Use of baicalein and its composition in the preparation of drugs for delaying ovarian senescence

Abstract

The present invention relates to the use of baicalein and its composition in the preparation of drugs for delaying ovarian senescence. The present invention provides the use of baicalein alone or in combination with taxifolin in the preparation of drugs for regulating follicular development, inhibiting ovulation and delaying ovarian senescence; the present invention has confirmed that the composition of baicalein alone or in combination with taxifolin can effectively inhibit ovarian angiogenesis, thereby inhibiting the development of growing follicles, and ultimately achieving the purpose of protecting female reproductive reserve, delaying ovarian senescence and protecting fertility.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to use of baicalein and a composition thereof in preparing a drug for delaying ovarian aging, and in particular to use of baicalein alone or in combination with taxifolin in preparing a drug for delaying ovarian aging. Background Art

[0002] With the rapid development of modern society, the average childbearing age of women has been continuously postponed. At the same time, the age-related infertility rate and spontaneous abortion rate continue to rise. The root cause of the above problems lies in the decline of female fertility. Unlike men who have constantly renewed spermatogonial stem cells, the reproductive reserve in the female ovaries, that is, the number of primordial follicles, no longer increases from birth. In the subsequent development process, most follicles become atretic, and only a small number of primordial follicles can develop into antral follicles and then ovulate. For women, fertility decreases with age, and the decrease in the number of follicles increases significantly after the age of 35. By the time of menopause, the reserve of primordial follicles has dropped from 1 to 2 million at birth to about 1,000. The exhaustion of follicles leads to menopause and ovarian aging in women, and ovarian aging is the most direct cause of female fertility loss.

[0003] Under physiological conditions, blood vessels in most organs of adult mammals remain quiescent, but the ovaries maintain active angiogenesis after adulthood. Recent studies have shown that treating adult mice with angiogenesis-inhibiting drugs can effectively inhibit angiogenesis in the ovaries and halt ovarian development, thereby preserving reproductive reserves and ultimately delaying ovarian aging and improving female fertility. Therefore, inhibiting adult angiogenesis may be an effective way to protect human primordial follicle reserves and delay ovarian aging.

[0004] Currently, most of the small molecule drugs used in clinical practice to inhibit angiogenesis are tyrosine kinase inhibitors, which are tumor suppressors with a variety of known side effects on the body. If they are used as drugs to protect human primordial follicle reserves and delay female reproductive aging, there may be many potential problems. Therefore, screening and developing safe, reliable and effective drugs to inhibit ovarian angiogenesis is a focus issue that needs to be solved urgently, which is of great significance for accelerating the effective implementation of work related to protecting female reproductive reserves and delaying reproductive aging.

[0005] In recent years, bioactive compounds extracted from plants have received increasing attention and research. Among them, flavonoids are the most abundant secondary metabolites in plants and have significant health-promoting effects. Baicalein is an active compound extracted from the traditional Chinese medicine Scutellaria baicalensis Georgi and is used for the prevention and treatment of various diseases. Taxifolin, also known as dihydroquercetin (also known as dithydroquercetin, distyliumletin, douglasin, taxifolin), is mainly present in conifers such as larch and is also widely present in olive oil, onions, and citrus fruits, etc.; dihydroquercetin is mainly used for its antioxidant properties and to improve the preventive and therapeutic properties in food, and can be used as a plant growth regulator in the agricultural field. Baicalein and dihydroquercetin belong to the flavonoid compounds and have a wide range of pharmacological effects such as anti-inflammatory, antioxidant, anti-angiogenic, and anti-cancer effects.

[0006] In the prior art, research reports related to baicalein have been disclosed, mainly proving that it can treat ovarian dysfunction, that is, the application of drugs related to polycystic ovary syndrome. For example, CN116832030A discloses that baicalein can improve the ovarian function disorder of PCOS by inhibiting the activation of the ferroptosis signaling pathway, reducing the inflammatory response, and weakening the lipid peroxidation of ovarian granulosa cells; CN103285021A discloses that baicalin can significantly reduce the high androgen level in the polycystic ovary syndrome rat model, promote follicle maturation, and induce ovulation.

[0007] However, polycystic ovary syndrome is an endocrine and metabolic disease. PCOS is mainly characterized by abnormally increased androgens, continuous anovulation, and the presence of multiple cysts on the ovaries. It is different from the problems of the present invention concerning the effects on female ovarian blood vessels, ovarian aging, and female fertility. Therefore, the prior art has not found relevant research on the effects of baicalein and its compositions on female ovarian blood vessels, ovarian aging, and female fertility. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide the related application of the combined drug of baicalein and / or taxifolin in delaying ovarian aging. Delaying ovarian aging refers to protecting the follicle reserve in the ovary by inhibiting physiological angiogenesis in the female ovary; or refers to slowing down the depletion of primordial follicles by inhibiting physiological angiogenesis in the adult female ovary. The above-mentioned female refers to mammals, further adult mice, or humans.

[0009] In the present invention, delaying ovarian aging and fertility protection include reducing the vascular density of growing follicles, regulating the development of growing follicles, and slowing down the consumption of primordial follicles.

[0010] The present invention also provides the application of baicalein and / or taxifolin in drugs for improving ovarian aging in female animals.

[0011] The present invention provides the use of baicalein and / or taxifolin in the preparation of a drug for inhibiting angiogenesis. In a preferred embodiment, the inhibition of angiogenesis further includes: reducing the follicular vascular density, reducing the growing follicular vascular density, regulating the development of growing follicles, and slowing down the consumption of primordial follicles.

[0012] The present invention also provides the use of baicalein and / or taxifolin in the preparation of a drug for inhibiting ovulation, delaying ovarian aging, and protecting fertility. In a preferred embodiment, the inhibition of ovulation includes: reducing the number of corpora lutea or slowing down the consumption of primordial follicles. That is, the present invention provides the use of baicalein as a single or combined drug composition with taxifolin in inhibiting follicular development, inhibiting corpus luteum development, and / or protecting female reproductive reserves.

[0013] The present invention provides a drug composition comprising baicalein and / or taxifolin, preferably as the only active pharmaceutical ingredient. Preferably, it consists of baicalein and / or taxifolin. More preferably, the drug composition further includes the pharmaceutically acceptable excipients, including carriers or excipients.

[0014] The pharmaceutically acceptable excipients are selected from one or a combination of two or more of diluents, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, bacteriostatic agents, solutes that make the preparation isotonic with the recipient's blood, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0015] In some specific embodiments of the present invention, the single or combined drug of baicalein with taxifolin has one or more of the following applications / uses for preparing drugs, including angiogenesis inhibitory activity, inhibiting ovarian angiogenesis, regulating the development and ovulation of growing follicles, protecting female reproductive reserves, delaying ovarian aging, and / or improving female fertility. Preferably, the female animal is an adult mouse or a human.

[0016] Preferably, the administration method includes intraperitoneal injection or dietary addition. The injection drug dose is 10 - 500 mg / kg, preferably 50 - 400 mg / kg, more preferably 60, 70, 80, 90, 100, 110, 120, 140, 150, 160, 180, 200, 220, 240, 250, 280, 300, 320, 360 mg / kg; administered 1 - 3 times a day for 2 - 8 weeks, preferably administered once a day for 2 - 4 weeks. The dietary addition method has a drug - food ratio of 0.2 - 5% for administration, and is taken continuously for 2 - 30 weeks. Preferably, the administration dose is 0.5 - 3% by weight ratio of the drug - food ratio. The weight ratio of baicalein to taxifolin is (0.5 - 5):1, preferably (0.5 - 2):1.

[0017] The technical solutions provided by the present invention include:

[0018] Provide the use of a single drug of baicalein or a combined drug of baicalein and taxifolin in the preparation of a drug for delaying ovarian aging and protecting female fertility.

[0019] Furthermore, delaying ovarian aging includes slowing down the consumption of primordial follicles.

[0020] On the other hand, provide the use of baicalein and taxifolin in the preparation of an angiogenesis inhibitor.

[0021] On the other hand, provide the use of a single drug of baicalein or a combined drug of baicalein and taxifolin in the preparation of a preparation for inhibiting the development of growing follicles.

[0022] On the other hand, provide the use of a single drug of baicalein or a combined drug of baicalein and taxifolin in the preparation of an ovulation-inhibiting drug.

[0023] Preferably, the drug in the above application further includes a single or combined drug based on the structures of baicalein and taxifolin and a pharmaceutically acceptable carrier.

[0024] On the other hand, the subject of administration of the above drug composition is a mammal. Preferably, the subject of administration is a human or a mouse. Preferably, the drug composition is administered by injection or orally, and does not cause adverse reactions in the recipient.

[0025] The present invention provides the use of an angiogenesis inhibitor in the preparation of a reagent for improving ovarian aging.

[0026] The baicalein and / or taxifolin of the present invention, or the above composition can be used in the application of preparing an angiogenesis-inhibiting drug. The prepared drug can interfere with angiogenesis in the adult ovary, protect ovarian reserve, and thus improve ovarian aging.

[0027] The present invention provides the use of baicalein and / or taxifolin, or the above composition for preparing a drug for interfering with angiogenesis in the adult ovary; for preparing a drug for protecting ovarian reserve; for preparing a drug for improving ovarian aging.

[0028] The beneficial effects of the present invention are as follows:

[0029] 1) It is first discovered that a single drug of the natural product baicalein or a combined drug of baicalein and taxifolin can be used as an angiogenesis inhibitor to regulate the development of growing follicles, thereby protecting reproductive reserve, delaying ovarian aging and protecting female fertility.

[0030] 2) The present invention also discloses the use of baicalein and taxifolin in the preparation of an ovulation-inhibiting drug.

[0031] 3) It is proved that the combined treatment of baicalein and taxifolin can regulate the development of growing follicles by inhibiting ovarian angiogenesis, reduce the activation of primordial follicles in the ovary, thereby blocking the development of adult ovaries and preserving ovarian reserve, delaying ovarian aging and being harmless to the health of female mice. Description of the Drawings

[0032] Figure 1 Shown is the screening of various flavonoids for inhibiting ovarian angiogenesis in mice. Specifically, they are acacetin (Aca), baicalein (Bai), syringic acid (Syr), and taxifolin (Tax).

[0033] Figure 2 Shown is that baicalein and taxifolin effectively inhibit ovarian angiogenesis in mice without affecting the health of mice and the vascular density of other tissues. Figure 2 In A, after 4 weeks of in vivo injection of baicalein or taxifolin (100 mg / kg), there was no significant difference in the body weight of the treated mice and the control group. Figure 2 In B, 2C, and 2D, the vascular density of follicles in the ovary was significantly reduced after treatment with baicalein and taxifolin. Figure 2 E - 2H confirmed that the treatment with baicalein and taxifolin did not affect the vascular density of the heart, kidneys, and liver in mice.

[0034] Figure 3 Shown is that baicalein inhibits ovulation in female mice, reduces the number of corpora lutea in the ovary, and slows down the consumption of primordial follicles in the ovary. Figure 3 A and Figure 3 In B, after 4 weeks of intraperitoneal injection of 100 mg / kg baicalein, the number of corpora lutea in the mouse ovary decreased. Figure 3 In C, the number of primordial follicles in the baicalein - treated group of mice was significantly higher than that in the control group of mice.

[0035] Figure 4 Shown is that the combined treatment of baicalein and taxifolin delays ovarian aging in female mice. Figure 4 In A, after feeding female mice with a diet containing 1% baicalein and taxifolin for 12 weeks, there was no significant difference in the body weight of the treated mice and the control group, and the health status of the mice was good. Figure 4 B and Figure 4 In C, the number of primordial follicles in the treated group of mice was significantly higher than that in the control group, and the consumption of primordial follicles during the treatment period was slowed down. Detailed Embodiments

[0036] The following content is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

[0037] The following further describes the use of baicalein alone or in combination with taxifolin provided by the present invention in inhibiting follicular angiogenesis, follicle and corpus luteum development and protecting female reproductive reserve.

[0038] Unless otherwise specified, the reagents used in the examples of the present invention can be purchased through regular commercial channels.

[0039] Baicalein, powder, was purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.

[0040] Taxifolin, powder, was purchased from Heilongjiang Ameigis Biotechnology Co., Ltd.

[0041] Example 1: Preliminary screening of substances that inhibit follicular angiogenesis

[0042] The experimental steps are as follows:

[0043] 1) Take age-appropriate Tek-CreER after tamoxifen treatment T2 ; mTmG female mice, and the vascular-follicle complex was isolated using a dissection needle in L15 medium.

[0044] 2) Different compounds, namely acacetin (Aca), baicalein (Bai), syringic acid (Syr), and taxifolin (Tax), were prepared at a drug concentration of 25 mM, and the control group was replaced with the same volume of 0.2% DMSO.

[0045] Take out from -20 refrigerator, dilute with complete culture medium to working concentration of 50μmol / L, that is, add 2μl drug / DMSO to every 998μl culture medium; add to culture dish after thorough mixing, and place in cell chamber (cell cultureinsert, Millicell organ culture standing culture chamber, hydrophilic PTFE membrane, 0.4um, 30mm, 50 / pk, item number PICM0RG50), avoid bubbles, and preheat at 37℃ for 5 minutes.

[0046] 3) After the separation of the blood vessel-follicle complex is completed, the separated follicles are transferred and inoculated into the cell chamber using a pipette and cultured for 48 hours.

[0047] 4) During the culture period, the angiogenesis was recorded using a fluorescence microscope every 24 hours.

[0048] 5) After 48 hours of drug treatment, the follicle diameter and vascular area after different drug treatments were analyzed.

[0049] The results are as follows Figure 1As shown, under these experimental conditions, acacetin (Aca) had poor inhibitory activity against angiogenesis in vitro, and the experimental data had a large variance, with the inhibitory effect being not significant. However, baicalein (Bai), syringic acid (Syr), and taxifolin (Tax) all had inhibitory activity against angiogenesis in vitro. In particular, the inhibitory effects of baicalein and taxifolin were more obvious compared to syringic acid. This was mainly manifested as a significant reduction in the follicular vascular area after adding the compounds to the in vitro follicle culture, without affecting the normal growth of the follicles.

[0050] Example 2: Baicalein and taxifolin effectively inhibit follicular angiogenesis

[0051] Baicalein and taxifolin were separately dissolved in the organic solvent DMSO to prepare working solutions with a concentration of 150 mg / ml, which were then aliquoted and stored in the dark at -20°C in the refrigerator.

[0052] The experimental operations were as follows:

[0053] 1) The experimental mice were divided into three groups: a control group, a baicalein group, and a taxifolin group, with 5 female mice at 6 weeks old of Tek-CreER T2 ; mTmG in each group.

[0054] 2) The 150 mg / ml working solutions were taken out from the -20°C refrigerator and diluted to the working concentration of 25 mg / ml with corn oil, and then thoroughly mixed for later use.

[0055] 3) Drug treatment was carried out by intraperitoneal injection, with a dosage of 100 mg / kg, once a day for 4 weeks.

[0056] 4) After drug treatment, the body weights of the mice were regularly measured to confirm their health status. If the body weight of the test mice significantly decreased, the drug dosage could be appropriately reduced.

[0057] 5) Four weeks after drug treatment, the ovaries and the main organs of the mice, namely the heart, kidneys, and liver, were collected, and the blood vessels in different tissues were analyzed by combining tissue clearing technology and high-resolution 3D imaging technology.

[0058] The results showed that the angiogenesis ability of the mouse ovaries was significantly reduced, mainly manifested as a significant decrease in the follicular vascular density in the ovaries after treatment with baicalein and taxifolin, without affecting the body weight of the mice and the vascular density of the heart, kidneys, and liver ( Figure 2 ). The experiment indicated that treatment with an appropriate amount of baicalein or taxifolin could be used as an angiogenesis inhibitor to inhibit ovarian angiogenesis and reduce the follicular vascular density in the ovaries.

[0059] Example 3: Baicalein inhibits ovulation in female mice and protects reproductive reserves

[0060] Reagent 1: DMSO

[0061] Reagent 2: Corn oil

[0062] Reagent 3: Baicalein (powder, purchased from Shanghai Aobox Biotechnology Co., Ltd.)

[0063] Reagent 4: Taxifolin (powder, purchased from Heilongjiang Amegis Biotechnology Co., Ltd.)

[0064] Dissolve baicalein and taxifolin separately in the organic solvent DMSO to prepare a working solution with a concentration of 150 mg / ml, and store it in aliquots in a -20°C refrigerator protected from light.

[0065] The experimental operations are as follows:

[0066] 1) Divide the experimental mice into three groups: a control group, a baicalein treatment group, and a taxifolin treatment group, with 5 female 6-week-old C57BL / 6N mice in each group.

[0067] 2) Take out the 150 mg / ml working solution from the -20°C refrigerator, dilute it with corn oil to a working concentration of 25 mg / ml, and mix well for later use.

[0068] 3) Administer the drug by intraperitoneal injection at a dose of 100 mg / kg once a day for 4 weeks.

[0069] 4) After the drug treatment, regularly weigh the mice to confirm their health status. If the test mice show a significant decrease in body weight, the drug dose can be appropriately reduced.

[0070] 5) After 4 weeks of drug treatment, collect the ovaries of the mice for histological staining and analysis.

[0071] The results showed that the number of corpora lutea in the ovaries of the mice was significantly lower than that in the control group, and the number of primordial follicles was significantly higher than that in the control group ( Figure 3 ). The experiment shows that appropriate treatment with baicalein can inhibit ovulation and effectively protect reproductive reserves.

[0072] Example 4: Combined treatment with baicalein and taxifolin delays ovarian aging

[0073] Reagent 1: H2O

[0074] Reagent 2: Purified feed (powder, purchased from Jiangsu Medison Biotechnology Co., Ltd.)

[0075] Reagent 3: Baicalein (powder, purchased from Shanghai Aobox Biotechnology Co., Ltd.)

[0076] Reagent 4: Taxifolin (powder, purchased from Heilongjiang Amegis Biotechnology Co., Ltd.)

[0077] Weigh 1 g of baicalein and taxifolin respectively, add them to 100 g of feed, that is, add them to the powder feed according to the amount of 2% (drug: (feed + water) = 1:100, weight ratio), mix the feed evenly and make it into a shape for feeding. Among them, baicalein and taxifolin are used as the only active drug ingredients, and the weight ratio is 1:1.

[0078] The experimental operations are as follows:

[0079] 1) Divide the experimental mice into two groups, namely the control group and the combined treatment group, with 9 female C57BL / 6N mice at 6 weeks of age in each group.

[0080] 2) Take out working reagents 2, 3 and 4 from the refrigerator, add reagents 1, 3 and 4 to reagent 2 according to the ratio, mix them evenly and make them into a shape for feeding the mice, and continuously feed them for 12 weeks.

[0081] 3) After starting to feed the medicated feed, regularly weigh the mice to confirm their health status. If the weight of the test mice decreases significantly, the drug dose can be appropriately reduced.

[0082] 4) At 4 weeks, 8 weeks and 12 weeks of drug treatment (medicated feed), collect the ovaries of 3 mice each, and perform statistical analysis on the number of primordial follicles.

[0083] The results showed that after 12 weeks of combined drug treatment, the body weight of the mice had no significant difference from that of the control group, and the number of primordial follicles in the ovaries of the mice was significantly higher than that of the control group, and the number of primordial follicles decreased slowly during the treatment ( Figure 4 ).

[0084] The above results indicate that the long-term combined treatment of baicalein and taxifolin can protect the female reproductive reserve and delay ovarian aging by inhibiting angiogenesis and regulating follicular development.

[0085] The present invention combines the vascular-specific endogenous fluorescence tracing mouse model Tek-CreER T2 ; mTmG, by injecting baicalein or taxifolin in vivo, detect the effects of the two compounds on ovarian follicular angiogenesis. It is proved that both baicalein and taxifolin can inhibit ovarian angiogenesis in vivo without affecting the vascular density and body weight of other multiple tissues and organs in adult female mice. At a dose of 100 mg / kg, baicalein can significantly reduce the follicular vascular area, thereby inhibiting ovulation in female mice and protecting the reproductive reserve.

[0086] Combined treatment was carried out by dietary supplementation to explore its long-term effects on ovarian primordial follicles. The results showed that during 12 weeks of combined treatment, the reproductive reserve, namely the number of primordial follicles in female mice, decreased slowly. It was proved that the combined treatment of baicalein and taxifolin could regulate the development of growing follicles by inhibiting ovarian angiogenesis, reduce the activation of primordial follicles in the ovary, thereby blocking adult ovarian development and preserving ovarian reserve, delaying ovarian aging and being harmless to the health of female mice. Therefore, compared with other chemical drugs, baicalein and taxifolin can achieve the purpose of delaying ovarian aging and protecting fertility within a reasonable dose range while ensuring the safety of the recipient.

[0087] In the specific embodiments of the present invention, except for the differences in experimental conditions, the remaining experimental materials, treatment methods, etc. are all kept consistent in each comparative experiment.

[0088] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The treatment strategies and their related applications described in the present invention have been described through examples, and those skilled in the art can obviously make changes or appropriate variations and combinations to the treatment strategies and their related applications described in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for delaying ovarian aging, characterized in that: The medicine composition consists of baicalein and taxifolin, wherein baicalein and taxifolin are the only effective medicine ingredients, and the weight ratio of the two is 1:1.

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