Use of linear diarylheptanoids in reproductive-related diseases

Abstract

This invention belongs to the field of biomedical technology and provides an application of linear diarylheptane compounds in reproductive-related diseases, including infertility, hyperandrogenemia, menstrual cycle disorders, ovulation disorders, and other reproductive disorders. These compounds can promote and restore ovulation, reduce hyperandrogenemia, and improve menstrual cycles, with good safety and no obvious toxic side effects. They can be used to prepare drugs for the prevention and treatment of reproductive disorders and for ovulation induction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a linear diarylheptane compound in the preparation of drugs for the prevention and treatment of reproductive-related diseases. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Modern society's changing dietary patterns, fast-paced lifestyles, and endocrine disorders have severely impacted women's reproductive health, leading to major reproductive disorders such as ovarian dysfunction and infertility, which have become a global public health issue. According to statistics from the National Health Commission, the incidence of infertility in my country has been rising steadily in recent years, reaching 18%, significantly impacting families and society. It is estimated that the annual cost of infertility treatment reaches hundreds of millions of yuan, creating a heavy socioeconomic burden.

[0004] The causes of female reproductive disorders are complex, and can be caused by a series of factors such as ovarian dysfunction and hormonal imbalances resulting from endocrine system disorders. Among them, ovarian dysfunction and ovulation disorders caused by hyperandrogenemia are common female reproductive disorders and a major cause of anovulatory infertility. Hyperandrogenemia can lead to menstrual cycle disorders, infrequent ovulation or persistent anovulation in women, and increases the risk of long-term complications such as endometrial cancer.

[0005] For the aforementioned patients who desire fertility, the current first-line clinical treatment involves lifestyle and menstrual cycle adjustments to alleviate hyperandrogenemia symptoms, followed by ovulation induction and assisted reproductive technologies (ART). However, the ovulation-inducing drugs routinely used in female reproductive diseases and ART are primarily steroid hormones, which are limited in type and have numerous side effects, potentially leading to serious complications such as ovarian hyperstimulation syndrome, ovarian cysts, bleeding, and fluid accumulation. Furthermore, both ovulation-inducing drugs and ART are time-consuming and expensive, placing a heavy economic and psychological burden on patients. Given these limitations in the treatment of reproductive disorders, there is an urgent need to discover more effective, safe, and economical drugs to address the increasing burden of reproductive disorders and the pressing needs of patients.

[0006] Natural products, due to their strong therapeutic effects, high safety, wide availability, and economic benefits, play a significant role in the prevention and treatment of various diseases. Alder ketone, a non-phenolic diarylheptane, is a natural plant-active compound derived from the traditional Chinese medicine cardamom and is found in many natural medicinal materials such as alder and turmeric. Previous studies have found that alder ketone possesses antitumor and antibacterial activities. Existing technologies also disclose related pharmaceutical uses of alder ketone: CN116019792A, CN115364080A, CN117159514A, and CN118217267A disclose the application of alder ketone in inhibiting and treating gastric cancer, disorders of glucose metabolism, atopic dermatitis, thrombocytopenia, and other conditions. However, whether this type of linear diarylheptane compound has a preventive or therapeutic effect on reproductive disorders and related endocrine hormone abnormalities, menstrual disorders, and ovulation disorders has not been previously reported. Summary of the Invention

[0007] This invention addresses the shortcomings of existing drugs for treating reproductive diseases by providing a novel pharmaceutical application of linear diarylheptane compounds. These compounds can promote and restore ovulation, reduce high androgen levels, and improve menstrual cycles. They can be used to prepare drugs for the prevention and treatment of reproductive diseases, as well as ovulation induction agents.

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

[0009] The application of a linear diarylheptane compound and its derivatives in the preparation of drugs for the prevention and treatment of reproductive-related diseases. These reproductive-related diseases include, but are not limited to, infertility, hormonal abnormalities, menstrual cycle disorders, ovulation disorders, and other reproductive disorders caused by various reasons.

[0010] The structural formula of the linear diarylheptane compound is:

[0011] ,

[0012] Among them, R1, R2, R3, and R4 are independently selected from -H, -OH, and -OCH3, respectively.

[0013] Preferably, the compound is selected from:

[0014] Compound 1: (alderne);

[0015] Compound 2: ;

[0016] Compound 3: ;or,

[0017] Compound 4: (Tsaokoarylone).

[0018] The derivatives of the linear diarylheptane compounds are physiologically acceptable salts formed by 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 by nitrogen-containing organic bases, such as sodium salts, potassium salts, calcium salts, and magnesium salts. The nitrogen-containing organic bases include, but are not limited to, trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, dibenzylamine, N-benzyl-β-phenylethylamine, and N,N'-dibenzylethylenediamine.

[0019] The derivative is a substituted product formed by the above-mentioned linear diarylheptane compounds and an inorganic acid or a physiologically acceptable ester formed by the above-mentioned linear diarylheptane compounds 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 acids.

[0020] The linear diarylheptane compounds can be obtained through chemical synthesis or biological extraction. Preferably, the biological extraction sources are selected from cardamom, alder, turmeric, and amomum tsao-ko.

[0021] Preferably, the above-mentioned linear diarylheptane compounds can be used in the preparation of ovulation-inducing agents. The applications of these ovulation-inducing agents include, but are not limited to, inducing ovulation through assisted reproductive technologies.

[0022] The present invention also provides a composition for treating reproductive disorders or inducing ovulation, comprising an effective dose of a linear diarylheptane compound and its derivatives, a plant containing a linear diarylheptane compound and its derivatives, or a plant extract containing a linear diarylheptane compound and its derivatives.

[0023] Preferably, the plant containing linear diarylheptane compounds is selected from at least one of cardamom, alder, turmeric, and amomum.

[0024] The above composition can be prepared according to methods known in the art or disclosed in the prior art.

[0025] The above compositions also include pharmaceutically or food-acceptable excipients for preparing the active ingredient into any dosage form suitable for human or animal use.

[0026] The above composition may also contain other active ingredients, such as estrogen and its analogues, luteinizing hormone and its analogues, follicle-stimulating hormone and its analogues, etc.

[0027] The composition can be administered orally, by injection, or via mucosal delivery. The composition can also be formulated as a sustained-release or controlled-release formulation. Dosage forms of the composition include, but are not limited to, tablets, capsules, granules, ointments, suppositories, pills, gels, injections, suspensions, oral solutions, or syrups.

[0028] The present invention has the following advantages:

[0029] This invention experimentally demonstrates that alder ketone has the effects of promoting and restoring ovulation, improving abnormal follicle development, reducing hyperandrogenemia, and restoring normal menstrual cycles in vivo. It also has good safety and no obvious toxic side effects, and can be used to prepare drugs for the prevention and treatment of reproductive disorders and for ovulation induction. Attached Figure Description

[0030] Figure 1 Alder ketone treatment restored ovarian morphology and promoted ovulation in mice with hyperandrogen infertility.

[0031] Figure 2 Treatment with alfalfa ketone significantly increased the number of corpus luteum in the ovaries of mice with hyperandrogenemia.

[0032] Figure 3 Alder ketone treatment improved abnormal follicular development in hyperandrogen-induced model mice;

[0033] Figure 4 Treatment with alfalfa ketone significantly reduced serum androgen levels in mice with hyperandrogenism.

[0034] Figure 5 The menstrual cycle disorder in mice with hyperandrogenemia was corrected after treatment with alder ketone. Detailed Implementation

[0035] Compounds 1-4 in this application can reduce hyperandrogenemia, promote ovulation, and improve menstrual cycle disorder symptoms in mice with reproductive dysfunction by injection or oral administration. The invention is further illustrated below with alder ketone as an example, in conjunction with embodiments and accompanying drawings; however, the invention is not limited to the following embodiments.

[0036] Example 1: Alderne promotes ovulation in mice with hyperandrogen-induced infertility.

[0037] 1. Construction of a mouse model of hyperandrogen infertility

[0038] Wild-type C57BL / 6J female mice (purchased from Beijing Vital River Pharmaceutical Co., Ltd.) were administered 60 mg / kg body weight of dehydroepiandrosterone (DHEA) dissolved in corn oil subcutaneously daily starting at 3 weeks of age. Control group mice received only the corresponding volume of corn oil solution. This injection model resulted in mice exhibiting reproductive disorders such as elevated serum androgen levels, estrous cycle irregularities, and anovulation, making it a commonly used mouse model for infertility.

[0039] 2. Alderone Dosing Regimen

[0040] Mice induced with hyperandrogenism were randomly divided into two groups: an alderne group (alderne powder, purchased from Shanghai Taoshu Biotechnology Co., Ltd., purity: 99.87%) dissolved in corn oil containing 5% DMSO, and administered intraperitoneally to C57 mice daily at a dose of 10 mg / kg); and a control group (mice with the same model were injected intraperitoneally daily with the same dose of corn oil containing 5% DMSO). Two weeks after administration, samples were collected to observe the therapeutic effects of alderne on the hyperandrogenism model mice.

[0041] 3. Isolation, fixation, embedding, and sectioning of mouse ovaries

[0042] After euthanasia via cervical dislocation and exsanguination, both ovaries of the mice were completely removed. Periosteal adipose tissue was removed under a microscope, and the tissues were washed with PBS, blotted dry with absorbent paper, and then fixed overnight in 4% paraformaldehyde on a shaker at 4°C. After washing three times with PBS, the tissues were transferred to an embedding cassette and dehydrated with ethanol and xylene. The dehydrated tissues were removed from the embedding cassette and embedded in paraffin at 65°C, then placed on an ice table at -20°C to allow the paraffin to solidify. The embedded tissues were sectioned: the paraffin blocks were serially sectioned using a microtome to a thickness of 5 μm. After thorough spreading, the tissue sections were retrieved onto a glass slide, baked, and stored.

[0043] 4. HE staining of mouse ovaries

[0044] Ovarian sections were subjected to the following treatments: 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, rinsed twice with distilled water, hematoxylin staining for 15 s, rinsed with running tap water for 2 min, rinsed twice with distilled water, eosin staining for 5 s, rinsed 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, and then mounted with a mounting medium made of neutral resin and xylene. The sections were then photographed using a panoramic microscope.

[0045] 5. Counting of corpus luteum and follicles at various stages in mouse ovaries.

[0046] Ovarian tissue structure was examined using HE staining, and follicles were counted. The number of primordial follicles, primary follicles, secondary follicles, and antral follicles was counted every three consecutive sections, and multiplied by 3 to obtain the total number of follicles in each ovary (to avoid double counting, only oocytes with visible nucleoli were counted). Figure 1 It was observed that in the normal control group, multiple follicles and corpora lutea at different developmental stages were visible in the ovaries of mice, with intact and neatly arranged granulosa cells. In the androgen-induced model group, the number of follicles at all stages in the ovaries was significantly increased, indicating abnormal follicular development; the corpora lutea were not visible, indicating anovulation; and the granulosa cells were loosely arranged, thinner in layers, and partially detached, with theca cell proliferation and disordered ovarian morphology, indicating ovarian dysfunction. After treatment with alderne, the number of follicles at different developmental stages decreased compared to the model group, while the number of corpora lutea significantly increased, indicating restored ovulation in the mice; the granulosa cells thickened and were more tightly arranged, with more layers, while theca cells were thinner, and the ovarian morphology was close to that of the normal control group. Furthermore, histological analysis of the ovaries showed no abnormal follicular structure or adverse reactions such as bleeding in the alderne-treated group, indicating that alderne has good safety and no obvious toxic side effects. Figure 1 ).

[0047] The corpus luteum count in the above mice revealed ( Figure 2 In the hyperandrogen-induced model group, the number of corpora lutea in the ovaries decreased sharply or even disappeared, indicating that ovulation had not occurred, thus confirming the successful establishment of a reproductive infertility model. In contrast, the number of corpora lutea in the ovaries of mice treated with alfalfa significantly increased, indicating that ovulation had resumed. Furthermore, the follicle counts at various stages in the ovaries of the above-mentioned mice also confirmed (…). Figure 3 Alder ketone can restore follicular development disorder induced by hyperandrogenemia in mice.

[0048] Data analysis following the above experiments showed that in a mouse model of infertility induced by hyperandrogenemia, treatment with alfalfa significantly improved ovarian morphology and increased the number of corpora lutea, indicating a recovery of ovulation capacity. The experimental data from the above examples demonstrate that alfalfa can effectively improve ovulation disorders in infertile animal models, promote and restore ovulation, and improve abnormal ovarian morphology, with no significant toxic side effects, providing sufficient in vivo biological evidence for the application of alfalfa in reproductive disorders.

[0049] Example 2: Alderne significantly reduced hyperandrogenemia in mice.

[0050] 1. Blood collection from model mice

[0051] Mice were given DHEA to induce hyperandrogenemia, followed by intraperitoneal injection of alfalfa for two weeks. The modeling and administration regimens were the same as in Example 1. After intervention, blood was collected from the mandibular vein of each group of mice. Serum was separated by centrifugation at 3000 rpm for 10 min at 4°C and stored at -80°C.

[0052] 2. Androgen determination

[0053] The serum from the mice collected above was used to measure the testosterone (T) content in 50 μL at the Beijing Northern Biotechnology Research Institute (Beijing, China) using radioimmunoassay.

[0054] Depend on Figure 4 The experimental results showed that, compared with the control group, the serum testosterone level in the DHEA model group was significantly increased, confirming the successful establishment of the hyperandrogenemia model. In contrast, the serum testosterone level in mice treated with alfalfa was significantly reduced, approaching the level of the normal control group, indicating that alfalfa can effectively reduce hyperandrogenemia and improve the corresponding reproductive symptoms.

[0055] Example 3: Alder significantly improved menstrual cycle disorders in mice.

[0056] 1. Vaginal cell smears from model mice

[0057] Mice were induced to have menstrual cycle disorders by continuous DHEA injections, followed by two weeks of intraperitoneal injections of alfalfa. The modeling and drug administration regimens were the same as in Example 1. Vaginal smears of mice in each group were examined for 14 consecutive days to observe changes in their estrous cycle. Samples were collected from mice at a fixed time each afternoon. The vaginal opening of the mice was exposed, and 20 μL of physiological saline was drawn up with a pipette and repeatedly aspirated from the vaginal opening. The liquid was then evenly spread onto a labeled clean glass slide to collect vaginal cells.

[0058] 2. Staining of mouse vaginal cell smears

[0059] After the smears air-dried, they were stained with hematoxylin and eosin (HE). The specific steps were: fixation with anhydrous ethanol → air-drying followed by hematoxylin staining for 5 min → rinsing with running tap water for 2 min → eosin staining for 1 min → rinsing with running tap water for 2 min and air-drying. The types and morphology of mouse vaginal cells were observed under a microscope, and the estrous cycle was recorded.

[0060] 3. Determination of the estrous cycle in mice

[0061] The estrous cycle in mice typically lasts 4-5 days and is divided into four phases: proestrus, estrus, metestrus, and diaestrus. The criteria for determining each phase are as follows: ① Proestrus: Nucleated epithelial cells dominate in vaginal smears, appearing singly or in patches, with a small number of leukocytes; ② Estrrus: Numerous anucleated keratinized squamous cells are present in the smear, large and flat with irregular edges, and few or no leukocytes or epithelial cells are visible in the field of view; ③ Metestrus: The number of keratinized epithelial cells in the vaginal cavity decreases, and many leukocytes and nucleated epithelial cells appear, with similar proportions of the three; ④ Diaestrus: Leukocytes predominate, and nucleated epithelial cells can be seen occasionally.

[0062] The statistical results of estrous cycles in mice in the androgen and aldosterone groups are shown below ( Figure 5 In the normal control group, the estrous cycle of mice was regular, while in the hyperandrogen model group, the estrous cycle was significantly disordered, with most mice in the post-estrous and inter-estrous phases. After treatment with aldosterone, the number of pro-estrous and estrous phases in mice increased significantly, and the estrous cycle returned to normal.

[0063] The experimental data in this embodiment demonstrate that alder ketone can effectively improve the estrous cycle in infertile animal models, promote and restore normal reproductive function, and provide in vivo biological evidence for the application of alder ketone in reproductive disorders.

[0064] The above studies, by constructing an animal model of infertility induced by hyperandrogenemia to simulate the pathophysiological processes of human reproductive-related diseases, revealed the novel functions of aldosterone in effectively promoting and restoring ovulation, reducing hyperandrogenemia, and improving the menstrual cycle, with good safety and no obvious toxic side effects. This indicates that aldosterone can serve as a novel and effective drug for the clinical prevention and treatment of reproductive-related diseases and as an ovulation induction agent.

[0065] Example 4: Preparation of alderne injection

[0066] Alder powder was dissolved in corn oil for injection to prepare an oil solution with a concentration of 50 mg / mL to 100 mg / mL, which was then dispensed into 1 mL ampoules to obtain the injection solution.

[0067] Example 5: Preparation of Tsaokoarylone tablets

[0068] Tsaokoarylone active pharmaceutical ingredient and excipients were pulverized and passed through a 100-mesh sieve to obtain a uniform, lumpy-free powder. Then, binder and active pharmaceutical ingredient were mixed, followed by the addition of filler and disintegrant, and finally lubricant. The mixture was then dry-compressed using a rotary tablet press to obtain tablets of 50mg-100mg / tablet. The binder was selected from dextrin, cyclodextrin, sodium carboxymethyl cellulose, povidone, polyethylene glycol, etc.; the filler was selected from sucrose, dextrin, starch, microcrystalline cellulose, etc.; the disintegrant was selected from sodium carboxymethyl cellulose, ethyl cellulose, hydroxypropyl cellulose, etc.; and the lubricant was selected from magnesium stearate, talc, micronized silica gel, hydrogenated vegetable oil, etc.

[0069] Example 6 Preparation of Compound 2 Capsules

[0070] Compound 2 powder and excipients were pulverized and passed through a 100-mesh sieve to obtain a uniform, lumpy powder. Then, the binder, filler, and active pharmaceutical ingredient were mixed and a small amount of ethanol solution was sprayed into the mixture to form a soft mass. The mass was then passed through a 15-mesh sieve to obtain wet granules, which were dried at about 50°C to obtain dry granules. These dry granules were then filled into gelatin hard capsule shells to obtain capsules with a specification of 100mg-200mg. The binder was selected from dextrin, cyclodextrin, sodium carboxymethyl cellulose, povidone, polyethylene glycol, etc. The filler was selected from sucrose, dextrin, starch, microcrystalline cellulose, etc.

[0071] Example 7 Preparation of Compound 3 Suppositories

[0072] Suppositories were prepared using a hot-melt method. First, compound 3 and borneol were mixed, pulverized, and passed through a 100-mesh sieve to obtain a uniform, lumpy-free powder. Glycerin was added to a small amount of water, followed by the addition of gelatin to swell the powder. Then, polyethylene glycol 400 and polyethylene glycol 4000 were added and heated in a water bath to about 65°C until completely melted to obtain the matrix. The powder was then mixed with liquid paraffin, added to the matrix, and stirred evenly. The mixture was then molded, cooled, and shaped to obtain suppositories of 1-2 g / pouch, with specifications of 100 mg-200 mg.

[0073] Example 8: Preparation of alder ketone topical thermosensitive gel

[0074] The formula ratio of poloxamer 407, poloxamer 188, chitosan, and hyaluronic acid is mixed evenly and added to a buffer solution with pH 4-5 to swell and dissolve, obtaining a gel matrix. Alder ketone is dissolved in DMSO. Hydroxypropyl-β-cyclodextrin is dissolved in water until saturated, and the cyclodextrin solution is added dropwise to the alder ketone solution. The mixture is stirred or sonicated to complete the inclusion, obtaining a suspension. The suspension is added to the gel matrix in proportion and stirred evenly to obtain a vaginal thermosensitive gel with a gel temperature of 32℃-35℃. It is then filled and stored at low temperature. After dilution with bodily fluids in the usage environment, the gel temperature is around 37℃.

[0075] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. The application of a linear diarylheptane compound in the preparation of an ovulation-inducing drug for treating ovulation disorders, characterized in that, The structural formula of the linear diarylheptane compound is: 。 2. The application according to claim 1, characterized in that, The linear diarylheptane compounds are obtained through chemical synthesis or biological extraction; the sources of biological extraction are selected from cardamom, alder, turmeric, or amomum.

3. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 1, characterized in that, The drug is administered orally, by injection, or via mucosal delivery. The dosage form of the drug is selected from tablets, capsules, granules, ointments, suppositories, pills, gels, injections, suspensions, oral liquids, or syrups.

5. The application according to claim 1, characterized in that, The drug is in the form of a sustained-release formulation or a controlled-release formulation.

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