Application of linear diaryl heptane compound in reproduction-related diseases

Abstract

The invention belongs to the technical field of biological medicines, and provides application of a linear diaryl heptane compound in reproductive related diseases, and the reproductive related diseases comprise infertility, hyperandrogenemia, menstrual cycle disorder, ovulation disorder and reproductive disorders caused by other reasons. The compound can promote and recover ovulation, reduce hyperandrogen and improve the menstrual cycle, is good in safety and free of obvious toxic and side effects, and can be used for preparing drugs for preventing and treating reproductive disorder diseases and promoting ovulation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a linear diarylheptane compound in the preparation of drugs for preventing and treating reproduction-related diseases. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] In modern society, changes in dietary structure, accelerated pace of life, endocrine disorders and other factors have seriously affected women's reproductive health. Major reproductive disorders such as ovarian dysfunction and infertility caused by these factors have become a global public health issue. According to statistics from the National Health Commission, the incidence of infertility in my country has continued to rise in recent years, climbing to 18%, which has had a huge impact on their families and society. According to statistics, the cost of infertility treatment is as high as hundreds of millions of yuan each year, causing a heavy social and economic burden.

[0004] The causes of female reproductive disorders are complex and can be caused by a series of factors such as ovarian dysfunction and hormone imbalance caused by endocrine system disorders. Among them, ovarian dysfunction and ovulation disorders caused by hyperandrogenism are common female reproductive disorders and the main cause of anovulatory infertility. Hyperandrogenism can lead to menstrual cycle disorders, infrequent ovulation or persistent anovulation in women, and will increase the risk of long-term complications such as endometrial cancer.

[0005] For the above patients who have fertility needs, the current first-line clinical treatment is to adjust lifestyle and menstrual cycle, relieve symptoms of hyperandrogenism, and on this basis, adopt ovulation induction and assisted reproductive technology to assist pregnancy. However, the ovulation induction drugs routinely used in clinical practice for female reproductive diseases and assisted reproductive technology are mainly steroid hormones, which are of a single type and have many side effects, and may cause ovarian hyperstimulation syndrome, ovarian cysts, bleeding, effusion and other serious complications. In addition, both ovulation induction drugs and assisted reproduction have long cycles and are expensive, and the economic and psychological burden on patients is heavy. In view of the above limitations in the treatment of reproductive disorders, there is an urgent need to discover more effective, safe and economical drugs to cope with the increasing burden of reproductive disorders and the urgent needs of patients.

[0006] Natural products have great advantages in the prevention and treatment of various diseases due to their strong therapeutic effects, high safety, wide sources, and good economy. Alnus ketone is a non-phenolic diaryl heptane, a natural plant active compound derived from the Chinese medicinal grass cardamom, and exists in many natural medicinal materials such as alnus and turmeric. Previous studies have found that alnus ketone has anti-tumor and antibacterial activities. In the prior art, the relevant medical uses of alnus ketone are also disclosed: CN116019792A, CN115364080A, CN117159514A, and CN118217267A respectively disclose the use of alnus ketone in inhibiting and treating gastric cancer, glucose metabolism disorder disease prevention, atopic dermatitis, thrombocytopenia and other diseases. However, whether this type of linear diaryl heptane compounds have a preventive and therapeutic effect in reproductive disorders and their related endocrine hormone abnormalities, menstrual disorders and ovulation disorders has not been reported in the past. Summary of the invention

[0007] In view of the shortcomings of existing drugs for treating reproduction-related diseases, the present invention provides a new medical application of linear diarylheptane compounds, which can promote and restore ovulation, reduce hyperandrogenism, and improve the menstrual cycle, and is used for preparing drugs for preventing and treating reproduction-related diseases and ovulation-promoting agents.

[0008] To achieve the above purpose, the present invention adopts the following technical solution.

[0009] A linear diarylheptane compound and its derivatives are used in the preparation of drugs for preventing and treating reproductive-related diseases, including but not limited to infertility, hormone abnormalities, menstrual cycle disorders, ovulation disorders and reproductive disorders caused by other reasons.

[0010] The structural formula of the linear diarylheptane compound is: , Wherein, R1, R2, R3, and R4 are independently selected from -H, -OH, and -OCH3.

[0011] Preferably, the compound is selected from: Compound 1: (Alderone); Compound 2: ; Compound 3: ;or, Compound 4: (Tsaokoarylone).

[0012] The derivatives of the linear diarylheptane compounds are physiologically acceptable salts formed by the linear diarylheptane compounds and inorganic or organic bases, including but not limited to alkali metal salts, alkaline earth metal salts, ammonium salts, and salts formed with nitrogen-containing organic bases, such as sodium salts, potassium salts, calcium salts, and magnesium salts. The nitrogen-containing organic bases are not limited to trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, dibenzylamine, N-benzyl-β-phenethylamine, and N,N'-dibenzylethylenediamine.

[0013] The derivative is a substitute formed by the above-mentioned linear diarylheptane compound and an inorganic acid or a physiologically acceptable ester formed by the above-mentioned linear diarylheptane compound and an organic acid; the inorganic acid is selected from sulfuric acid, phosphoric acid, carbonic acid, and hydrochloric acid; the organic acid is selected from formic acid, acetic acid, propionic acid, butyric acid, maleic acid, oxalic acid, methanesulfonic acid, succinic acid or fatty acid.

[0014] The linear diarylheptane compound can be obtained by chemical synthesis or biological extraction. Preferably, the biological extraction source is selected from cardamom, alnus sibiricum, turmeric, tsaoko and the like.

[0015] Preferably, the linear diarylheptane compounds can be used in the preparation of ovulation-promoting agents. The applications of the ovulation-promoting agents include but are not limited to ovulation induction in assisted reproductive technology.

[0016] The present invention also provides a composition for treating reproductive disorders or promoting ovulation, comprising an effective dose of linear diarylheptane compounds and their derivatives, plants containing linear diarylheptane compounds and their derivatives, or plant extracts containing linear diarylheptane compounds and their derivatives.

[0017] Preferably, the plant containing linear diarylheptane compounds is at least one selected from the group consisting of Alnus fragrans, Alnus truncatula, Curcuma longa and Amomum villosum.

[0018] The above composition can be prepared according to methods well known in the art or disclosed in the prior art.

[0019] The above composition also includes pharmaceutically or food acceptable auxiliary materials for preparing the active ingredients into any dosage form suitable for human or animal use.

[0020] The above composition may also contain other effective ingredients, such as estrogen and its analogs, luteinizing hormone and its analogs, follicle-stimulating hormone and its analogs, etc.

[0021] The composition can be taken orally, by injection or by mucosal administration. The composition can also be prepared as a sustained-release preparation or a controlled-release preparation. The dosage form of the composition includes, but is not limited to, tablets, capsules, granules, ointments, suppositories, pills, gels, injections, suspensions, oral liquids or syrups.

[0022] The present invention has the following advantages: The present invention verifies through experiments that alder ketone has the effects of promoting and restoring ovulation, improving abnormal follicular development, reducing hyperandrogenism, and restoring normal menstrual cycle in vivo, and has good safety and no obvious toxic and side effects, and can be used to prepare drugs for preventing and treating reproductive disorders and promoting ovulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Giving alderone to mice modeling hyperandrogenism infertility restored ovarian morphology and promoted ovulation function; Figure 2 The number of corpora lutea in the ovaries of mice with high androgen levels was significantly increased after treatment with alderone. Figure 3 Treatment of hyperandrogenism model mice with alderone improved abnormal follicular development; Figure 4 After the hyperandrogenism model mice were treated with alder ketone, the serum hyperandrogenism level was significantly reduced; Figure 5 Menstrual cycle disturbances in mice modeling hyperandrogenism were corrected after treatment with alderone. DETAILED DESCRIPTION

[0024] Compounds 1-4 in the present application can reduce hyperandrogenism, promote ovulation and improve menstrual cycle disorder symptoms in reproductive disorder infertility model mice by injection or oral administration. The present invention is further described below with reference to the examples and drawings using alnus ketone as an example, but the present invention is not limited to the following examples.

[0025] Example 1 Alnus ketone promotes ovulation in high androgen infertility mice 1. Construction of the hyperandrogenism infertility mouse model Wild-type C57BL / 6J female mice (purchased from Beijing Weitong Lihua Company) were given 60 mg / kg body weight of dehydroepiandrosterone (DHEA) dissolved in corn oil by subcutaneous injection every day from 3 weeks of age, while the control group mice were only injected with the corresponding volume of corn oil solvent for 21 days. This model mouse can show reproductive disorder phenotypes such as increased blood androgen levels, estrous cycle disorders, and anovulation, and is a commonly used infertility mouse model.

[0026] 2. Alnus ketone dosing regimen The mice induced by hyperandrogen were randomly divided into two groups: the alderone group: the alderone powder (purchased from Shanghai Taoshu Biotechnology Co., Ltd., purity: 99.87%) was dissolved in corn oil containing 5% DMSO, and the C57 mice were intraperitoneally injected at a dose of 10 mg / kg every day; the control group: the same model mice were intraperitoneally injected with the same dose of corn oil solvent containing 5% DMSO every day. After two weeks of administration, the samples were collected to observe the treatment results of alderone on hyperandrogen model mice.

[0027] 3. Isolation, Fixation, Embedding, and Sectioning of Mouse Ovaries After bleeding from the eyeballs and killing by dislocating the neck, the bilateral ovaries of the mice were completely removed, and the fat tissue around the ovaries was removed under a microscope. After washing with PBS, the water was dried with absorbent paper, and the mice were immersed in 4% paraformaldehyde and fixed overnight in a shaker at 4°C. After washing with PBS three times, they were transferred to an embedding box and dehydrated with ethanol and xylene. The dehydrated tissue was taken out of the embedding box, embedded in 65°C paraffin, and then placed on a -20°C ice table to cool and solidify the paraffin. The embedded tissue was sliced: the tissue wax block was sliced ​​continuously with a slicer, and the slice thickness was 5μm. After fully spreading the slice, the tissue slice was picked up with a slide, and the slice was baked and stored.

[0028] 4. HE staining of mouse ovaries Ovarian sections were treated with xylene I for 10 min, xylene II for 10 min, xylene III for 10 min→anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, 95% ethanol for 2 min→rinse the sections twice with distilled water→stain with hematoxylin for 15 s, rinse with running water for 2 min→rinse twice with distilled water→stain with eosin for 5 s, rinse twice with distilled water→95% ethanol for 30 s, anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min→xylene I for 10 min, xylene II for 10 min→seal the sections with a sealing agent prepared with neutral gum and xylene, and then photographed with a panoramic microscope.

[0029] 5. Counting of Corpus Luteum and Follicles of Different Stages in Mouse Ovaries HE staining was used to detect the ovarian tissue structure and count the follicles. The number of primordial follicles, primary follicles, secondary follicles, and antral follicles was counted once every three consecutive sections and multiplied by 3 to obtain the total number of follicles in each ovary (to avoid repeated counting, only oocytes with visible nucleoli were counted). Figure 1It can be seen that: multiple follicles and corpora lutea at different developmental stages can be seen in the ovaries of mice in the normal control group, and the granulosa cells are complete in morphology and neatly arranged. The number of follicles at all levels in the ovaries of mice in the androgen modeling group increased significantly, indicating abnormal follicular development; the corpus luteum of the ovary was not visible, indicating that ovulation did not occur; and the granulosa cells were loosely arranged, the layers became thinner, and some fell off, the theca cells proliferated, and the morphological structure in the ovaries was disordered, indicating ovarian dysfunction. After treatment with alder ketone, it can be seen that the number of follicles at different stages of development was reduced compared with the modeling group, and the number of corpora lutea increased significantly, indicating that the ovulation of mice was restored; the granulosa cells were thickened and arranged more tightly, with more layers, the theca cells were thinner, and the ovarian morphology was close to that of the normal control group. In addition, from the histological structure of the ovaries, it can be seen that the ovaries of mice in the alder ketone treatment group did not show abnormal follicle structure and bleeding and other adverse reactions, indicating that the use of alder ketone is safe and has no obvious toxic and side effects ( Figure 1 ).

[0030] The corpus luteum counts of the above mice revealed that ( Figure 2 ), the corpus luteum in the ovaries of mice in the high androgen modeling group decreased sharply or even disappeared, indicating that the mice did not ovulate, confirming the successful modeling of reproductive infertility. In contrast, the number of corpus luteum in the ovaries of mice treated with alder ketone increased significantly, indicating that the mice resumed ovulation. In addition, the results of the follicle counts at all levels of the ovaries of the above mice also confirmed ( Figure 3 ), Alnus ketone can restore the high androgen-induced follicular development disorder in mice.

[0031] The data analysis results after the above experiment show that in the high androgen-induced infertility mouse model, the ovarian morphological phenotype of the mice was significantly improved and the number of corpora lutea increased significantly after treatment with alnus ketone, indicating the recovery of ovulation ability. The experimental data of the above embodiment show that alnus ketone can effectively improve the ovulation disorder of the infertility animal model, promote and restore ovulation, and improve the abnormal ovarian morphology without obvious toxic and side effects, providing sufficient in vivo biological evidence for the application of alnus ketone in reproductive disorders.

[0032] Example 2 Alnus ketone significantly reduces hyperandrogenism in mice 1. Blood collection from model mice Mice were given DHEA to induce hyperandrogenism, and then alderone was intraperitoneally injected for two weeks. The modeling and administration scheme were the same as in Example 1. After the intervention, blood was collected from the submandibular vein of the mice in the above groups. Serum was separated by centrifugation at 3000 rpm for 10 minutes at 4°C and stored at -80°C.

[0033] 2. Determination of androgen After the above blood collection, 50 μL of the mouse serum was taken and measured for the testosterone (T) content in the serum using radioimmunoassay at Beijing North Institute of Biotechnology (Beijing, China).

[0034] Depend on Figure 4 The experimental results show that compared with the control group, the serum testosterone content of mice in the DHEA modeling group increased significantly, confirming that the hyperandrogenemia modeling was successful. In contrast, the serum testosterone level of mice treated with alder ketone decreased significantly, close to the level of the normal control group, indicating that alder ketone can effectively reduce hyperandrogenemia and improve the corresponding reproductive symptoms.

[0035] Example 3 Alnus ketone significantly improves menstrual cycle disorders in mice 1. Vaginal cell smear of model mice Mice were given continuous DHEA injections to induce menstrual cycle disorders, and then alder ketone was intraperitoneally injected for two weeks. The modeling and administration scheme were the same as in Example 1. Vaginal smears were examined for 14 consecutive days for the above groups of mice to observe the changes in their estrous cycle. The mice were sampled at a fixed time every afternoon, the vaginal opening of the mice was exposed, 20 μL of physiological saline was drawn with a pipette and placed in the vaginal opening of the mice for repeated suction, and then the liquid was evenly applied on a labeled clean slide to collect vaginal cells.

[0036] 2. Mouse vaginal cell smear staining After the smear is air-dried, HE staining is performed. The specific steps are: fix with anhydrous ethanol → air-dry naturally and then stain with hematoxylin for 5 minutes → rinse with running water for 2 minutes → stain with eosin for 1 minute → rinse with running water for 2 minutes and air-dry naturally. Observe the types and morphology of mouse vaginal cells under a microscope and record the estrous cycle.

[0037] 3. Determination of the estrous cycle of mice The estrous cycle of mice usually lasts 4-5 days and is divided into four periods: proestrus, estrus, metestrus and diestrus. The criteria for determining each estrus period are as follows: ① Proestrus: Nucleated epithelial cells predominate in vaginal smears, which may be single or in sheets, and there are a small number of white blood cells; ② Estrus: There are many anucleated keratinized squamous cells in the smear. The cells are large and flat with irregular edges. There are no or very few white blood cells and epithelial cells in the field of vision; ③ Metestrus: The number of keratinized epithelial cells in the vaginal cavity decreases, and many white blood cells and nucleated epithelial cells appear, and the proportions of the three are similar; ④ Diestrus: White blood cells account for the vast majority, and nucleated epithelial cells can occasionally be seen.

[0038] The statistical results of the estrous cycle of mice in the androgen and alderone groups showed that ( Figure 5 ), the estrous cycle of mice in the normal control group was regular, while the estrous cycle of mice in the high androgen modeling group was significantly disordered, with most of them in late estrus and interestrus. After treatment with alder ketone, the number of mice in proestrus and estrus increased significantly, and the estrous cycle returned to normal.

[0039] The experimental data of this example show that alnus ketone can effectively improve the estrous cycle of infertility animal models, promote and restore normal reproductive function, and provide in vivo biological evidence for the application of alnus ketone in reproductive disorders.

[0040] The above studies, by constructing an animal model of infertility induced by hyperandrogenism to simulate the pathophysiological process of human reproductive-related diseases, revealed the new function of alnus ketone by effectively promoting and restoring ovulation, reducing hyperandrogenism, and improving the menstrual cycle, with good safety and no obvious toxic side effects. This shows that alnus ketone can be used as a new and effective drug for the clinical prevention and treatment of reproductive-related diseases and ovulation induction.

[0041] Example 4 Preparation of Alnus ketone injection The alnus ketone powder is dissolved in corn oil for injection to prepare an oil solvent of 50 mg / mL-100 mg / mL, and the oil solvent is packaged into 1 mL / ampoule bottles to obtain an injection solution.

[0042] Example 5 Preparation of Tsaokoarylone Tablets The Tsaokoarylone API and excipients are crushed and passed through a 100-mesh sieve to obtain a uniform powder without lumps; then, a filler and a disintegrant are added after mixing the binder and the API, and finally a lubricant is added, and a rotary tablet press is used for dry tableting to obtain tablets of 50 mg to 100 mg per tablet; the binder is selected from dextrin, cyclodextrin, sodium carboxymethyl cellulose, povidone, polyethylene glycol, etc.; the filler is selected from sucrose, dextrin, starch, microcrystalline cellulose, etc.; the disintegrant is selected from sodium carboxymethyl cellulose, ethyl cellulose, hydroxypropyl cellulose, etc.; the lubricant is selected from magnesium stearate, talc, micropowder silica gel, hydrogenated vegetable oil, etc.

[0043] Example 6 Preparation of Compound 2 Capsules The powder of compound 2 and auxiliary materials are crushed and passed through a 100-mesh sieve to obtain a uniform powder without lumps; then the binder, filler and raw material are mixed and sprayed with a small amount of ethanol solution to form a soft material, which is then passed through a 15-mesh sieve to obtain wet granules, which are dried at about 50°C to obtain dry granules, which are then filled into gelatin hard capsule skins to obtain capsules with a specification of 100mg-200mg; the binder is selected from dextrin, cyclodextrin, sodium carboxymethyl cellulose, povidone, polyethylene glycol, etc.; the filler is selected from sucrose, dextrin, starch, microcrystalline cellulose, etc.

[0044] Example 7 Preparation of Compound 3 Suppository The hot melt method is used to prepare suppositories. First, compound 3 and borneol are mixed and crushed and passed through a 100-mesh sieve to obtain a uniform powder without lumps; a small amount of water is added to glycerin, and then gelatin is added to swell, and then polyethylene glycol 400 and polyethylene glycol 4000 are added and heated to about 65°C in a water bath to completely melt to obtain a matrix; then the powder is mixed with liquid paraffin, added to the matrix and stirred evenly, and then injection molding and cooling are performed to obtain 1-2g / tablet suppositories with a specification of 100mg-200mg.

[0045] Example 8 Preparation of Alnus ketone external thermosensitive gel Poloxamer 407, poloxamer 188, chitosan and hyaluronic acid in a formula ratio are uniformly mixed and added to a buffer solution of pH 4-5 for swelling and dissolution to obtain a gel matrix; alnus ketone is dissolved in DMSO; hydroxypropyl-β-cyclodextrin is dissolved in water to saturation, the cyclodextrin solution is dropped into the alnus ketone solution, and the inclusion is completed by stirring or ultrasound to obtain a suspension; the suspension is added to the gel matrix in proportion and stirred to obtain a vaginal thermosensitive gel with a gel temperature of 32°C-35°C, which is filled and stored at low temperature; the gel temperature is about 37°C after being diluted with environmental body fluids.

[0046] The above is only the embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Use of a linear diarylheptane compound and its derivatives in the preparation of drugs for preventing and treating reproductive-related diseases, It is characterized in that The reproductive-related disease is selected from reproductive disorders caused by infertility, ovulation disorders, hormone abnormalities or menstrual cycle disorders; The structural formula of the linear diarylheptane compound is: ， Among them, R 1 , R 2 , R 3 , R 4 are independently selected from -H, -OH and -OCH 3 ; The derivative is a physiologically acceptable salt formed by the linear diarylheptane compound and an inorganic or organic base; Or, the linear diarylheptane compound is a substitute formed by an inorganic acid or a physiologically acceptable ester formed by an organic acid.

2. The use according to claim 1, It is characterized in that The linear diarylheptane compound is selected from ; ; ;or, ; The salt is selected from alkali metal salts, alkaline earth metal salts, ammonium salts or salts formed with nitrogen-containing organic bases; the inorganic acid is selected from sulfuric acid, phosphoric acid, carbonic acid or hydrochloric acid; the organic acid is selected from formic acid, acetic acid, propionic acid, butyric acid, maleic acid, oxalic acid, methanesulfonic acid, succinic acid or fatty acids.

3. The use according to claim 2, It is characterized in that The salt is selected from sodium salt, potassium salt, calcium salt or magnesium salt; the nitrogen-containing organic base is selected from trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, dibenzylamine, N-benzyl-β-phenethylamine or N,N'-dibenzylethylenediamine.

4. The use according to claim 1, It is characterized in that The linear diarylheptane compounds can be obtained by chemical synthesis or biological extraction; preferably, the source of biological extraction is selected from cardamom, alnus truncatula, turmeric or amomum villosum.

5. The use according to claim 1, It is characterized in that The drug is an ovulation inducing agent.

6. A composition for treating reproductive disorders or promoting ovulation, It is characterized in that The invention comprises an effective dose of the linear diarylheptane compound and its derivatives as claimed in any one of claims 1 to 5; a plant containing the linear diarylheptane compound and its derivatives as claimed in any one of claims 1 to 5; or a plant extract containing the linear diarylheptane compound and its derivatives as claimed in any one of claims 1 to 5.

7. The composition according to claim 6, It is characterized in that The plant is selected from at least one of Alnus fragrans, Alnus truncatus, Turmeric and Amomum villosum.

8. The composition according to claim 6, It is characterized in that It also contains other active ingredients and / or pharmaceutically or food acceptable excipients, such as estrogen and its analogues, luteinizing hormone and its analogues, follicle stimulating hormone and its analogues.

9. The composition according to claim 6, It is characterized in that The composition is taken orally, by injection or by mucosal administration; the composition is in the form of tablets, capsules, granules, ointments, suppositories, pills, gels, injections, suspensions, oral liquids or syrups; preferably, the composition is in the form of sustained-release preparations or controlled-release preparations.

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