Use of notoginseng saponin Ft1 in promoting blood supply to ovarian follicles and preparing assisted ovulation-inducing drugs

Abstract

The present invention provides a use of notoginseng saponin Ft1 in promoting the blood supply to ovarian follicles and preparing an auxiliary ovulation-inducing drug; the present invention confirms that notoginseng saponin Ft1 can effectively improve the blood supply to ovarian follicles, can significantly increase the number of ovulations in female mice, and can promote the angiogenesis of follicles; when a clinical verification test on mice was carried out by injecting notoginseng saponin Ft1, it was found that the pregnancy rate and the number of litters of mice treated with notoginseng saponin Ft1 were significantly higher than those of the control group.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to use of notoginseng saponin Ft1 and its derivatives and structural analogs in the field of assisted reproduction. Background Art

[0002] The ovary is the core reproductive organ of female mammals, and it undertakes the important mission of releasing mature eggs and reproducing offspring. As an endocrine organ, the ovary secretes hormones such as E2 and P4 through granulosa cells and interstitial cells to maintain the body's endocrine homeostasis. It also responds to FSH and LH secreted by the pituitary gland and transported to the ovary through blood vessels to promote granulosa cell proliferation, maintain the normal development of follicles and the formation of the corpus luteum. Therefore, a complete and functional vascular system in the ovary is very important for maintaining ovarian function.

[0003] Previous studies have suggested that the decline in the number of follicles with age is the main cause of ovarian aging. However, it is worth noting that even at the end of a woman's reproductive life, around 40 years old, there are still tens of thousands of follicles in the ovaries, far more than the number of eggs needed to eventually reproduce. One of the reasons for not being able to conceive at this time is that the follicles' ability to respond to hormones decreases, causing them to be unable to develop to the pre-ovulatory follicle stage.

[0004] Active angiogenesis is one of the unique characteristics of the ovaries in the adult body, and is also an important channel for regulating blood supply, hormone transport, and ultimately the development of follicles. Therefore, promoting angiogenesis in the ovaries of older women is expected to become a new strategy to improve ovarian aging. At the same time, since a variety of angiogenic diseases, such as tumors, rheumatoid arthritis, and diabetic retinopathy, can produce excessive angiogenesis, safe and effective drugs to promote angiogenesis are still to be developed.

[0005] With the development of social economy, the pace of human life has gradually accelerated, which has gradually delayed the childbearing age of women. In addition, endocrine disorders caused by ovarian aging also bring health hazards to women themselves. At the same time, the commonly used assisted reproductive strategy in clinical practice is in vitro fertilization. For older women, not only is the success rate low, but multiple egg retrievals also bring great pain to patients physically and mentally. Therefore, it is urgent to develop new methods to promote follicle development in older women and improve fertility.

[0006] Notoginsenoside Ft1 was discovered in 2006. It is a new type of saponin extracted from the leaves of Panax notoginseng, with a CAS number of 80418-24-2. Notoginsenoside Ft1 has a more effective function of promoting endothelial cell proliferation than other saponins in Panax notoginseng, such as notoginsenoside R1, ginsenoside Rb1 and Re. Studies have shown that Ft1 promotes the transfer of hypoxia-inducible factor Hif1α from the cytoplasm to the nucleus and promotes the binding of Hif1α to the Vegfa promoter, thereby increasing the transcription level of Vegfa and promoting angiogenesis. In addition, the PI3K / AKT / mTOR and Raf / MEK / ERK signaling pathways are involved in the regulation of angiogenesis by Ft1. Its chemical formula is as follows:

[0007] .

[0008] Although the prior art has made some research progress on notoginsenoside Ft1, including CN107625778A disclosing the use of notoginsenoside Ft1 in the preparation of drugs for treating Parkinson's disease; CN115317496A disclosing the use of ginsenoside Rg3, notoginsenoside Ft1 and their combination in the preparation of drugs for treating prostatic hyperplasia; CN115252631A disclosing that notoginsenoside Fc and notoginsenoside Ft1 can be used in the preparation of drugs for treating diabetic nephropathy; CN108014118A disclosing that notoginsenoside Ft1 is used as an active ingredient in the preparation of TGR5 agonists, and in the preparation of drugs or health care products for preventing and / or treating diseases such as abnormal glucose metabolism, abnormal lipid metabolism or / and abnormal insulin sensitivity. Food; CN104771408A discloses the use of notoginseng saponin Ft1, prodrugs and pharmaceutically acceptable salts thereof or plant extracts containing notoginseng saponin Ft1 in the preparation of drugs or health products for preventing or treating ulcerative colitis; CN103908459A discloses that notoginseng saponin Ft1 can be used to prevent and treat various hemorrhagic diseases such as platelet dysfunction and coagulation dysfunction; CN107625778A discloses the use of compound notoginseng saponin Ft1 in the preparation of drugs for treating Parkinson's disease PD. In the above-mentioned prior art disclosures, although the therapeutic or auxiliary therapeutic effects of notoginseng saponin Ft1 on various types of diseases and mechanisms are disclosed, the prior art has not studied notoginseng saponin Ft1 in ovaries and assisted reproduction, and improving ovarian function. It is still unknown what effect notoginseng saponin Ft1 has on the reproductive function and follicular development of the ovaries. Summary of the invention

[0009] In view of this, the object of the present invention is to provide notoginseng saponin Ft1 (C 47 H 80 O 17), specifically, using notoginseng saponin Ft1 as a drug for improving ovarian blood supply, improving ovulation efficiency, and / or improving ovarian function, improving female reproductive health and female fertility. The notoginseng saponin Ft1 referred to in the present invention has the following chemical formula:

[0010] .

[0011] The present invention provides the use of notoginseng saponin Ft1 in the preparation of a drug for improving ovarian function, increasing female fertility and promoting female health; in a preferred embodiment, the improvement of ovarian function includes: promoting angiogenesis of ovarian follicles and enhancing blood supply to the ovary.

[0012] In another specific embodiment, the improved fertility includes increasing the pregnancy rate and offspring birth rate of older females.

[0013] The present invention also aims to provide the use of notoginseng saponin Ft1 in the method for preparing assisted ovulation induction drugs. In a preferred embodiment, the assisted ovulation induction includes increasing the number of oocytes after superovulation.

[0014] In some specific embodiments of the present invention, notoginseng saponin Ft1 was used as an experimental object to verify that it has the effect of enhancing ovarian vascular supply, thereby improving ovarian function and fertility in elderly females. At the same time, it was verified that notoginseng saponin Ft1 can assist in ovulation and improve ovulation efficiency. However, notoginseng total saponins do not have the above functions.

[0015] Specifically, the technical solution provided by the present invention includes: providing a use of notoginseng saponin Ft1 in the preparation of a drug for improving ovarian function, increasing female fertility and improving female reproductive health. Further, improving fertility includes increasing the ovulation rate of young females and increasing the pregnancy rate and litter size of older females.

[0016] On the other hand, the present invention provides the use of notoginseng saponin Ft1 in the preparation of a drug for promoting ovarian fertility in adolescent or elderly mammals. Preferably, the mammal is a mouse or a primate, and preferably the mammal is a mouse, a rat or a human.

[0017] On the other hand, the present invention provides the use of notoginseng saponin Ft1 in the preparation of a drug for improving fertility. Preferably, improving fertility refers specifically to improving the pregnancy rate of older females and the birth rate of offspring.

[0018] On the other hand, the present invention provides the use of notoginseng saponin Ft1 in the preparation of a drug for improving ovarian function, increasing female fertility and / or enhancing female health. Preferably, female health is female reproductive health, preferably increasing female fertility and reducing fertility-related symptoms.

[0019] On the other hand, the invention provides the use of notoginsenoside Ft1 in preparing a preparation for enhancing the blood supply to the ovary.

[0020] On the other hand, the invention provides the use of notoginseng saponin Ft1 in the preparation of ovulation-inducing auxiliary drugs.

[0021] Preferably, the assisted ovulation induction includes increasing the number of oocytes after superovulation and natural ovulation in older females.

[0022] More preferably, the preparation or medicine in the above application further includes a pharmaceutically acceptable carrier. Preferably, the preparation or medicine in the above application further includes a pharmaceutically acceptable excipient, which is selected from 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 agent, a solubilizer, a thickener, a stabilizer, a sweetener, and a combination of one or more of two or more of the spices. It also includes a pharmaceutically acceptable excipient, including a carrier or an excipient.

[0023] Preferably, in the above-mentioned pharmaceutical products or pharmaceutical compositions and applications, notoginseng saponin Ft1 is used as the only active pharmaceutical ingredient.

[0024] The beneficial effects of the present invention are:

[0025] 1) For the first time, a new application of the natural small molecule compound Panax notoginseng saponin Ft1 was discovered, specifically an application in enhancing the blood supply to the ovaries to improve ovarian aging, increase the pregnancy rate of older females and the birth rate of their offspring, and improve female health.

[0026] 2) The present invention also discloses the use of notoginseng saponin Ft1 in the preparation of ovulation-inducing auxiliary drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results show that notoginseng saponin Ft1 promotes angiogenesis in follicles of aged mice, which is mainly manifested in that during the in vitro culture of follicles, after adding notoginseng saponin Ft1 (50 μM), the growth rate of follicular blood vessels is significantly accelerated within 24 hours, and the vascular network is more densely constructed, while notoginseng total saponins cannot promote the construction of follicular vascular network;

[0028] Figure 2 The results show that notoginseng saponin Ft1 promotes follicular vascular permeability in aged mice, which is mainly manifested by the fact that after one week of in vivo injection of notoginseng saponin Ft1 (50 mg / kg.BW), mouse follicles were isolated 15 minutes after tail vein injection of Evans blue. Notoginseng saponin Ft1 can significantly promote follicular vascular permeability, while there is no significant change in follicular vascular permeability after treatment with notoginseng total saponins.

[0029] Figure 3The figure shows that notoginseng saponin Ft1 improves the efficiency of gonadotropin-induced ovulation, which is mainly manifested as follows: after prepubertal mice were injected with notoginseng saponin Ft1 (50 mg / kg.BW) and given superovulation treatment, the number of ovulations of the mice increased significantly (A and B), and there was no significant change in the weight of the mice (C), while notoginseng total saponins could not increase the number of ovulations of mice;

[0030] Figure 4 The results show that notoginseng saponin Ft1 significantly improves the fertility of aged mice, which is mainly manifested by the increase in pregnancy rate (A) and the number of offspring born (B) after in vivo injection of notoginseng saponin Ft1 (50 mg / kg.BW) for one week and co-cage with normal fertile adult male mice for 5 days. However, after treatment with notoginseng total saponins, the pregnancy rate and the number of offspring born of mice were not significantly different from those of the control group (A and B). DETAILED DESCRIPTION

[0031] The above are only preferred embodiments of the present invention. It should be pointed out 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 scope of protection of the present invention.

[0032] The following further describes the use of notoginsenoside Ft1 provided by the present invention in improving ovarian aging, increasing the pregnancy rate of elderly women and the birth rate of their offspring, and improving female health, as well as the use of notoginsenoside Ft1 in the preparation of ovulation-inducing auxiliary drugs.

[0033] Example 1: Notoginseng saponin Ft1 promotes ovarian angiogenesis

[0034] Reagent 1: DMSO

[0035] Reagent 2: Panax notoginseng total saponins

[0036] Reagent 3: Notoginsenoside Ft1 (powder)

[0037] The reagent of the present invention can package Panax notoginseng total saponins, Panax notoginseng saponin Ft1 and organic solvent separately. When used, Panax notoginseng total saponins and Panax notoginseng saponin Ft1 are dissolved in the organic solvent DMSO to prepare a working solution with a concentration of 1 mM, and placed in a -20°C refrigerator to be stored away from light. 50 μM is used in specific applications.

[0038] The experimental operation is as follows:

[0039] 1) The experimental mice were divided into three groups: control group, Panax notoginseng total saponin group and Panax notoginseng saponin Ft1 group. Ten 12-month-old mice were isolated from each group. Tek-CreER T2 ;mTmG Ovarian follicles from female mice;

[0040] 2) Take the working reagent out of the -20 ℃ refrigerator and place it at room temperature until it is fully dissolved before use;

[0041] 3) 50 μM notoginseng saponin Ft1, notoginseng total saponins and an equal amount of DMSO were added into the culture medium of the follicles of the notoginseng total saponins group, notoginseng saponin Ft1 group and control group, respectively, and cultured continuously in vitro for 24 hours and performed real-time imaging;

[0042] 4) After imaging, the follicular vascular area was calculated every 4 hours using ImageJ software and the follicular vascular growth rate curve within 24 hours was obtained. The results are as follows: Figure 1 As shown, notoginseng saponin Ft1 significantly accelerated the growth rate of follicular blood vessels, while notoginseng total saponins did not have this effect.

[0043] This indicates that notoginseng saponin Ft1 treatment can significantly promote ovarian follicle angiogenesis, while notoginseng total saponin does not have the function of promoting ovarian follicle angiogenesis at the same concentration.

[0044] Example 2: Notoginseng saponin Ft1 improves ovarian blood supply

[0045] Reagent 1: DMSO

[0046] Reagent 2: Panax notoginseng total saponins (powder)

[0047] Reagent 3: Notoginsenoside Ft1 (powder)

[0048] The reagent of the present invention can package notoginsenoside Ft1 and organic solvent separately. When used, notoginsenoside Ft1 is dissolved in organic solvent DMSO to prepare a working solution with a concentration of 20-40 mg / ml, and placed in a -20°C refrigerator to be packaged and stored away from light. In specific applications, 10 mg / ml is used.

[0049] The experimental operation is as follows:

[0050] 1) The experimental mice were divided into three groups: control group, Panax notoginseng total saponin group and Panax notoginseng saponin Ft1 group, each group consisted of 5 12-month-old female mice with ICR background;

[0051] 2) Take the working reagent out of the -20 ℃ refrigerator and place it at room temperature until it is fully dissolved before use;

[0052] 3) Drug treatment was performed by intraperitoneal injection at a dose of 50 mg / kg.BW, once a day for 1 week;

[0053] 4) After drug treatment, the health status of the experimental mice is determined by regularly weighing the test mice. If the weight of the test mice remains unchanged or increases at a slow rate, it indicates that the test mice are in good condition. If the weight of the test mice decreases significantly, the drug dosage can be appropriately reduced;

[0054] 5) After 1 week of drug treatment, mice were injected with Evans blue (0.05 mg / kg.BW) via the tail vein. Fifteen minutes later, the ovaries were collected and individual follicles were separated. The follicle morphology was recorded under a fluorescence microscope and its fluorescence intensity was counted. The results showed that notoginseng saponin Ft1 significantly improved the blood supply to the follicles of aged mice, while notoginseng total saponins did not have the above effect ( Figure 2 ).

[0055] It shows that after treatment with an appropriate amount of Panax notoginseng saponin Ft1, the blood supply to the follicles can be significantly improved, thereby enhancing the working efficiency of gonadotropin and other drugs that are beneficial to follicle development, thereby promoting follicle development.

[0056] Example 3: Notoginseng saponin Ft1 improves superovulation efficiency in young mice

[0057] Reagent 1: DMSO

[0058] Reagent 2: Panax notoginseng total saponins (powder)

[0059] Reagent 3: Notoginsenoside Ft1 (powder)

[0060] The reagent of the present invention can package Panax notoginseng total saponins, Panax notoginseng saponin Ft1 and organic solvent separately. When used, Panax notoginseng total saponins and Panax notoginseng saponin Ft1 are dissolved in the organic solvent DMSO to prepare a working solution with a concentration of 20-40 mg / ml, and placed in a -20°C refrigerator to be packaged and stored away from light. 10 mg / ml is used in specific applications.

[0061] The experimental operation is as follows:

[0062] 1) The experimental mice were divided into three groups: control group, Panax notoginseng total saponin group and Panax notoginseng saponin Ft1 group, each group consisted of 5 23-day-old female mice on ICR background;

[0063] 2) Take the working reagent out of the -20 ℃ refrigerator and place it at room temperature until it is fully dissolved before use;

[0064] 3) Drug treatment was performed by intraperitoneal injection at a dose of 50 mg / kg.BW, once a day for 1 week; after drug withdrawal, superovulation was performed by administering gonadotropin (0.5 IU / g), followed by human chorionic gonadotropin (0.5 IU / g) 46 hours later, and oocytes from the ampulla of the mouse oviduct were collected 16 hours later for morphological examination and counting;

[0065] 4) The results showed that after mice were injected with notoginseng saponin Ft1 (50 mg / kg.BW) and given superovulation treatment, the number of ovulations in mice increased by about 38.04% ( Figure 3 A, 3B, mean 20.25 / 31.75), and there was no significant change in the weight of mice ( Figure 3 C), while after treatment with Panax notoginseng total saponins, the number of ovulation in mice did not increase significantly ( Figure 3 A, 3B, mean 20.25 / 23.00);

[0066] The results showed that after treatment with notoginseng saponin Ft1, the number of ovulations in mice increased significantly and the morphology of follicular cells was normal, and the weight of mice was not significantly different from that of the control group, while notoginseng total saponins did not have this effect.

[0067] This shows that an appropriate amount of Panax notoginseng saponin Ft1 can significantly improve the efficiency of ovulation induction in young mice.

[0068] Example 4: Notoginseng saponin Ft1 improves pregnancy rate and litter size in aged mice

[0069] Reagent 1: DMSO

[0070] Reagent 2: Panax notoginseng total saponins (powder)

[0071] Reagent 3: Notoginsenoside Ft1 (powder)

[0072] The reagent of the present invention can package Panax notoginseng total saponins, Panax notoginseng saponin Ft1 and organic solvent separately. When used, Panax notoginseng total saponins and Panax notoginseng saponin Ft1 are dissolved in the organic solvent DMSO to prepare a working solution with a concentration of 20-40 mg / ml, and placed in a -20°C refrigerator to be packaged and stored away from light. 10 mg / ml is used in specific applications.

[0073] The experimental operation is as follows:

[0074] 1) The experimental mice were divided into three groups: the control group of Panax notoginseng total saponins and the Panax notoginseng saponins Ft1 group, each group consisted of 5 12-month-old female mice with ICR background;

[0075] 2) Take the working reagent out of the -20 ℃ refrigerator and place it at room temperature until it is fully dissolved before use;

[0076] 3) The drug was administered by intraperitoneal injection at a dose of 50 mg / kg.BW, once a day, for 1 week; after drug withdrawal, the mice were co-housed with healthy male mice for 5 days (one estrus cycle), and the pregnancy and littering of the mice were monitored and recorded;

[0077] 4) The results are shown in Table 1:

[0078] Table 1 Pregnancy rate and litter size of mice after intraperitoneal injection

[0079]

[0080] Compared with the control group, the pregnancy rate and litter size of mice in the notoginseng saponin Ft1 group increased significantly, while notoginseng total saponins did not have this effect ( Figure 4 and Table 1). The average pregnancy rate increased by approximately 81.48% ( Figure 4 A), the number of offspring born increased by about 255.56% ( Figure 4 B), however, after treatment with Panax notoginseng total saponins, the pregnancy rate and number of offspring born of mice were not significantly different from those of the control group.

[0081] That is, the pregnancy rate and the number of offspring in the mice in the notoginseng saponin Ft1 group were significantly increased, while notoginseng total saponins did not have this effect.

[0082] It shows that an appropriate amount of Panax notoginseng saponin Ft1 can significantly enhance the reproductive capacity of elderly female mammals, increase the pregnancy rate and the number of offspring born, and improve reproductive aging.

[0083] Experimental conclusion: The present invention adopts notoginseng saponin Ft1, whose molecular structure is C 47 H 80 O 17 Through in vitro culture and in vivo injection, it was shown that notoginseng saponin Ft1 treatment can significantly increase the number of ovulations in female mice and promote angiogenesis of follicles. The above results prove that notoginseng saponin Ft1 is involved in follicular angiogenesis, follicle development and ovulation-related physiological processes.

[0084] Adolescent mice and aged mice were further selected and intraperitoneal injection was used to explore the effect of notoginseng saponin Ft1 on ovarian fertility.

[0085] When using adolescent mice for clinical trials, it was found that the number of ovulations in mice treated with Panax notoginseng saponin Ft1 was significantly higher than that in the control group. When using aged mice for clinical validation trials, it was found that the pregnancy rate and litter size of mice treated with Panax notoginseng saponin Ft1 were significantly higher than those in the control group.

[0086] In the specific embodiment of the present invention, a comparative test is involved. Except for the necessary differences between the test groups, the rest of the test environment and raw materials remain the same.

[0087] 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.

[0088] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. Application of Panax notoginseng saponin Ft1 in the preparation of ovulation-inducing auxiliary drugs.

2. The use according to claim 1, characterized in that: The application is to promote ovulation in advanced-age females.

3. The use according to claim 1 or 2, characterized in that: The application is to promote female pregnancy rate and offspring birth rate.

4. The use according to claim 1, characterized in that: The ovulation induction assistance is to increase the number of oocytes released after superovulation.

5. Use of notoginseng saponin Ft1 in the preparation of a drug for improving female fertility, characterized in that: The application is to improve ovarian function and female reproductive health.

6. The use according to claim 5, characterized in that: The improving female fertility includes improving the ovulation rate of young females and improving the pregnancy rate and litter size of older females.

7. The use according to any one of claims 1 to 6, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that The auxiliary materials include excipients.

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