Pharmaceutical composition for treating endometriosis

Abstract

The invention discloses a medicine composition for treating endometriosis. The pharmaceutical composition for treating the endometriosis, disclosed by the invention, is prepared from the following raw materials: curcumin and epigallocatechin gallate co-delivery liposome and pterostilbene nano-structure liposome. The curcumin and the EGCG are prepared into the co-delivery liposome, so that the solubility of the curcumin in water can be remarkably improved; meanwhile, the co-delivery liposome can protect the two from being influenced by light, oxygen, temperature and pH value, and the chemical stability, the antioxidant activity and the drug effect of the liposome are improved. According to the pterostilbene nanostructure liposome prepared by the invention, the water solubility of pterostilbene is improved, the in-vitro drug release of the pterostilbene is promoted, the in-vivo stability of the pterostilbene is improved, the full absorption of drugs is facilitated, and the bioavailability of the pterostilbene is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition for treating endometriosis, belonging to the technical field of medicine. BACKGROUND

[0002] Endometriosis (EMs) is a disease in which endometrial tissue (glands and stroma) appears, grows and infiltrates in the uterine cavity covered with endometrium and other parts of the uterus. It is a common disease in women of childbearing age, with extensive lesions, diverse morphology, high invasiveness and recurrence, and sex hormone dependence.

[0003] The occurrence of EMs is related to sex hormones, immunity, inflammation, and genetics, but its pathogenesis is not clear. There are currently several theories of pathogenesis, of which the retrograde menstruation theory is the dominant theory. The endometrium in the pelvic cavity is implanted and grown through adhesion, invasion, and angiogenesis. The in-situ endometrium theory emphasizes the characteristics of the patient's in-situ endometrium (such as abnormal inflammatory response, high hormone sensitivity, strong invasion / angiogenesis ability) as the determining factor for the onset, making the exfoliated cells easy to ectopic colonization. Other pathogenesis theories, including the body cavity epithelialization theory, vascular and lymphatic metastasis theory, and stem cell theory, also suggest that the occurrence and development of ectopic endometrial tissue are closely related to inflammation levels, hormone stimulation, immune escape, or over-enhanced proliferation and differentiation ability. These theories provide key theoretical directions for the diagnosis and treatment of EMs to varying degrees, and the specific mechanisms are summarized as follows: First, EMs is a hormone-dependent disease, and estrogen can promote its occurrence and development, which has been widely accepted. Multiple estrogen-related genes such as STAR, CYP19A, and 17β-HSD2 are closely related to its development. Progesterone and its different subtypes of receptors (PR-A and PR-B) are also related to the occurrence of EMs. Progesterone resistance is one of the important characteristics of the pathogenesis of endometriosis, and gene expression analysis results in EMs patients suggest that there are different subtypes of progesterone resistance in EMs patients, and their susceptibility to disease is related to FOXO1A, MIG6, CYP26A1, etc.

[0004] Similarly, EMs is also a systemic autoimmune disease, characterized by local inflammation and immune imbalance in endometrial ectopic lesions, inflammation and its secondary fibrosis can cause a series of clinical manifestations including pelvic pain, digestive system, urinary system symptoms and infertility in patients. Studies have found that the levels of inflammatory factors such as TNF-α, IL-4, IL-6, IL-10, and IL-1b are significantly increased in the peritoneal fluid of patients. Among them, the expression of RANTES, IL-1b, MIF, TNF-α, etc. in ectopic lesions also increases. The abnormal expression of these factors may cause local immune imbalance in the lesion, form immune tolerance and promote the growth of ectopic lesions. Macrophages, thymus-dependent lymphocytes and other immune cells and their secreted inflammatory mediators are involved in the pathogenesis.

[0005] In addition, stem cell factors are also important factors affecting the pathogenesis of EMs. Uterine bleeding in the neonatal period leads to ectopic endometrial mesenchymal stem cells and endometrial epithelial progenitor cells, which proliferate and differentiate under the activation of estradiol, promoting the occurrence and development of ectopic endometrial lesions; after puberty, the menstrual period, the stem cells or progenitor cells of the endometrium reverse flow to the pelvis with the shedding of the endometrium, adhere to the pelvic mesothelial cells and grow, which leads to the classic "menstrual blood reflux theory" of EMs. Bone marrow-derived stem cell populations, including bone marrow mesenchymal stem cells, hematopoietic stem cells and endothelial progenitor cells, may be beneficial to endometrial regeneration. Bone marrow-derived stem cell populations can migrate to the endometrium or its ectopic lesions and differentiate into endometrial cells, so bone marrow-derived stem cell populations are also one of the sources of ectopic endometrium.

[0006] The goal of EMs treatment is to reduce and eliminate lesions, reduce and eliminate pain, improve and promote fertility, and reduce and avoid recurrence. Treatment strategies can be roughly divided into surgical resection of lesions and drug therapy. The international guidelines recommend surgical resection of all subtypes of endometrial ectopic tissue based on evidence that laparoscopic treatment improves disease-related pain compared to a diagnostic laparoscopy alone at 6 months, and surgical resection is generally considered the best treatment for ovarian EMs. Drug treatment for EMs-related infertility usually follows two strategies: one is to induce amenorrhea by suppressing follicular growth, thereby inhibiting the development and growth of endometrial ectopic lesions to improve subsequent fertility; the other is to stimulate follicular growth and ovulation. The initial treatment is usually the use of compound oral contraceptives, which can effectively reduce pain and prevent postoperative recurrence. Since there is evidence that steroids play a key role in the pathophysiology of EMs, hormone suppression therapy is routinely prescribed. In general, if a young woman is suspected of having endometriosis before surgery confirms the lesion, treatment can be considered, and if symptoms do not improve or the disease recurs, treatment will also be provided after surgery.

[0007] Although surgery and hormone therapy are widely used in the treatment of EMs, their limitations are increasingly evident. First, surgical resection can remove lesions in the short term, but it cannot block the recurrence of stem cell-driven sources, with a 5-year recurrence rate of up to 40-50%. Second, hormone suppression therapy (such as oral contraceptives and GnRH agonists) relies too much on the progesterone receptor pathway, and its effectiveness is significantly reduced for patients with progesterone resistance. Long-term use can cause osteoporosis, vasomotor symptoms and other side effects. In addition, few existing drugs in the immune / inflammatory adjustment program target the local immune escape microenvironment of the lesion (such as M2 macrophage polarization and abnormal activation of Treg cells). More importantly, there is a lack of multi-mechanism synergistic intervention between existing treatment strategies, and traditional single therapy is difficult to reverse the vicious cycle of hormone imbalance-immune disorder-stem cell activation at the same time. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a pharmaceutical composition for treating endometriosis.

[0009] Technical scheme: To solve the above technical problems, the present application provides a pharmaceutical composition for treating endometriosis, which contains Cur, EGCG and Pte.

[0010] Preferably, the pharmaceutical composition comprises co-delivery liposomes containing curcumin and EGCG, and nanostructured liposomes containing pterostilbene.

[0011] Preferably, the molar ratio of curcumin to EGCG is 1-2:1-2.

[0012] Preferably, the molar ratio of curcumin to EGCG is 1-2:1-2.

[0013] Preferably, the molar ratio of curcumin to EGCG in the co-delivery liposomes is 1:1.

[0014] Preferably, the molar ratio of curcumin to EGCG in the co-delivery liposomes is 1:1.

[0015] Preferably, the concentration ratio of EGCG, pterostilbene and curcumin is 1-2:4-8:5-10.

[0016] Preferably, the concentration ratio of EGCG, pterostilbene and curcumin is 1-2:4-8:5-10.

[0017] Preferably, the concentration ratio of EGCG, pterostilbene and curcumin is 1-2:4-8:5-10. (1) Mix curcumin and EGCG and dissolve in anhydrous ethanol, and completely dissolve; (2) Cholesterol and egg yolk phospholipid are weighed and dissolved in ethyl acetate, which is added to step (1), and after being mixed thoroughly, the organic solvent is removed to form a uniform film, and then a poloxamer aqueous solution is added, and after ultrasonic treatment, the co-delivery liposome containing curcumin and EGCG is obtained.

[0018] (3) The stable suspension system is obtained by adding distilled water for stirring and suspension, and passing through a 0.45 μm microporous filter.

[0019] The molar ratio of the total mass of curcumin and EGCG, cholesterol and egg yolk phospholipid is 1: (1.5-3.5): (1-3).

[0020] Preferably, the molar ratio of curcumin, EGCG, cholesterol and egg yolk phospholipid is 1:1:7:5.

[0021] The amount of added anhydrous ethanol is 25-40 times the total mass of curcumin and EGCG.

[0022] The amount of added ethyl acetate is 20-30 times the total mass of curcumin and EGCG.

[0023] The amount of added poloxamer aqueous solution is 20-45 times the total mass of curcumin and EGCG.

[0024] The volume of added distilled water is equal to the volume of the poloxamer aqueous solution.

[0025] The preparation steps of the nanostructured liposome containing pterostilbene are: (1) Pterostilbene, glyceryl tristearate and peanut oil are weighed and dissolved in ethyl acetate as an organic phase; (2) The poloxamer 188 aqueous solution is used as an aqueous phase, which is added dropwise to the organic phase under magnetic stirring, and then ultrasonic treatment is performed to remove the organic solvent.

[0026] The drug-lipid molar ratio of pterostilbene to glyceryl tristearate and peanut oil is 1:15-1:25.

[0027] The solid-liquid-lipid molar ratio of glyceryl tristearate and peanut oil is 3:1-5:1.

[0028] The amount of added poloxamer 188 aqueous solution is 1-5 times the amount of ethyl acetate.

[0029] Preferably, the drug-lipid molar ratio of pterostilbene to glyceryl tristearate and peanut oil is 1:15, the solid-liquid-lipid molar ratio of glyceryl tristearate and peanut oil is 5:1, and the amount of added poloxamer aqueous solution is 1 times the amount of ethyl acetate in step (1).

[0030] The drug composition further comprises a solvent, which includes but is not limited to pure water or physiological saline.

[0031] The application further provides application of the drug composition in the field of biological medicine.

[0032] Since curcumin is a hydrophobic polyphenol, it has low water solubility, poor stability, high metabolic rate, fast clearance rate and limited oral bioavailability, which limits its further clinical application. EGCG is a catechin monomer extracted from green tea, and is the main active and water-soluble component of green tea. In vitro, the phenolic hydroxyl group of EGCG is easily oxidized to form o-quinone under the influence of light, oxygen, temperature and pH value, and o-quinone is unstable, which causes degradation problems in storage, transportation and processing. In vivo, EGCG has a short retention time in the gastrointestinal tract, a low absorption rate, and is easily degraded by body fluids, and has a low oral bioavailability. Therefore, it is novel and beneficial to develop a delivery system that can simultaneously encapsulate hydrophilic and hydrophobic substances to better exert the synergistic effect of the two.

[0033] Liposomes are microcapsules with lipid bilayer structures, which can encapsulate hydrophilic and hydrophobic drug functional factors, reduce the influence of the encapsulated substances on the external environment, improve the bioavailability, and enable the encapsulated functional factors to more effectively reach the target site.

[0034] The application adopts a thin film ultrasonic method to construct a liposome system for simultaneously encapsulating curcumin and EGCG. The formed inclusion compound can significantly improve the solubility and stability of the two, and improve the therapeutic effect on endometriosis. By forming an inclusion compound with cholesterol and egg yolk phospholipids, the solubility of curcumin in water can be significantly improved; at the same time, the formation of the inclusion compound can also protect curcumin and EGCG from light, heat and oxygen, and improve their chemical stability, antioxidant activity and drug efficacy.

[0035] The solubility of pterostilbene in water is only 0.03 mg / mL, which is much lower than that of curcumin and EGCG, and seriously affects the dissolution thereof, and in the body, pterostilbene is easily affected by various enzymes, and the oral absorption bioavailability is only 11.9%, which is not conducive to clinical application. Therefore, the application separately prepares a pterostilbene nanostructured lipid carrier. Since liquid lipid carriers are introduced, compared with solid lipid nanoparticles, the nanostructured lipid carrier has higher encapsulation efficiency, drug loading capacity and stability. After being prepared into a nanostructured lipid carrier, the water solubility of pterostilbene is improved, the in vitro drug release is promoted, the pterostilbene can stably exist in the gastrointestinal fluid for a certain period of time, and the influence of various enzymes in the gastrointestinal tract on the stability of the drug is reduced, which is conducive to the full absorption of the drug and improves the bioavailability thereof. At the same time, the surfactant poloxamer 188 in the prescription also has a positive effect on the in vivo absorption of the drug.

[0036] Epigallocatechin gallate has multiple effects such as inhibiting cell migration and invasion, anti-cancer, anti-oxidation and anti-inflammation. In the present application, the effect of inhibiting cell migration and invasion is mainly played.

[0037] Pterostilbene has multiple effects such as inhibiting cell proliferation, anti-oxidation, anti-inflammation, anti-cancer, anti-diabetes and anti-obesity. In the present application, the effect of inhibiting cell proliferation is mainly played.

[0038] Curcumin has multiple effects such as anti-inflammation, anti-oxidation, anti-proliferation and anti-angiogenesis. In the present application, the effects of anti-angiogenesis, reducing endometrial cell estrogen sensitivity and reducing estrogen synthesis are mainly played.

[0039] The present application discloses that the formula (EPC05) composed of the three components of epigallocatechin gallate, pterostilbene and curcumin in a specific ratio has effects in treating endometriosis in three different directions of invasion and migration, cell proliferation, estrogen synthesis and sensitivity, and the process optimization significantly improves the bioavailability and absorption efficiency. The research is more detailed, accurate and comprehensive.

[0040] The present application firstly studies the in-vitro treatment effect and mechanism of epigallocatechin gallate on human endometriosis cells. In the aspect of treating endometriosis with pterostilbene, the existing related research is only carried out in a cell model, and the present application firstly studies the treatment effect of pterostilbene on a mouse model of endometriosis. In the aspect of treating endometriosis with curcumin, the existing related research only mentions that curcumin extract can reduce estradiol to inhibit endometriosis, and there is no public data to disclose the relationship among curcumin, estrogen receptor and endometriosis. The present application firstly proves that curcumin reduces the sensitivity of endometriosis cells by inhibiting the level of estrogen receptor β, thereby inhibiting endometriosis.

[0041] To break through the above limitations, the present application innovatively designs a synergistic formula (EPC05) of curcumin (Cur), epigallocatechin gallate (EGCG) and pterostilbene (Pte), which aims to target inhibit estrogen synthesis (CYP19A1 / STAR) and regulate estrogen sensitivity of ectopic cells by curcumin, target inhibit cell migration and invasion ability by EGCG, and target inhibit cell proliferation ability by pterostilbene. On this basis, the best effective ratio and dose are further explored by means of animal experiments, and the bioavailability and absorption efficiency are further improved by means of process, so as to provide a new treatment strategy with high efficiency and low toxicity for EMs.

[0042] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: 1. This invention significantly improves the solubility of curcumin in water by preparing co-delivery liposomes of curcumin and EGCG. At the same time, the co-delivery liposomes can also protect both from the effects of light, oxygen, temperature and pH, thereby improving their chemical stability and antioxidant activity.

[0043] 2. The nanostructured liposomes prepared by this invention improve the water solubility of pterostilbene, promote its in vitro drug release, increase its in vivo stability, facilitate the full absorption of the drug, improve its bioavailability, and thus enhance its efficacy.

[0044] 3. This invention is the first in China and abroad to combine curcumin, pterostilbene, and EGCG in a specific ratio to improve EMs symptoms in mice with ectopic endometrial cells in vitro and allogeneic endometrial transplantation. It provides a rigorous and comprehensive demonstration of the mechanism by which this drug combination improves key symptoms of EMs. Attached Figure Description

[0045] Figure 1 The effects of different concentrations of EGCG, Cur, and Pte on the viability of 12Z cells were investigated as follows: A. Effect of different concentrations of EGCG on cell viability; B. IC50 of EGCG after 1 day of administration; C. Effect of different concentrations of Pte on cell viability; D. IC50 of Pte after 1 day of administration; E. Effect of different concentrations of Cur on cell viability; F. IC50 of Cur after 1 day of administration. Figure 2 The effects of EGCG on cell migration and invasion: A. Effects of different concentrations of EGCG on cell migration; B. Statistical analysis of cell migration rate results; C. Effects of different concentrations of EGCG on cell invasion; D. Statistical analysis of cell invasion rate results; Figure 3 The effect of Cur on proteins in the estrogen synthesis pathway; Figure 4 The effect of Pte on cell proliferation; Figure 5 The effect of different component ratios of EPC05 on cell proliferation; Figure 6 The effect of different component ratios of EPC05 on cell migration; Figure 7 The effect of different component ratios of EPC05 on cell invasion; Figure 8 The effects of different component ratios of EPC05 on estrogen sensitivity and angiogenic proteins; Figure 9 The effects of different component ratios of EPC05 on proteins in estrogen synthesis and conversion pathways; Figure 10The effect of EPC05 on EMs mice treatment (n=3): A, the modeling of EMs mice; B, the analysis of ectopic lesion infection level; C, the morphological results of lesion section; D, the detection results of CD206 in lesion section; Figure 11 The in vitro drug release curve of pterostilbene (n=3). DETAILED DESCRIPTION

[0046] The technical solutions of the present application are further described below in combination with the drawings.

[0047] Table 1 Analysis instrument

[0048] 2、Reagents: Normal endometrial cells (HEE, Cat. No. IM-H405) were purchased from Xiamen Yimeng Biotechnology Co., Ltd.; Ectopic endometrial cells (12Z, Cat. No. CL-0976) were purchased from Wuhan Punuo Life Science Co., Ltd.; Curcumin (Cur, Cat. No. HY-N0005), Epigallocatechin gallate (EGCG, Cat. No. HY-13653) were purchased from Shanghai Haoyuan Biotechnology Co., Ltd.; Pterostilbene (Pte, Cat. No. Z-014-210914) was purchased from Shanghai Shidand Standard Technology Service Co., Ltd.; Capric and caprylic acid triglyceride (Cat. No. 20201003) was purchased from Nanjing Dongfangzhuzhu Industry and Trade Co., Ltd.; Tristearin (Cat. No. 20200509) was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; Sephadex gel (Cat. No. Sephadex-G50) was purchased from GE Company of the United States; CCK8 detection kit (Cat. No. C0039) was purchased from Biyun Tian Biotechnology Co., Ltd.; Transwell chamber (Cat. No. 353097) was purchased from Corning-Costar Technology Co., Ltd.; PBS (Cat. No. G4202), crystal violet (Cat. No. G1014), 4% paraformaldehyde (Cat. No. G1101), electrophoresis buffer (Cat. No. G2142), transfer buffer (Cat. No. G2028), hematoxylin (Cat. No. G1004), eosin (Cat. No. G1001) were purchased from Saivier Biotechnology Co., Ltd.; Trypsin (Cat. No. C0201) was purchased from Biyun Tian Biotechnology Co., Ltd.; Complete medium (Cat. No. CM-0976), serum-free medium (Cat. No. PM150312) were purchased from Nanjing Wingsnow Biotechnology Co., Ltd.; Lysis solution (Cat. No. P0013), BCA kit (Cat. No. P0012) were purchased from Biyun Tian Biotechnology Co., Ltd.; ECL luminous liquid (Cat. No. 180-501) was purchased from Shanghai Tian Neng Life Science Co., Ltd.; Xylene (Cat. No. A530011), ethanol (Cat. No. A500737) were purchased from Shengong Biotech Co., Ltd.; Neutral gum (Cat. No. 10004160) was purchased from Shanghai Hushi Co., Ltd.; VEGFA (Cat. No. 66828-1-Ig) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; ER-β (Cat. No. ab187291) was purchased from Abways Trading Co., Ltd.; CYP19A1 (Cat. No. 16554-1-AP), STAR (Cat. No. 12225-1-AP), GAPDH (Cat. No. 60004-1-Ig), CD206 (Cat. No. 18704-1-AP), CoraLite488-conjugated Goat Anti-Rabbit IgG (H+L) (Cat. No. SA00013-2) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; Horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Cat. No. A0216) was purchased from Biyun Tian Biotechnology Co., Ltd. of Shanghai. Example 1: Optimal effective concentration of each component

[0049] EGCG selection 5 μM, 10 μM, 20 μM, 40 μM, 80 μM five gradient alone administration, pterostilbene selection 10 μM, 20 μM, 40 μM, 80 μM, 160 μM five gradient alone administration, curcumin selection 5 μM, 10 μM, 20 μM, 40 μM, 80 μM five gradient alone administration, using CCK8 detection cell viability.

[0050] 1、CCK8 detection: (1) in 96 well plates inoculated with HEE or 12Z cell suspension (100 μL / well), 5000 cells per well, the culture plate was placed in a 37℃, 5% CO2 incubator for 24 h pre-culture; (2) to the culture plate was added to the above different drugs to be tested; (3) the culture plate was placed in an incubator; (4) to each well was added 10 μL of CCK8 solution; (5) the culture plate was placed in an incubator for 1-4 h; (6) the absorbance at 450 nm was measured by microplate reader.

[0051] The results of the implementation: as Figure 1 A shown, 12Z cells alone after adding epigallocatechin gallate, the first day, 0-40 μM cell viability was not significantly decreased, 80 μM cell viability decreased significantly, the second day, 0-20 μM cell viability was not significantly decreased, 40-80 μM cell viability decreased significantly, the half inhibitory concentration of cells was 115.1 μM ( Figure 1 B); alone after adding pterostilbene, the first day, 0-40 μM cell viability was not significantly decreased, 80-160 μM cell viability decreased significantly, the second day, 0-40 μM cell viability was not significantly decreased, 80-160 μM cell viability decreased significantly ( Figure 1 C), the half inhibitory concentration of cells was 100.9 μM ( Figure 1 D); alone after adding curcumin, the first day, 0-20 μM cell viability was not significantly decreased, 40-80 μM cell viability decreased significantly, the second day, 0-10 μM cell viability was not significantly decreased, 20-80 μM cell viability decreased significantly ( Figure 1 E), the half inhibitory concentration of cells was 50 μM ( Figure 1 F). Therefore, we will control the final concentration of epigallocatechin gallate within 20 μM, select 5-15 μM for experiment; the concentration of pterostilbene control within 80 μM, select 10-40 μM for experiment; the concentration of curcumin control within 20 μM, select 2.5-15 μM for experiment, to ensure the safety of the drug.

[0052] As Figure 4 shown, to detect the addition of pterostilbene, 12Z and HEE cell proliferation, 12Z alone pterostilbene, found in HEE cell proliferation ability is significantly weaker than 12Z cells, and in 80 μM and 160 μM concentration Pte effect, 12Z cell proliferation was significantly inhibited, to play pterostilbene anti-proliferative effect, pterostilbene drug concentration increased to 40-80 μM.

[0053] 2, the change of cell migration rate analysis (wound healing assay method): (1) line: first with a needle in the back of the 6-hole plate, evenly cross line, about 2-3 cm a, across the hole, each hole 2 lines; (2) plate: take a full dish cell in example 1, trypsin digestion into a single cell suspension, inoculated in 6-hole culture plate; inoculation principle is that the fusion rate reaches 100% after overnight, the final total amount of medium per hole is 2 mL; (3) scratch: the next day with 200 microliter gun head, parallel or perpendicular to the back of the cross line scratch, gun head must be vertical, can not be inclined; (4) washing: stick to the wall into PBS, shake the cell 1 time, remove the cells under the scratch; (5) under the microscope to take pictures, ensure that the scratch is in the middle and vertical, pay attention to the background is consistent, can be sampled at 0, 24, 48 h time point, take pictures, each group 8 photos.

[0054] As Figure 2 A- Figure 2 B shown, 12Z cell migration rate is significantly higher than HEE cells, 20 μM of EGCG concentration after 1 day, the cell migration rate decreased significantly, 5, 10 μM of EGCG concentration no significant difference; 2 days later, 10 and 20 μM of EGCG concentration cell migration rate decreased significantly, 5 μM of EGCG concentration no significant difference. Proved in 10-20 μM concentration, EGCG can inhibit cell migration.

[0055] 3, the change of cell invasion ability analysis (Transwell method): (1) preparation of cell suspension: the cells in example 1 to be tested were cultured to the logarithmic growth phase, digested with 0.25% trypsin for 2-3 min, centrifuged the cells at 1000 rpm for 3 min, discarded the supernatant after centrifugation, washed the cells with PBS for 1-2 times, then suspended the cells with serum-free medium; (2) cell inoculation: add 600 μL complete medium to the lower chamber of the 24-well plate, then place the Transwell chamber in the 24-well plate with forceps, take 1 × 10 5Cell suspension of 2000 cells / mL, add serum-free medium to 200 μL, add to the upper chamber, and finally put into the incubator for 48 h; (3) Cell fixation: The next day, carefully remove the chamber with tweezers, wash the chamber with PBS for 3 times, put the chamber into a new well, add 400 μL of 4% paraformaldehyde, and fix for 20-30 min after putting the chamber; (4) Cell staining: Remove the chamber, wash the chamber with PBS for 3 times, put the chamber into a new well, add 400 μL of crystal violet staining solution, and stain for 15-30 min. Remove the crystal violet that is not combined with the cells, and gently wipe the upper side of the chamber with a cotton swab to remove the dye that is not specifically combined with the upper surface of the chamber for subsequent microscopic counting; (5) Cell counting: Remove the chamber, gently soak and wash with water several times, carefully wipe off the cells on the surface of the membrane at the bottom of the upper chamber with a wet cotton swab, put the chamber into a new well, add a little hot water, take photos under a microscope, count the purple positive cells, and count the results.

[0056] Implementation results: As shown in Figure 2 C- Figure 2 D, the invasion rate of 12Z as ectopic endometrial cells is significantly higher than that of HEE normal endometrial cells in the natural state, and the cell migration rate is significantly reduced at an EGCG concentration of 5-20 μM. It is proved that EGCG can inhibit cell invasion at a concentration of 5-20 μM.

[0057] Therefore, in order to make EGCG play a role in inhibiting cell migration and invasion in EPC05, the concentration of EGCG selected in the follow-up experiment is 10-20 μM.

[0058] 4. Analysis of changes in related proteins (Western blotting): (1) Protein extraction: Take the treated cells, wash with PBS once, add lysis buffer, scrape the cells with a spatula, centrifuge at 4°C, and collect the supernatant into a 1.5 ml EP tube; (2) Protein concentration determination: Use the BCA kit, prepare the working solution, add the sample, add 200 μl of working solution to each well, incubate at 37°C for 30 min, detect the absorbance with a microplate reader, calculate the protein concentration, and denature the protein at 95°C for 5 min; (3) Western Blot: prepare electrophoresis buffer, transfer buffer, gel, calculate the loading amount according to the sample concentration (30 μg per well); MARKER (3 μL), 85 V constant voltage electrophoresis until the MARKER is completely separated, about 30 min; change to 125 V constant voltage electrophoresis until the lowermost band of the MARKER approaches the bottom of the gel, about 1 h. PVDF membrane (6x9 cm): soak in methanol for 30 s (wake up the membrane), wash with dd H2O for 3 times, cut the corner as a mark, soak in the transfer buffer, place according to the order from bottom to top: white clip-sponge-filter paper-PVDF membrane-gel-filter paper-sponge-black clip, exhaust air; 300 mA transfer for 100 min, 5% skim milk blocking at room temperature for 2 h, cut the required band, wash the membrane with TBST for 3 times, each for 5 min; prepare the primary antibody according to the instructions, incubate overnight at 4°C; wash the membrane with TBST for 3 times, each for 5 min; prepare the secondary antibody according to the instructions, incubate at room temperature for 2 h; wash the membrane with TBST for 3 times, each for 5 min; (4) Expose the developer to the film, and use the gel imaging system to expose and save the image.

[0059] As shown in Figure 3 FIG. 2, the ER-β and VEGFA protein levels of 12Z cells are higher than those of HEE cells, but the ER-β and VEGFA protein levels of 12Z cells decrease after adding 5-15 μM curcumin. Therefore, 5-15 μM curcumin is selected for subsequent experiments.

[0060] Example 2: Efficacy evaluation of EPC05 at different proportions Epigallocatechin gallate, pterostilbene and curcumin are prepared in different proportions and grouped as follows: group 1: 20 μM, 80 μM, 10 μM; group 2: 10 μM, 80 μM, 10 μM; group 3: 20 μM, 40 μM, 10 μM; group 4: 10 μM, 40 μM, 10 μM; group 5: 20 μM, 80 μM, 5 μM; group 6: 20 μM, 80 μM, 15 μM. The specific proportions are shown in Table 2.

[0061] Table 2

[0062] In Table 2, HEE is normal endometrial cells, and 12Z is ectopic endometrial cells.

[0063] Cell culture method: the cells are cultured in a 37°C, 5% CO2 incubator, and the drug is administered when the cell fusion density is 80%, and the analysis experiment is performed 24 hours after administration.

[0064] The results are shown in Figure 5As shown, after adding different EPC05 proportions, the cell viability of groups 2 and 6 decreased significantly on the first day, and the cell viability of each group was significantly different on the second day. To ensure drug safety, groups 1, 2 and 6 were excluded, and groups 3, 4 and 5 with the least drug toxicity were selected for subsequent experimental research.

[0065] The effect of different component proportions on cell migration was determined according to the method of Example 1. The results are shown in Figure 6 As shown, the cell migration rate of the EPC05-4 administration group was significantly slower than that of the other groups, proving that the fourth group of drug concentration ratios had the most obvious inhibitory effect on cell migration.

[0066] The effect of different component proportions on cell invasion was determined according to the method of Example 1. As shown in Figure 7 As shown, the cell invasion rate of the EPC05-4 administration group was significantly slower than that of the other groups, proving that the fourth group of drug concentration ratios had the most obvious inhibitory effect on cell invasion.

[0067] The effect of different component proportions on estrogen sensitivity and angiogenic protein was determined according to the method of Example 1. As shown in Figure 8 As shown, the ER-β and VEGFA protein levels of the EPC05-4 administration group were significantly lower than those of the other groups. ER-β is an estrogen receptor closely related to estrogen action, and VEGFA is an angiogenic-related protein, proving that the fourth group of drug concentration ratios had the most obvious inhibitory effect on estrogen action and angiogenesis.

[0068] The effect of different component proportions on estrogen synthesis and transformation-related pathway proteins was determined according to the method of Example 1. As shown in Figure 9 As shown, the STAR and CYP19A1 protein levels of the EPC05-4 administration group were significantly lower than those of the other groups. STAR protein regulates estrogen synthesis, and CYP19A1 protein can convert androgens into estrogens, proving that the fourth group of drug concentration ratios had the most obvious inhibitory effect on estrogen synthesis and transformation.

[0069] Example 3 EPC05 efficacy evaluation (allogeneic transplantation animal experiment) Animal experiment modeling: SPF grade BALB / C strain 7~8 week old female mice were intraperitoneally injected with allogeneic mouse uterine fragments (1 mg / mouse) Figure 10 A).

[0070] 1. Animal experiment scheme:

[0071] ​​​​​​The mice with successful modeling were divided into 3 groups: control group, model group (MOD) and EPC05 group (epicatechin gallate 10 μM, pterostilbene 40 μM, curcumin 10 μM), 6 in each group. Among them, the EPC05 group was weighed at a fixed time every day and administered intragastrically once, for 1 week of continuous intragastric administration. The EPC05 group was administered intragastrically with epicatechin gallate 20 mg / kg + pterostilbene 80 mg / kg + curcumin 20 mg / kg daily. The other groups were weighed at a fixed time every day and administered intragastrically with the same amount of normal saline. The samples were taken on the 8th day after administration, and the occurrence site and size of ectopic lesions were recorded, the tissue was photographed, and was fixed in 4% PFA.

[0072] The implementation results are as shown in Figure 10 B, the number of ectopic lesions in the EPC05 group was significantly reduced, and the volume of ectopic lesions was also significantly smaller than that in the model group, and the EMs symptoms of the mice were treated.

[0073] Observation of histological changes of endometrial lesions (HE staining method): (1) The ectopic endometrial lesion tissue section was placed in a 65°C oven for 2 hours of dewaxing; (2) The section was immersed in 37°C xylene twice for 15 min each time, and the section was immersed in 100% ethanol twice for 2 min each time, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and water for 10 min; (3) Hematoxylin staining for 5 min; water flushing for 10 min; (4) 1% hydrochloric acid aqueous solution differentiation for 30 s, water flushing for 10 min; (5) Eosin staining for 2.5 min; (6) The section was immersed in 70% ethanol for 2 min; 80% ethanol for 2 min; 90% ethanol for 2 min; 100% ethanol twice for 2 min each time; xylene, incubated twice for 15 min each time; neutral resin mounting.

[0074] The implementation results are as shown in Figure 10 C, the ectopic lesions of the model group and the EPC05 group can be observed to have one or more cystic structures, and the structure of the ectopic lesions is similar to that of the in-situ endometrial structure. The epithelial cell layer grows in a ring shape with columnar epithelial cells; a large number of blood vessels can be seen in the interstitial cell layer, but the number of glands is relatively small, and even disappears, proving that the abdominal injection modeling is successful.

[0075] 3. Detection of changes in M2 macrophage marker CD206 (immunofluorescence method) Operation example:

[0076] (1) The ectopic endometrial lesion tissue section was placed in a 65°C oven for 2 hours of dewaxing; (2) Slice immersion in 37℃ xylene twice, 15 min each time, slice immersion in 100% ethanol twice, 2 min each time, 90% ethanol 2 min, 80% ethanol 2 min, 70% ethanol 2 min, 10 min of water flow; (3) 0.5% Triton, 37℃ 15 min, PBS wash 3 times, 5 min each time; (4) 2.5% BSA room temperature blocking 2h; (5) Primary antibody 4℃ incubation overnight, PBS wash 3 times, 5 min each time; (6) Secondary antibody room temperature incubation for 2h, PBS wash 3 times, 5 min each time; (7) Use DAPI to mount the slice.

[0077] Experimental results: as shown in Figure 10 D, the CD206 fluorescence intensity of the control group (NC) is weak, and the protein expression level is low; the CD206 fluorescence intensity of the model group is strong, and the protein expression level is high; the CD206 fluorescence intensity of the EPC05 group is weak, and the protein expression level is low. CD206 is a marker of M2 macrophages, which has high specificity and is closely related to cell proliferation and metastasis. The results show that the endometrial tissue in normal mice has weak proliferation and metastasis ability, while the endometrial tissue in EMs mice has strong proliferation and metastasis ability, which is weakened after taking EPC05.

[0078] Example 4 Preparation of co-delivery liposomes of curcumin and EGCG by thin film ultrasonic method Precisely weigh curcumin 0.74g (0.002mol) and EGCG 0.46g (0.001mol), mix them together and dissolve in 25ml of anhydrous ethanol, ultrasonic until completely dissolved. Weigh cholesterol 2.3g and egg yolk phospholipid 1.55g, dissolve in 15ml of ethyl acetate, mix well with the former, then place in a 40℃ constant temperature water bath and rotary evaporate to remove the organic solvent, so that the film forming material forms a uniform film on the bottle wall. Add 1.0% poloxamer 188 aqueous solution 20ml, ultrasonic treatment for 40 min, then get the co-delivery liposomes of curcumin and EGCG, place in the refrigerator at 4℃ for solidification, save for later use. When used, add distilled water to 20ml, pass through 0.45μm microporous filter membrane, get stable suspension system.

[0079] Example 5 Preparation of co-delivery liposomes of curcumin and EGCG by thin film ultrasonic method Precisely weigh curcumin 0.37 g (0.001 mol) and EGCG 0.46 g (0.001 mol), mix them and dissolve in 30 ml of anhydrous ethanol, and ultrasonically dissolve until completely dissolved. Weigh cholesterol 1.55 g and egg yolk phospholipid 0.77 g, dissolve in 20 ml of ethyl acetate, mix well with the former, and then place in a 40°C constant temperature water bath to remove the organic solvent by rotary evaporation, so that the film-forming material forms a uniform thin film on the bottle wall. Then add 30 ml of 1.0% poloxamer 188 aqueous solution, ultrasonically treat for 40 min, and then obtain the co-delivery liposome of curcumin and EGCG. Place in a refrigerator at 4°C to solidify, and store for later use. When used, add distilled water to 30 mL, and pass through a 0.45 μm microporous filter to obtain a stable suspension system.

[0080] Example 6 Preparation of co-delivery liposome of curcumin and EGCG by thin film ultrasonic method

[0081] Precisely weigh curcumin 0.37 g (0.001 mol) and EGCG 0.46 g (0.001 mol), mix them and dissolve in 30 ml of anhydrous ethanol, and ultrasonically dissolve until completely dissolved. Weigh cholesterol 1.55 g and egg yolk phospholipid 0.77 g, dissolve in 20 ml of ethyl acetate, mix well with the former, and then place in a 40°C constant temperature water bath to remove the organic solvent by rotary evaporation, so that the film-forming material forms a uniform thin film on the bottle wall. Then add 30 ml of 1.0% poloxamer 188 aqueous solution, ultrasonically treat for 40 min, and then obtain the co-delivery liposome of curcumin and EGCG. Place in a refrigerator at 4°C to solidify, and store for later use. When used, add distilled water to 30 mL, and pass through a 0.45 μm microporous filter to obtain a stable suspension system.

[0082] Example 7 Preparation of pterostilbene nanostructured liposome by solvent evaporation method

[0083] Weigh pterostilbene raw material 0.2 g, peanut oil 1.25 g, and glyceryl tristearate 3.75 g, dissolve in 20 mL of ethyl acetate as the organic phase; weigh poloxamer 188 to make an aqueous solution of 1.0% concentration of 100 mL as the water phase (40°C), and add the water phase to the organic phase under magnetic stirring, ultrasonically treat (power 250 W, work for 2 s every 2 s interval) for 20 min, and then remove the organic solvent under vacuum on a 40°C rotary evaporator, concentrate to 20 mL, and then place in a refrigerator to solidify. When used, add distilled water to 60 mL, and pass through a 0.45 μm microporous filter to obtain a stable suspension system.

[0084] Example 8 Preparation of pterostilbene nanostructured liposome by solvent evaporation method

[0085] Take 0.81g of the raw material of pterostilbene, 3.24g of peanut oil and 12.96g of glycerol tri-stearate, dissolve them in 40mL of ethyl acetate as the organic phase; take an appropriate amount of poloxamer 188 to make a 1.1% concentration of aqueous solution 100mL as the water phase (40℃), drop the water phase into the organic phase under magnetic stirring, after dropping, ultrasonic treatment (power 250W, work for 2s every 2s interval) for 20min, remove the organic solvent under vacuum on a rotary evaporator at 40℃, concentrate to 40mL, solidify in a refrigerator, add distilled water to 60mL when needed, pass through a 0.45μm microporous filter, and a stable suspension system is obtained.

[0086] Example 9 Preparation of pterostilbene nanostructured liposome by solvent evaporation method

[0087] Take 0.81g of the raw material of pterostilbene, 3.24g of peanut oil and 12.96g of glycerol tri-stearate, dissolve them in 40mL of ethyl acetate as the organic phase; take an appropriate amount of poloxamer 188 to make a 1.1% concentration of aqueous solution 100mL as the water phase (40℃), drop the water phase into the organic phase under magnetic stirring, after dropping, ultrasonic treatment (power 250W, work for 2s every 2s interval) for 20min, remove the organic solvent under vacuum on a rotary evaporator at 40℃, concentrate to 40mL, solidify in a refrigerator, add distilled water to 60mL when needed, pass through a 0.45μm microporous filter, and a stable suspension system is obtained.

[0088] Example 10

[0089] A composition for treating endometriosis, comprising the following raw materials: curcumin and EGCG co-delivery liposome, pterostilbene nanostructured lipid carrier, pure water; The molar ratio of curcumin and EGCG in the curcumin and EGCG co-delivery liposome is 1:1; The amount of pterostilbene in the pterostilbene nanostructured lipid carrier is 4 times that of curcumin (molar ratio).

[0090] The curcumin and EGCG co-delivery liposome used is obtained from Example 5; the pterostilbene nanostructured liposome used is obtained from Example 9.

[0091] Example 11

[0092] A composition for treating endometriosis, comprising the following raw materials: curcumin and EGCG co-delivery liposome, pterostilbene nanostructured lipid carrier, pure water; The molar ratio of curcumin and EGCG in the curcumin and EGCG co-delivery liposome is 2:3; The amount of pterostilbene in the pterostilbene nanostructured lipid carrier is 2 times that of curcumin (molar ratio).

[0093] The curcumin and EGCG co-delivery liposome used is obtained from Example 5; the pterostilbene nanostructured liposome used is obtained from Example 8.

[0094] Example 12 A composition for treating endometriosis, comprising the following raw materials: curcumin and EGCG co-delivery liposome, pterostilbene nanostructured liposome, pure water; The molar ratio of curcumin and EGCG in the curcumin and EGCG co-delivery liposome is 1:4; The amount of pterostilbene in the pterostilbene nanostructured liposome is 1 times that of curcumin.

[0095] The curcumin and EGCG co-delivery liposome used is obtained from Example 4; the pterostilbene nanostructured liposome used is obtained from Example 7.

[0096] Example 13 Performance test 1. The curcumin and EGCG co-delivery liposome prepared in Examples 4-6 is subjected to the following tests: (1) Curcumin and EGCG embedding rate and drug loading test: HPLC method is used to determine the content of curcumin and EGCG in the compound liposome.

[0097] Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-C 18 (250 mm x 4.6 mm i.d., 5 μm) and Agilent Zorbax SB-C 18 guard column (12.5 mm x 4.6 mm, i.d., 5 μm). Mobile phase: acetonitrile-0.3% glacial acetic acid aqueous solution (80:20), column temperature: 30°C; flow rate: 1.0 ml / min, EGCG content is detected at 280 nm wavelength, curcumin content is detected at 425 nm wavelength, sample injection volume is 10 μL.

[0098] Respectively, 0.5 ml of each liposome suspension system prepared in Examples 4-6 is precisely taken and subjected to dextran gel G50 column, eluted with 50 ml of distilled water in a 100 ml volumetric flask, added with methanol to constant volume, to obtain sample 1. Another 0.5 ml of each liposome suspension system in Examples 4-6 containing equal amount of curcumin and EGCG is taken, dissolved with methanol and constant volume in a 100 ml volumetric flask, to obtain sample 2. After filtration, 10 μL of each sample is injected, the total content of curcumin and EGCG in sample 1 and sample 2 is calculated by external standard method, and is respectively recorded as W 样1 and W 样2 , the embedding rate is calculated according to the following formula: Embedding rate = W 样1 / W 样2 x 100% The drug loading amount was calculated according to the following formula: Drug loading amount = W 样1 / (W 蛋黄磷脂 +W 胆固醇 ) x 100%.

[0099] Wherein, W 蛋黄磷脂 and W 胆固醇 respectively refer to the mass of egg yolk phospholipid and cholesterol.

[0100] The specific test results are shown in Table 3.

[0101] Table 3. Co-delivery liposome property parameters of curcumin and EGCG

[0102] (2) Stability: The liposome suspension system in Example 6 was stored in a sealed container at 4°C for 15 days, and samples were taken at 0, 1, 3, 7, 11, and 15 days, respectively, to measure the particle size, polydispersity index, and Zeta potential at each time point, with 3 parallel measurements. The results are shown in Table 4. The change in the liposome was also observed, and it was found that the liposome system changed little over time, and there was no significant change in appearance after storage, indicating good stability.

[0103] Table 4. Storage stability of co-delivery liposomes of curcumin and EGCG

[0104] 2. The following tests were performed on the pterostilbene nanostructured liposomes of Examples 7-9: (1) Pterostilbene entrapment efficiency and drug loading amount test: HPLC method was used to determine the content of pterostilbene in the pterostilbene nanostructured liposomes.

[0105] Chromatographic conditions: Agilent ZORBAX SB-C 18 (250 mm x 4.6 mm i.d., 5 μm) and Agilent Zorbax SB-C 18 guard column (12.5 mm x 4.6 mm, i.d., 5 μm). Mobile phase acetonitrile-water (60:40), volume flow rate 1.0 mL / min; column temperature 30°C; detection wavelength 306 nm.

[0106] Take 1 mL of nanostructured lipid carrier suspension, treat with 10 mL of acetonitrile for 5 min, then dilute to 100 mL, mix well, take 5 mL into a 10 mL volumetric flask, dilute to the mark with the mobile phase, and prepare the test solution to determine the total amount of pterostilbene m 总药量; another 1 mL was taken into an ultrafiltration tube (molecular weight cut-off 8000-12000 Da), placed in a 4 ℃ high-speed centrifuge, centrifuged at 8500 r / min for 10 min, and the filtrate was taken to measure the free amount of tanshinone II A 游离 , the encapsulation efficiency and drug loading were calculated, and the formulas were respectively encapsulation efficiency = [(m 总药量 -m 游离 ) / m 总药量 ] × 100%, and drug loading = [(m 总药量 -m 游离 ) / m 总 ] × 100%, wherein m 总 was the total amount of drug and lipid, and the results were shown in Table 5.

[0107] Table 5. Shape parameters of tanshinone II A nanostructured liposomes

[0108] (2) In vitro drug release study: tanshinone II A, physical mixture (drug and excipient ratio same as nanostructured liposomes), and nanostructured liposomes were taken in an appropriate amount and added to distilled water (tanshinone II A mass concentration was 10 mg / mL), 2 mL was taken into a dialysis bag (molecular weight cut-off 8000-14000 Da), the drug release medium was set to 1000 mL 1.0% SDS solution, the medium temperature was (37±1) ℃, the stirring paddle speed was 75 r / min, and 4 mL was taken at 0, 0.5, 1, 1.5, 2, 4, 8, 16, 24, 36, 48 h, immediately supplemented with 4 mL 1.0% SDS solution, filtered with a 0.45 μm water microporous filter, and the filtrate was taken, injected and measured under the above chromatographic conditions, the cumulative release was calculated, and the results were shown in Table 5. Figure 11 It was found that the in vitro drug release of the raw material and the physical mixture was basically the same, while the cumulative release of the nanostructured liposome lyophilized powder at different time points was significantly higher than that of the two, reaching 79.8% within 48 h.

Claims

1. A pharmaceutical composition for treating endometriosis, characterized by, It contains curcumin, EGCG and pterostilbene.

2. The pharmaceutical composition according to claim 1, wherein The curcumin and EGCG are co-delivery liposomes containing curcumin and EGCG; the pterostilbene is nanostructured liposomes containing pterostilbene.

3. The pharmaceutical composition of claim 1, wherein, The molar ratio of the curcumin and EGCG is 1-4:1-4.

4. The composition of claim 1, wherein, The molar ratio of the curcumin and pterostilbene is 1-4:1-16.

5. The pharmaceutical composition of claim 1, wherein The preparation steps of the co-delivery liposomes of curcumin and EGCG are: (1) mixing curcumin and EGCG, dissolving in anhydrous ethanol, and completely dissolving; (2) weighing cholesterol and egg yolk phospholipid, dissolving in ethyl acetate, adding to step (1), mixing thoroughly, removing the organic solvent to form a uniform film, adding a poloxamer aqueous solution, and ultrasonic treatment to obtain co-delivery liposomes containing curcumin and EGCG.

6. The pharmaceutical composition according to claim 5, wherein The molar ratio of the total mass of curcumin and EGCG, cholesterol and egg yolk phospholipid is 1:(1.5-3.5):(1-3).

7. The pharmaceutical composition of claim 1, wherein, The preparation steps of the nanostructured liposomes containing pterostilbene are: (1) weighing pterostilbene, glyceryl tristearate and peanut oil, dissolving in ethyl acetate as an organic phase; (2) using a poloxamer 188 aqueous solution as an aqueous phase, adding the aqueous phase to the organic phase under magnetic stirring, and then ultrasonic treatment to remove the organic solvent.

8. The pharmaceutical composition according to claim 7, wherein The drug-lipid molar ratio of pterostilbene, glyceryl tristearate and peanut oil is 1:15-1:

25.

9. The composition of claim 7, wherein, The solid-liquid-lipid molar ratio of glyceryl tristearate and peanut oil is 3:1-5:

1.

10. The pharmaceutical composition of claim 7, wherein, The addition amount of the poloxamer 188 aqueous solution is 1-5 times of ethyl acetate.

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