Application of Dantai capsule in preparation of product for treating endometriosis

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of a Dantai capsule in preparation of a product for treating endometriosis. The invention finds that the Dantai capsule and progestational hormone are combined to have a synergistic effect, and the intervention effect on endometrial interstitial cells is superior to that of a single component. Furthermore, the invention provides a traditional Chinese medicine drug serum, the main medicinal components of the traditional Chinese medicine drug serum are quercetin and luteolin, and the traditional Chinese medicine drug serum can effectively inhibit growth and migration of endometrial interstitial cells and induce apoptosis of the endometrial interstitial cells. Animal experiments find that the Dantai capsule can inhibit expression of endometrial tissue AKT1, and cell experiments find that the traditional Chinese medicine drug serum can induce apoptosis of endometrial interstitial cells. It is prompted that the intervention effect of the traditional Chinese medicine Dantai capsule and the traditional Chinese medicine serum thereof on the endometrial interstitial cells is related to AKT1 expression, and an important thought is provided for clarification of related mechanisms.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of Danbie capsule in preparing a product for treating endometriosis. Background Art

[0002] Endometriosis (EMS) is a common estrogen-dependent chronic inflammatory disease characterized by periodic bleeding, proliferation and fibrosis caused by the growth of endometrial glands and stroma outside the uterus. About 6-10% of women of childbearing age are affected by it, which seriously affects women's health, quality of life, and work productivity, and imposes a serious economic burden on society. Endometriosis is closely related to a variety of symptoms, including severe dysmenorrhea, chronic pelvic inflammatory pain, menorrhagia, intestinal and bladder symptoms, fatigue and depression, etc. Therefore, it is more of a "syndrome". Due to the high heterogeneity of endometriosis symptoms and the obvious complexity of its pathogenesis, its diagnosis lacks specific symptoms and markers, and the diagnosis may be delayed by 4-11 years. The clinical treatment of endometriosis mainly includes surgical resection of the lesion and drug therapy for ovarian suppression. However, the recurrence rate after surgical treatment is high, and drug therapy has obvious side effects. The treatment of endometriosis is still a major challenge in clinical practice today. Therefore, further exploration is needed to understand the potential pathogenesis of endometriosis and design new treatment strategies.

[0003] Danbie Capsule (National Medicine Standard Z20040037) is a national third-class new Chinese medicine developed and produced by Guangzhou Baiyunshan Pan Gaoshou Pharmaceutical Co., Ltd. It is composed of Danshen, Panax notoginseng, Trillium, Curcuma, peach kernel (peeled), Angelica, Carapace of the Chinese herb, Sargassum, Eucommia (fried with salt), Atractylodes (fried), Scutellaria barbata, and Cinnamomum cassia. It has the effects of promoting blood circulation and removing blood stasis, softening and dispersing nodules. It is mainly used to treat qi stagnation and blood stasis, and symptoms (uterine fibroids, pelvic inflammatory masses) caused by blood stasis in the uterus and lower abdomen. Hu Sisi et al. found that Danbie Capsule can reduce the concentration of PGE2 and TNF-α in the blood of EMs rats, which may be one of its effective mechanisms for treating endometriosis. Liu Zhenming et al. showed that Danbie Capsule has a certain therapeutic effect on endometriosis, and studied its target and specific mechanism of action based on its active ingredients. Zhang Chunhong et al. found that Danbie Capsule 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; it plays a role in the treatment of endometriosis through its anti-inflammatory, analgesic, and improvement of blood rheology and microcirculation functions. Summary of the invention

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

[0005] The purpose of the first aspect of the present invention is to provide the use of Danbie Capsule combined with progesterone in the preparation of a product for the treatment of endometriosis.

[0006] The second aspect of the present invention aims to provide a composition.

[0007] The third aspect of the present invention aims to provide a medicine.

[0008] The fourth aspect of the present invention aims to provide a drug-containing serum.

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

[0010] The purpose of the sixth aspect of the present invention is to provide the use of the drug-containing serum of the fourth aspect of the present invention.

[0011] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides the use of Danbie Capsule combined with progesterone in the preparation of a product for treating endometriosis.

[0013] Preferably, the product comprises a medicament.

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

[0015] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0016] Preferably, the auxiliary materials include at least one of a carrier, an excipient, a stabilizer, a filler, a binder, a lubricant, a coating material, and a flavoring agent.

[0017] Preferably, the carrier comprises a solid carrier or a liquid carrier

[0018] Preferably, the liquid carrier comprises: saline, buffer, glucose solution, aqueous solution, glycerol, 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, Elagolix, Abarelix; selective progesterone receptor modulators, such as Mifepristone (RU486), Ulipristal acetate; selective estrogen receptor modulators, such as tamoxifen (TAM), Raloxifene, Bazedoxifene (BZA); aromatase inhibitors, such as Letrozole (LZ); immunomodulatory factors, such as tumor necrosis factor α (TNF-α); anti-angiogenic drugs, such as angiostatin, endostatin, cyclooxygenase 2 (COX-2), dopamine receptor agonists, EGFR tyrosine kinase inhibitors, etc.

[0021] In the first aspect of the present invention, Danbie Capsule combined with progesterone has the effect of inhibiting the growth and migration of endometrial stromal cells and inducing apoptosis of endometrial stromal cells. The combined effect of the two components is better than that of a single component, and the two components synergize to improve the therapeutic effect for endometriosis.

[0022] The second aspect of the present invention provides a composition comprising Danbie capsule and progesterone.

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

[0024] The third aspect of the present invention provides a medicine 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 capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0026] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0027] Preferably, the auxiliary materials include at least one of a carrier, an excipient, a stabilizer, a filler, a binder, a lubricant, a coating material, and a flavoring agent.

[0028] Preferably, the carrier comprises a solid carrier or a liquid carrier

[0029] Preferably, the liquid carrier comprises: saline, buffer, glucose solution, aqueous solution, glycerol, 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 drug may also include other active ingredients for treating endometriosis, and the relevant active ingredients are the same as those in the first aspect of the present invention.

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

[0033] 1) Danbie capsule;

[0034] 2) The composition according to the second aspect of the present invention and / or the drug according to the third aspect of the present invention.

[0035] Preferably, the metabolism refers to the products of the active ingredient after absorption and processing after oral administration or injection of an effective dose of the pharmaceutical ingredient.

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

[0037] Preferably, the drug-containing serum includes progesterone and / or a metabolic product of progesterone.

[0038] Preferably, the animal comprises at least one of mice, cats, dogs, rabbits, monkeys and humans.

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

[0040] In the fourth aspect of the present invention, the present invention extracts drug-containing serum from rats, and finds that the serum can significantly inhibit the growth and migration of human endometrial stromal cells and induce apoptosis of human endometrial stromal cells. The serum obtained by combining Danbie Capsule and progesterone can exert a better effect. The present invention further identifies that the substances that play a role in the drug-containing serum include quercetin and luteolin. However, it does not mean that other components in the drug-containing serum do not play a therapeutic effect in endometriosis. Other metabolites of Danbie serum after metabolism in animals cannot be a limitation of the present invention, and drug-containing serum containing other components also belongs to the protection scope of the present invention.

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

[0042] Administering at least one of 1) to 2) to an animal, and collecting serum;

[0043] 1) Danbie capsule;

[0044] 2) The composition according to the second aspect of the present invention and / or the drug according to the third aspect of the present invention.

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

[0046] Preferably, the administration is administration of an effective dose of the pharmaceutical ingredient.

[0047] Preferably, the animal comprises at least one of mice, cats, dogs, rabbits, monkeys and humans.

[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 present invention, the administration includes administration for multiple consecutive days, including but not limited to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or longer. The serum collection time is 30 to 180 minutes after the last administration.

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

[0051] Preferably, the product comprises a medicament.

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

[0053] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0054] Preferably, the auxiliary materials include at least one of a carrier, an excipient, a stabilizer, a filler, a binder, a lubricant, a coating material, and a flavoring agent.

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

[0056] Preferably, the liquid carrier comprises: saline, buffer, glucose solution, aqueous solution, glycerol, 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, and the relevant active ingredients are the same as those in the first aspect of the present invention.

[0059] In the above aspects of the present invention, the Danbie Capsule is a national Class III new Chinese medicine developed and produced by Guangzhou Baiyunshan Pan Gaoshou Pharmaceutical Co., Ltd., and is composed of Salvia miltiorrhiza, Panax notoginseng, Trillium, Curcuma, peach kernel (peeled), Angelica sinensis, Carapace of the turtle, Sargassum, Eucommia ulmoides (fried with salt), Atractylodes macrocephala (fried), Scutellaria barbata, and Cinnamon twig. It has the effects of promoting blood circulation and removing blood stasis, softening and dispersing nodules, and is mainly used to treat qi stagnation and blood stasis, and symptoms (uterine fibroids, pelvic inflammatory mass) caused by blood stasis in the uterus and lower abdomen. The national medicine standard number is Z20040037.

[0060] Among them, although Danbie Capsule is a capsule dosage form, it does not mean that other dosage forms cannot achieve the technical effects of the present invention. For example, the active ingredients in Danbie Capsule can be converted into other dosage forms such as solvents, granules, tablets, etc., which can also have their due effects. The term "Danbie Capsule" refers to the drug with the national medicine standard Z20040037, and "capsule" does not constitute a limitation of the dosage form. Other dosage form products developed based on the drug with the national medicine standard Z20040037 should also be included in the protection scope of the present invention.

[0061] The beneficial effects of the present invention are:

[0062] The present invention finds that Danbie Capsule combined with progesterone has a synergistic effect, and the intervention effect on endometrial stromal cells is better than that of a single component. Further, the present invention provides a Chinese medicine-containing serum, the main medicinal ingredients of which are quercetin and luteolin, which can effectively inhibit the growth and migration of endometrial stromal cells and induce apoptosis of endometrial stromal cells. Traditional surgical treatment and hormone therapy often have greater invasiveness and more side effects. The Chinese medicine-containing serum provided by the present invention provides a new idea for the subsequent development of drugs for the treatment of endometriosis. The present invention provides a Chinese medicine-containing serum, which is derived from Danbie Capsule. Animal experiments have found that Danbie Capsule can inhibit the expression of AKT1 in endometrial tissue, and cell experiments have found that the Chinese medicine-containing serum can induce apoptosis of endometrial stromal cells, suggesting that the intervention effect of Chinese medicine Danbie Capsule and its Chinese medicine-containing serum on endometrial stromal cells may be related to the expression of AKT1, which provides an important idea for the elucidation of related mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0064] Figure 1 The chromatograms of quercetin and luteolin are as follows: (1) chromatogram of the mixed standard of quercetin and luteolin; (2) chromatogram of the serum containing Danbie capsule; A: quercetin; B: luteolin.

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

[0066] Figure 3 Figure 2 shows the migration status 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 OD values ​​and cell inhibition rates of each group in the CCK8 experiment; a combination group; b Danbie capsule group; c progesterone group; d saline control group; e cell control group; f culture medium blank control; * indicates that the difference is statistically significant compared with the control group (P < 0.05).

[0069] Figure 6 Comparison of cell apoptosis rates between the control group and the Danbie capsule group. DETAILED DESCRIPTION

[0070] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0071] Example 1 Preparation of a Chinese medicine-containing serum

[0072] 1. Preparation of experimental animals

[0073] SPF-grade SD rats were purchased, female, 40 in total, weighing 210-230 g, from Zhejiang Weitong Lihua Company, license number: SCXK (Zhejiang) 2019-0001. The relative humidity of the animal room was 40-70%, the room temperature was 20-25 °C, and the use of experimental animals was approved by the Animal Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University. The rats were randomly divided into 4 groups (10 in each group), namely, saline group, progesterone group, Danbie capsule group, and progesterone + Danbie capsule combined group. The rats entered the animal room of the Sixth Affiliated Hospital of Sun Yat-sen University and were observed for 3 days before the quarantine was passed before the experiment began.

[0074] 2. Preparation of drug-containing serum

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

[0076] After 7 days of drug intervention, rats were anesthetized 90 minutes after the last administration of the drug, and inhaled isoflurane was used for anesthesia. Blood was collected through the abdominal aorta, and whole blood was collected using a disposable non-anticoagulated vacuum blood collection tube. After being placed at 37°C for 3 hours, it was placed at 4°C overnight, and centrifuged at 2500 / min for 20 minutes using a centrifuge. The supernatant was taken and sterilized in a 56°C water bath for 30 minutes, and then filtered with a bacterial filter, and stored in a -80°C refrigerator after being packaged in EP tubes for later use.

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

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

[0079] High performance liquid chromatography was used to detect and verify the main medicinal ingredients in Chinese medicine-containing serum.

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

[0081] 2. Results

[0082] The results of HPLC showed that the main medicinal ingredients in the serum containing Chinese medicines were quercetin and luteolin, such as Figure 1 shown.

[0083] Example 3 Danbie Capsule reduces the expression of AKT1

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

[0085] After blood was drawn, the uterine tissue of each group of rats was collected, and wax blocks were made after being soaked in formalin. The tissue made into slides was then baked at 60°C for 2 hours. Dewaxing was performed with xylene for 15 minutes, repeated three times. The tissue was dehydrated with anhydrous, 95%, 90%, 80%, 70% ethanol and distilled water for 5 minutes respectively. Washing with PBS for 5 minutes, repeated three times. Repair in a citrate buffer boiling water bath for 15 minutes and naturally cool to room temperature. Washing with PBS for 5 minutes, repeated three times. Blocking with 3% hydrogen peroxide for 20 minutes, washing with PBS for 5 minutes, repeated three times. Blocking with normal fetal bovine serum at 37°C for 20 minutes. The primary antibody was incubated at 4°C overnight, rewarmed for 20 minutes after overnight, washed with PBS for 5 minutes, repeated three times; secondary antibody incubation, incubated at 37°C for 20 minutes, washed with PBS for 5 minutes, repeated three times. Use DAB for color development, observe the results under a microscope, and terminate in time. Stain with hematoxylin for 10 seconds, rinse with tap water for 5 minutes to turn blue. Dehydrate with 70%, 80%, 90%, 95% and anhydrous ethanol for 5 minutes each, dehydrate with xylene for 15 minutes, repeat three times. Slices are sealed with neutral gum. Finally, the gray value of protein bands is analyzed using ImageJ software.

[0086] 2. Results

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

[0088] Example 4: Danbie capsule combined with progesterone inhibits the migration of endometrial stromal cells

[0089] 1. Cell scratch assay to detect cell migration

[0090] hESC human endometrial stromal cells were purchased from Guangzhou Huatuo Biotechnology Co., Ltd., product number: HTX2487. DMEM / F12 culture medium containing 10% fetal bovine serum was used and placed at 37°C and 5% CO 2 The cells were cultured in an incubator. When the cell fusion rate reached about 80%, they were digested with trypsin and subcultured. hESC human endometrial stromal cells in the logarithmic growth phase were taken at a cell number of 2×10 5 Each well was inoculated in a culture plate and incubated at 37°C with 5% CO 2 After 24 hours of conditioned culture, the cells were streaked with a 1000 μL pipette tip, washed 3 times with PBS, and replaced with a medium containing 10% serum. The cells were divided into a cell control group (10 v / v% fetal bovine serum), a saline control group (10 v / v% saline intervention group rat serum), a progesterone group (10 v / v% dydrogesterone intervention group rat serum), a Danbie capsule group (10 v / v% Danbie capsule intervention group rat serum), and a combined group (10 v / v% combined group rat serum). Photos were taken before, 24 hours after, and 48 hours after drug addition. The number of cells in the scratch area of ​​each group was calculated.

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

[0092] The scratch test was used to detect the migration ability of endometrial stromal cells in the cell control group, saline group, progesterone group, Danbie capsule group and combined group. After measurement, the scratch areas of the five groups of cells were similar in the initial state. After 24 hours and 48 hours of scratching, the cells in the scratch area of ​​each group were counted. The number of cells in the scratch area of ​​the combined group was the least (12 in 24 hours and 8 in 48 hours). The others were the progesterone group (47 in 24 hours and 23 in 48 hours), Danbie capsule group (45 in 24 hours and 50 in 48 hours), saline control group (53 in 24 hours and 42 in 48 hours) and cell control group (30 in 24 hours and 89 in 48 hours). The migration ability of endometrial stromal cells in the combined group was weaker than that of the other four groups. Danbie capsule combined with progesterone serum had a tendency to inhibit cell migration, such as Figure 3 , 4 shown.

[0093] Example 5 Effect of Danbie Capsule on Cell Proliferation

[0094] 1. CCK-8 method to detect cell proliferation

[0095] hESC human endometrial stromal cells were collected at logarithmic stage at 1×10 5 The cells were inoculated into 96-well culture plates at a density of 100 μL per well and incubated at 37°C with 5% CO 2 Incubate for 24 hours. Replace with a culture medium containing 10% serum, including a cell control group (10v / v% fetal bovine serum), a saline control group (10v / v% saline intervention group rat serum), a progesterone group (10v / v% dydrogesterone intervention group rat serum), a Danbie capsule group (10v / v% Danbie capsule intervention group rat serum), a combined group (10v / v% combined group rat serum), and a culture medium control group (no cells inoculated). Continue to culture for 6 hours (6h group) and 48 hours (48h group). After 6 / 48 hours, add 10μL of CCK-8 reagent to each well, incubate at 37°C for 1 hour, measure the absorbance (OD) value at 450nm with an enzyme reader, and calculate the cell inhibition rate. (Inhibition rate = [(control group - experimental group) / (control group - blank group)].

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

[0097] There were no significant differences in the OD values ​​and cell inhibition rates of each group in the short term (6 hours) after intervention, but after 48 hours of intervention, the groups showed different changes. The OD value of the combined group was the lowest (P < 0.05), followed by the Danbie capsule group. The progesterone group did not show a significant inhibitory effect on the cells. The inhibition rates of the 6-hour combined group, Danbie capsule group, progesterone group and saline group were 18.25%, 23.54%, 21.69% and 18.52%, respectively. The inhibition rates of the 48-hour combined group, Danbie capsule group, progesterone group and saline group were 79.61%, 32.04%, -9.11% and 13.59%, respectively. Figure 5 The combined group and Danbie capsule group showed more obvious inhibitory effects when intervening cells for a long time.

[0098] Example 6 Effect of Danbie Capsule on Cell Apoptosis

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

[0100] Cell culture method: Cells from the control group (10 v / v% fetal bovine serum) and Danbie capsule group (10 v / v% rat serum from Danbie capsule intervention group) were collected and washed twice with PBS. The cells were centrifuged at 1000 rpm for 5 min to precipitate the cells. The cells were resuspended in 1× Binding Buffer to a cell concentration of 1×10 6 / mL; 100μL of the corresponding cell suspension was placed in a labeled sterile flow tube, 5μL Annexin V-FITC was added, and 10μL PI was immediately added after incubation at room temperature in the dark for 10min, and finally 400ul of PBS was added, and the tube was immediately placed in a flow cytometer for detection. We used the Annexin-V and PI double staining method, and apoptotic cells and necrotic cells can be stained with Annexin-V and PI. Early apoptotic cells are located in the lower right quadrant, and late apoptotic cells are located in the upper right quadrant, so the sum of the apoptotic rates in the lower right quadrant and the upper right quadrant is the total cell apoptosis rate.

[0101] 2. Results

[0102] The total apoptosis rate of the cell control group was 18.68%, and the total apoptosis rate of the Danbie capsule group was 23.14%. The serum containing Danbie capsule has a tendency to induce apoptosis of endometrial stromal cells. Figure 6 shown.

Claims

1. Application of Danbie Capsule combined with progestin in the preparation of products for the treatment of endometriosis.

2. The use according to claim 1, characterized in that: The products include pharmaceuticals; Preferably, the dosage form of the drug includes at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

3. A composition, characterized in that: The composition comprises Danbie capsule and progestogen.

4. A drug, characterized in that: The medicine comprises the composition according to claim 3.

5. The drug according to claim 4, characterized in that: The drug includes pharmaceutically acceptable excipients; Preferably, the auxiliary materials include at least one of a carrier, an excipient, a stabilizer, a filler, a binder, a lubricant, a coating material, and a flavoring agent.

6. A drug-containing serum, characterized in that: The drug-containing serum is the serum after the animal metabolizes at least one of 1) to 2); 1) Danbie Capsule; 2) The composition according to claim 3 and / or the medicament according to any one of claims 4 to 5.

7. The drug-containing serum according to claim 6, characterized in that: The drug-containing serum includes quercetin and / or luteolin.

8. The drug-containing serum according to claim 7, characterized in that: The animal includes at least one of mice, cats, dogs, rabbits, monkeys and humans.

9. The method for preparing the drug-containing serum according to any one of claims 6 to 8, comprising the following steps: Administering at least one of 1) to 2) to an animal, and collecting serum; 1) Danbie Capsule; 2) The composition according to claim 3 and / or the drug according to any one of claims 4 to 5; Preferably, the administration comprises at least one of oral administration and injection.

10. Use of the drug-containing serum according to any one of claims 6 to 8 in the preparation of a product for treating endometriosis.

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