Application of naringenin in preparation of medicine for treating uterine fibroids or sarcoma

Abstract

The application belongs to the field of medicine, and particularly relates to application of naringenin in preparation of medicines for treating uterine fibroids or uterine sarcoma. It is found through exploration that naringenin has obvious inhibitory effect on extracellular matrix synthesis of human primary uterine fibroid cells hUL and rat uterine fibroid cell line ELT-3, thereby inhibiting cell proliferation and migration; meanwhile, naringenin can significantly inhibit expression of cell cycle related enzymes of human uterine sarcoma cell lines SKN, SKUT-1 and MES-SA, thereby inhibiting cell proliferation and promoting cell apoptosis. The application lays a research foundation for exploring medicines for treating uterine fibroids or uterine sarcoma.

Description

Technical Field

[0001] This application belongs to the field of medicine, specifically involving the fields of biology and pharmaceutical technology, and relates to the application of naringenin in the preparation of drugs for treating uterine fibroids or uterine sarcomas. Background Technology

[0002] Uterine leiomyoma (UL), also known as uterine leiomyomas, is characterized by the proliferation of smooth muscle cells in the myometrium, interspersed with a small amount of fibrous connective tissue. Uterine fibroids are the most common benign tumors of the female reproductive system, with an incidence rate as high as 70-80% in women of reproductive age. Although benign uterine fibroids are generally mild and have a low risk of malignancy, they are characterized by rapid growth, large size, and multiple occurrences. Therefore, approximately 50% of patients experience significant clinical symptoms, such as abnormal uterine bleeding, dysmenorrhea, pelvic pain, infertility, pregnancy complications, and other comorbidities, seriously endangering physical and mental health and constituting a major public health issue of widespread social concern. Currently, treatment methods for uterine fibroids mainly include surgical treatment (fibroid removal, hysterectomy, uterine artery embolization), hormone therapy (gonadotropin-releasing hormone agonists, aromatase inhibitors, combined oral contraceptives, etc.), and non-hormonal therapy (tranexamic acid, nonsteroidal anti-inflammatory drugs, etc.). However, these conventional treatments are currently not very effective, prone to recurrence, and have certain side effects.

[0003] Uterine sarcoma is a rare malignant tumor, accounting for approximately 1% of all female genital tumors and 3%-7% of all uterine malignancies. The most common pathological type is carcinosarcoma (malignant mixed mesodermal tumor), accounting for up to 50%. Less common types include endometrial stromal sarcoma (15%) and undifferentiated sarcoma (5%). While both uterine fibroids and uterine sarcomas are tumors occurring in the uterus, they differ significantly in tumor nature, pathogenesis, and treatment. Uterine sarcoma is a malignant tumor, while uterine fibroids are benign tumors with completely different cellular properties. Although uterine fibroids can potentially become cancerous, the probability is relatively low. The occurrence of uterine sarcoma is related to factors such as pelvic radiotherapy and gene mutations. The occurrence of uterine fibroids is related to factors such as genetics, obesity, and childbirth. Unlike uterine fibroids, uterine sarcoma is a malignant tumor. Once diagnosed, timely surgical removal of the lesion is necessary, such as abdominal hysterectomy. If cancer cells infiltrate or metastasize, radiotherapy, chemotherapy, and targeted therapy may also be required. According to WHO criteria, even when lesions are confined to the uterus, leiomyosarcoma has a poor prognosis, with a recurrence rate as high as 53%-71%. Common clinical treatments for uterine sarcoma include: early adjuvant therapy (hormone therapy, adjuvant chemotherapy, adjuvant radiotherapy, and combined radiotherapy and chemotherapy); and advanced-stage treatment (surgery, chemotherapy, immunotherapy, and targeted therapy). Due to the low incidence of uterine sarcoma, optimizing its diagnosis and treatment is challenging. Although numerous clinical attempts have been made to improve the treatment outcomes of uterine sarcoma through systemic therapy, the treatment effects and prognosis remain unsatisfactory for both patients with simple uterine sarcoma and those with metastatic disease.

[0004] Therefore, how to solve this technical problem and effectively stop the progression of uterine fibroids / sarcomas is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the aforementioned problems, the purpose of this application is to provide the application of naringenin in the preparation of drugs for treating uterine fibroids or uterine sarcomas. Naringenin can be used as an effective drug for treating uterine fibroids and uterine sarcomas, with few side effects and high safety.

[0006] This application provides the use of naringenin or a pharmaceutically acceptable salt of naringenin in the preparation of drugs for uterine fibroids and / or uterine sarcomas.

[0007] The drug can inhibit the proliferation of human primary uterine fibroid cells, rat uterine fibroid cell lines, or human uterine sarcoma cell lines.

[0008] Furthermore, the rat uterine fibroid cells are ELT-3; the human uterine sarcoma cells are SKUT-1, SKN, or MES-SA.

[0009] Furthermore, the concentration range of the naringenin or the medicinal salt of naringenin is 100μM-700μM (micromolar), preferably 200μM-700μM, more preferably 300μM-700μM, and most preferably 400μM-700μM.

[0010] This application also provides that the naringin or its pharmaceutically usable salt has the effect of inhibiting the synthesis of extracellular matrix in human primary uterine fibroid cells and rat uterine fibroid cell lines, thereby inhibiting cell migration, or that the naringin or its pharmaceutically usable salt has the effect of inhibiting the synthesis of cell cycle-related enzymes in human uterine sarcoma cell lines, thereby inhibiting cell proliferation and promoting cell apoptosis.

[0011] The drug described in this application is a pharmaceutical composition containing naringenin or a pharmaceutically usable salt of naringenin.

[0012] Furthermore, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0013] Furthermore, the pharmaceutical composition is formulated into any pharmaceutically acceptable dosage form.

[0014] The pharmaceutically usable dosage forms described in this application are selected from tablets, capsules, solutions, granules, powders, ointments, pills, suspensions, suppositories, liniments, emulsions, ointments, patches, or sprays.

[0015] Furthermore, the dosage form is in unit dose form, and each unit dose contains naringenin or a pharmaceutically acceptable salt of naringenin: 1mg-10mg, or 10mg-100mg, or 100mg-1000mg, or 1000mg-10000mg.

[0016] The following is an explanation and description of the terms and concepts used in this application:

[0017] Naringenin is the aglycone of naringin, widely found in plants of the Rutaceae family. It is a natural dihydroflavonoid compound with a molecular weight of 272.25 Da. It is a hydrolytic derivative of naringin and is mainly found in the form of glycosides in Rutaceae plants such as grapefruit, tomato, grape, and citrus fruits. It is also one of the main active ingredients in traditional Chinese medicines such as Citrus aurantium and Astragalus membranaceus. Its common name is Naringenin; its chemical name is 4”,5,7-trihydroxyflavone; its trade names are naringinogen and hesperidin; its CAS registration number is 480-41-1; and its molecular formula is C 15 H 12 O5; relative molecular weight: 272.25. Its chemical structural formula is as follows:

[0018]

[0019] Naringin can be purchased from the market or prepared using methods disclosed in existing literature.

[0020] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0021] The pharmaceutical composition described in this application can be any reusable pharmaceutical formulation, such as oral, injectable, or topical forms. Oral dosage forms include, but are not limited to, tablets, capsules, oral liquids, granules, pills, and suspensions. Injectable formulations are selected from water-based injections or powder injections. All formulations can be prepared according to conventional pharmaceutical techniques. For example, using any one of the compounds of this application, or their stereoisomers, or their pharmaceutically acceptable salts as the active pharmaceutical ingredient, and adding a pharmaceutically acceptable carrier if necessary, the above-mentioned pharmaceutical dosage forms suitable for oral administration can be prepared. The unit dose of the active pharmaceutical ingredient can be 0.1 mg to 1000 mg, such as each tablet containing 0.1 mg to 1000 mg, preferably 5 to 500 mg of the active pharmaceutical ingredient.

[0022] The terms "salt" and "pharmaceutical salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds or their stereoisomers with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may be collected by filtration after precipitating in solution, recovered after solvent evaporation, or freeze-dried after reaction in an aqueous medium. The salts described in this application can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compounds.

[0023] In some embodiments, one or more compounds of this application may be used in combination with each other. Alternatively, the compounds of this application may be combined with any other active reagent to prepare drugs or pharmaceutical compositions for regulating cell function or treating diseases. If a group of compounds is used, these compounds may be administered to the test subject simultaneously, separately, or sequentially. Obviously, based on the above description of this application, and in accordance with ordinary technical knowledge and common practice in the art, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of this application.

[0024] The beneficial technical effects of this application are as follows:

[0025] 1. This application, at the cellular level, administered a certain dose of naringenin or a combination of naringenin (such as salts of naringenin) to primary uterine fibroid cells / uterine fibroid cell lines and uterine sarcoma cell lines, and then detected the synthesis of extracellular matrix and cell cycle-related enzymes, as well as the cell proliferation, migration, and apoptosis capabilities. The study found that, based on the broad biological activity and pharmacological effects of the active ingredient naringenin from natural plant extracts, naringenin also has therapeutic efficacy for uterine fibroids / sarcomas. On the one hand, naringenin and its pharmaceutically usable salts inhibit the synthesis of extracellular matrix in uterine fibroid cells, thereby inhibiting cell proliferation and migration; on the other hand, naringenin can inhibit the synthesis of cell cycle-related enzymes in uterine sarcoma cells, thereby inhibiting cell proliferation and promoting cell apoptosis. Simultaneously, due to the high bioavailability and safe dosage of the active ingredient naringenin from natural plant extracts and its pharmaceutical preparations, the potential side effects during treatment are small. Therefore, naringenin and its pharmaceutical combinations may be a new generation of highly promising and widely applicable therapeutic drugs for uterine fibroids / sarcomas.

[0026] 2. This application compared the effects of naringenin and its pharmaceutical composition with the active ingredients of three other natural plant extracts (epistemolide, β-sitosterol, and stigmasterol) on uterine fibroids and uterine sarcoma cells. The results showed that only naringenin could effectively inhibit the proliferation of uterine fibroids and uterine sarcoma cells.

[0027] 3. The inhibitory effect of naringenin and its pharmaceutical composition provided in this application on uterine fibroids and uterine sarcoma cells is concentration-dependent. In human primary uterine fibroid cells and rat uterine fibroid cell lines, the pharmaceutically usable salts of naringenin are more effective than naringenin. Different unit doses or different compositional forms can be prepared to exert different therapeutic effects. Attached Figure Description

[0028] Figure 1: Results of the inhibitory effect of naringenin on uterine sarcoma cell lines SKN, SKUT-1, and MES-SA in this application;

[0029] Figure 2 The results of this application on the extracellular matrix synthesis and migration ability of naringenin and naringenin sulfate on human primary uterine fibroid cells (hUL) and uterine fibroid cell line (ELT-3);

[0030] Figure 3 This application presents the results of tests on the ability of naringenin to inhibit the synthesis, proliferation, and apoptosis of cell cycle-related enzymes in uterine sarcoma cell lines SKN, SKUT-1, and MES-SA.

[0031] in, Figure 2B represents the immunofluorescence experiment. Green fluorescence indicates the expression of extracellular matrix-related molecules in fibroid cells. A stronger average intensity indicates enhanced synthesis of these molecules. The control group showed a strong average green fluorescence intensity in fibroid cells, demonstrating enhanced extracellular matrix synthesis. After intervention with naringenin and naringenin sulfate, the average green fluorescence intensity of fibroid cells (right image) decreased, indicating that naringenin and naringenin sulfate can inhibit extracellular matrix synthesis in fibroid cells.

[0032] in, Figure 2 C is the scratch test. Fibroid cells have the ability to migrate and can migrate to the blank scratch in the middle, thus healing the scratch. However, after 24 hours of intervention with naringenin and naringenin sulfate, the migration ability of fibroid cells decreased, and the scratch still existed, proving that naringenin and naringenin sulfate can inhibit the migration of fibroid cells.

[0033] in, Figure 3 C represents the EDU experiment. The stronger the average red fluorescence intensity, the stronger the cell's proliferative capacity. The strong average red fluorescence intensity of the sarcoma cells in the control group proves that the sarcoma cells have a strong proliferative capacity. After naringenin intervention, the average red fluorescence intensity of the sarcoma cells in the right figure weakens, indicating that naringenin can inhibit the proliferation of sarcoma cells.

[0034] in, Figure 3 D represents the TUNEL assay; a stronger average red fluorescence intensity indicates increased cell apoptosis. The right-hand graph shows a higher average red fluorescence intensity than the left-hand graph, demonstrating that naringenin can promote apoptosis in sarcoma cells. Detailed Implementation

[0035] The following examples further illustrate this application, but are not intended to limit the scope of this application.

[0036] Currently, the causes of uterine fibroids and sarcomas are unknown, but they are likely closely related to multiple factors, such as gene modification, immune inflammation levels, pelvic radiotherapy, epigenetic modifications, and lifestyle. Although both are uterine tumors, they differ significantly in tumor nature, pathogenic factors, and treatment methods. Clinical treatment of uterine sarcomas is unsatisfactory, characterized by a lack of long-term effective management after surgical treatment, significant side effects and high recurrence rates with drug therapy, and a tendency for tumor progression and even malignant transformation after treatment. Therefore, effectively preventing the progression of uterine fibroids / sarcomas is a pressing issue.

[0037] Naringenin is a natural dihydroflavonoid compound with a molecular weight of 272.25 Da. It is a derivative of naringin hydrolysis and is widely found, primarily in the form of glycosides, in plants of the Rutaceae family such as grapefruit, tomato, grape, and citrus fruits. It is also one of the main active ingredients in traditional Chinese medicines such as Citrus aurantium and Astragalus membranaceus. In animal models and human studies, naringenin, after absorption through the digestive tract, is mainly distributed in the gastrointestinal tract, liver, kidneys, lungs, and trachea. After consumption of citrus fruits or oral administration, naringin is hydrolyzed into naringenin by the intestinal microbiota of the mouth and small intestine. After absorption, naringenin is metabolized into glucuronic acid and sulfate metabolites through phase I and II metabolic reactions. These metabolites and unabsorbed flavonoids are further metabolized by microorganisms, producing phenolic catabolites. These metabolites have been detected in urine and fecal samples from both animals and humans. In the United States, naringenin is an edible flavoring approved by the Flavor and Extract Manufacturers Association (FEMA) and is widely used in the food industry as a food additive, with high application value.

[0038] In recent years, with the deepening research on naringenin by scientists at home and abroad, its application in disease prevention and treatment has developed rapidly. Naringenin is a natural substance used to treat or prevent various diseases such as obesity, heart disease, diabetes, and metabolic syndrome. This application, by comparing the effects of naringenin and the active ingredients of three other natural plant extracts (hederine, β-sitosterol, and stigmasterol) on uterine fibroids and uterine sarcoma cells, unexpectedly discovered that only naringenin could effectively inhibit the proliferation of uterine fibroids and uterine sarcoma cells, while other plant extracts had no significant effect. This application, by administering a certain dose of naringenin or a combination of naringenin (a pharmaceutically usable salt of naringenin) to primary uterine fibroid cells / uterine fibroid cell lines and uterine sarcoma cell lines at the cellular level, and then detecting their extracellular matrix synthesis and the synthesis, proliferation, migration, and apoptosis abilities of cell cycle-related enzymes, found that, based on the broad biological activity and pharmacological effects of the natural plant extract active ingredient naringenin, naringenin also has therapeutic effects on uterine fibroids / sarcomas. Specifically, naringenin and its pharmaceutical composition inhibit extracellular matrix synthesis in human primary uterine fibroid cells (hUL) and rat uterine fibroid cell line ELT-3, thereby inhibiting cell proliferation and migration. On the other hand, naringenin and its pharmaceutical composition inhibit cell proliferation and promote apoptosis by suppressing the expression of cell cycle-related enzymes in human uterine sarcoma cell lines SKN, SKUT-1, and MES-SA. Therefore, due to the high bioavailability and safe dosage of the active ingredient naringenin from natural plant extracts and its pharmaceutical preparations, the potential side effects during treatment are minimal. Thus, naringenin and its pharmaceutical composition may be a promising new generation of highly versatile therapeutic drugs for uterine fibroids / sarcomas. Furthermore, the inhibitory effect of naringenin and its pharmaceutical composition on uterine fibroids and uterine sarcoma cells provided in this application exhibits a concentration-dependent effect, allowing for the preparation of different unit doses to achieve different therapeutic effects. The specific research process of this application is illustrated in the following specific examples. The chemical raw materials used in the following examples include any materials conventional in the art.

[0039] Example 1

[0040] Laboratory animals and materials:

[0041] 1. Experimental Cells: Human primary uterine leiomyoma cells (hUL), rat-derived ELT-3 uterine leiomyoma cell line, and human-derived uterine sarcoma cell lines SKUT-1, SKN, and MES-SA; Source, strain, and variety: Primary uterine leiomyoma cells were selected from patients diagnosed with uterine leiomyoma at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, who had not undergone drug treatment and required myomectomy. Informed consent was obtained from all patients, and the samples were reviewed by the ethics committee of Tongji Hospital. All other cell lines were ordered from the American Type Culture Collection (ATCC).

[0042] 2. Experimental Materials and Reagents: Naringenin (MCE, Shanghai, China); Ivy extract (MCE, Shanghai, China); β-sitosterol (MCE, Shanghai, China); Stigmasterol (MCE, Shanghai, China); Type I collagenase (Google Biotechnology Co., Ltd., Wuhan, China); DMEM / F12, DMEM, McCoy's 5A medium (Gibco, USA); Fetal bovine serum (Gibco, USA); 70μm cell filter (Biosharp Biotechnology Co., Ltd., Shanghai, China); 0.25% trypsin (Google Biotechnology Co., Ltd.); CCK8 solution (Abbkine, Wuhan, China); TUNEL kit (Abbkine, Wuhan, China); EDU kit (Abbkine, Wuhan, China); PBS (Google Biotechnology Co., Ltd., Wuhan, China); Penicillin-streptomycin mixture (Google Biotechnology Co., Ltd., Wuhan, China); Red blood cell lysis buffer (Google Biotechnology Co., Ltd., Wuhan, China); Formalin / 4% paraformaldehyde (Google Biotechnology Co., Ltd., Wuhan, China); Triton... X100 (Google Biotechnology Co., Ltd., Wuhan, China); BSA / skim milk (Google Biotechnology Co., Ltd., Wuhan, China); HRP / FITC-labeled goat anti-rabbit / mouse IgG (Google Biotechnology Co., Ltd., Wuhan, China); DAPI staining solution (Google Biotechnology Co., Ltd., Wuhan, China); Antibodies COL1A1, MMP2, CDC25A, CDC25C, Cyclin A2, Cyclin D1, P53, β-actin (ABclonal, Wuhan, China); 2xChemQSYBRqPCRmix / Primers α-SMA, TGFβ1, COL1A1, COL3A1, MMP1, MMP2, MMP9 (Qingke Biotechnology Co., Ltd., Beijing, China); Tris-Base, glycine, SDS (Google Biotechnology Co., Ltd., Wuhan, China); Protein marker, 5x protein loading buffer, transfer buffer, TBS buffer, exposure solution (Google Biotechnology Co., Ltd., Wuhan, China); Optical microscope: Olympus, Japan; Fluorescent PCR thermal cycler: Bio-Rad, USA; Vertical electrophoresis tank (Mini-PROTEAN) electrophoresis transfer tank (Mini Trans-Blot): Bio-Rad, USA.

[0043] 3. Experimental methods:

[0044] Step 1: Extraction of primary human uterine fibroid cells

[0045] Patients diagnosed with uterine leiomyomas at Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, who had not received drug treatment and required myomectomy, were randomly selected. Fresh tumor tissue obtained from the sterile operating room was placed in pre-cooled sterile saline, and the procedure was performed in a biosafety cabinet. The fibroid tissue was rinsed three times with sterile saline, and the outermost capsule and the innermost central ischemic zone were peeled off. The ovarian specimen was divided into 1cm×1m×1m tissue blocks using scissors. Each small tissue block was placed in 200μL of (1x) IV collagenase solution (prepared by dissolving type IV collagenase powder in 100ml DMEM medium + 0.1% penicillin-streptomycin mixture). The ovarian tissue was minced to a paste-like consistency using scissors. The container was rinsed three times with 3mL of type IV collagenase solution and transferred to a 4mL sterile EP tube. 15μL of DNase (5μL / mL ratio) was added to prevent tissue adhesion. After mixing thoroughly, the tube opening was sealed with disposable adhesive tape and placed inside a disposable PE glove. The tube was then shaken horizontally and evenly in a 37℃ constant temperature shaker to ensure thorough mixing of the tissue and collagenase solution. After 16 hours (depending on the tissue digestion progress, i.e., when the tissue blocks become smaller, paler, and more adherent between tissues), the mixture was filtered through a sterile 70μm cell filter, and the filtrate was collected. Centrifuge the filtrate (1200 rpm, 5 min 30 s, room temperature), discard the supernatant, and add 10 mL of sterile red.

[0046] Resuspend the cell pellet in each EP tube with cell lysis buffer, incubate for 15 min, then centrifuge (1200 rpm, 5 min 30 s, room temperature). Discard the supernatant after centrifugation, wash once with 10 mL PBS, and centrifuge again (1200 rpm, 5 min 30 s, room temperature). Resuspend in DMEM / F12 medium and seed in 6-well plates (ensuring high seeding density to guarantee cell viability). Passage using trypsin digestion.

[0047] Step 2: Cell line digestion and passage

[0048] When the density of uterine fibroid / sarcoma cells reaches 80%-90%, they can be digested and passaged. Sterile cells are rinsed once with PBS solution. Discard the PBS solution, add 0.25% trypsin, and incubate at 37°C for 3-5 minutes. Observe under a microscope whether the cells become round and bright. Add twice the volume of complete culture medium to stop digestion, thoroughly pipette the undigested cells, and collect the cell suspension into a sterile EP tube. Centrifuge to collect the cell pellet: 800 rpm, 5 min 30 s. Discard the supernatant, resuspend with an appropriate amount of culture medium, mix thoroughly, and then seed into culture flasks or plates. Incubate at 37°C in a cell culture incubator until ready for use.

[0049] Step 3: CCK8 Experiment

[0050] Human primary uterine fibroid cells and uterine fibroid / sarcoma cell lines were seeded at a suitable density of 100 μL / well in 96-well plates. After the cells reached a suitable density, naringenin and a combination of naringenin (naringenin sulfate) were added for intervention (concentration gradient 0-700 μM, gradient interval 100 μM, for 48 hours). After intervention, 10 μL of CCK8 solution was added to each well, and the cells were incubated at 37℃ for 2-4 hours. The absorbance was measured using a microplate reader. According to Tables 1 and 2, naringenin and naringenin sulfate significantly inhibited the proliferation of human primary uterine fibroid cells (hUL) and uterine fibroid cell line (ELT-3), with naringenin sulfate showing a better inhibitory effect than naringenin. Furthermore, the drug effect of naringenin sulfate was concentration-dependent; as the concentration increased, the effect of naringenin sulfate decreased. The survival rate of uterine fibroid cells gradually decreased. As shown in Tables 3 to 5 and Figure 1 of the specification, naringenin significantly inhibited the proliferation of human uterine sarcoma cell lines (SKN, SKUT-1, MES-SA), and the drug effect was concentration-dependent. In specific embodiments, this application mainly investigated the inhibitory activity of naringenin or its pharmaceutically usable salts at concentrations ranging from 100 μM to 700 μM. Clearly, as the concentration continued to increase, the inhibitory activity further increased until it exhibited an inhibitory effect of approximately 100%. Increasing the concentrations of the other three natural plant extract active ingredients (ependylene, β-sitosterol, and stigmasterol) had no effect on the survival rate of uterine fibroids and uterine sarcoma cells. When the intervention concentration of naringenin or its pharmaceutically usable salts was within the IC50 range... 50 At that time, its inhibition rate on the proliferation of human primary uterine fibroid cells (hUL), uterine fibroid cell line (ELT-3), and uterine sarcoma cell line (SKN, SKUT-1, MES-SA) was 40%-60%, while the active ingredients of the other three natural plant extracts had no inhibitory effect on uterine fibroids and uterine sarcoma cells. See Tables 1-5 for details.

[0051] Table 1 Comparison of mean survival rates of human primary uterine fibroid cells (hUL) after intervention at different concentrations

[0052]

[0053] * This indicates that the value was <0.05 compared to the control group. # P < 0.001 compared to the control group; Comparative Example 1 used ivy extract, Comparative Example 2 used β-sitosterol, Comparative Example 3 used stigmasterol, and the control group was a blank control without any added active ingredients.

[0054] Table 2 Comparison of mean survival rates of uterine fibroid cell line (ELT-3) after different concentrations of intervention.

[0055]

[0056] * This indicates that the value was <0.05 compared to the control group. # P < 0.001 compared to the control group; Comparative Example 1 used ivy extract, Comparative Example 2 used β-sitosterol, Comparative Example 3 used stigmasterol, and the control group was a blank control without any added active ingredients.

[0057] Table 3 Comparison of mean survival rates of uterine sarcoma cell line SKN after different concentrations of intervention.

[0058]

[0059] * This indicates that the value was <0.05 compared to the control group. # P < 0.001 compared to the control group; Comparative Example 1 used ivy extract, Comparative Example 2 used β-sitosterol, Comparative Example 3 used stigmasterol, and the control group was a blank control without any added active ingredients.

[0060] Table 4 Comparison of mean survival rates of SKUT-1 uterine sarcoma cell line after different concentrations of intervention.

[0061]

[0062] * This indicates that the value was <0.05 compared to the control group. # P < 0.001 compared to the control group; Comparative Example 1 used ivy extract, Comparative Example 2 used β-sitosterol, Comparative Example 3 used stigmasterol, and the control group was a blank control without any added active ingredients.

[0063] Table 5 Comparison of mean survival rates of uterine sarcoma cell line MES-SA after different concentrations of intervention.

[0064]

[0065] * This indicates that the value was <0.05 compared to the control group. # P < 0.001 compared to the control group; Comparative Example 1 used ivy extract, Comparative Example 2 used β-sitosterol, Comparative Example 3 used stigmasterol, and the control group was a blank control without any added active ingredients.

[0066] As shown in Table 3 of this application, when the concentration of naringenin reaches 600 μM or higher, its inhibition rate on the proliferation of the uterine sarcoma cell line SKN is below 50%; as shown in Table 4 of this application, when the concentration of naringenin reaches 600 μM or higher, its inhibition rate on the proliferation of the uterine sarcoma cell line SKUT-1 is also below 50%; as shown in Table 5 of this application, when the concentration of naringenin reaches approximately 500 μM, its inhibition rate on the proliferation of the uterine sarcoma cell line MES-SA is approximately 50%. This indicates that when the intervention concentration of naringenin is within the IC50 range... 50 At the same time, it showed a good rate of inhibition of proliferation of uterine sarcoma cell lines (SKN, SKUT-1, MES-SA).

[0067] Step 4: Real-time quantitative PCR (RT-PCR)

[0068] Human primary uterine fibroid cells and rat uterine fibroid cell lines were seeded at appropriate densities in 6-well plates. After the cells reached the appropriate density, naringenin and naringenin sulfate were added. After 48 hours of intervention, RNA was extracted from the cells. RNA was reverse transcribed into cDNA and then subjected to RT-PCR. Primer preparation: The reaction system was prepared in a single PCR tube according to the table below. Specific raw materials and quantities are shown in Table 6 below:

[0069] Table 6

[0070]

[0071] Mix thoroughly using a vortex mixer and centrifuge using a handheld centrifuge. Spot the samples according to the pre-designed plate layout. Then, transfer the samples to a PCR cycler for real-time quantitative PCR. Perform the qPCR reaction according to the requirements in Table 7 below.

[0072] Table 7

[0073]

[0074] From the attached diagram in the instruction manual Figure 2As shown in Figure A, after intervention with naringenin and naringenin sulfate, the expression of mRNAs (α-SMA, TGFβ1, COL1A1, COL3A1, MMP1, MMP2, MMP9) of extracellular matrix synthesis-related molecules in human primary uterine fibroid cells and rat uterine fibroid cell lines was lower than that in the control group, and the effect of naringenin sulfate was better than that of naringenin, indicating that naringenin and naringenin sulfate can significantly inhibit extracellular matrix synthesis in fibroid cells.

[0075] Step 5: Cell immunofluorescence staining

[0076] Human primary uterine fibroid cells and rat uterine fibroid cell lines were seeded at appropriate densities in 24-well plates. After the cells reached the appropriate density, naringenin and a naringenin combination (naringenin sulfate) were added for intervention for 48 hours. The treated cells were then washed three times with 1x cell culture PBS solution for 3 min each time. Fixed with 4% paraformaldehyde at room temperature for 15 min, followed by three washes with PBS solution. Permeabilization with 0.5% Triton X100 solution was added, and the cells were permeabilized at room temperature for 20 min, followed by three washes with PBS solution. Blocking was performed for 1 h in a 37°C incubator with 5% BAS or 10% goat anti-rabbit serum. The blocking solution was aspirated, and the cells were not washed. The primary antibody was diluted with blocking solution according to the target antibody dilution ratio, and an appropriate volume of primary antibody was added to each well. The cells were incubated overnight at 4°C. The next day, the cell culture plates were removed and allowed to warm to room temperature for 15-30 min. Wash three times with PBS solution. Dilute the secondary antibody with PBS according to the dilution ratio. Add FITC-labeled goat anti-rabbit / mouse IgG corresponding to the source of the primary antibody to each well. Incubate at 37°C in the dark for 1 hour. Wash three times with PBS solution. Add an appropriate volume of ready-to-use DAPI solution to each well and incubate at room temperature for 15 minutes. Then wash three more times with PBS solution. Observe the expression location of the target molecule's fluorescence under an inverted fluorescence microscope (DAPI blue light, cell nucleus blue staining; FITC green light, target protein green staining). See the attached diagram in the instruction manual. Figure 2 B indicates that after intervention with naringenin and naringenin sulfate, the average green fluorescence intensity of extracellular matrix synthesis-related molecules in human primary uterine fibroid cells and rat uterine fibroid cell lines was lower than that in the control group. In the control group, extracellular matrix synthesis was enhanced, and there was excessive accumulation of extracellular matrix between cells, indicating that naringenin and naringenin sulfate can significantly inhibit extracellular matrix synthesis in fibroid cells.

[0077] Step Six: Scratch Migration Experiment

[0078] Human primary uterine fibroid cells and rat uterine fibroid cell lines were seeded at appropriate densities in 6-well plates. When the cells reached 90% confluence or completely covered the wells, a horizontal line was drawn perpendicularly to the bottom of the well using a sterile 1 mL pipette tip. Exfoliated cells were gently washed away with PBS. 2 mL of culture medium (with naringin and naringin sulfate added to the experimental group, IC50 concentration) was added to each well. The cells were incubated at 37°C in a constant temperature cell culture incubator. Images were taken under a light microscope at 0 and 24 hours. (Refer to the accompanying diagram in the instruction manual.) Figure 2 As is known, naringenin and naringenin sulfate have a significant inhibitory effect on the migration of human primary uterine fibroid cells (hUL) and uterine fibroid cell line (ELT-3).

[0079] Step 7: Western Blot Assay

[0080] Human uterine sarcoma cell lines were seeded at an appropriate density in 6-well plates. After the cells reached the appropriate density, naringenin and naringenin sulfate were added. After 48 hours of intervention, cell proteins were extracted for immunoblotting experiments. Electrophoresis: Electrophoresis buffer was prepared according to the method shown in Table 8 below.

[0081] Table 8

[0082]

[0083] Thoroughly mix the electrophoresis buffer and let it stand at room temperature. Determine the loading volume according to the protein concentration (20 μg protein sample per well), and the protein marker loading volume is 5 μL. Adjust the voltage to 60 V, and the electrophoresis time is approximately 40 min to 1 h. Once the markers of different molecular weights are clearly separated, adjust the voltage to 90 V. Stop electrophoresis when the protein markers drop vertically to the ideal position. Prepare the transfer buffer according to the method shown in Table 9 below, mix the prepared transfer buffer thoroughly, and pre-cool it on ice. After electrophoresis, begin the transfer process under the following conditions: 280 mA, 1 h, 40 min.

[0084] Table 9

[0085]

[0086] After the transfer, the membrane is blocked: Prepare a 5% BSA-TBST solution or a 5% skim milk-TBST solution. After the transfer, remove the PVDF membrane and place it in the blocking solution. Block it on a shaker at room temperature for 2 hours. Specific raw materials and dosages are shown in Table 10.

[0087] Table 10

[0088]

[0089] After blocking, following the protein marker's instructions, add the appropriate primary antibody dilution buffer according to the target protein's molecular weight and incubate overnight at 4°C. The next day, remove the bands from 4°C, allow them to warm to room temperature for 30 min, and wash thoroughly in TBST buffer (3 times, 10 min each time). Select the secondary antibody corresponding to the primary antibody source and incubate at room temperature for 1 h. Wash the membrane again with TBST buffer, washing thoroughly on a shaker (3 times, 10 min each time). Exposure: Protect from light throughout. Mix ECL exposure solutions A and B in a 1:1 ratio and use immediately after preparation. Immerse the bands in the exposure solution for 20 s, then acquire images using the ChemiDoc MP imaging system (Blot mode). See attached diagram in the instruction manual. Figure 3 B indicates that after naringenin intervention, the expression of cell cycle-related enzymes (CDC25A, CDC25C, Cyclin A2, Cyclin D1, P53) in human uterine sarcoma cells was lower than that in the control group, while the synthesis of cell cycle-related enzymes in the control group increased, indicating that naringenin can significantly inhibit the cell cycle of sarcoma cells.

[0090] Step 8: EdU Proliferation Experiment

[0091] Human uterine sarcoma cell lines were seeded at an appropriate density in 96-well plates, and an equal volume of preheated (37°C) 2×EdU solution was added to a solution containing naringenin intervention (naringenin, IC50). 50 Cells were cultured in a medium containing 1× BSAWash Solution (1×) to achieve a final EdU concentration of 1× in the 96-well plate. Cells were incubated for 2 hours under optimal conditions. After incubation, the medium was removed, and 0.1 mL of fixative (PBS containing 3.7% formaldehyde) was added to each well. Cells were incubated at room temperature for 15 minutes. After removing the fixative, cells were washed with 0.1 mL of BSAWash Solution (1×) for 5 minutes, repeated three times. After removing the washing buffer, 0.1 mL of permeabilizer (PBS containing 0.5% Triton X-100) was added to each well. Cells were incubated at room temperature for 15 minutes. After removing the permeabilizer, cells were washed with 0.1 mL of BSAWash Solution (1×) for 5 minutes, repeated twice. 100 μL of Click-iT reaction mixture was added to each sample, and cells were incubated at room temperature in the dark for 30 minutes. After removing the reaction mixture, cells were washed with 0.1 mL of BSAWash Solution (1×) for 5 minutes, and the washing buffer was removed. Nuclear staining (1×Hoechst 33342) or antibody labeling was performed. Finally, the labeled DNA in the sample was analyzed using a fluorescence microscope (Ex / Em = 546 / 565nm), and the cell nucleus was detected using an Ex / Em = 360 / 460nm microscope. (Refer to the accompanying diagram in the instruction manual.) Figure 3As is known, after intervention with naringenin, the average fluorescence intensity of uterine sarcoma cells was reduced compared with that of the control group, indicating that naringenin has a significant inhibitory effect on the proliferation of the uterine sarcoma cell line (SKN).

[0092] Step Nine: TUNEL Apoptosis Assay

[0093] Human uterine sarcoma cell lines were seeded at an appropriate density in 96-well plates and incubated for at least 24 hours. The cells were then analyzed using naringenin (naringenin, IC50). 50 Cells were treated with a concentration of FITC, while uninduced control cells were cultured. The culture medium was removed, and cells were fixed with 50 μL of 4% paraformaldehyde for 30 min at room temperature. The fixative was removed, and cells were washed three times with 200 μL of PBS for 5 min each time. After fixation, 50 μL of 0.3% Triton X-100 was added to each well, and the cells were incubated at room temperature for 30 min. The permeabilizer was removed, and cells were washed 2-3 times with 50 μL of BSA Working Solution. 50 μL of the reaction mixture was added to each sample, and the cells were incubated in a humidified chamber at 37°C in the dark for 2 h. The samples were washed three times with PBS for 5 min each time. The samples were counterstained with 1×DAPI for 10 min. The samples were washed three times with an appropriate amount of PBS for 5 min each time. Finally, the cells were submerged in an appropriate amount of PBS, and then observed under a fluorescence microscope. FITC channels (Ex / Em = 490 nm / 520 nm) are shown in the accompanying diagram. Figure 3 As is known, after intervention with naringenin, the average fluorescence intensity of uterine sarcoma cells was higher than that of the control group, proving that naringenin can promote apoptosis in the uterine sarcoma cell line (MES-SA).

[0094] In summary, the naringenin drug protected in this application inhibits the synthesis of extracellular matrix in uterine fibroid cells, thereby inhibiting the proliferation and migration of fibroid cells; on the other hand, naringenin can inhibit the synthesis of cell cycle-related enzymes in uterine sarcoma cells, thereby inhibiting sarcoma cell proliferation and promoting sarcoma cell apoptosis.

[0095] The following examples further explore the use of naringenin in combination with conventional pharmaceutically acceptable carriers to formulate any pharmaceutically acceptable dosage form and investigate the inhibitory effects of these drugs on human uterine sarcoma cells (SKN).

[0096] Example 2 Tablet Preparation

[0097] Prescription: 400g naringenin, 40g starch, 24g 10% starch slurry, 23g dry starch, 3g magnesium stearate. Make 1000 tablets. Pass the naringenin through an 80-mesh sieve, mix it with the starch, add starch slurry to make a soft mass, extrude it through a 14-mesh sieve to granulate, dry it at 70℃-80℃, granulate it through a 12-mesh sieve, add dry starch and magnesium stearate, mix well, and then compress into tablets.

[0098] Example 3: Tablet Preparation

[0099] Prescription: 100g naringenin, 50g starch, 20g sucrose, 10g microcrystalline cellulose, 5g magnesium stearate, and an appropriate amount of 0.5% CMC solution. Prepare 1000 tablets, granulate using conventional wet granulation, compress, and package.

[0100] Example 4: Capsule Preparation

[0101] Prescription: 330g naringenin, 250g microcrystalline cellulose, 16g croscarmellose sodium, 60g microcrystalline silica, 4g magnesium stearate, and an appropriate amount of 5% polyvinylpyrrolidone aqueous solution. Pass the microcrystalline cellulose and excipients in the prescription through an 80-mesh sieve. Weigh the prescribed amount of schisandrin and mix it evenly with all the excipients. Add the 5% polyvinylpyrrolidone aqueous solution to prepare a soft mass, granulate, dry, sizing, add talc powder, mix well, and fill into capsules to obtain 1000 capsules.

[0102] Example 5: Capsule Preparation

[0103] Prescription: 100g naringenin, 40g lactose, 5g starch, 5g sucrose, 10g microcrystalline cellulose, and an appropriate amount of 1% HPMC solution. Make 1000 capsules using conventional wet granulation, fill the capsules after granulation, and package them.

[0104] The preparations from Examples 2 to 5 were used to inhibit human uterine sarcoma cells (SKN) according to the procedure in Example 1, and the cell survival rate was found to be below 50%.

[0105] In summary, naringenin, an active ingredient in natural plant extracts, can effectively inhibit the progression of uterine fibroids / sarcomas, specifically by inhibiting the proliferation and migration of uterine fibroid / sarcoma cells and promoting apoptosis. Therefore, naringenin and its pharmaceutical preparations have high bioavailability and safe dosage, resulting in fewer potential side effects during treatment. Naringenin may be a promising new generation of highly versatile drugs for the treatment of uterine fibroids / sarcomas.

[0106] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. Application of naringenin or its pharmaceutically usable salts in the preparation of drugs for treating uterine sarcoma; the concentration range of naringenin or its pharmaceutically usable salts is 400 μM-700 μM.

2. The application according to claim 1, characterized in that, The drug has an inhibitory effect on the proliferation of human uterine sarcoma cell lines.

3. The application according to claim 1 or 2, characterized in that, The human uterine sarcoma cells include any one or more of SKUT-1, SKN, or MES-SA.

4. The application according to claim 2, characterized in that, The naringenin or its pharmaceutically usable salt has the effect of inhibiting the synthesis of cell cycle-related enzymes in human uterine sarcoma cell lines, thereby inhibiting cell proliferation and migration and promoting cell apoptosis.

5. The application according to claim 1 or 2, characterized in that, The drug is a pharmaceutical composition containing naringenin or a pharmaceutically usable salt of naringenin.

6. The application according to claim 5, characterized in that, The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

7. The application according to claim 6, characterized in that, The pharmaceutical composition is formulated into a pharmaceutically acceptable dosage form.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable dosage form is selected from tablets, capsules, solutions, granules, or suspensions.

9. The application according to claim 8, characterized in that, The dosage form is in unit dose form, and each unit dose contains naringenin or a pharmaceutically acceptable salt of naringenin: 1mg-10mg, or 10mg-100mg, or 100mg-1000mg, or 1000mg-10000mg.

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