration for gavage before use to ensure a consistent drug delivery system. Animal modeling, grouping, drug administration, and serum collection were performed in the same manner as in Experiment 1. Serum isolated from mice in each group was collected, and serum malondialdehyde (MDA) levels were measured using a commercially available MDA detection kit. Sample addition, incubation, and colorimetric determination were strictly performed according to the kit instructions. MDA is used to evaluate the degree of lipid peroxidation damage in the body.

[0108] The results of serum MDA detection in each group of mice are shown in Table 2.

[0109] Table 2. Results of MDA detection in mouse serum in each group.

[0110]

[0111] Test Results: Compared to the normal control group, the serum MDA level in the model group mice was significantly increased, indicating that cyclophosphamide modeling induced significant lipid peroxidation damage in mice. Example 6, a CPJP basic granule formulation without additional active additives, significantly reduced serum MDA levels in model mice, demonstrating that the CPJP complex itself has the ability to alleviate lipid peroxidation damage. Comparative Example 6 As can be seen from Figure 8, the addition of 3,8-dimethylamine to the CPJP complex... dihydroxy 6H Dibenzo[B,D]pyran 6 Following the addition of ketones, the formulation's effect in reducing MDA was further enhanced, and the improvement effect showed a gradually increasing trend with the increase of the active ingredient ratio; Example 9 involved the simultaneous compounding of 3,8 dihydroxy 6H Dibenzo[B,D]pyran 6 Following the addition of ketones and valerian root extract, the effect of reducing lipid peroxidation damage was the best among all granule formulations in this group. Further investigation was conducted in Comparative Example 5. 7. Comparative Example 5 showed a weaker effect in reducing MDA compared to Examples 7 and 8, indicating that insufficient addition of the active ingredient cannot fully realize the beneficial effects of the compounding process; Comparative Example 6 used L Ascorbic acid replacement 3,8 dihydroxy 6H Dibenzo[B,D]pyran 6 The effect of ketones in improving oxidative damage was not as good as that in Example 7 with the same formulation; in Comparative Example 7, licorice extract was used instead of valerian root extract, and its overall effect was weaker than that in Example 9. Based on the above trends, it can be seen that although the CPJP basic complex itself has antioxidant properties, selecting specific active ingredients for compounding can bring further enhancements; replacing it with other common antioxidant raw materials in the art or reducing the proportion of characteristic active ingredients makes it difficult to achieve the effect of Example 7 of the present invention. The overall effect of the modified granule formulation corresponding to 9. This proves that the modified granule formulation of the present invention is not simply achieved by mixing ordinary antioxidants with the CPJP complex. The selection of specific active substances and the range of their proportions play a key role in the final anti-oxidative damage effect of the composition.

[0112] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0113] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A complex for improving diminished ovarian reserve, characterized in that: Including kudzu root extract, cistanche extract, jujube seed extract, and pyrroloquinoline quinone; The kudzu root extract uses puerarin as the quality control component, and the puerarin content is labeled as 95%~99%. The Cistanche deserticola extract uses echinacoside as the quality control component, with a labeled echinacoside content of 70%~80%. The jujube seed extract uses jujube seed saponins as the quality control component, and the jujube seed saponin content is labeled as 8%~10%.

2. The complex according to claim 1, characterized in that: The mass ratio of kudzu root extract to cistanche extract is 7.5~8.5:9.0~11.0, the mass ratio of kudzu root extract to jujube seed extract is 7.5~8.5:75.0~85.0, and the mass ratio of kudzu root extract to pyrroloquinoline quinone is 7.5~8.5:1.5~2.

0.

3. A compound formulation, characterized in that: The formulation includes the complex according to any one of claims 1-2, and the formulation includes one or more of suspensions, tablets or granules.

4. The complex formulation according to claim 3, characterized in that: The suspension is prepared by adding the complex to physiological saline, stirring and mixing, vortexing for 2-4 minutes, and then sonicating: ultrasonic power 180-220W, single ultrasonic working time 25-35s, interval 15-25s, cycle 2-4 times to obtain the suspension.

5. The complex formulation according to claim 3, characterized in that: The method for preparing the granules is as follows: the complex is mixed with lactose, dextrin and steviol glycosides to obtain a mixed powder; purified water is added to the mixed powder to prepare a soft material; the soft material is fed into a swing granulator for wet granulation to obtain wet granules; the wet granules are granulated and then placed in a hot air circulating oven for drying; after drying, they are granulated again and sieved to obtain granules.

6. The complex formulation according to claim 5, characterized in that: The mixed powder comprises 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one.

7. The complex formulation according to claim 6, characterized in that: The mass ratio of the complex to 3,8-dihydroxy-6H-dibenzo[B,D]pyran-6-one is 100:3~10.

8. The preparation for improving diminished ovarian reserve according to claim 5, characterized in that: The mass ratio of the complex to lactose is 4~6:5~7, the mass ratio of the complex to dextrin is 3~5:0.8~1.2, and the mass ratio of the complex to steviol glycosides is 110~140:

1.

9. The use of the complex of any one of claims 1-2 or the complex formulation of any one of claims 3-8 in the preparation of a drug for improving chemotherapy-induced ovarian reserve decline.

10. The use of the complex of any one of claims 1-2 or the complex formulation of any one of claims 3-8 in the preparation of products that simultaneously improve ovarian reserve endocrine indicators and / or oocyte maturation capacity and / or cytoplasmic function, and / or subsequent embryonic development capacity.

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