Application of Danbie Capsules in the Preparation of Products for Treating Endometriosis

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of Danbie capsules in the preparation of products for treating endometriosis. This invention discovers that the combined use of Danbie capsules and progestins has a synergistic effect, with a superior intervention effect on endometrial stromal cells compared to a single component. Furthermore, this invention provides a traditional Chinese medicine-containing serum, the main medicinal components of which are quercetin and luteolin, which can effectively inhibit the growth and migration of endometrial stromal cells and induce apoptosis. This traditional Chinese medicine-containing serum is derived from Danbie capsules. Animal experiments have shown that Danbie capsules can inhibit the expression of AKT1 in endometrial tissue, and cell experiments have shown that the traditional Chinese medicine-containing serum can induce apoptosis of endometrial stromal cells, suggesting that the intervention effect of the traditional Chinese medicine Danbie capsules and its containing serum on endometrial stromal cells is related to AKT1 expression, providing an important insight into the relevant mechanism.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Danbie capsules in the preparation of products for treating endometriosis. Background Technology

[0002] Endometriosis (EMS) is a common estrogen-dependent chronic inflammatory disease characterized by the extrauterine growth of endometrial glands and stroma, resulting in cyclical bleeding, proliferation, and fibrosis. It affects approximately 6-10% of women of reproductive age, severely impacting their health, quality of life, and work productivity, thus imposing a significant economic burden on society. Endometriosis is closely associated with a variety of symptoms, including severe dysmenorrhea, chronic pelvic inflammatory disease pain, menorrhagia, bowel and bladder symptoms, fatigue, and depression; therefore, it is more often described as a "syndrome." Due to the high heterogeneity of endometriosis symptoms and the significant complexity of its pathogenesis, diagnosis lacks specific symptoms and biomarkers, and confirmation may be delayed by 4-11 years. Clinical treatment of endometriosis mainly includes surgical resection of the lesions and ovarian suppression medication. However, the recurrence rate after surgery is high, and drug treatment has significant side effects; therefore, the treatment of endometriosis remains a major clinical challenge. Therefore, further exploration is needed to understand the potential pathogenesis of endometriosis and to design new treatment strategies.

[0003] Danbie Capsules (National Drug Approval Number Z20040037) are a Class III new traditional Chinese medicine developed and produced by Guangzhou Baiyunshan Pangao Shou Pharmaceutical Co., Ltd. They are composed of Salvia miltiorrhiza, Panax notoginseng, Sparganium stoloniferum, Curcuma zedoaria, Prunus persica (peeled), Angelica sinensis, Carapax Trionycis, Sargassum, Eucommia ulmoides (salt-fried), Atractylodes macrocephala (fried), Scutellaria barbata, and Cinnamomum cassia. They have the effects of promoting blood circulation, removing blood stasis, softening and dispersing masses, and are mainly used to treat masses (uterine fibroids, pelvic inflammatory masses) caused by qi stagnation and blood stasis, with blood stasis accumulating in the uterus and lower abdomen. Studies by Hu Sisi et al. have found that Danbie Capsules can reduce the concentrations of PGE2 and TNF-α in the blood of EMs rats, which may be one of the effective mechanisms for treating endometriosis. Studies by Liu Zhenming et al. have shown that Danbie Capsules have a certain therapeutic effect on endometriosis, and their active ingredients were studied to determine their target and specific mechanism of action. Studies by Zhang Chunhong et al. have found that Danbie capsules can inhibit the growth of ectopic endometrial tissue by reducing the levels of PGE-2 and TNF in the serum and peritoneal fluid of model rats; and play a role in the treatment of endometriosis by having anti-inflammatory, analgesic, and hemorheological and microcirculation-improving functions. Summary of the Invention

[0004] Therefore, based on the above background, the purpose of this invention is to provide the application of Danbie capsules in combination with estrogen in the preparation of endometriosis products, and further to provide a drug-containing serum, providing a reference for the development of drugs related to the treatment of endometriosis.

[0005] The first objective of this invention is to provide the application of Danbie capsules in combination with progestins in the preparation of products for treating endometriosis.

[0006] A second aspect of the present invention is to provide a composition.

[0007] A third aspect of the present invention is to provide a drug.

[0008] The fourth aspect of this invention is to provide a drug-containing serum.

[0009] The fifth aspect of this invention is to provide a method for preparing drug-containing serum according to the fourth aspect of this invention.

[0010] The sixth aspect of this invention aims to provide the application of the drug-containing serum of the fourth aspect of this invention.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0012] In a first aspect, the present invention provides the use of Danbie capsules in combination with progestins in the preparation of products for treating endometriosis.

[0013] Preferably, the product includes a drug.

[0014] Preferably, the dosage form of the drug includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0015] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0016] Preferably, the excipients include at least one of the following: carrier, excipient, stabilizer, filler, binder, lubricant, coating material, and flavoring agent.

[0017] Preferably, the carrier includes a solid carrier or a liquid carrier.

[0018] Preferably, the liquid carrier includes: saline, buffer solution, glucose solution, aqueous solution, glycerol, and ethanol.

[0019] Preferably, the solid carrier comprises at least one of low molecular weight dextran, polyethylene glycol 400, polyethylene glycol 6000, cyclodextrin, mannitol, lactose, sucrose, sodium chloride, and sorbitol.

[0020] Preferably, the product may also include other active ingredients for treating endometriosis, including but not limited to: gonadotropin-releasing hormone antagonists, such as Ganirelix, cetrorelix, ellagolix, and abarelix; selective progesterone receptor modulators, such as mifepristone (RU486) and ullipristal acetate; selective estrogen receptor modulators, such as tamoxifen (TAM), raloxifene, and bazedoxifene (BZA); aromatase inhibitors, such as letrozole (LZ); immunomodulatory factors, such as tumor necrosis factor-α (TNF-α); and anti-angiogenic drugs, such as angiostatin, endostatin, cyclooxygenase-2 (COX-2), dopamine receptor agonists, and EGFR tyrosine kinase inhibitors.

[0021] In a first aspect of this invention, the combination of Danbie capsules and progesterone inhibits the growth and migration of endometrial stromal cells and induces apoptosis of endometrial stromal cells. The combined effect of the two components is superior to that of a single component, and the synergistic effect between them improves the therapeutic effect on endometriosis.

[0022] A second aspect of the present invention provides a composition comprising a turtle capsule and a progestin.

[0023] Preferably, the composition may further include other active ingredients for treating endometriosis, the relevant active ingredients being the same as those in the first aspect of the invention.

[0024] A third aspect of the present invention provides a medicament comprising the composition described in the second aspect of the present invention.

[0025] Preferably, the dosage form of the drug includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0026] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0027] Preferably, the excipients include at least one of the following: carrier, excipient, stabilizer, filler, binder, lubricant, coating material, and flavoring agent.

[0028] Preferably, the carrier includes a solid carrier or a liquid carrier.

[0029] Preferably, the liquid carrier includes: saline, buffer solution, glucose solution, aqueous solution, glycerol, and ethanol.

[0030] Preferably, the solid carrier comprises at least one of low molecular weight dextran, polyethylene glycol 400, polyethylene glycol 6000, cyclodextrin, mannitol, lactose, sucrose, sodium chloride, and sorbitol.

[0031] Preferably, the medicament may further include other active ingredients for treating endometriosis, the relevant active ingredients being the same as those in the first aspect of the invention.

[0032] In a fourth aspect, the present invention provides a drug-containing serum, wherein the drug-containing serum is serum obtained after an animal has metabolized at least one of 1) to 2);

[0033] 1) Danbie Capsules;

[0034] 2) The composition described in the second aspect of the present invention and / or the medicament described in the third aspect of the present invention.

[0035] Preferably, the metabolism refers to the product of the active ingredient after absorption and processing following oral or injection of an effective dose of the drug ingredient.

[0036] Preferably, the drug-containing serum includes quercetin and / or luteolin.

[0037] Preferably, the drug-containing serum includes progestin and / or products of progestin metabolism.

[0038] Preferably, the animal includes at least one of a mouse, cat, dog, rabbit, monkey, and human.

[0039] In some embodiments of the present invention, the animal is a rat, and the serum is rat serum.

[0040] In a fourth aspect of this invention, drug-containing serum was extracted from rats, and it was found that this serum could significantly inhibit the growth and migration of human endometrial stromal cells and induce apoptosis of human endometrial stromal cells. Serum obtained by combining *Dendrobium nobile* capsules with progesterone showed even better effects. This invention further identified quercetin and luteolin as the active substances in this drug-containing serum. However, this does not mean that other components in the drug-containing serum do not have a therapeutic effect on endometriosis. Other metabolites of *Dendrobium nobile* serum after metabolism in animals are not a limitation of this invention, and drug-containing serums containing other components are also within the scope of protection of this invention.

[0041] A fifth aspect of the present invention provides a method for preparing drug-containing serum according to the fourth aspect of the present invention, comprising the following steps:

[0042] Administer at least one of 1) to 2) to the animal and collect serum;

[0043] 1) Danbie Capsules;

[0044] 2) The composition described in the second aspect of the present invention and / or the medicament described in the third aspect of the present invention.

[0045] Preferably, the administration includes at least one of oral administration and injection.

[0046] Preferably, the application refers to the administration of an effective dose of the drug component.

[0047] Preferably, the animal includes at least one of a mouse, cat, dog, rabbit, monkey, and human.

[0048] In some embodiments of the present invention, the animal is a rat, and the serum is rat serum.

[0049] In some embodiments of the invention, the administration includes administration over multiple consecutive days, including but not limited to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer. Serum collection is performed 30–180 minutes after the last administration.

[0050] A sixth aspect of the present invention provides the use of the drug-containing serum of the fourth aspect of the present invention in the preparation of a product for treating endometriosis.

[0051] Preferably, the product includes a drug.

[0052] Preferably, the dosage form of the drug includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0053] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0054] Preferably, the excipients include at least one of the following: carrier, excipient, stabilizer, filler, binder, lubricant, coating material, and flavoring agent.

[0055] Preferably, the carrier includes a solid carrier or a liquid carrier.

[0056] Preferably, the liquid carrier includes: saline, buffer solution, glucose solution, aqueous solution, glycerol, and ethanol.

[0057] Preferably, the solid carrier comprises at least one of low molecular weight dextran, polyethylene glycol 400, polyethylene glycol 6000, cyclodextrin, mannitol, lactose, sucrose, sodium chloride, and sorbitol.

[0058] Preferably, the product may also include other active ingredients for treating endometriosis, the relevant active ingredients being the same as those in the first aspect of the invention.

[0059] In the above aspects of the present invention, the Danbie Capsule is a Class III new Chinese medicine developed and produced by Guangzhou Baiyunshan Pangao Shou Pharmaceutical Co., Ltd. It is composed of Danshen, Sanqi, Sanleng, Ezhu, Taoren (peeled), Danggui, Aojia, Haizao, Duzhong (salt-fried), Baizhu (fried), Banzhilian, and Guizhi. It has the effects of promoting blood circulation, removing blood stasis, softening and dispersing masses. It is mainly used to treat masses (uterine fibroids, pelvic inflammatory masses) caused by qi stagnation and blood stasis, and blood stasis in the uterus and lower abdomen. The National Drug Approval Number is Z20040037.

[0060] Although Danbie Capsules are in capsule form, this does not mean that other dosage forms cannot achieve the technical effects of this invention. For example, the active ingredients in Danbie Capsules can be modified into solvents, granules, tablets, or other dosage forms, and will still have the desired effects. The term "Danbie Capsules" refers to the drug with the national drug approval number Z20040037. "Capsule" does not constitute a limitation on the dosage form, and other dosage form products developed based on the drug with national drug approval number Z20040037 should also be included in the scope of protection of this invention.

[0061] The beneficial effects of this invention are:

[0062] This invention discovers that the combined use of Danbie capsules and progestins has a synergistic effect, exhibiting superior intervention on endometrial stromal cells compared to single-component treatments. Furthermore, this invention provides a traditional Chinese medicine-containing serum, whose main medicinal components are quercetin and luteolin, which can effectively inhibit the growth and migration of endometrial stromal cells and induce apoptosis. Traditional surgical and hormone therapies often involve significant invasiveness and numerous side effects. The traditional Chinese medicine-containing serum provided by this invention offers a new approach for the development of subsequent drugs for treating endometriosis. This invention provides a traditional Chinese medicine-containing serum derived from Danbie capsules. Animal experiments have shown that Danbie capsules can inhibit AKT1 expression in endometrial tissue, and cell experiments have shown that the traditional Chinese medicine-containing serum can induce apoptosis in endometrial stromal cells. This suggests that the intervention effect of Danbie capsules and its medicated serum on endometrial stromal cells may be related to AKT1 expression, providing an important insight into the relevant mechanism. Attached Figure Description

[0063] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0064] Figure 1 Chromatograms of quercetin and luteolin: (1) Chromatogram of mixed standard of quercetin and luteolin; (2) Chromatogram of serum containing Danbie capsules; A: quercetin; B: luteolin.

[0065] Figure 2 The expression of AKT1 was observed in the Danbie capsule group and the saline control group.

[0066] Figure 3 The migration of endometrial stromal cells in each group.

[0067] Figure 4 Quantitative statistical results of endometrial stromal cell migration: a) cell control group; b) saline group; c) progesterone group; d) Danbie capsule group; e) combined group.

[0068] Figure 5 The following figures represent the OD values ​​and cell inhibition rates of each group in the CCK8 experiment: a) combined group; b) Danbie capsule group; c) progesterone group; d) saline control group; e) cell control group; f) culture medium blank control. * indicates that the differences were statistically significant compared with the control group (P < 0.05).

[0069] Figure 6 This study compares the apoptosis rates of cells in the control group and the Danbie capsule group. Detailed Implementation

[0070] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0071] Example 1 Preparation of Traditional Chinese Medicine-containing Serum

[0072] 1. Preparation of experimental animals

[0073] Purchase 40 female SPF-grade SD rats with a body weight of 210 - 230 g from Zhejiang Vital River Co., Ltd., license number: SCXK(Zhe)2019 - 0001. The relative humidity in the animal room is 40 - 70%, and the room temperature is 20 - 25°C. The use of experimental animals has been approved by the Animal Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University. The rats are randomly divided into 4 groups (10 rats in each group), namely the saline group, the progesterone group, the Danbie Capsule group, and the progesterone + Danbie Capsule combined group. After the rats enter the animal room of the Sixth Affiliated Hospital of Sun Yat-sen University and pass the 3-day quarantine observation, the experiment begins.

[0074] 2. Preparation of medicine-containing serum

[0075] The dydrogesterone group is given 1 mL of dydrogesterone tablet solution (3.02 mg / kg). The dosage of the Danbie Capsule group is 1 mL (0.71 g / kg). The control group is given 1 mL of normal saline by gavage. The progesterone + Danbie Capsule combined group is given 1 mL of dydrogesterone tablet solution (3.02 mg / kg) + 1 mL of Danbie Capsule with a dosage of (0.71 g / kg). Gavage is performed once a day for 7 consecutive days.

[0076] After drug intervention for 7 days, the rats are anesthetized 90 minutes after the last administration, using inhaled isoflurane for anesthesia. Blood is collected from the abdominal aorta, and whole blood is collected using a disposable non-anticoagulant vacuum blood collection tube. After being placed at 37°C for 3 hours, it is left to stand overnight at 4°C. Then, it is centrifuged at a speed of 2500 / min for 20 minutes using a centrifuge. The supernatant is taken, placed in a water bath at 56°C for 30 minutes for sterilization, and then filtered through a bacterial filter. After being分装 into EP tubes, it is stored in a -80°C refrigerator for standby.

[0077] Example 2 Identification of active ingredients in medicine-containing serum

[0078] 1. Verification of the main active ingredients in medicine-containing serum:

[0079] Use high performance liquid chromatography to detect and verify the main medicinal ingredients in traditional Chinese medicine-containing serum.

[0080] Chromatographic conditions: Platisil C18 column (250 mm × 4.6 mm × 5 μm); mobile phase: methanol-0.2% trifluoroacetic acid (55:45, V / V); flow rate: 1.0 mL / min; detection wavelength: 350 nm; column temperature: 25℃; injection volume: 20 μL. Standards quercetin, luteolin, and β-sitosterol were purchased from Taizhou Danding Biotechnology Co., Ltd., Guangzhou Weigu Technology Co., Ltd., and Lemeitian Pharmaceutical Co., Ltd., respectively. The column, methanol, and trifluoroacetic acid were all purchased from Beijing Dicoma Technology Co., Ltd. The high-performance liquid chromatography (HPLC) instrument used was a Shimadzu LCMS-8040CL.

[0081] 2. Results

[0082] The results of high-performance liquid chromatography (HPLC) showed that the main medicinal components in the serum containing traditional Chinese medicine contained quercetin and luteolin, such as... Figure 1 As shown.

[0083] Example 3: Danbie capsules reduce AKT1 expression

[0084] 1. Immunohistochemical semi-quantitative analysis of AKT1 expression in the Danbie capsule group and the control group.

[0085] After blood collection, uterine tissue from each group of rats was collected, soaked in formalin, and then used to prepare paraffin blocks. The tissue slides were then baked at 60°C for 2 hours. Dewaxing was performed with xylene for 15 minutes, repeated three times. The tissues were then dehydrated for 5 minutes each with anhydrous, 95%, 90%, 80%, and 70% ethanol and distilled water, respectively. Washing with PBS for 5 minutes was repeated three times. Repair was performed in a citrate-buffered boiling water bath for 15 minutes, followed by natural cooling to room temperature. Washing with PBS for 5 minutes was repeated three times. Blocking with 3% hydrogen peroxide for 20 minutes, followed by washing with PBS for 5 minutes, was repeated three times. Blocking with normal fetal bovine serum was performed at 37°C for 20 minutes. The first antibody incubation was performed overnight at 4°C, followed by warming for 20 minutes and washing with PBS for 5 minutes, repeated three times. The second antibody incubation was performed at 37°C for 20 minutes, followed by washing with PBS for 5 minutes, repeated three times. DAB was used for staining, and the results were observed under a microscope, with timely termination of the incubation process. Stain with hematoxylin for 10 seconds, rinse with tap water for 5 minutes until bluish. Dehydrate with 70%, 80%, 90%, 95% ethanol and anhydrous ethanol for 5 minutes each, then dehydrate with xylene for 15 minutes, repeating three times. Seal the sections with neutral resin. Finally, analyze the grayscale values ​​of the protein bands using ImageJ software.

[0086] 2. Results

[0087] The AKT1 positivity rate in the Danbie capsule group was 66.67% (2 weak positives + 1 negative); the AKT1 positivity rate in the saline control group was 100% (1 weak positive + 1 positive). The AKT1 positivity rate in the uterus of rats in the Danbie capsule group was lower than that in the control group, indicating that Danbie capsules can reduce AKT1 expression in the rat uterus. Figure 2 As shown.

[0088] Example 4: Danbie capsules combined with progestin inhibit the migration of endometrial stromal cells.

[0089] 1. Cell scratch assay for detecting cell migration

[0090] hESC human endometrial stromal cells were purchased from Guangzhou Huatuo Biotechnology Co., Ltd., product number: HTX2487. They were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37℃ in a 5% CO2 incubator. When the cell confluence reached approximately 80%, they were digested with trypsin and passaged. hESC human endometrial stromal cells in the logarithmic growth phase were collected at a cell number of 2 × 10⁶ cells / year. 5 Cells were seeded per well in culture plates and incubated at 37°C and 5% CO2 for 24 h. Cells were streaked with a 1000 μL pipette tip, washed three times with PBS, and then replaced with medium containing 10% serum. Cell control groups were defined as follows: 10 v / v% fetal bovine serum; saline control group: 10 v / v% saline intervention group rat serum; progesterone group: 10 v / v% dydrogesterone intervention group rat serum; Danbie capsule group: 10 v / v% Danbie capsule intervention group rat serum; and combined group: 10 v / v% combined group rat serum. Photos were taken before drug administration, 24 hours after drug administration, and 48 hours after drug administration. The number of cells within the scratched area was counted in each group.

[0091] The results of this step are as follows:

[0092] The migration ability of endometrial stromal cells in the cell control group, saline group, progesterone group, Danbie capsule group, and combined group was detected by scratch assay. Measurements showed that the scratch areas were similar across the five groups at the initial stage. Cell counts were performed on the scratched areas at 24 and 48 hours. The combined group had the fewest cells (12 at 24 hours, 8 at 48 hours), followed by the progesterone group (47 at 24 hours, 23 at 48 hours), the Danbie capsule group (45 at 24 hours, 50 at 48 hours), the saline control group (53 at 24 hours, 42 at 48 hours), and the cell control group (30 at 24 hours, 89 at 48 hours). The migration ability of endometrial stromal cells in the combined group was weaker than the other four groups. The combination of Danbie capsule and progesterone serum showed a tendency to inhibit cell migration. Figure 3 , 4 As shown.

[0093] Example 5: Effect of Danbie Capsules on Cell Proliferation

[0094] 1. CCK-8 assay for cell proliferation

[0095] Log-phase hESC human endometrial stromal cells were collected at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with medium containing 10% serum. Cell control groups were established (10 v / v% fetal bovine serum), saline control groups (10 v / v% saline intervention group rat serum), progesterone groups (10 v / v% dydrogesterone intervention group rat serum), Danbie capsule groups (10 v / v% Danbie capsule intervention group rat serum), and combined groups (10 v / v% combined group rat serum). A separate medium control group (no cells seeded) was also established. Cells were cultured for 6 h (6h group) and 48 h (48h group). After 6 / 48 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance (OD) at 450 nm was measured using a microplate reader, and the cell inhibition rate was calculated. (Inhibition rate = [(control group - experimental group) / (control group - blank group)]).

[0096] The results of this step are as follows:

[0097] Short-term (6h) after intervention, there were no significant differences in OD values ​​and cell inhibition rates among the groups. However, significant differences emerged after 48 hours of intervention. The combined treatment group had the lowest OD value (P < 0.05), followed by the Danbie capsule group. The progesterone group did not show a significant inhibitory effect on cells. At 6 hours, the inhibition rates of the combined treatment group, Danbie capsule group, progesterone group, and saline group were 18.25%, 23.54%, 21.69%, and 18.52%, respectively. At 48 hours, the inhibition rates of the combined treatment group, Danbie capsule group, progesterone group, and saline group were 79.61%, 32.04%, -9.11%, and 13.59%, respectively. Figure 5 As shown, the combined group and the Danbie capsule group exhibited more significant inhibitory effects when cells were intervened for a long time.

[0098] Example 6: Effect of Danbie Capsules on Cell Apoptosis

[0099] 1. Flow cytometry analysis of apoptosis in model cells

[0100] Cell passage was used to collect cells from the control group (10 v / v% fetal bovine serum) and the Danbie capsule group (10 v / v% rat serum from the Danbie capsule intervention group). Cells were washed twice with PBS, centrifuged at 1000 rpm for 5 min, and the cells were pelleted. Cells were resuspended in 1× Binding Buffer to a cell concentration of 1×10⁻⁶. 6 / mL; 100μL of the corresponding cell suspension was taken from each group and placed in a labeled sterile flow cytometry tube. 5μL Annexin V-FITC was added, and the cells were incubated at room temperature in the dark for 10 min. Immediately after incubation, 10μL PI was added, followed by 400ul of PBS. The cells were then immediately placed in a flow cytometer for detection. We used Annexin-V and PI double staining to stain apoptotic and necrotic cells. Early apoptotic cells were located in the lower right quadrant, and late apoptotic cells were located in the upper right quadrant. Therefore, the sum of the apoptosis rates in the lower right and upper right quadrants was the total apoptosis rate.

[0101] 2. Results

[0102] The total apoptosis rate in the control group was 18.68%, while the total apoptosis rate in the Danbie capsule group was 23.14%. Serum containing Danbie capsules showed a tendency to induce apoptosis in endometrial stromal cells, such as... Figure 6 As shown.

Claims

1. Application of Danbie capsules combined with progestins in the preparation of drugs for treating endometriosis; The progestin mentioned is dydrogesterone; The drug uses dydrogesterone and Danbie capsules as its main active ingredients.

2. The application according to claim 1, characterized in that: The dosage form of the drug includes one of the following: capsules, tablets, microcapsules, suppositories, powders, pills, granules, honey-based ointments, sustained-release preparations, or oral liquid preparations.

Images