Application of licorice extract in preparation of medicine for treating premature ovarian failure

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a licorice extract in preparation of a medicine for treating premature ovarian failure. On the basis of network pharmacology prediction and animal experiment verification, through experimental exploration, it is clear that diammonium glycyrrhizinate can treat premature ovarian failure by improving ovarian tissue energy metabolism, reducing the oxidative stress level and inhibiting ovarian cell apoptosis, and also can treat premature ovarian failure by improving the ovarian growth and development microenvironment. According to the present invention, the growth and development rate of ovarian granular cells and membrane cells is improved, the sufficient quantity of granular cells and membrane cells are ensured to secrete female progestational hormone, the damaged ovarian tissue is further protected, the new strategy and hope are provided for the treatment of premature ovarian failure, and the pharmaceutical composition is expected to become the safe and effective treatment drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of licorice extract in the preparation of drugs for treating premature ovarian failure. Background Technology

[0002] In the field of traditional Chinese medicine, licorice plays an important role in the treatment of many diseases due to its unique effects such as clearing heat and detoxifying, relieving pain, and harmonizing other medicines. Modern scientific research has further revealed the rich chemical components of licorice, mainly including triterpenoid saponins, flavonoids, and polysaccharides. These components endow licorice with a wide range of biological activities, giving it various pharmacological effects such as antioxidation, immunomodulation, antiviral, anti-inflammatory, and hepatoprotective properties.

[0003] With the deepening of research on licorice, modern medicine has discovered that licorice extract and its active ingredients, such as glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin A, and total licorice flavonoids, have shown significant effects in regulating glucose and lipid metabolism, reducing oxidative stress, altering gut microbiota, and protecting the intestinal barrier, providing new approaches and ideas for the treatment of metabolic-related diseases.

[0004] Diammonium glycyrrhizate (DG), as a third-generation extract of the active ingredients in licorice, exhibits significant advantages over traditional glycyrrhizic acid preparations in terms of stability, solubility, and bioactivity. DG specifically inhibits 11β-hydroxysteroid dehydrogenase (11β-HSD1), precisely regulating the body's steroid effects through glucocorticoid and mineralocorticoid receptors, thereby exerting broad-spectrum anti-inflammatory, antioxidant, and immunomodulatory biochemical and pharmacological activities. This mechanism of action allows DG to fundamentally correct the oxidative-antioxidant imbalance, effectively inhibiting inflammatory responses and providing a potential drug option for the treatment of various diseases.

[0005] Premature ovarian failure (POF), a common disease in the field of gynecological reproductive endocrinology, seriously threatens women's reproductive health and quality of life. Its typical characteristics include reduced ovarian reserve, accompanied by low estrogen and high gonadotropin levels, ultimately leading to ovarian failure. POF not only accelerates the decline in ovarian function but also causes premature menopause, triggering menstrual irregularities, infertility, menopausal symptoms, and a series of discomforts caused by estrogen deficiency, placing a significant physical and psychological burden on patients.

[0006] From the perspective of human female physiology, a woman's fertility gradually declines with age, and this decline becomes more pronounced after age 35. Premature ovarian failure (POF) is a specific pathological condition characterized by an irreversible decline in ovarian function beyond the normal range for a woman's age. Its development typically progresses gradually from a latent phase to a biochemically abnormal phase. Currently, treatment options for POF are limited and their effectiveness is unsatisfactory. Therefore, finding a safe and effective treatment method has become an urgent problem to be solved in the medical field.

[0007] Based on the pharmacological properties of DG, this invention proposes its application in the treatment of premature ovarian failure. By utilizing DG to improve the ovarian growth and development microenvironment, increasing the growth and development rate of ovarian granulosa cells and membrane cells, and ensuring a sufficient number of granulosa cells and membrane cells secrete estrogen and progesterone, this further protects damaged ovarian tissue, providing a new strategy and hope for the treatment of premature ovarian failure. Summary of the Invention

[0008] The purpose of this invention is to provide an application of licorice extract in the preparation of a drug for treating premature ovarian failure. DG has significant technical effects in improving ovarian tissue energy metabolism, reducing oxidative stress levels, and inhibiting ovarian cell apoptosis, providing a new strategy and hope for the treatment of premature ovarian failure, and is expected to become a safe and effective therapeutic drug.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] This invention provides the application of licorice extract in the preparation of a drug for treating premature ovarian failure, wherein the licorice extract is diammonium glycyrrhizate.

[0011] Furthermore, the licorice extract treats premature ovarian failure by improving energy metabolism in ovarian tissue, reducing oxidative stress levels, and inhibiting ovarian cell apoptosis.

[0012] Furthermore, the licorice extract can also improve the ovarian growth and development microenvironment, increase the growth and development rate of ovarian granulosa cells and membrane cells, and ensure that a sufficient number of granulosa cells and membrane cells secrete estrogen and progesterone, thereby further protecting damaged ovarian tissue.

[0013] The present invention also provides a drug for treating premature ovarian failure, the drug comprising diammonium glycyrrhizate.

[0014] Furthermore, the diammonium glycyrrhizate is the sole active ingredient.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Beneficial effects:

[0017] This invention, using the luciferase assay to detect ATP content in rat ovarian tissue, found that compared to the model group (CTX group), the ATP content in the ovarian tissue of rats treated with different doses of DG combined with CTX was significantly increased. The dose group (M-DG+CTX group) approached or reached the level of the normal control group (Control group). This indicates that DG can improve energy metabolism in ovarian tissue, providing sufficient energy supply to ovarian granulosa cells and membrane cells, laying the necessary material basis for their normal growth, development, and function, and contributing to the restoration of normal ovarian physiological function.

[0018] This invention utilizes immunofluorescence to determine the expression of reactive oxygen species (ROS) in the ovarian tissue of rats in different groups. The results showed that ROS expression was significantly increased in the ovarian tissue of the model group, while ROS expression was significantly decreased in the DG combined with CTX treatment groups and the positive control group (femostone + CTX group). Simultaneously, ELISA analysis of SOD, GSH-PX, and MDA levels in rat ovarian tissue indicated that SOD and GSH-PX activities were decreased and MDA content was increased in the ovarian tissue of the model group, suggesting elevated oxidative stress levels. After DG combined with CTX treatment, SOD and GSH-PX activities significantly increased, and MDA content significantly decreased, with some dosage groups showing effects similar to the positive control group. These results indicate that DG can effectively scavenge excess free radicals in the body by increasing antioxidant enzyme activity, reducing oxidative stress damage to ovarian tissue, protecting ovarian cells from oxidative stress, and maintaining the normal structure and function of ovarian tissue.

[0019] This invention used Western blotting to determine the expression levels of Bax, Bcl-2, Caspase-3, and Caspase-9 proteins in the ovarian tissues of rats in each group. The results showed that the expression of pro-apoptotic proteins Bax, Caspase-3, and Caspase-9 was significantly increased, while the expression of the anti-apoptotic protein Bcl-2 was significantly decreased in the ovarian tissues of the model group, indicating increased ovarian cell apoptosis. After treatment with DG combined with CTX, the expression of pro-apoptotic proteins decreased, while the expression of anti-apoptotic proteins increased, and the effects in some dosage groups were similar to those in the positive control group. Further TUNEL assay was used to detect ovarian cell apoptosis in each group, confirming that DG could significantly reduce the number of apoptotic ovarian cells. This indicates that DG can inhibit ovarian cell apoptosis by regulating the expression of apoptosis-related proteins, protecting the number of cells in ovarian tissue and maintaining normal ovarian function.

[0020] In summary, DG has significant technical effects in improving ovarian tissue energy metabolism, reducing oxidative stress levels, and inhibiting ovarian cell apoptosis, providing new strategies and hope for the treatment of premature ovarian failure, and is expected to become a safe and effective therapeutic drug. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a graph showing the effect of DG on ATP content in rat ovarian tissue in Example 1 of the present invention, where x±s,n=6;

[0023] Figure 2 This is a graph showing the effect of DG on ROS in rat ovarian tissue in Example 2 of the present invention, where x±s,n=4;

[0024] Figure 3 The figure shows the effect of DG on rat ovarian cell apoptosis in Example 5 of the present invention, where x±s,n=6. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Unless otherwise specified, the test methods used in the following implementations are all commonly used test methods in this field;

[0031] Unless otherwise specified, the test materials used in the following implementations are all commonly used test materials in this field.

[0032] Example 1: Detection of ATP content in rat ovarian tissue using luciferase method

[0033] In this embodiment, rat ovarian tissue was thoroughly homogenized with lysis buffer, centrifuged at 12000g / min for 5 min at 4°C, and the supernatant was collected. The procedure was strictly followed according to the instructions of the ATP luciferase assay kit (Beijing Bio-Rad Laboratories Co., Ltd.). An appropriate amount of ATP detection working solution was prepared, and the ATP detection working solution, sample, or standard was added sequentially to a 96-well plate. The optical density was measured using a microplate reader, and the concentration of ATP in the sample was calculated based on the standard curve. The final ATP content in rat ovarian tissue of different groups is shown below. Figure 1 As shown.

[0034] Depend on Figure 1 It was found that, compared with the model group (CTX group, model established by injecting CTX at an initial dose of 50 mg / kg and a maintenance dose of 8 mg / kg for 14 consecutive days), the ATP content in the ovarian tissue of rats treated with different doses of DG (L-DG 50 mg / kg / d; M-DG 100 mg / kg / d; H-DG 150 mg / kg / d) combined with CTX (L-DG+CTX, M-DG+CTX, H-DG+CTX) was significantly increased. Furthermore, the ATP content in the M-DG+CTX group was close to or reached the level of the normal control group (Control group), and the ATP content in the femostone+CTX group was also close to that of the normal control group. This indicates that DG can improve the energy metabolism of ovarian tissue, increase the energy supply to ovarian granulosa cells and membrane cells, provide the necessary material basis for normal cell growth, development, and function, and help restore normal ovarian physiological function.

[0035] Example 2: Immunofluorescence assay to determine the expression of ROS in the ovarian tissue of rats in each group.

[0036] This embodiment strictly followed the instructions of the chemiluminescence assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). An appropriate amount of rat ovarian tissue was taken, added to the enzyme-containing buffer in the kit, filtered, and the resulting solution had a concentration of 5 × 10⁻⁶. 6Cell suspension was centrifuged at 3000 r / min for 5 min at 4℃. The supernatant was discarded, and cells were washed with PBS. The DCFH-DA probe was added, and flow cytometry was used to detect and analyze the cells, calculating the mean fluorescence intensity (MFI). The final MFI results in rat ovarian tissue from different groups are shown below. Figure 2 As shown.

[0037] Depend on Figure 2 It can be seen that ROS expression in the ovarian tissue of rats in the model group (CTX group) was significantly increased, while ROS expression in the DG combined with CTX treatment group and the femostone+CTX group was significantly decreased.

[0038] Example 3: ELISA method for detecting the levels of SOD, GSH-PX and MDA in rat ovarian tissue.

[0039] This embodiment uses an ELISA kit (Shanghai Boyan Biotechnology Co., Ltd.) to detect the levels of oxidative factors such as SOD, GSH-PX, and MDA in rat ovarian tissue. The experiment was conducted according to the kit instructions. Rat ovarian tissue samples were thawed at room temperature. Standards were serially diluted and added to the test solution and working solution A, then incubated for 1 hour. After washing with washing buffer, working solution B was added, followed by washing again. Substrate solution was added for color development in the dark, and finally, stop solution was added. The optical density (OD value) of each well was measured at 450 nm using a microplate reader. Finally, a curve was fitted, and the sample concentration was calculated. The final results are shown in Table 1.

[0040] Table 1. Effects of DG on GSH-Px, MDA, and SOD levels in rat ovarian tissue (x±s, n=6)

[0041]

[0042] Note: Compared with the Control group, aP < 0.05, bP < 0.01; compared with the Model group, cP < 0.05, dP < 0.01.

[0043] Table 1 shows that in the model group rats, the activities of SOD and GSH-PX were decreased, while the content of MDA was increased, indicating an elevated level of oxidative stress. However, after treatment with DG combined with CTX, the activities of SOD and GSH-PX were significantly increased, and the content of MDA was significantly decreased. Furthermore, some dosage groups showed effects similar to the femostone + CTX group. These results indicate that DG can reduce the damage of oxidative stress to ovarian tissue by increasing the activity of antioxidant enzymes, scavenging excess free radicals in the body, protecting ovarian cells from oxidative stress, and maintaining the normal structure and function of ovarian tissue.

[0044] Example 4: Western blotting was used to determine the expression levels of Bax, Bcl-2, Caspase-3, and Caspase-9 proteins in the ovarian tissues of rats in each group.

[0045] This embodiment uses a full protein extraction kit. Protein lysis buffer was prepared according to the kit's instructions. An appropriate amount of ovarian tissue was added to an EP tube containing the protein lysis buffer, and after cryogenic grinding, the protein concentration of rat ovarian tissue was determined according to the kit's instructions. Protein sample and marker were added, and electrophoresis was performed at a constant voltage of 120V. Protein transfer was performed using a constant current of 300mA. The transferred bands were blocked in a protein-free rapid blocking buffer, washed with TBST, and incubated overnight with primary antibody at concentrations of (Bax: 1:8000; Bcl-2: 1:1000; Caspase-3: 1:1000; Caspase-9: 1:1000). Immunoblotting was performed using an immunoblotting imaging system, and the band grayscale values ​​were measured using ImageJ and statistical analysis was performed. The final results are shown in Table 2.

[0046] Table 2. Effects of DG on Bax, Bcl-2, Caspase-3, and Caspase-9 in rat ovarian tissue (x±s, n=3)

[0047]

[0048]

[0049] Note: Compared with the Control group, aP < 0.05, bP < 0.01; compared with the Model group, cP < 0.05, dP < 0.01.

[0050] As shown in Table 2, the expression of pro-apoptotic proteins Bax, Caspase-3, and Caspase-9 was significantly increased and the expression of anti-apoptotic protein Bcl-2 was significantly decreased in the ovarian tissue of rats in the model group, indicating increased ovarian cell apoptosis. After treatment with DG combined with CTX, the expression of pro-apoptotic proteins decreased and the expression of anti-apoptotic proteins increased, and the effects of some dose groups were similar to those of the femostone + CTX group.

[0051] Example 5: TUNEL assay for ovarian cell apoptosis in rats of different groups.

[0052] In this embodiment, the apoptosis of rat ovarian cells in each group was detected by TUNEL assay. Before TUNEL staining, the sections were baked in a 65°C oven for 30 min, dewaxed, rehydrated, and rinsed. Proteinase K working solution was added, and the sections were permeabilized in a 37°C incubator for 20 min (1× DNase I buffer was added to the positive control group). Then, the sections were incubated with DNase I working solution for 10 min, rinsed with PBS solution, and then TdT incubation buffer was added. Finally, the sections were incubated under light-protected conditions. After nuclear staining with X-100 and 5 mg / ml BSA and DAPI, slides were mounted with anti-fluorescence quenching mounting medium and observed and analyzed under a fluorescence microscope. Green fluorescence was observed under fluorescence at 520±20 nm; blue DAPI was observed at 460 nm. DAPI stained all cells blue. Bright Green incorporation into apoptotic cell nuclei produced green fluorescence. The final effect of DG on rat ovarian cell apoptosis was as follows: Figure 3 As shown, by Figure 3 As can be seen, this embodiment further confirms that DG can significantly reduce the number of apoptotic ovarian cells by detecting ovarian cell apoptosis in each group of rats using the TUNEL assay. This indicates that DG can inhibit ovarian cell apoptosis by regulating the expression of apoptosis-related proteins, protecting the number of cells in ovarian tissue, and maintaining normal ovarian function.

[0053] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a licorice extract in the preparation of a drug for treating premature ovarian failure, characterized in that, The licorice extract is diammonium glycyrrhizate.

2. The application according to claim 1, characterized in that, The licorice extract treats premature ovarian failure by improving energy metabolism in ovarian tissue, reducing oxidative stress levels, and inhibiting ovarian cell apoptosis.

3. The application according to claim 1, characterized in that, The licorice extract can also improve the ovarian growth and development microenvironment, increase the growth and development rate of ovarian granulosa cells and membrane cells, and ensure that a sufficient number of granulosa cells and membrane cells secrete estrogen and progesterone, thereby further protecting damaged ovarian tissue.

4. A drug for treating premature ovarian failure, characterized in that, The drug includes diammonium glycyrrhizate.

5. The drug according to claim 4, characterized in that, The diammonium glycyrrhizate is the only active ingredient.

6. The drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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