New medical application of icariin

Abstract

The invention relates to application of icariin in preparation of a medicine for treating ovarian reserve function decline diseases. The ovarian reserve function decline disease is infertility caused by ovarian reserve function decline. It is found through experiments that icariin improves the number of DOR mouse sinus follicles; the effect of icariin on the hormone level of a DOR mouse; the influence of icariin on DOR mouse ovarian apoptosis; the influence of icariin on the ovary mitochondrial function of the DOR mouse; icariin (ICA) can be used for increasing the proliferation of ovarian granular cells induced by CTX; icariin can improve the mitochondrial function; the icariin can improve the mitochondrial membrane potential and reduce the generation of active oxygen. The traditional Chinese medicine monomer icariin has a considerable clinical application prospect in improvement of mouse ovarian reserve function decline.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to a new medical application of icariin. Background Art

[0002] DOR (poor ovarian reserve) is due to a decrease in the number and / or quality of oocytes, resulting in insufficient ovarian function and decreased fertility, accompanied by decreased AMH levels, decreased AFC, and increased basal FSH levels.

[0003] Icariin is a compound extracted from the dried leaves of the Berberidaceae plants Epimedium, Epimedium sagittatum, Epimedium pubescens or Epimedium koreana. This product is a light yellow needle-shaped crystalline powder with a molecular formula of C 33 H 40 O 15 , molecular weight is 676.65.

[0004] Chinese patent document CN109528980A discloses a traditional Chinese medicine composition for treating diseases of diminished ovarian reserve function, characterized in that it is made of the following raw materials in parts by weight: 4-30 parts of Rehmannia root, 3-25 parts of Polygonatum sibiricum, 3-20 parts of Viscum album, 3-20 parts of Dipsacus asper, 3-20 parts of Ligustrum lucidum, 3-20 parts of Lycium barbarum, 3-25 parts of Salvia miltiorrhiza, and 2-20 parts of Curcuma aromatica.

[0005] Chinese patent document CN 101129396A discloses the use of icariin in the preparation of drugs for treating Alzheimer's disease and the product thereof. The icariin is a flavonol glycoside compound extracted from the genus Epimedium of the Berberidaceae family. The pharmaceutical preparation of the present invention is a single prescription preparation made from icariin as a raw material. Compared with the prior art, the raw materials and preparation process used in the present invention are simple, the cost is low, the efficacy is significant, the dosage is small, and there is no toxic side effect, which provides a reliable drug for the treatment of Alzheimer's disease.

[0006] Chinese patent document CN 101590071A discloses an application of icariin as a drug for the preparation of a drug for the treatment of acute cerebral infarction. The present invention successfully converts icariin into a drug for the treatment of acute cerebral infarction, utilizing the anti-oxidative stress effect and affecting the expression and activity of downstream peroxisome proliferator-activated receptors, thereby allowing the multifunctional co-activator PGC-1A to participate in regulating mitochondrial oxidative metabolism and, as a key link in regulating mitochondrial biosynthesis, mediate mitochondrial biosynthesis under certain physiological and pathological conditions, thereby playing an important protective role in ischemic brain damage, including cerebral infarction.

[0007] However, there are no reports on the Chinese medicine monomer icariin improving the ovarian reserve function impairment in mice. Summary of the invention

[0008] The purpose of the present invention is to provide a new medical use of icariin in view of the deficiencies in the prior art.

[0009] In a first aspect, the present invention provides the use of icariin in preparing a medicine for the disease of diminished ovarian reserve function.

[0010] As a preferred example, the disease of diminished ovarian reserve function is infertility caused by diminished ovarian reserve function.

[0011] As another preferred example, the drug is added with pharmaceutically acceptable excipients and carriers to prepare a pharmaceutical preparation, and the excipients include at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener and a colorant.

[0012] The invention has the advantages that: the invention experimentally found that: the icariin prescription improves the number of antral follicles in DOR mice; the effect of icariin on the hormone level of DOR mice; the effect of icariin on ovarian apoptosis in DOR mice; the effect of icariin on ovarian mitochondrial function in DOR mice; icariin (ICA) can increase the proliferation of ovarian granulosa cells after CTX induction; icariin can improve mitochondrial function; icariin can increase mitochondrial membrane potential and reduce the generation of reactive oxygen. The Chinese medicine monomer icariin improves the ovarian reserve function of mice and has considerable clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : HE staining of ovaries and the number of antral follicles in each group of mice (compared with the model group, *P<0.05,

[0014] ***P<0.001, ****P<0.0001).

[0015] Figure 2 : The expression levels of AMH, E2 and FSH in serum of mice in each group (compared with the model group, *P<0.05,

[0016] **P<0.01, ***P<0.001, ****P<0.0001).

[0017] Figure 3 : TUNEL staining of ovaries of mice in each group (compared with the model group, *P<0.05, **P<0.01, ***P<0.001).

[0018] Figure 4 : Ovarian mitochondrial morphology and serum ROS content of mice in each group (compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0019] Figure 5: CCK8 cell proliferation ability.

[0020] Figure 6 : Mitochondrial morphology of each group.

[0021] Figure 7 :Expression of reactive oxygen species and mitochondrial membrane potential in each group. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0023] Example 1 Animal Experiment

[0024] 1. Animals and Grouping

[0025] This experiment used 50 6-week-old SPF C57BL / 6 female mice, weighing 20-23g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. (License No.: 2024-0001). The use and operation strictly followed the corresponding requirements and regulations of the Animal Experiment Center Management Committee of Shanghai Institute of Biomedical Technology (Ethics No.: PJ2021-49). The 50 mice were divided into 5 groups by body weight random block method, namely the control group, model group, icariin low-dose group, icariin medium-dose group, and icariin high-dose group, with 10 mice in each group.

[0026] 1. Drug Preparation

[0027] 1.1 Icariin:

[0028] Icariin (purity: 99.06%, HY-N0014) was purchased from MCE. The oral dose for each mouse was 12.33 times that of a 60 kg human body weight, and the amount of Chinese medicine solution for each oral administration was 0.15 ml.

[0029] 1.2 Cyclophosphamide solution:

[0030] Cyclophosphamide (CTX) was purchased from MCE and prepared into 18.5 mg / mL cyclophosphamide solution.

[0031] 2. Modeling and intervention;

[0032] Except for the control group, the mice in the four groups were injected with CTX at 90 mg / kg / d (the control group was injected with normal saline) for 7 days. The mice in the low, medium and high dose groups of icariin were treated by gavage for 14 days after injection of CTX (high dose: 200 mg / kg / d; medium dose: 100 mg / kg / d; low dose: 50 mg / kg / d), and the control group and the model group were gavaged with normal saline. One day after the intervention, the mice were anesthetized with isoflurane and blood was collected from the eyeballs. The serum was obtained by centrifugation and stored in a refrigerator at -80 degrees. After being killed by cervical dislocation, the ovaries were removed by laparotomy, and one ovary was placed in 4% paraformaldehyde, and the other ovary was placed in electron microscopy fixative.

[0033] 3. HE staining:

[0034] The sections were dewaxed and hydrated, placed in xylene, anhydrous ethanol, and 75% ethanol in sequence, then washed with distilled water and dried. Stained with hematoxylin, differentiated with 1% hydrochloric acid alcohol, blued with 0.6% ammonia water, stained with eosin, dehydrated with ethanol, placed in xylene until transparent, and sealed with neutral glue. The number of mouse ovarian antral follicles was observed using an optical microscope.

[0035] 4.ELISA test;

[0036] ELISA was used to detect anti-Mullerian hormone, estradiol, follicle-stimulating hormone, and reactive oxygen species in the serum of each group of mice (Shanghai ELISA Biological Co., Ltd.). Dual-wavelength detection was performed using an ELISA reader (Thermo Fisher Scientific), and the OD value of each well was measured at 450 nm.

[0037] 5. TUNEL assay to detect apoptosis:

[0038] The one-step TUNEL in situ apoptosis detection kit (Elabscience) was used for detection. The sections were dewaxed and hydrated, 1× proteinase K working solution was added, and the labeling working solution was added after washing with PBS and protected from light for 1 hour. After adding DAPI working solution and incubating for 5 minutes, the sections were washed and sealed to observe cell apoptosis. The sections were observed under a fluorescence microscope. TUNEL-positive cells usually show green fluorescence, while DAPI-labeled nuclei show blue fluorescence. Five fields of view were randomly selected for counting. In each field of view, TUNEL-positive cells (apoptotic cells) and the total number of cells (DAPI-labeled nuclei) were counted respectively. The percentage of apoptotic cells = (number of TUNEL-positive cells / total number of cells) × 100%, and the percentage of apoptosis in each field of view was calculated, and then the average value was taken. Relative apoptosis rate = apoptosis rate of each group / control group.

[0039] 6. Transmission electron microscopy observation of mitochondrial morphology:

[0040] Mouse ovarian tissue was quickly cut into 1-2 mm blocks to avoid damage to mitochondrial structure. The tissue blocks were immediately fixed in ice-cold 2.5% glutaraldehyde solution and washed with 0.1M phosphate buffer. The fixed samples were fixed in 1% osmium tetroxide solution. The samples were placed in 50%, 70%, 90%, 95% and 100% ethanol in sequence, and finally treated twice in acetone for gradient alcohol dehydration. The dehydrated samples were soaked in mixed epoxy resin overnight. Then they were polymerized at 60°C for 48 hours to completely cure for resin embedding. The cured samples were cut into ultrathin sections of 60-90 nm thickness using an ultrathin slicer. The sections were placed on a grid and stained with 2% uranyl acetate and 0.2% lead citrate in sequence, each staining for 5-10 minutes. The stained ultrathin sections were observed using a transmission electron microscope (TEM).

[0041] 7. Research Results:

[0042] 7.1 Icariin improves the number of antral follicles in DOR mice:

[0043] The number of antral follicles in the ovaries of the mice in the model group was significantly lower than that in the control group (P<0.05). The number of antral follicles in the ovaries of the mice after oral administration of low, medium and high doses of Chinese medicine was higher than that in the model group (P<0.05, P<0.05, P<0.05). Figure 1 )

[0044] 7.2 Effects of icariin on hormone levels in DOR mice:

[0045] Compared with the control group, the model group had lower AMH and E2 expression in serum (P<0.05), and higher FSH expression (P<0.05). After intragastric administration of low, medium, and high doses of icariin, the expression of AMH and E2 in serum of mice was higher than that in the model group (P<0.05), and FSH was lower than that in the model group (P<0.05, P<0.05, P<0.05) (e.g. Figure 2 )

[0046] 7.3 Effect of icariin on ovarian apoptosis in DOR mice:

[0047] The apoptosis of ovarian granulosa cells in the model group was more obvious than that in the control group (P<0.05). The apoptosis of ovarian granulosa cells in the mice after oral administration of low, medium and high doses of icariin was gradually reduced compared with that in the model group (P<0.05, P<0.05, P<0.05) (e.g. Figure 3 )

[0048] 7.4 Effects of icariin on ovarian mitochondrial function in DOR mice:

[0049] Transmission electron microscopy showed that the mitochondrial morphology of the ovarian granulosa cells of the mice in the model group was significantly damaged compared with that in the control group, with mitochondrial swelling, damaged outer membrane, and broken inner cristae. After oral administration of low, medium, and high doses of icariin, the mitochondrial morphology of the ovarian granulosa cells of the mice gradually recovered. Elisa detected the ROS content in the serum. The ROS content in the model group was significantly higher than that in the control group (P<0.05). After oral administration of low, medium, and high doses of icariin, the serum reactive oxygen species of the mice gradually decreased compared with the model group (P<0.05, P<0.05, P<0.05) (e.g. Figure 4 )

[0050] Example 2 In vitro cell experiment

[0051] 1. Cell culture:

[0052] Human ovarian granulosa cells (KGN) were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C, 5% CO2, and 95% humidity.

[0053] 2. Cell proliferation:

[0054] 8000 cells per well in a 96-well plate were added to CTX (50 μmol / L) for 12 hours, and then different concentrations of icariin (5, 10, 20 μmol / L) were added and cultured for another 24 hours. Cell proliferation was evaluated by measuring OD450 values ​​using a cell counting kit-8 (Beyotime).

[0055] 3. Mitochondrial membrane potential and reactive oxygen species detection:

[0056] The cells were placed in a 6-well plate until 80% of the cells were confluent. After adding CTX (50umol / L) for 12 hours, icariin (10umol / L) was added and cultured for another 24 hours. The culture medium was removed and 1ml of TMRE staining working solution (1:1000) or DCFH-DA (10umol / L) was added. The cells were incubated at 37°C in a cell culture incubator for 30 minutes. After the incubation at 37°C, the supernatant was removed and washed. 2ul of hoest staining solution was added and the cells were incubated at 37°C and observed under a fluorescence microscope.

[0057] 4. Observation of mitochondrial morphology by transmission electron microscopy: same as above.

[0058] 5. Research results:

[0059] 5.1 Icariin (ICA) can increase the proliferation of ovarian granulosa cells induced by CTX:

[0060] After CTX induction, the proliferation of KGN cells decreased, while ICA could increase the proliferation activity.

[0061] ( Figure 5 )

[0062] 5.2 Icariin can improve mitochondrial function:

[0063] Transmission electron microscopy observations revealed that ICA can alleviate mitochondrial swelling, outer membrane damage, and inner membrane rupture caused by CTX. Figure 6 )

[0064] 5.3 Icariin can increase mitochondrial membrane potential and reduce the generation of reactive oxygen species:

[0065] After CTX induction, the mitochondrial membrane potential of KGN cells decreased and the reactive oxygen species increased. ICA can increase the mitochondrial membrane potential and reduce the generation of reactive oxygen species ( Figure 7 ).

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Application of icariin in the preparation of drugs for the treatment of decreased ovarian reserve function.

2. The use according to claim 1, characterized in that: The decreased ovarian reserve disease is infertility caused by decreased ovarian reserve function.

3. The use according to claim 1 or 2, characterized in that: The drug is added with pharmaceutically acceptable excipients and carriers to prepare a drug preparation, wherein the excipients include at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener and a colorant.

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