CN118903105A - Application of baicalein and composition thereof in preparation of medicines for delaying ovarian aging - Google Patents

Abstract

The invention relates to application of baicalein and a composition thereof in preparation of a medicament for delaying ovarian aging. The invention provides application of baicalein singly or in combination with taxifolin in preparation of medicines for regulating follicular development, inhibiting ovulation and delaying ovarian aging; the invention proves that the single baicalein or the composition of the single baicalein and the taxifolin can effectively inhibit the angiogenesis of the ovaries, thereby inhibiting the growth of follicles, and finally achieving the purposes of protecting female reproductive reserves, delaying the aging of the ovaries and protecting fertility.

Description

Use of baicalin and its composition in preparing drugs for delaying ovarian aging

Technical Field

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

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.

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.

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.

In recent years, bioactive compounds extracted from plants have received increasing attention and research, among which flavonoids are the most abundant secondary metabolites in plants and have significant health-promoting effects. Baicalin is an active compound extracted from the traditional Chinese medicine Scutellaria baicalensis, which is used to prevent and treat a variety of diseases. Taxifolin, also known as dihydroquercetin (also known as dihydroquercetin, quercetin, quercetin, and taxol), is mainly found in coniferous trees such as larch, and is widely found in olive oil, onions, and citrus; dihydroquercetin is mainly used for its antioxidant properties and to improve its preventive and therapeutic properties in food. It can be used as a plant growth regulator in the agricultural field. Baicalin and dihydroquercetin belong to the same flavonoid class of compounds, and have a wide range of pharmacological effects such as anti-inflammatory, antioxidant, anti-angiogenic, and anti-cancer.

The prior art discloses research reports related to baicalin, which mainly prove that it can treat ovarian dysfunction, that is, the application of drugs related to polycystic ovary syndrome. For example, CN116832030A discloses that baicalin improves PCOS ovarian dysfunction by inhibiting the activation of ferroptosis signaling pathway, alleviating inflammatory response, and weakening 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.

However, polycystic ovary syndrome is an endocrine metabolic disease, and PCOS is characterized by abnormal increase in androgen, persistent anovulation, and the presence of multiple cysts on the ovaries. This is different from the effects of the present invention on female ovarian blood vessels, ovarian aging, and female fertility. Therefore, the prior art has not found any relevant research on the effects of baicalin and its composition on female ovarian blood vessels, ovarian aging, and female fertility.

Summary of the invention

In view of this, the purpose of the present invention is to provide the application of baicalein and/or a combination drug with taxifolin in delaying ovarian aging. Delaying ovarian aging refers to protecting the follicle reserve in the ovary by inhibiting the physiological angiogenesis in the female ovary; or refers to slowing down the loss of primordial follicles by inhibiting the physiological angiogenesis in the female adult ovary. The female mentioned above refers to a mammal, further an adult mouse, or a human.

The delay of ovarian aging and fertility protection in the present invention include reducing the blood vessel density of growing follicles, regulating the development of growing follicles and slowing down the consumption of primordial follicles.

The present invention also provides the use of baicalein and/or taxifolin in medicine for improving ovarian aging of female animals.

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

The present invention also provides the use of baicalein and/or taxifolin in the preparation of ovulation inhibition drugs, delaying ovarian aging and fertility protection drugs. In a preferred embodiment, the ovulation inhibition 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 pharmaceutical composition with taxifolin in inhibiting follicle development, inhibiting corpora lutea development and/or protecting female reproductive reserves.

The present invention provides a pharmaceutical composition, comprising baicalein and/or taxifolin, preferably as the only effective pharmaceutical ingredient. Preferably, the pharmaceutical composition is composed of baicalein and/or taxifolin, and more preferably, the pharmaceutical composition further comprises the pharmaceutically acceptable excipients, including carriers or excipients.

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

In some specific embodiments of the present invention, a drug using baicalein alone or in combination with taxifolin has one or more of the following applications/uses for preparing drugs, including angiogenesis inhibition activity, inhibition of ovarian angiogenesis, regulation of growth follicle development and ovulation, protection of female reproductive reserves, delay of ovarian aging and/or improvement of female fertility. Preferably, the female animal is an adult mouse or a human.

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

The technical solution provided by the present invention includes:

Provided is a drug/drug composition using baicalein alone or in combination with taxifolin in the preparation of drugs for delaying ovarian aging and protecting female fertility.

Furthermore, delaying ovarian aging includes slowing the consumption of primordial follicles.

On the other hand, the invention provides the use of baicalein and taxifolin in preparing angiogenesis inhibitors.

In another aspect, the invention provides use of baicalein alone or in combination with taxifolin in preparing a preparation for inhibiting the development of growing follicles.

In another aspect, the invention provides use of baicalein alone or in combination with taxifolin in preparing an ovulation inhibiting drug.

Preferably, the drug in the above application also includes a single or combined drug based on baicalein and taxifolin and a pharmaceutically acceptable carrier.

On the other hand, the subject of administration of the above-mentioned pharmaceutical composition is a mammal, preferably, a human or a mouse. Preferably, the pharmaceutical composition is administered by injection or oral administration, and does not cause adverse reactions in the receptor.

The present invention provides the use of angiogenesis inhibitor in preparing a reagent for improving ovarian aging.

The baicalein and/or taxifolin of the present invention, or the above-mentioned combination can be used for preparing angiogenesis inhibitory drugs. The prepared drugs can interfere with adult ovarian angiogenesis, protect ovarian reserve, and thus improve ovarian aging.

The present invention provides the use of baicalein and/or taxifolin, or the above-mentioned combination for preparing a drug for interfering with adult ovarian angiogenesis; for preparing a drug for protecting ovarian reserve; and for preparing a drug for improving ovarian aging.

The beneficial effects of the present invention are:

1) For the first time, it was discovered that the natural product baicalein alone or in combination with taxifolin can act as an angiogenesis inhibitor to regulate the development of growing follicles, thereby protecting reproductive reserves, delaying ovarian aging and protecting female fertility.

2) The present invention also discloses the use of baicalein and taxifolin in the preparation of ovulation inhibition drugs.

3) It was demonstrated 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 ovaries, thereby blocking the development of adult ovaries and preserving ovarian reserves, delaying ovarian aging and being harmless to the health of female mice.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the screening of various flavonoid compounds for inhibiting ovarian angiogenesis in mice, including acacetin (Aca), baicalein (Bai), syringic acid (Syr), and taxifolin (Tax).

Figure 2 shows that baicalein and taxifolin effectively inhibited angiogenesis in mouse ovaries without affecting the health of mice and the vascular density of other tissues. In Figure 2A, after 4 weeks of in vivo injection of baicalein or taxifolin (100 mg/kg), there was no significant difference in the body weight of mice in the treatment group and the control group; in Figures 2B, 2C, and 2D, the vascular density of follicles in the ovaries was significantly reduced after treatment with baicalein and taxifolin; Figures 2E-2H confirmed that baicalein and taxifolin treatment did not affect the vascular density of the mouse heart, kidney, and liver.

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

Figure 4 shows that the combined treatment of baicalein and taxifolin delayed the ovarian aging of female mice. In Figure 4A, after 1% baicalein and taxifolin were added to the mouse feed for 12 weeks, there was no significant difference in the weight of the mice in the treatment group and the control group, and the mice were in good health; in Figures 4B and 4C, the number of primordial follicles in the ovaries of the mice in the treatment group was significantly higher than that in the control group, and the consumption of primordial follicles slowed down during the treatment period.

DETAILED DESCRIPTION

The following contents are preferred embodiments of the present invention. It should be noted that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

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.

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

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

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

Example 1: Preliminary screening of substances that inhibit follicular angiogenesis

The experimental steps are as follows:

1) Take Tek-CreER ^T2 ; mTmG female mice of appropriate age treated with tamoxifen, and use a separation needle to isolate the blood vessel-follicle complex in L15 culture medium.

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.

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.

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.

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

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

The results are shown in Figure 1. Under this experimental condition, acacetin (Aca) has poor inhibitory activity on in vitro angiogenesis, and the experimental data has a large variance, and the inhibitory effect is not significant. However, baicalein (Bai), syringic acid (Syr) and taxifolin (Tax) all have in vitro angiogenesis inhibitory activity, especially baicalein and taxifolin have more obvious inhibitory effects than syringic acid, which is mainly manifested in that the follicular vascular area is significantly reduced after adding compounds to in vitro follicle culture, and it does not affect the normal growth of follicles.

Example 2: Baicalein and Taxifolin Effectively Inhibit Follicular Angiogenesis

The baicalein and taxifolin were dissolved in the organic solvent DMSO to prepare a working solution with a concentration of 150 mg/ml, and stored in a -20°C refrigerator away from light.

The experimental operation is as follows:

1) The experimental mice were divided into three groups: a control group, a baicalein group, and a taxifolin group, each group consisting of 5 6-week-old female Tek-CreER ^T2 ; mTmG mice.

2) Take out the 150 mg/ml working solution from the -20°C refrigerator and dilute it to a working concentration of 25 mg/ml using corn oil. Mix thoroughly and set aside.

3) Drug treatment was performed by intraperitoneal injection at a dose of 100 mg/kg, once a day for 4 weeks.

4) After drug treatment, mice were weighed regularly to confirm their health. If the mice showed significant weight loss, the drug dosage could be appropriately reduced.

5) After 4 weeks of drug treatment, the mouse ovaries and major organs such as heart, kidney and liver were collected, and the blood vessels of different tissues were analyzed by combining tissue clearing technology with high-resolution 3D imaging technology.

The results showed that the angiogenesis capacity of the mouse ovaries was significantly reduced, mainly manifested by a significant decrease in the vascular density of the ovarian follicles after treatment with baicalein and taxifolin, without affecting the weight of the mice and the vascular density of the heart, kidneys and liver (Figure 2). The experiment showed that appropriate amounts of baicalein or taxifolin treatment can act as an angiogenesis inhibitor to inhibit ovarian angiogenesis and reduce the vascular density of the ovarian follicles.

Example 3: Baicalin inhibits ovulation in female mice and protects reproductive reserves

Reagent 1: DMSO

Reagent 2: Corn oil

Reagent 3: Baicalin (powder, purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.)

Reagent 4: Taxifolin (powder, purchased from Heilongjiang Ameigis Biotechnology Co., Ltd.)

The baicalein and taxifolin were dissolved in the organic solvent DMSO to prepare a working solution with a concentration of 150 mg/ml, and stored in a -20°C refrigerator away from light.

The experimental operation is as follows:

1) The experimental mice were divided into three groups: control group, baicalein and taxifolin treatment groups, each group consisted of 5 6-week-old female C57BL/6N mice.

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, mix thoroughly and set aside.

3) Drug treatment was performed by intraperitoneal injection at a dose of 100 mg/kg, once a day for 4 weeks.

4) After drug treatment, mice were weighed regularly to confirm their health. If the mice showed significant weight loss, the drug dosage could be appropriately reduced.

5) After 4 weeks of drug treatment, the mouse ovaries were collected for histological staining and analysis.

The results showed that the number of corpora lutea in the mouse ovaries 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 showed that appropriate amount of baicalin treatment can inhibit ovulation and effectively protect reproductive reserves.

Example 4: Combined treatment of baicalein and taxifolin delays ovarian aging

Reagent 1: _H2O

Reagent 2: Purified feed (powder, purchased from Jiangsu Medison Biopharmaceutical Co., Ltd.)

Reagent 3: Baicalin (powder, purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.)

Reagent 4: Taxifolin (powder, purchased from Heilongjiang Ameigis Biotechnology Co., Ltd.)

Weigh 1g of baicalein and taxifolin, add them to 100g of feed, that is, add them to the powdered feed at a weight ratio of 2% (drug: (feed + water) = 1:100), mix the feed and make it into a shape for feeding. Baicalein and taxifolin are the only effective drug ingredients, and the weight ratio is 1:1.

The experimental operation is as follows:

1) The experimental mice were divided into two groups: a control group and a combined treatment group, each group consisted of 9 6-week-old C57BL/6N female mice.

2) Take working reagents 2, 3 and 4 out of the refrigerator, add reagents 1, 3 and 4 to reagent 2 in proportion, mix thoroughly to make a shape, and then feed it to mice for 12 weeks.

3) After starting to feed the medicated feed, the mice were weighed regularly to confirm their health. If the mice showed significant weight loss, the drug dosage could be appropriately reduced.

4) After 4, 8 and 12 weeks of drug treatment (medicated feed), ovaries of 3 mice were collected and the number of primordial follicles was statistically analyzed.

The results showed that after 12 weeks of combined drug treatment, there was no significant difference in the body weight of mice compared with the control group, the number of primordial follicles in the mouse ovaries was significantly higher than that in the control group, and the number of primordial follicles decreased slowly during the treatment period (Figure 4).

These results indicate that long-term combined treatment with baicalein and taxifolin can protect female reproductive reserves and delay ovarian aging by inhibiting angiogenesis and regulating follicle development.

The present invention combines the vascular-specific endogenous fluorescent tracing mouse model Tek-CreER ^T2 ; mTmG, and detects the effects of baicalin or taxifolin on ovarian follicle angiogenesis by in vivo injection of baicalin or taxifolin. It is proved that both baicalin and taxifolin can inhibit ovarian angiogenesis in vivo, and do not affect the vascular density and body weight of other multiple tissues and organs of adult female mice. At a dose of 100 mg/kg, baicalin can significantly reduce the follicular vascular area, thereby inhibiting ovulation in female mice and protecting reproductive reserves.

Combined treatment was performed by dietary addition to explore its long-term effects on ovarian primordial follicles. The results showed that within 12 weeks of combined treatment, the reproductive reserve of female mice, that is, the number of primordial follicles, decreased slowly. It was 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 ovaries, thereby blocking the development of adult ovaries and preserving ovarian reserves, delaying ovarian aging and being harmless to the health of female mice. Therefore, compared with other chemical drugs, baicalein and taxifolin can be used within a reasonable dose range under the premise of ensuring the safety of receptors to achieve the purpose of delaying ovarian aging and protecting fertility.

In the specific embodiments of the present invention, except for the experimental conditions, the other experimental materials and processing methods of each comparative experiment are the same.

Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The processing strategy and related applications of the present invention have been described through embodiments, and relevant personnel can obviously modify or appropriately change and combine the processing strategy and related applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims (10)

1. Use of a pharmaceutical composition in the preparation of a drug for delaying ovarian aging, characterized in that the pharmaceutical composition comprises baicalein and/or taxifolin. 2. The use according to claim 1, characterized in that baicalein and/or taxifolin are used as the only effective medicinal ingredients. 3. Use of a pharmaceutical composition in the preparation of a drug for inhibiting angiogenesis and delaying ovarian aging, characterized in that the pharmaceutical composition comprises baicalein and/or taxifolin. 4. The use according to claim 3, characterized in that the inhibition of angiogenesis is the inhibition of follicular angiogenesis. 5. The use according to claim 3 or 4, characterized in that the inhibition of angiogenesis is the inhibition of follicular angiogenesis, and does not affect the vascular density and body weight of other tissues and organs. 6. The use according to claim 5, characterized in that the other tissues and organs are heart, kidney and liver. 7. A pharmaceutical composition, characterized in that it contains baicalein and/or taxifolin as the only effective pharmaceutical ingredients. 8. The pharmaceutical composition according to claim 7, further comprising the pharmaceutically acceptable excipients, including carriers or excipients. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a diluent, a lubricant, a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an antibacterial agent, a solute that makes the preparation isotonic with the blood of the recipient, a suspending agent, a suspending aid, a solubilizer, a thickener, a stabilizer, a sweetener and a fragrance. 10. Use of the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of a drug for the following purposes, characterized in that the purposes include one or more of the following: a) Angiogenesis inhibitory activity; b) inhibit ovarian angiogenesis; c) Regulate the development of growing follicles and ovulation; d) protect the female reproductive reserve; e) Delaying ovarian aging and/or improving female fertility.