CN105194550A - Compound traditional Chinese medicine composition and application thereof to resistance to prostate cancer - Google Patents

Abstract

The invention discloses a compound traditional Chinese medicine composition with an anti-prostate cancer effect and an application thereof. The compound traditional Chinese medicine composition comprises the following traditional Chinese medicine raw materials in parts by weight: 4-12 parts of Ailanthus sinensis bark, 12-30 parts of Hedyotis diffusa, 3-15 parts of curcuma, 15-30 parts of salvia miltiorrhiza, 5-15 parts of Prunella vulgaris, 4-12 parts of licorice. The traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention, according to the theory of traditional Chinese medicine and the pathogenesis of prostate cancer, selects the raw material compatibility formula, the proportion of each component is scientific and reasonable, and the results of pharmacological experiments and preclinical experiments show that the present invention provides The traditional Chinese medicine compound composition has a good anti-prostate cancer effect, not only can significantly inhibit the proliferation and growth of prostate cancer, but also inhibit the migration, invasion and metastasis of prostate cancer, significantly improve the quality of life of tumor-bearing mice, and improve the quality of life of tumor-bearing mice. Various immune indicators in mice, and the route of administration is convenient and simple, without obvious adverse reactions. It can be developed into a new generation of anti-prostate cancer drugs, and it is expected to become a good prescription for the treatment of prostate cancer.

Description

A compound composition of traditional Chinese medicine and its application in anti-prostate cancer

technical field

The invention relates to the technical field of medicine, in particular to a traditional Chinese medicine compound composition and its application in preparing anti-prostate cancer.

Background technique

Prostate cancer (PCa), as the most common malignant tumor of the male reproductive system, is a disease that occurs frequently in elderly men, and its incidence increases with age. The incidence of prostate cancer is unevenly distributed around the world. Every year, there are more than 900,000 new cases worldwide, of which more than 260,000 cases die due to the progression of prostate cancer. The incidence of prostate cancer in European and American countries is significantly higher than that in Asia and Africa. In the United States, prostate cancer accounts for the first incidence rate and the second death rate of male tumors in the United States. In my country, the incidence of prostate cancer has increased by 70% compared with the past, ranking first in the incidence rate of malignant tumors of the male reproductive system in my country. million male population, and is still climbing at an annual growth rate of 10%. According to the literature report on the analysis of the current situation and epidemic trend of prostate cancer in China in the 04th issue of "Journal of Clinical Oncology" in 2013, the incidence of prostate cancer in my country shows an obvious and continuous growth trend, and prostate cancer is becoming a major urinary disease that seriously affects the health of men in my country. Systemic malignancies. In the next 10 years, with the increasingly serious phenomenon of aging society in our country, the incidence of prostate cancer in my country may more than double, and the incidence of prostate cancer may enter a peak period. The common clinical manifestation of prostate cancer is dysuria, which gradually occurs with frequent urination, urgency, hematuria and other symptoms accompanied by pain or burning during urination and continuous pain in the lower back, upper thigh or pelvis. Prostate cancer tumors do not have any clinical manifestations when they are young, and metastasis symptoms are often the earliest reason for seeing a doctor. Therefore, it is difficult to diagnose early based on clinical symptoms alone. Due to the long incubation period of prostate cancer, it is difficult to detect. When it is discovered, it has generally developed into advanced prostate cancer, causing serious adverse consequences to patients.

The effect of traditional Chinese medicine in preventing and treating tumors has long been clinically proven. The treatment of prostate cancer in traditional Chinese medicine has been discussed as early as Zhang Zhongjing's "Synopsis of the Golden Chamber" in the Han Dynasty and Chao Yuanfang's "On the Origin and Symptoms of Various Diseases" in the Sui Dynasty. According to the main clinical manifestations and signs of this disease, traditional Chinese medicine often classifies prostate cancer into disease categories such as "leaching syndrome", "hematuria", "accumulation", and "mass in the abdomen". Deficiency of kidney qi, blood stasis and spermatorrhea, and accumulation of lower energy are the main etiology and pathogenesis of prostate cancer. "Jingyue Quanshu" by Zhang Jingyue in the Ming Dynasty comprehensively suggested the treatment methods of clearing heat and detoxification, promoting blood circulation and removing blood stasis, softening hard masses and eliminating knots, warming yang and diuresis. In a large number of clinical practices, modern medical scientists have determined the pattern of syndrome differentiation and treatment, attacking evil and strengthening the body, which can increase the efficiency and reduce toxicity and adverse reactions in the process of cooperating with surgery, radiotherapy, chemotherapy, and endocrine therapy, and treat prostate cancer based on syndrome differentiation. Wide range of clinical applications. Aiming at the general etiology and pathogenesis of prostate cancer, four syndrome types have been adopted in clinical research, namely damp-heat betting, phlegm and blood stasis, liver and kidney yin deficiency, and qi and blood deficiency. Many scholars have used some clinical prescriptions and other unilateral and compound treatments or combined Western medicine to treat prostate cancer, which can improve the curative effect and quality of life of patients to a certain extent, relieve symptoms, improve physical strength, and reduce serum prostate specific antigen (Prostate specificantigen, PSA) levels. The level of androgen-independent resistance after anti-androgen treatment is delayed, and the quality of life of patients is significantly improved. In recent years, it has been reported in the literature that in the research on the role of traditional Chinese medicine in prostate cancer, there have been many studies on cell lines, and new mechanisms of action and regulatory pathways have been continuously elucidated, which has deepened the mechanism of traditional Chinese medicine in the treatment of prostate cancer to a certain extent and clarified the Efficacy of medical treatment of prostate cancer. Patients with advanced stage lose the opportunity of surgery, radiotherapy, and chemotherapy, and simply use traditional Chinese medicine to relieve pain, prolong survival, and improve the quality of life. Due to its remarkable clinical efficacy, traditional Chinese medicine compound plays an irreplaceable role in the treatment of prostate cancer and many other cancers, and has great application potential and development prospects in improving the quality of life of patients and delaying tumor metastasis and progression. It is of far-reaching clinical significance to seek effective and low-toxic traditional Chinese medicine, especially for patients with advanced refractory prostate cancer. Therefore, under the guidance of traditional Chinese medicine theory, it is very necessary to research and develop a Chinese medicine compound preparation with good anti-prostate cancer effect, low adverse reactions, reasonable price and easy popularization and application.

The growth and metastasis of prostate cancer are closely dependent on androgen. About 90% of androgen secretion comes from the testes, and about 10% comes from the adrenal glands. Androgen (such as dihydrotestosterone, Dihydrotestosterone, DHT) is secreted into cells and can bind to androgen receptor (Androgenreceptor, AR). The androgen receptor is an important nuclear transcription factor that regulates the expression of numerous genes and the proliferation of prostate cancer cells. When androgen binds to the androgen receptor, the androgen receptor enters the nucleus to initiate gene expression (such as an important indicator for screening prostate cancer—PSA), thereby promoting the growth and metastasis of prostate cancer. Therefore, androgen deprivation therapy (including castration therapy that removes the testicles and drugs to suppress androgen levels) has a significant effect on most prostate cancers. However, the control of tumor by androgen deprivation therapy can generally only be maintained for 1.5 to 4.0 years, and almost all prostate cancer patients eventually turn into androgen-independent prostate cancer (AIPC), and then develop into hormone-refractory prostate cancer Cancer (Hormonerefractoryprostatecancer, HRPC), also known as castration-resistant prostate cancer (Castrationresistantprostatecancer, CRPC). At this point, androgen deprivation therapy loses its effect. CRPC is an important cause of death in prostate cancer patients. There are many mechanisms of CRPC, including androgen receptor mutation, loss of androgen receptor expression, and expansion of androgen-insensitive cell populations in a low-androgen environment. In other words, although androgen deprivation therapy has no effect, the androgen receptor also plays an important role in most castration-resistant prostate cancers, so the androgen receptor is the current treatment for general prostate cancer and castration-resistant prostate cancer. important target of cancer.

In today's global medical community, there is no single drug that can cure prostate cancer alone. The main treatment methods for prostate cancer include surgery, radiotherapy, chemotherapy, and biological therapy. Not only do patients have to experience a near-death experience, but the family’s financial pressure is huge and the debts are heavy. Experience the pain of parting from life and death. The latest drugs approved by the U.S. Food and Drug Administration (FDA) for the treatment of CRPC include Abiraterone (Abiraterone) and MDV3100 (Enzalutamide). The former inhibits the synthesis of androgen, and the latter is an antagonist of the androgen receptor. Both of these drugs target the androgen receptor signaling pathway, and have achieved significant curative effects in the treatment of castration-resistant prostate cancer. However, after the above-mentioned endocrine therapy and androgen receptor-targeted drug treatment, the patient still develops drugs. Resistance, the treatment effect is very limited, and the cost is expensive, and the patient's economic ability is poor. In addition, the side effects caused by radiotherapy and chemotherapy are also the bottleneck of clinical treatment. As one of the important means of comprehensive treatment of prostate cancer, traditional Chinese medicine plays an irreplaceable role in the treatment of prostate cancer and many other cancers because of its remarkable clinical efficacy. Combining my country's abundant resources of traditional Chinese medicinal materials and the long-standing theory of traditional Chinese medicine, the development of a new drug combination that targets prostate cancer drug resistance and has my country's independent intellectual property rights has very important clinical significance for the treatment of prostate cancer.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, to provide a compound Chinese medicine composition with anti-prostate cancer effect, a preparation method and its application in the field of prostate cancer treatment, which has remarkable curative effect, little toxic and side effects, and is convenient to take. application.

The present invention proposes a traditional Chinese medicine compound composition, which includes the following Chinese medicinal materials in parts by weight: 4-12 parts of Ailanthus bark, 12-30 parts of Hedyotis diffusa, 3-15 parts of Curcuma, 15-30 parts of Salvia miltiorrhiza, summer Subtilis 5-15 parts, licorice 4-12 parts.

Among them, preferably, the Chinese medicine compound composition includes the following Chinese medicinal materials in parts by weight: 6-9 parts of Ailanthis sinensis, 15-24 parts of Hedyotis diffusa, 4-12 parts of Curcuma, 18-21 parts of Salvia miltiorrhiza, summer Subtilis 6-10 parts, licorice 6-9 parts.

The present invention also proposes a preparation method of the above-mentioned traditional Chinese medicine compound composition, comprising the following steps:

(1) Soaking: take the six-flavored Chinese medicinal material described in claim 1 or 2, add distilled water for soaking, and obtain the crude drug distilled water mixture;

(2) decocting: decocting the crude drug distilled water mixture described in step (1), to obtain the crude extract of traditional Chinese medicine;

(3) concentrating: concentrating the Chinese medicine crude extract prepared in step (2) to obtain a Chinese medicine concentrate;

(4) Sterilization: take the concentrated Chinese medicine solution prepared in step (3) and filter it through a microporous membrane to obtain the compound Chinese medicine composition.

The invention also proposes the application of the traditional Chinese medicine compound composition in the preparation of medicines for treating malignant tumors.

Wherein, the malignant tumor is caused by abnormal amplification and mutation of androgen receptor, including prostate cancer, breast cancer, lung cancer, colon cancer, brain cancer, skin cancer, bladder cancer, and kidney cancer.

The present invention further proposes the application of the traditional Chinese medicine compound composition in the preparation of medicaments for treating prostate cancer.

The present invention further proposes the application of the traditional Chinese medicine compound composition in inhibiting the growth, proliferation, metastasis, migration, invasion of prostate cancer cells and promoting the apoptosis of prostate cancer cells.

Wherein, the prostate cancer is castration-resistant prostate cancer or androgen-dependent prostate cancer.

The present invention further proposes the use of the traditional Chinese medicine compound composition for inhibiting dihydrotestosterone-induced transcriptional activity of the androgen receptor and the transcriptional activity of the androgen receptor lacking the ligand domain in vitro, or for inhibiting the transcriptional activity of the androgen receptor in vitro Androgen receptor activity.

The present invention also proposes to make the Chinese medicine compound composition and pharmaceutically acceptable carrier into injectable fluid, aerosol, cream, gel, pill, syrup, transdermal patch, capsule, Drugs in the form of microcapsules, tablets, granules or oral liquids.

The traditional Chinese medicine compound composition of the present invention can be used as a medicine alone or in combination with other medicines.

The present invention also proposes the use of the traditional Chinese medicine compound composition for inhibiting the proliferation of the androgen receptor mutated prostate cancer cell line 22RV1 in vitro, and inhibiting the proliferation of the androgen-dependent prostate cancer cell line LNCaP.

The present invention also proposes the use of the traditional Chinese medicine compound composition for inhibiting the metastasis of prostate cancer cells in vitro, or for inhibiting the migration of prostate cancer cells PC-3, LNCaP and 22RV1 in vitro, or for inhibiting dihydrotestosterone in vitro Induced transcriptional activity of androgen receptor and use of transcriptional activity of androgen receptor lacking a ligand domain, or inhibiting androgen receptor activity in vitro, inhibiting growth, migration and invasion of prostate cancer cells, and promoting the growth, migration and invasion of prostate cancer cells The use of apoptosis, or the use of inhibiting the proliferation of androgen receptor mutant cell line 22RV1 cells ineffective in bicalutamide or MDV3100 treatment in vitro.

The present invention also proposes to use the traditional Chinese medicine compound composition for inhibiting the proliferation of prostate cancer cell line 22RV1 with androgen receptor mutation in vivo, inhibiting the proliferation of androgen-dependent prostate cancer cell line LNCaP, or for inhibiting Metastasis of prostate cancer cells, or for inhibiting the growth and metastasis of castration-resistant prostate cancer in vivo, or for inhibiting androgen receptor activity, inhibiting growth, migration and invasion of prostate cancer cells, and promoting apoptosis of prostate cancer cells , or for inhibiting the proliferation of androgen receptor mutant cell line 22RV1 cells ineffective in bicalutamide or MDV3100 treatment, or for inhibiting the growth of prostate cancer cell line 22RV1 in mice, or for inhibiting prostate cancer in mice Transfer of cells 22RV1.

The invention also proposes the application of the traditional Chinese medicine compound composition in the preparation of medicines for treating malignant tumor diseases related to the mutation and abnormal amplification of the androgen receptor.

Wherein, the malignant tumor is an application in the proliferation, growth, migration and invasion of malignant tumor cells caused by abnormal amplification and mutation of the androgen receptor; wherein, the malignant tumor includes prostate cancer, breast cancer, lung cancer, colon cancer , brain cancer, skin cancer, bladder cancer, kidney cancer.

The traditional Chinese medicine compound composition mentioned in the present invention is made of the following Chinese medicinal materials in parts by weight: 4-12 parts of Ailanthus sinensis, 12-30 parts of Hedyotis diffusa, 3-15 parts of Curcuma, 15-30 parts of Salvia miltiorrhiza, summer Subtilis 5-15 parts, licorice 4-12 parts.

Wherein, preferably, the Chinese medicine compound composition is made of the following Chinese medicinal materials in parts by weight: 6-9 parts of Ailanthis sinensis bark, 15-24 parts of Hedyotis diffusa, 4-12 parts of Curcuma zedoary, 18-21 parts of Salvia miltiorrhiza , 6-10 parts of Prunella vulgaris, 6-9 parts of licorice.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the transcriptional activity of the androgen receptor. The provided traditional Chinese medicine compound composition inhibits the proliferation of prostate cancer cell lines with androgen receptor mutation. It inhibits the proliferation of androgen receptor-mutated prostate cancer cell line 22RV1 in vitro and in vivo, and inhibits the proliferation of androgen-dependent prostate cancer cell line LNCaP.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the metastasis of prostate cancer cells in vitro and in vivo.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the transcriptional activity of the androgen receptor and the growth of prostate cancer cells.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the proliferation of primary isolated prostate cancer cells derived from patient's tumor tissue.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the colony formation of prostate cancer cells.

In the application of the present invention, the provided traditional Chinese medicine compound composition induces the apoptosis of cell lines LNCaP and 22RV1 with positive expression of androgen receptor.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the growth and metastasis of castration-resistant prostate cancer cells in a mouse model in vivo.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the proliferation of the androgen receptor mutant cell line 22RV1 whose treatment with bicalutamide or MDV3100 is ineffective.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the migration of prostate cancer cell lines PC-3 and LNCaP cells in vitro and the metastasis of 22RV1 cells in vivo.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the growth of prostate cancer cell lines LNCaP, 22RV1, PC-3 and primary isolated prostate cancer cell lines derived from patient's tumor tissue.

In the application of the present invention, the application and method of inhibiting the androgen receptor by the compound Chinese medicine composition are provided. The provided traditional Chinese medicine compound composition significantly down-regulates the transcriptional activity of the androgen receptor and inhibits its downstream gene expression by blocking the protein expression level of the androgen receptor.

In the application of the present invention, the provided traditional Chinese medicine compound composition inhibits the activity of the luciferase reporter gene regulated by the androgen receptor in vitro, can strongly inhibit the proliferation and migration of prostate cancer cells in vitro and in vivo, and can effectively block the Hormone receptor signaling pathway.

In the application of the present invention, the provided traditional Chinese medicine compound composition can not only efficiently inhibit the proliferation of the androgen-dependent prostate cancer cell line LNCaP, but also inhibit the prostate cancer cell line 22RV1 with androgen receptor mutation (the cell line expresses a continuously activated mutated androgen receptor, and resistant to clinical drug androgen receptor antagonists, such as bicalutamide and MDV3100, etc.). In addition, the present invention proves that the provided traditional Chinese medicine compound composition inhibits the metastasis of prostate cancer in vitro and in vivo. Further experiments have proved that the prostate cancer cell lines expressing androgen receptor have better sensitivity to the traditional Chinese medicine compound composition provided by the present invention. The traditional Chinese medicine compound composition inhibits the protein expression level of the androgen receptor, thereby inhibiting the transcriptional activity of the androgen receptor and the growth of prostate cancer cells. The traditional Chinese medicine compound composition provided by the invention can not only treat early androgen-dependent prostate cancer, but also treat late castration-resistant prostate cancer.

The application of the Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention can make the extract of the Chinese medicine compound composition and a pharmaceutically acceptable carrier into injectable fluid, aerosol, cream, gel, Drugs in the form of pills, syrups, transdermal patches, capsules, microcapsules, tablets, granules or oral liquids.

When the Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention is made into a tablet, the Chinese medicine compound composition with anti-prostate cancer effect and carrier lactose or cornstarch are added when necessary, and the lubricant magnesium stearate is added, mixed uniformly, Then compressed into tablets. When the Chinese medicine compound composition with anti-prostate cancer effect provided by the invention is made into capsules, the Chinese medicine compound composition with anti-prostate cancer effect and carrier lactose or cornstarch are uniformly mixed, granulated, and then packed into capsules to make capsules. When the anti-prostate cancer compound provided by the present invention is made into granules, the traditional Chinese medicine compound composition with anti-prostate cancer and diluent lactose or cornstarch are uniformly mixed, granulated and dried to make granules.

The invention also discloses the application of the provided traditional Chinese medicine compound composition with anti-prostate cancer effect or its pharmaceutically acceptable preparation in the preparation of medicines for treating malignant tumor diseases related to mutation and abnormal amplification of androgen receptor .

In order to achieve the above object, the Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention is made of the following raw materials in parts by weight: 4 to 12 parts of Ailanthus sinensis, 12 to 30 parts of Hedyotis diffusa, 3 parts of Curcuma ～15 parts, 15～30 parts of Salvia miltiorrhiza, 5～15 parts of Prunella vulgaris, 4～12 parts of licorice.

As a preferred option, the above-mentioned Chinese medicine compound composition with anti-prostate cancer effect is made of the following raw materials in parts by weight: 6 to 9 parts of Ailanthus sinensis, 15 to 24 parts of Hedyotis diffusa, zedoary 4-12 parts, 18-21 parts of Salvia miltiorrhiza, 6-10 parts of Prunella vulgaris, 6-9 parts of licorice.

Under the guidance of the theory of traditional Chinese medicine, the present invention applies dialectical treatment, combines the pathogenesis and symptoms of prostate cancer, and treats prostate cancer with traditional Chinese medicine to nourish qi and nourish blood, invigorate the kidney and detoxify, dissipate blood stasis and relieve pain, promote blood circulation and stop bleeding, regulate endocrine, diuresis and diuresis. The overall treatment concept of strengthening the body and anti-cancer of stranguria, eliminating blood stasis and dissipating stagnation is the treatment principle to carry out compatible prescriptions. Based on the above treatment principles, through a large number of literature searches and experimental verifications, a Chinese medicine compound composition with an anti-prostate cancer effect according to the present invention was screened out. Formulated with 6 traditional Chinese medicines of licorice: Ailanthus bark in the prescription, bitter, astringent, cold in nature, has the functions of clearing away heat and dampness, reducing astringency and stopping bleeding, and contains anti-tumor ingredients. Hedyotis diffusa, bitter, sweet, cold in nature, clears away heat and detoxifies, promotes dampness and relieves stranguria, has a strong effect of clearing away heat and detoxification, and is often used for heat-toxin and other syndromes. It contains oleanolic acid, triterpene acid, coumarin Active ingredients and effective parts such as ketones and polysaccharides all have anti-tumor effects, among which triterpenoids have the strongest anti-tumor effects. Combining with Ailanthus bark complements each other and has a higher effect than Leehom, and the two are the king medicine of the prescription. Curcuma, bitter in taste and warm in nature, has outstanding functions of promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, promoting qi and blood circulation, relieving pain and removing blood stasis. The active ingredient is curcuma oil, which is recognized as a good anti-cancer medicine. Salvia miltiorrhiza, bitter in taste, slightly cold in nature, promotes blood circulation and removes blood stasis, nourishes blood and calms nerves, cools blood and eliminates carbuncle, mainly contains fat-soluble diterpenoids and water-soluble phenolic acids. The diterpenoid component Tanshinone ⅡA is currently the most reported antitumor active ingredient in Danshen. Curcuma and Danshen both have the effects of dispelling blood stasis and relieving pain, promoting blood circulation and nourishing blood, and they are ministerial drugs together. Prunella vulgaris, bitter, pungent, cold in nature, is a commonly used heat-clearing medicine, which has the effects of clearing the liver and improving eyesight, dispelling stagnation and detoxification; in this prescription, it has a significant effect of softening hard masses and dispelling stagnation, and the auxiliary medicine further softens the tumor site and eliminates it. The effect of carbuncle poison is an adjuvant drug. Licorice, sweet in taste, flat in nature, invigorates the spleen and Qi, clears away heat and detoxifies, and harmonizes various medicines. In this prescription, it mainly plays the role of promoting the adjustment of the body's immune function and restoring righteousness. The 6 herbs are strictly compatible according to the principle of monarch, minister, assistant, and envoy, and they have the effects of nourishing qi and blood, invigorating the kidney and detoxifying, dispelling blood stasis and relieving pain, promoting blood circulation and stopping bleeding, regulating endocrine, diuresis and treating stranguria, and eliminating stasis and stagnation.

The preparation method of the traditional Chinese medicine compound composition of the present invention: take the above-mentioned weight parts of the traditional Chinese medicine compound composition, and each dose of the six-flavored Chinese medicinal material crude drug dry product several grams, add 10 to 15 times the volume of the traditional Chinese medicine compound composition, soak in distilled water for 1 to 3 hours ; decoct in an automatic Chinese medicine decoction device, the number of decoctions is 1 to 3 times, and each decoction is 0.5 to 1 hour; the prepared crude extract of Chinese medicine is evaporated and concentrated on the automatic Chinese medicine decoction device until the final volume is 5% to 10% of the volume of the initial extract; filter and sterilize the obtained Chinese medicine concentrate through a 0.22 micron microporous membrane to obtain a Chinese medicine water decoction mother liquor, and store it in a refrigerator at 4 degrees Celsius for later use.

Compared with the prior art, the traditional Chinese medicine compound composition provided by the invention has the following advantages:

(1) The traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention is made by selecting raw materials according to the theory of traditional Chinese medicine and the pathogenesis of prostate cancer. Clearing heat and detoxifying, promoting qi and blood circulation, diuresis and dredging stranguria, softening and dispelling stagnation, relieving stasis and relieving pain, nourishing qi and nourishing blood. The growth and metastasis of prostate cancer in mice can improve the quality of life and immune function, prolong the survival period, improve biochemical indicators, and reduce the scope of lesions. Combined with the chemotherapy drug cyclophosphamide, it has the effect of synergizing and reducing toxicity, and has no adverse reactions. It has a very good anti-cancer and righting effect.

(2) The traditional Chinese medicine compound composition provided by the present invention has anti-prostate cancer effect, by down-regulating the protein level of androgen receptor, inhibiting the expression of its downstream genes, and blocking the androgen receptor signaling pathway, thereby realizing the inhibition of prostate cancer Objective: The drug has definite curative effect and clear mechanism of action, and has made new breakthroughs in the treatment of prostate cancer, which can to a certain extent fill the gap in my country's current field of prostate cancer treatment drugs that basically rely on imports and lack independent intellectual property rights drugs.

(3) The traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention has a low-cost, environmentally-friendly preparation method, and can realize large-scale industrial production.

(4) The traditional Chinese medicine compound composition with anti-prostate cancer effect provided by the present invention can be prepared into various dosage forms with pharmaceutically acceptable carriers, which is convenient for clinical use and is expected to be developed into a new anti-prostate cancer drug.

Terms and Definitions

Castration-resistant prostate cancer (CRPC): Prostate cancer patients relapse after castration therapy (removal of testicles or chemical castration to reduce androgen). Prostate cancer that is ineffective in castration therapy is called castration-resistant prostate cancer. Cancer is the biggest killer of prostate cancer patients.

Prostate cancer cell PC-3: a poorly differentiated, androgen-independent prostate cancer cell isolated from bone metastases of human prostate cancer, does not express endogenous androgen receptors, and has a moderate degree of metastatic potential. Exhibits prominent epithelial cell carcinoma features and is widely used in the study of androgen-resistant prostate cancer.

Prostate cancer cells LNCaP: established by Horoszewicz et al. in 1980 from the lymph node metastases of prostate cancer patients, it retains the characteristics of prostate cancer tumor cytology and early differentiation function, has significant epithelial cell characteristics, and expresses androgen receptor, is typical of androgen-dependent prostate cancer cells.

Prostate cancer cell 22RV1: a human prostate cancer epithelial cell line isolated from androgen-dependent prostate cancer CWR22 cells xenografted into mice for serial passage. This cell line expresses prostate specific antigen, grows dependent on androgen stimulation, and has strong tumorigenic ability in nude mice.

Cell Proliferation: The process by which cells divide to produce new individuals and offspring. Single-celled organisms that produce new individuals by cell division. Multicellular organisms produce new cells by cell division to replenish aging and dead cells in the body; at the same time, multicellular organisms can develop from a fertilized egg to a new multicellular organism after cell division and differentiation individual. Through cell division, the duplicated genetic material can be equally distributed among the two daughter cells. Cell proliferation is the basis of organism growth, development, reproduction and heredity, and is an important life feature of organisms.

Apoptosis: In order to maintain a stable internal environment, cells are autonomously, orderly, and programmed cell death controlled by genes. Apoptosis involves the activation, expression and regulation of a series of genes, and is a process of cell death that is actively fought for in order to adapt to the living environment.

Cell migration: also known as cell crawling, cell movement or cell movement, refers to the movement of cells after receiving migration signals or feeling the gradient of certain substances. Cell migration is an alternate process in time and space of the elongation of the pseudopodia of the cell head, the establishment of new adhesions, and the contraction of the tail of the cell body. Cell migration is one of the basic functions of normal cells, a physiological process of normal growth and development of the body, and a common form of movement in living cells. Cell migration is involved in processes such as embryonic development, angiogenesis, wound healing, immune response, inflammatory response, atherosclerosis, and cancer metastasis.

Cell invasion: the ability of tumor cells to migrate across the basement membrane to the interstitium or other tissues for distant metastasis. Basement membrane is a specialized extracellular matrix, which is the physical barrier that tumor cells must cross during metastasis. Components of the basement membrane interact with cell surface receptors integrins. Tumor cells destroy the tissue structure of the basement membrane by secreting matrix-degrading enzymes, and at the same time regulate the expression of extracellular matrix receptor integrins, creating favorable conditions for passing through the basement membrane and adhesion and proliferation in target organs.

Tumor growth: The in situ tumor volume increases due to division and proliferation of tumor cells and invades the surrounding tissue space, lymphatic vessels or blood vessels, and is closely connected with adjacent normal tissues without obvious boundaries.

Tumor metastasis: the process in which tumor cells invade lymphatic vessels, blood vessels or body cavities from their original site, and carry them to another site or organ along with the blood flow and lymph flow to continue to grow and form a tumor of the same type as the primary tumor.

Prostate cancer growth and metastasis: In the process of malignant transformation of prostate cancer, tumor cell population dynamics, tumor cell apoptosis rate, tumor angiogenesis, tumor evolution and heterogeneity deterioration are caused by the enhanced ability of tumor cell proliferation, migration and invasion the process of. Prostate cancer becomes more and more aggressive in the process of growth and metastasis, eventually resulting in accelerated tumor growth, invasion of surrounding tissues and distant metastasis.

Androgen Receptor (AR): The androgen receptor belongs to the steroid receptors in the nuclear receptor superfamily. The human androgen receptor is a single-copy gene with a molecular weight of about 110 kilodaltons. It generally consists of four domains: N-terminal transcription activation domain (N-terminal domain, NTD), DNA binding domain (DNAbinding domain, DBD), Hinge region and ligand binding region (Ligandbindingdomain, LBD). Androgen receptor is closely related to the occurrence and development of prostate cancer. After androgen such as dihydrotestosterone (Dihydrotestosterone, DHT) binds to the androgen receptor, the androgen receptor can enter the nucleus and bind to the transcription activation domain to initiate downstream gene expression. Currently clinically used androgen receptor antagonists such as Bicalutamide (Bicalutamide) and MDV3100 (Enzalutamide) bind to the ligand-binding domain (LBD) of androgen receptor to inhibit its transcriptional activity.

Transcriptional activity of androgen receptor: Androgen receptor is a typical nuclear receptor transcription factor, which regulates the expression of various eukaryotic genes by mediating the action of androgen. Its specific function is to regulate the transcription and expression of its downstream target genes through transcriptional activation, and can exert transcriptional activity in both ligand-dependent and ligand-independent pathways.

Androgen receptor lacking the ligand domain: The androgen receptor LBD region consists of 12 discontinuous α-helices, forming a ligand-binding pocket, which has the function of binding ligand. The androgen receptor may lack the LBD region in the case of mutations, and the androgen receptor with the deletion of the LBD region has the same constitutive transcriptional activation function as the intact androgen receptor.

Luciferase reporter gene detection (Luciferase assay): Luciferase (Luciferase, Luc) reporter gene detection system is a reporter system that uses luciferin as a substrate to detect firefly luciferase (Fireflyluciferase) activity. Luciferase can catalyze the oxidation of Luciferin to Oxy-luciferin, and during the oxidation of Luciferin, bioluminescence will be emitted. The bioluminescence released during the oxidation of Luciferin can then be measured by a fluorometer, also known as a chemiluminometer (Luminometer) or a liquid scintillation analyzer. The bioluminescent system of luciferin and luciferase can detect gene expression extremely sensitively and efficiently, and is a detection method for detecting the interaction between transcription factors and target gene promoter region DNA.

Description of drawings

Fig. 1 shows the inhibitory effect of the compound Chinese medicine composition of the present invention on the proliferative activity of various prostate cancer cell lines.

Fig. 1(A) shows the inhibition of the proliferation of various prostate cancer cell lines and the selective effect on normal prostate epithelial cells by the traditional Chinese medicine compound composition of the present invention.

Fig. 1(B) shows that the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the proliferation of LNCaP cells is time-gradient and concentration-gradient dependent.

Figure 1(C) shows that the inhibitory effect of the compound Chinese medicine composition of the present invention on the proliferation of 22RV1 cells is time-gradient and concentration-gradient dependent.

FIG. 1(D) shows that the anti-proliferation effect of the compound Chinese medicine composition of the present invention on PC-3 cells is time-gradient and concentration-gradient dependent.

Fig. 1(E) shows that the anti-proliferation effect of the traditional Chinese medicine compound composition of the present invention on the primary isolated prostate cancer cells derived from the patient's tumor tissue is time-gradient and concentration-gradient dependent.

Fig. 2 shows the inhibitory effect of the compound Chinese medicine composition of the present invention on the colony formation of various prostate cancer cell lines.

The left side of Fig. 2 shows the white light photos of the inhibitory effect of the compound Chinese medicine composition of the present invention on the colony formation of LNCaP and 22RV1 respectively.

The right side of Fig. 2 shows the statistical results of the inhibitory effect of the compound Chinese medicine composition of the present invention on the clone formation of LNCaP and 22RV1 respectively.

Figure 3 shows the apoptosis-inducing effect of the compound Chinese medicine composition of the present invention on prostate cancer cells.

Fig. 3(A) shows the apoptosis-inducing effect of the compound Chinese medicine composition of the present invention on LNCaP cells.

Fig. 3(B) shows the apoptosis-inducing effect of the compound Chinese medicine composition of the present invention on 22RV1 cells.

Fig. 3(C) shows the statistical results of the apoptosis-inducing effect of the compound Chinese medicine composition of the present invention on LNCaP, 22RV1.

Fig. 4 shows the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the migration and invasion of prostate cancer cells.

Figure 4 shows the inhibitory effect of the compound Chinese medicine composition of the present invention on the lateral migration of PC-3 cells.

Figure 4 shows the inhibitory effect of the compound Chinese medicine composition of the present invention on the longitudinal migration of LNCaP cells.

Figure 4 below shows the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the invasion process of 22RV1 cells.

Fig. 5 shows the molecular mechanism of the Chinese medicine compound composition of the present invention inhibiting prostate cancer by affecting the androgen receptor signaling pathway.

Fig. 5(A) shows the inhibitory effect of the Chinese medicine compound composition of the present invention on the expression of the luciferase reporter gene for detecting androgen receptor transcriptional activity in LNCaP cells induced by dihydrotestosterone (DHT).

Fig. 5(B) shows the inhibitory effect of the Chinese medicine compound composition of the present invention on the expression of luciferase reporter gene for detection of androgen receptor transcriptional activity in 22RV1 cells induced by dihydrotestosterone (DHT).

Fig. 5 (C) shows that the Chinese medicine compound composition of the present invention detects the fluorescence of androgen receptor transcriptional activity after the exogenous transfection of wild-type androgen receptor (AR) under the induction of dihydrotestosterone (DHT) in PC-3 cells Inhibitory effect of primease reporter gene expression.

Figure 5(D) shows that the compound Chinese medicine composition of the present invention does not affect the transcription level of the androgen receptor (AR) gene itself in LNCaP cells.

Fig. 5(E) shows that the compound Chinese medicine composition of the present invention down-regulates the expression level of androgen receptor (AR) protein in LNCaP cells.

Fig. 5(F) shows that the traditional Chinese medicine compound composition of the present invention inhibits the transcriptional activation of androgen receptor (AR) on its three classic downstream target genes TMPRSS2, FKBP5 and PSA.

Fig. 6 shows that the traditional Chinese medicine compound composition of the present invention inhibits the growth of 22RV1 xenograft tumors in the subcutaneous tumor-bearing in vivo animal model of nude mice.

Fig. 6(A) shows the white light photo of the inhibitory effect of the compound Chinese medicine composition of the present invention on the tumor volume stripped in the 22RV1 xenograft tumor growth model.

Fig. 6(B) shows the statistical results of the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the growth of tumor volume with days in the 22RV1 xenograft tumor growth model.

Fig. 6(C) shows the white light photo of the inhibitory effect of the compound Chinese medicine composition of the present invention on the tumor weight stripped in the 22RV1 xenograft tumor growth model.

Fig. 7 shows that the traditional Chinese medicine compound composition of the present invention inhibits the growth and metastasis of 22RV1-luc xenograft tumor in the in vivo animal model of orthotopic xenograft tumor growth and metastasis in nude mice.

Fig. 7(A) shows the in vivo imaging results of the inhibitory effect of the Chinese medicine compound composition of the present invention on tumor fluorescence intensity in the 22RV1-luc xenograft tumor growth and metastasis model.

Fig. 7(B) shows the statistical results of the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the growth and metastasis of orthotopic tumors in the 22RV1-luc xenograft tumor growth and metastasis model.

Figure 7(C) shows the statistical results of the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the growth and metastasis of the whole body tumor fluorescence intensity in the 22RV1-luc xenograft tumor growth and metastasis model.

Fig. 7(D) shows the effect of the traditional Chinese medicine compound composition of the present invention on the body weight of nude mice in the way of intragastric administration.

Detailed ways

The technology of the present invention will be further described in detail in conjunction with the following specific embodiments and accompanying drawings. The protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope. The specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in the claims. The process, conditions, reagents, experimental methods, etc. for implementing the present invention are general knowledge and common knowledge in the art except for the content specifically mentioned below, and the present invention has no special limitation content.

Embodiment 1: the preparation of Chinese medicine compound composition of the present invention

The traditional Chinese medicine compound composition with anti-prostate cancer effect in this example is made from the following raw materials: 9 parts of Ailanthus sinensis bark, 30 parts of Hedyotis diffusa, 15 parts of Curcuma zedoary, 30 parts of Salvia miltiorrhiza, 15 parts of Prunella vulgaris, and 9 parts of licorice , adding 15 times the volume of the traditional Chinese medicine compound composition in distilled water and soaking for 3 hours; decocting in an automatic Chinese medicine decoction device, the number of decoctions is 2 times, and decocting for 1 hour each time; Open the cover on the automatic decoction device and evaporate to concentrate until the final volume is 70 ml; the final concentration of the mother liquor of Chinese medicine decoction obtained by concentration is about 1.5 g of crude drug/ml. Filter and sterilize the obtained concentrated Chinese medicine through a 0.22-micron microporous membrane to obtain a Chinese medicine water decoction mother liquor, which is stored in a refrigerator at 4 degrees Celsius for later use.

The compound Chinese medicine composition adopted in the following examples 2-7 is prepared by Example 1

Example 2: Inhibition of the proliferative activity and clone formation of the compound Chinese medicine composition of the present invention on different prostate cancer cells and the induction of apoptosis on different prostate cancer cells

Technical method:

1. Cell Culture

The cells (LNCaP, 22RV1, PC-3, PNT1A) used in this experiment were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; the other cell line came from primary isolated prostate cancer cells derived from tumor tissue of prostate cancer patients in Shanghai Changhai Hospital ( PCa primary cells), the cells were cultured in a constant temperature incubator at 37 degrees Celsius (humidity 95%, _CO2 concentration 5%), and the medium was RPMI-1640 (Gibco) containing 10% fetal bovine serum (Front).

2. Determination of cell proliferation by SRB (sulfonyl rhodamine) method

Different prostate cancer cell lines were inoculated into 96-well plates (Corning) at a density of 5×10 ³ cells/well. After 24 hours, different concentrations of the traditional Chinese medicine compound composition of the present invention were added, and the same amount of PBS was added to the control group. multiple holes. After 48 hours of drug treatment, pre-cooled TCA (trichloroacetic acid, 50%, w/v) was added and incubated at 4°C for 60 minutes to fix the cells. After fixing, rinse with running water 5 times and air dry. Add 50 microliters of SRB staining solution (4%, w/v) to each well and incubate at room temperature for 10 minutes for staining. Aspirate the dye solution, add 100 microliters of 1% acetic acid to each well and wash 5 times to remove unbound dye. After air-drying, add 100 microliters of Tris solution with a concentration of 10 mmol to each well, and shake to dissolve the bound SRB dye. The 96-well plate was placed in a microplate reader (SPECTRAMAX190), and the OD value was measured at a wavelength of 515 nm. The effect of the traditional Chinese medicine compound composition on the cell proliferation level was statistically analyzed. In order to detect whether the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on prostate cancer cell lines is concentration-gradient-dependent and time-concentration-gradient-dependent, different prostate cancer cell lines were inoculated into 96-well plates at a density of 5×10 ³ cells/well, After 24 hours, different concentrations of the traditional Chinese medicine compound composition of the present invention were added, and the same amount of PBS was added to the control group, and 6 replicate wells were set up in each group. After continuing to culture for 24 hours, 48 hours and 72 hours respectively, the influence of the Chinese medicine compound composition on the cell proliferation level was detected by the same method as above.

3. Colony formation experiment

Colonyformation can be used to detect the proliferation and tumorigenic ability of tumor cell clones formed in vitro. Tumor cells can divide and proliferate in vivo to finally form solid tumors visible to the naked eye. In vitro experiments, the ability of tumor cells to form solid tumors in vivo can be reflected according to their ability to form solid tumors. Insert 1000 prostate cancer cells into a 6-well plate, add different concentrations of the traditional Chinese medicine compound composition of the present invention after the cells adhere to the wall, fix the cells with paraformaldehyde one week later, then stain with crystal violet, take pictures and count the number of clones , and each experiment was repeated three times.

4. Detection of cell apoptosis by flow cytometry

In normal cells, phosphatidylserine (PS) is only distributed on the inner side of the lipid bilayer of the cell membrane, but in the early stage of apoptosis, the phosphatidylserine (PS) in the cell membrane is turned from the inside to the outside of the lipid membrane. AnnexinV is a Ca ²⁺ -dependent phospholipid-binding protein with a molecular weight of 35-36kD. It has a high affinity with phosphatidylserine, so it can bind to the cell membrane of early apoptosis cells through phosphatidylserine exposed on the outside of the cell. Therefore, AnnexinV is regarded as one of the sensitive indicators for detecting early apoptosis of cells. The AnnexinV is labeled with fluorescein (EGFP, FITC, etc.), and the labeled AnnexinV is used as a fluorescent probe to detect the occurrence of apoptosis by using a fluorescence microscope or flow cytometer. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the complete cell membrane, but for cells in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane and stain the nucleus red. Therefore, the matching of AnnexinV and PI can distinguish cells in different apoptotic stages. The specific operation steps are as follows: after different prostate cancer cells were treated with different concentrations of the Chinese medicine compound composition of the present invention for 36 hours, the suspended cells were collected and centrifuged (2000 rpm, centrifuged for 5 minutes); the adherent cells were treated with EDTA-free pancreas Enzymatic digestion and collection; wash cells twice with PBS (2000 rpm, centrifuge for 5 minutes) to collect 1~ ⁵ ×105 cells; add 500 microliters of Bindingbuffer to suspend cells; add 5 microliters of AnnexinV-FTIC and mix well, add 5 microliters of PI, mixed well; room temperature, protected from light, reacted for 5-15 minutes; within 1 hour, carried out flow cytometry detection on the machine.

Experimental results:

As shown in Figure 1, the traditional Chinese medicine compound composition of the present invention has a very significant inhibitory effect on each strain of prostate cancer cells, including the proliferation of primary prostate cancer cells derived from patient tissues, and its 48-hour half inhibitory concentration is about 5 micrograms/day. ml. In addition to the significant inhibitory effect on the proliferation of commonly used prostate cancer cell lines and the primary prostate cancer cells derived from patient tissues, the traditional Chinese medicine compound composition of the present invention also has a high concentration (100 micrograms/ml) on normal prostate epithelial cell line PNT1A. Slight inhibition, but little inhibition at low concentrations, showing good selectivity between normal cells and prostate cancer cells. At the same time, the inhibitory effect of the compound Chinese medicine composition of the present invention on the proliferation of prostate cancer cells exhibits a concentration gradient dependence and a time gradient dependence. In addition, as shown in Fig. 2, the traditional Chinese medicine compound composition of the present invention also strongly inhibits the colony formation of each prostate cancer cell line. Fig. 3 shows that the traditional Chinese medicine compound composition of the present invention significantly promotes the apoptosis of prostate cancer cells LNCaP, 22RV1. The experimental results suggest that the traditional Chinese medicine compound composition of the present invention can be used as a potential drug against the growth of prostate cancer.

Example 3: Inhibition of the compound Chinese medicine composition of the present invention on the migration and invasion of prostate cancer cells

Technical method:

1. Streak Migration Experiment

In order to study whether the Chinese medicine compound composition of the present invention inhibits the lateral migration of prostate cancer cells, PC-3 cells were inoculated into a 6-well plate, and conventionally cultivated in a 5% _CO2 incubator at 37 degrees Celsius for 24 hours until the cells grew to 100% full . Replace the serum-free medium and continue to culture for 12 hours. In the culture wells full of cells, scratch with a 200 microliter sterile pipette tip, wash the cells twice with PBS after scratching, absorb the floating cells, and add 1 ml of complete medium to each well. Add different concentrations of traditional Chinese medicine compound compositions to the cell culture wells respectively, put the culture plate into a _CO2 incubator, and continue conventional culture at 37 degrees Celsius for 24 hours. Observe the movement of the cells to the lined part under a microscope and take pictures. Statistically analyze the number of cells migrating into the streaked area in different doses of drug groups to determine the effect of drugs on cell migration.

2. Transwell chamber migration experiment

The permeable polycarbonate film on the bottom of the Transwell chamber has a large number of micropores with a diameter of 8 microns. During the experiment, the Transwell chamber was placed in a 24-well plate, and the polycarbonate film divided each hole of the 24-well plate into upper and lower holes. 1, the lower two chambers, the Transwell chamber is called the upper chamber, and the culture plate is called the lower chamber. The upper chamber is filled with the upper layer culture solution, and the lower chamber is filled with the lower layer culture solution. The upper and lower layer culture solutions are separated by polycarbonate membranes. The cells are seeded in the upper chamber, and because the polycarbonate membrane is permeable, the components in the lower culture medium can induce the cells in the upper chamber to migrate to the lower chamber, so that the cells move from the upper chamber surface of the film to the lower chamber surface, so This experiment is a classic experiment for studying cell movement. In order to study whether the Chinese medicine compound composition of the present invention inhibits the longitudinal migration of prostate cancer cells, a Transwell chamber is used to insert excessive LNCaP cells in the upper chamber, so that the cells will migrate to the lower chamber; while adding different concentrations of the Chinese medicine compound composition, After 12 hours, photos were taken to count the cell migration rate.

3. Transwell assay for cell invasion

Put the Transwell chamber into a 24-well plate, the upper chamber is filled with the upper culture medium, the lower chamber is filled with the lower culture medium, and the upper and lower culture medium are separated by a polycarbonate membrane, the upper chamber of the Transwell chamber is pre-laid with a layer of collagen matrix Matrigel, and then the cells Inoculate it. Because the polycarbonate membrane is permeable, the components in the culture medium of the lower layer can induce the cells in the upper chamber to migrate downward, so that the cells move from the upper chamber surface of the membrane to the lower chamber surface. However, if tumor cells want to move to the lower chamber, they must first use various chemical substances released by themselves to dissolve the collagen matrix, and then move through the dissolved cavity. This process is similar to the infiltration process of tumor cells in the body. To reflect the invasion and infiltration ability of tumor cells. Therefore, this experiment is a classic experiment for studying cell invasion. The tumor cells in the logarithmic growth phase were inoculated into the upper chamber of the Transwell chamber at 5×10 ⁴ cells/well. The drug-dosed group was added with the corresponding concentration of traditional Chinese medicine compound composition, and the control group was added with the same amount of PBS. Add complete culture medium to each of the lower chambers. Incubate for 12 hours at 37 °C in a 5% CO ₂ incubator. Take out the Transwell chamber, wipe the upper side of the Transwell chamber with a cotton swab dipped in water, and wipe off the cells that do not penetrate the membrane. The Transwell chamber was fixed in 4% paraformaldehyde for 10 minutes at room temperature, stained with 1% crystal violet for 10 minutes, and rinsed with tap water. Take pictures under a microscope and count the number of cells in 5 fields of view in each well. An average of 3 replicate wells were set in each group. Statistically compare the number of transmembrane cells in groups with different doses of the compound Chinese medicine composition, and determine the effect of the compound Chinese medicine composition on the cell invasion ability.

Experimental results:

The results are shown in Figure 4, the traditional Chinese medicine compound composition of the present invention can effectively inhibit the lateral migration of PC-3, the vertical migration of LNCaP and the invasion ability of 22RV1 at a lower dose (2.5 μg/ml). The left side of Figure 4 is the experimental white light photo results of the three types of cells, and the right side of Figure 4 is the statistical results of the three types of cells. The experimental results suggest that the traditional Chinese medicine compound composition of the present invention can be used as a potential drug against prostate cancer metastasis.

Example 4: The compound Chinese medicine composition of the present invention inhibits the transcriptional activity of the androgen receptor in vitro

Technical Method: Dual Luciferase Reporter Gene Assay

The plasmids used in this experiment are MMTV-LUC and Renila. MMTV-LUC is a reporter gene plasmid with androgen receptor transcription activation elements (ARresponseelements) and luciferase. After the combination of androgen receptor and androgen, the plasmid can be activated to express luciferase. The substrate luciferin emits fluorescence, and the degree of androgen receptor activation can be detected by detecting the intensity of the fluorescence; the internal reference reporter gene of Renila has the promoter region of renilla luciferase, which can express renilla luciferase, and through the plasmid It can be determined whether the transfection efficiency of each group of cells is consistent, and androgen has no effect on the Renila internal control reporter gene plasmid. In order to initially explore whether the Chinese medicine compound composition of the present invention can inhibit the transcriptional activity of Androgen receptor (AR), we transfected MMTV-LUC and Renila two plasmids, in addition, exogenous wild-type AR was transformed into PC-3 cells that do not express AR; 24 hours after cell transfection, the androgen dihydrotestosterone (Dihydrotestosterone, DHT) was added to activate the expression of MMTV-LUC reporter gene Firefly Luciferase; add different concentrations of traditional Chinese medicine compound composition while adding DHT, lyse the cells after 12 hours of treatment, and measure the fluorescence expression intensity of the two reporter genes. The final result is compared with the activity of the internal reference plasmid Renila Each group of experiments was repeated three times.

Experimental results:

As shown in Figures 5A, 5B, and 5C, the traditional Chinese medicine compound composition of the present invention can dose-dependently reduce the activity of the MMTV-LUC reporter gene induced by androgen (DHT) in different prostate cancer cell lines, and at low concentrations ( About 2.5 μg/mL) can reach the half inhibitory rate (IC ₅₀ ), which proves that the traditional Chinese medicine compound composition of the present invention can inhibit the transcriptional activity of androgen receptor in vitro.

Example 5: The traditional Chinese medicine compound composition of the present invention significantly down-regulates the level of androgen receptor protein and the transcription level of its target gene

Technical method:

1. Polymerase chain reaction (Polymerase chain reaction, PCR) experiment

After the LNCaP cells were treated with different concentrations of traditional Chinese medicine compound compositions, the RNA was isolated and extracted with TRIzol, and the RNA was reverse-transcribed to obtain cDNA. The mRNA expression level of the AR gene was qualitatively detected by reverse transcription-PCR (RT-PCR) with AR-specific primers ; Different AR downstream target gene-specific primers (TMPRSS2, FKBP5 and PSA) were used to detect the mRNA expression levels of the three by fluorescent real-time quantitative PCR (Q-PCR).

2. Western blot experiment

After the LNCaP cells were treated with different concentrations of traditional Chinese medicine compound compositions, the cells were lysed to extract proteins, the proteins were boiled and denatured, and the protein samples were separated by polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. AR antibody was incubated for two hours, and then incubated with fluorescently labeled secondary antibody for one hour, and finally the influence of the traditional Chinese medicine compound composition of the present invention on the expression level of AR protein was detected by LicorOdyssey two-color infrared laser imaging system.

Experimental results:

As shown in Figure 5E, the Western blot experiment (Westernblot) proves that the Chinese medicine compound composition of the present invention can significantly inhibit the protein expression level of the androgen receptor; with the increase of the concentration of the drug, the protein expression of the AR is significantly reduced. It is suggested that the inhibitory effect of the traditional Chinese medicine compound composition of the present invention on the proliferation and migration of prostate cancer tumor cells may be realized by inhibiting the androgen receptor signaling pathway. RT-PCR experiment found that at a certain concentration, the traditional Chinese medicine compound composition of the present invention did not affect the mRNA level of AR ( FIG. 5D ), but significantly inhibited the transcriptional activation of its downstream target genes ( FIG. 5F ). Q-PCR experiments showed that the transcription levels of three classic representative target genes TMPRSS2, FKBP5 and PSA downstream of AR were significantly inhibited by the traditional Chinese medicine compound composition of the present invention. The above experimental results prove from multiple perspectives that the inhibition of the growth and metastasis of prostate cancer by the compound Chinese medicine composition of the present invention is achieved by inhibiting the activity of the androgen receptor signaling pathway.

Example 6: Inhibitory effect of the traditional Chinese medicine compound composition of the present invention in nude mouse prostate cancer subcutaneous tumor-bearing growth model and prostate cancer orthotopic growth and metastasis model

Technical method:

1. Inhibitory effect of Chinese medicine compound composition of the present invention on nude mouse prostate cancer subcutaneous tumor-bearing growth model

A subcutaneous tumor-bearing growth model of prostate cancer in nude mice was established using human prostate cancer cell line 22RV1. Subcutaneously inject 5 million 22RV1 cells into the right back of 7-week-old male immunodeficiency mice (BLAB/c-nude, nude mice). When the subcutaneous tumor grows to about 100 cubic millimeters, the mice are divided into 8 groups (the average tumor volume is the same), followed by the model group, the control group, the Chinese medicine compound (without Ailanthus bark) group, the Chinese medicine compound low-dose group, the Chinese medicine compound high-dose group, the compound Sophora flavescens injection group, cyclophosphamide (Cyclophosphamide, CTX) group, CTX+ Chinese medicine compound high-dose group. The route of administration and the dosage of administration are as follows in turn: the mice in the model group were not treated, the mice in the control group were given an equivalent amount of PBS for intragastric administration (0.1 ml) every day, and the mice in the Chinese medicine compound (without Ailanthus bark) group mice were treated every day. Give an equal amount of Chinese medicine compound (0.1 milliliters) that does not contain Ailanthus bark (0.1 milliliter, the specific decoction method is consistent with the Chinese medicine compound of the present invention, without adding Ailanthus bark crude drug, correspondingly reduce the concentrated final volume, and the actual dose is 15 grams/kg/day), The mice in the low-dose Chinese medicine compound group were given 1/2 amount of Chinese medicine compound mother liquor for intragastric administration (0.05 milliliters, actual dose 7.5 g/kg/day), and the mice in the Chinese medicine compound high-dose group were given Chinese medicine compound mother liquor for intragastric administration (0.1 milliliters) every day. , mother liquor concentration 1.5 gram crude drug/ml, actual dose 15 g/kg/day), compound Kushen injection group mouse is given 0.1 milliliter compound Kushen injection intraperitoneal injection (actual dose 0.6 g/kg/day) every day, CTX Group mice were given intraperitoneal injection of 0.05 milliliters of CTX every day (actual dose 0.05 g/kg/day), and mice in the CTX+Chinese medicine compound high-dose group were given intraperitoneal injection of 0.05 milliliters of CTX and 0.1 milliliters of Chinese medicine compound mother liquor for intragastric administration (actual dose 0.05 g/kg/day). kg/day + 15 g/kg/day). The tumor volume of the subcutaneous tumor bearing tumor on the back of the mouse was measured every 2 days, calculated according to the formula volume=length×width ² ×0.52, and the tumor volume was counted. The body weight of the mice was measured every 2 days. The mice were sacrificed 14 days after the drug administration, and the subcutaneous tumors were taken and photographed.

2. Inhibition of the compound Chinese medicine composition of the present invention on the growth and metastasis model of nude mouse prostate cancer in situ

Transfer the metastatic prostate cancer cell line 22RV1 into a firefly luciferase (Luciferase) expression plasmid (the plasmid has a G418 resistance selection marker), and use G418 to select a stable cell line, the stable cell can express luciferase, luciferase After contacting with its substrate, fluorescein, it will emit fluorescence, and the range and intensity of fluorescence are positively correlated with the location and volume of the tumor, so that the number and location of tumor cells can be detected by the animal live imaging system. The constructed stable cell line was named 22RV1-luc, and the 22RV1-luc cells were mixed with matrigel and injected into the dorsal lobe of the mouse prostate. After the tumor xenograft was successfully transplanted, 22RV1 in the mouse body could be detected by in vivo imaging technology. -Growth and metastasis of luc cells. The specific operation process is as follows: after 7-week-old male immunodeficiency mice were anesthetized, the abdominal cavity was dissected to expose the prostate, and 500,000 22RV1-luc cells were mixed with Matrigel and injected into the dorsal lobe of the mouse prostate. From day 0, the fluorescence value was detected by the in vivo imaging system and the mice were randomly divided into groups and administered. The body weight of the mice was measured every 2 days. When the experiment was carried out to the 15th day, the experiment was terminated, the mice were sacrificed, and the organs of the mice were removed to count the number of metastatic nodules.

Experimental results:

The results are shown in Figure 6. In the subcutaneous tumor-bearing growth model of prostate cancer in nude mice, the size of the subcutaneous tumors in the eight groups of mice was significantly different (Figure 6A). Figure 6B shows that the tumor volume in the model group and the control group was the largest according to the measurement of the growth of the tumor volume with the number of days of the experiment, and there was no significant difference between the two groups, indicating that the modeling was successful. Compared with the control group, the final volume of the tumor in the compound Chinese medicine (without Ailanthus cortex) group was slightly reduced, indicating that there was a certain effect, but the effect was not obvious; the effect of the low-dose Chinese medicine compound group was lower than that of the experiment without The group effect is good, but still not too significant; The effect of the Chinese medicine compound high-dose group is more remarkable, illustrates that the Chinese medicine compound of the present invention can effectively inhibit the growth of prostate cancer tumors; In this experiment, a good tumor suppressing effect has also been shown, but from the experimental results, the suppressing effect is not as strong as that of the Chinese medicine compound of the present invention; the CTX group, as the positive control of traditional chemotherapeutics, has a significant effect on tumors. Inhibition effect; CTX+ Chinese medicine compound high dose group has the most obvious effect on tumor suppression, and is the group with the smallest tumor volume in all experimental groups, illustrating that the Chinese medicine compound composition of the present invention can enhance the tumor suppression effect of traditional chemotherapy drugs. Weight statistics of exfoliated tumors also illustrate similar experimental results (Fig. 6C).

In addition, as shown in Figure 7, in the growth and metastasis model of prostate cancer in situ in nude mice, Figure 7A is the therapeutic effect on the tumor in situ of the prostate in mice after 14 days of medication (n≥5), through the animal live imaging system Take a picture, the colored area in the picture represents the fluorescent signal, indicating that there are tumor cells gathered in this area, and the darker the fluorescence intensity, the larger the tumor. The experimental results are similar to the results of the previous animal experiment, and the effect of the high-dose Chinese medicine compound group is more significant, indicating that the Chinese medicine compound composition of the present invention can effectively inhibit the growth (Fig. 7C) and metastasis (Fig. 7B) of prostate cancer tumors, and this The inhibitory effect was stronger than that of the positive traditional Chinese medicine compound—Compound Kushen Injection, which was equivalent to the inhibitory effect of the traditional chemotherapy drug CTX; the treatment of the traditional Chinese medicine compound composition in the above groups had no significant effect on the body weight of the mice (Figure 7D), which preliminarily shows that this The invented traditional Chinese medicine compound composition has less toxic and side effects on the body under the effective dose; the body weight of the mice under CTX treatment decreased. Combined with the experimental results of the mouse body weight, CTX, as a chemotherapeutic drug, has certain toxicity to the body, causing a slight decrease in the body weight of the mice, and compared with the body weight of the mice in the CTX group, the compound Chinese medicine composition of the present invention is comparable to the body weight of the mice. When CTX is used in combination, the inhibitory effect on tumor growth and metastasis is the most significant among all groups, and the body weight of the mice can be restored (Figure 7D), which shows that the Chinese medicine compound composition of the present invention can not only enhance the anti-tumor effect of traditional chemotherapy drugs It can also improve the damage to the body caused by the toxicity of traditional chemotherapeutic drugs, thus proving that the traditional Chinese medicine compound composition of the present invention has the effect of synergizing and reducing toxicity of traditional chemotherapeutic drugs.

Example 7: The effect of the Chinese medicine compound composition of the present invention on the synergistic and attenuating effect of tumor suppression in tumor-bearing nude mice and the influence of immune function indicators

Technical method:

1. The influence of Chinese medicine compound composition of the present invention on the spleen index of nude mice

After observing the differences in the quality of life and living conditions of the nude mice in the model group, the control group, and the drug-administered group, especially the tumor-bearing mice in the chemotherapy drug CTX group showed varying degrees of rough skin, listlessness, sluggishness, unresponsiveness and weight loss. However, the mice in the compound Chinese medicine combination medication group of the present invention have smooth skin, good spirits, active activities, normal body weight, and small tumor masses in situ. Therefore, it is speculated that the compound Chinese medicine composition of the present invention can enhance the effect of chemotherapeutic drugs. The effective attenuation effect may be achieved by improving the immune function of mice and improving various immune indicators. Therefore, the nude mice of the orthotopic tumor growth and metastasis model in Example 6 were sacrificed after the experiment, and the spleen weight and body weight were weighed to calculate the spleen index.

2. The influence of Chinese medicine compound composition of the present invention on the number of serum leukocytes in nude mice

After the nude mice of the orthotopic tumor growth and metastasis model in Example 6 were sacrificed, blood was taken from the eye sockets, and 20 microliters of blood was accurately sucked with a pipette gun. There was no blood on the outer wall of the pipette tip, and the blood was quickly and gently put into a container containing 380 microliters WBC diluent, shake well. According to the method of counting red blood cells, drop the white blood cell suspension into the counting pool, let it stand for 2 to 3 minutes, and count after the white blood cells sink. Under low magnification, the white blood cells are round, the pulp is translucent, and the nuclei are purple-black with a slight refraction, which can be distinguished from impurities. The number of white blood cells = the total number of 5 large grids × 500, and the differences between the groups were counted after counting.

3. The influence of the Chinese medicine compound composition of the present invention on the T cell proliferation response induced by concanamycin A (ConcanamycinA, ConA)

After the nude mice of the orthotopic tumor growth and metastasis model in Example 6 were killed after the experiment, the spleen was weighed and put into a pre-prepared test tube containing pre-cooled PBS, and the spleen and washing solution in the test tube were poured into a plate , put the spleen into the cell strainer and squeeze the spleen with tweezers to obtain a cell suspension. Afterwards, collect the cell suspension into a test tube, centrifuge at 1500 rpm for 6 minutes, pour off the washing solution, add red blood cell lysate for 3 minutes, and add the washing solution PBS after observing that the color of the liquid becomes lighter, and filter with a filter membrane In another test tube, centrifuge again at 1500 rpm for 6 minutes, pour off the washing solution and cell culture solution, add ConA (working concentration 5 μg/ml) to stimulate the proliferation of T lymphocytes, and place in a constant temperature incubator at 37 degrees Celsius (humidity 95%, CO ₂ concentration 5%) for 72 hours. The SRB method measures the absorbance value (A value) of T cells on a microplate reader to detect their proliferation.

Experimental results:

The results are shown in Table 1. The synergistic and attenuating effect of the Chinese medicine compound composition of the present invention on tumor inhibition in tumor-bearing nude mice and three representative indicators of the immune function of tumor-bearing nude mice——spleen index, white blood cell number and sword bean The effects of ConcanamycinA (ConA)-induced T cell proliferation were significantly different.

Table 1 shows the statistical results of three typical indicators of mouse immune function. Compared with the control group of the model group, there was no significant difference in the three indexes between each administration group of the compound Chinese medicine composition. Compared with the model group, the three indexes of the CTX group were significantly different, and the toxicity of the chemotherapeutic drug CTX to the body was verified again, and the immune system was damaged to a certain extent, and the Chinese medicine compound composition of the present invention was combined with it. After that, it can significantly restore its toxic effect on mice, and significantly improve the quality of life of tumor-bearing mice. Experimental results prove that compared with CTX, the Chinese medicine compound composition of the present invention has less toxic and side effects, does not cause immune organ atrophy, and has obvious synergistic and attenuating effects when used in combination with CTX, suggesting that the Chinese medicine compound composition of the present invention has anti-inflammatory effects. There is a direct correlation between tumor activity and immunomodulatory effects. In summary, the traditional Chinese medicine compound composition of the present invention not only has the function of inhibiting the growth and metastasis of prostate cancer, but also enhances the antitumor effect of traditional chemotherapeutic drugs, and improves the damage to the body caused by the toxicity of traditional chemotherapeutic drugs, thereby It is proved that the traditional Chinese medicine compound composition of the present invention has the effect of synergizing and reducing toxicity of traditional chemotherapy drugs.

Table 1 The effect of the traditional Chinese medicine compound composition of the present invention on the synergistic attenuation and immune function indicators of tumor suppression in tumor-bearing nude mice

Note: Compared with the model group and the control group, *P<0.05, **P<0.01

Compared with CTX group, ^^P<0.01

Embodiment 8: Verification of the preferred scheme of the compound Chinese medicine composition of the present invention

In order to verify the preferred effect of the ratio of each component of the Chinese medicine compound composition of the present invention, take the dry product of the six-flavored Chinese medicinal material combined in different proportions as shown in Table 2, make a Chinese medicinal water decoction according to the method in Example 1 and compare with the implementation The Chinese medicine compound composition in the example 1 carries out the parallel experiment of embodiment 2-7, finds that in the present embodiment different Chinese medicine compound compositions 8a-8g drug effect is the same as the Chinese medicine compound composition in the embodiment 1, all can reach implementation The same technical effect of example 2-7 has therefore verified the preferred scheme of each component proportioning in the Chinese medicine compound composition of the present invention.

The Chinese medicine compound composition of table 2 different proportioning

The above-mentioned embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the essence of the content of the present invention shall fall within the protection scope of the present invention.

Claims (15)

1. a Traditional Chinese medicine compound composition, is characterized in that, comprises the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 4 ~ 12 parts, Herba Hedyotidis Diffusae 12 ~ 30 parts, Rhizoma Curcumae 3 ~ 15 parts, Radix Salviae Miltiorrhizae 15 ~ 30 parts, Spica Prunellae 5 ~ 15 parts, 4 ~ 12 parts, Radix Glycyrrhizae.

2. Traditional Chinese medicine compound composition according to claim 1, is characterized in that, comprises the Chinese crude drug of following parts by weight: ailanthus altissima (mill.) swingle skin 6 ~ 9 parts, Herba Hedyotidis Diffusae 15 ~ 24 parts, Rhizoma Curcumae 4 ~ 12 parts, Radix Salviae Miltiorrhizae 18 ~ 21 parts, Spica Prunellae 6 ~ 10 parts, 6 ~ 9 parts, Radix Glycyrrhizae.

3., as the preparation method of the Traditional Chinese medicine compound composition of claim 1 or 2, it is characterized in that, comprise the following steps:

(1) soak: get Six-element Chinese crude drug described in claim 1 or 2, add distilled water immersion, obtain crude drug distilled water mixture;

(2) decoct: the crude drug distilled water mixture described in step (1) is decocted, obtains Chinese medicine crude extract;

(3) concentrated: Chinese medicine crude extract step (2) prepared concentrates, and obtains the concentrated liquid of tradition Chinese medicine;

(4) degerming: to get the concentrated liquid of tradition Chinese medicine that step (3) prepares degerming by filtering with microporous membrane, namely obtain described Traditional Chinese medicine compound composition.

4. Traditional Chinese medicine compound composition according to claim 1 is applied to the application of preparation treatment malignant tumor medicine.

5. application according to claim 4, is characterized in that, described malignant tumor comprises carcinoma of prostate, breast carcinoma, pulmonary carcinoma, colon cancer, the brain cancer, skin carcinoma, bladder cancer, renal carcinoma.

6. Traditional Chinese medicine compound composition according to claim 1 is applied to the application of preparation treatment by the medicine of the propagation of the abnormal amplification of androgen receptor and the malignant cell caused that suddenlys change, growth, migration and infiltration.

7. Traditional Chinese medicine compound composition as claimed in claim 1 is applied to the purposes of preparation treatment carcinoma of prostate medicine.

8. Traditional Chinese medicine compound composition according to claim 1 is applied to the purposes of apoptosis suppressing the growth of prostate gland cancer cell, propagation, transfer, migration, invasion and attack and promotion prostate gland cancer cell.

9. purposes as claimed in claim 7, it is characterized in that, described carcinoma of prostate is castration-resistant prostate cancer or androgen-dependent prostate cancer.

10. Traditional Chinese medicine compound composition according to claim 1 is applied to the purposes of the transcriptional activity of the transcriptional activity of the androgen receptor of vitro inhibition dihydrotestosterone induction and the androgen receptor of disappearance ligand domain, or for vitro inhibition estrogen receptor activity.

Traditional Chinese medicine compound composition according to claim 1 is applied to the propagation of Prostatic cancer cell lines 22RV1 of vitro inhibition androgen receptor sudden change, the purposes of the propagation of suppression androgen-dependent prostate JEG-3 LNCaP by 11..

12. purposes Traditional Chinese medicine compound composition according to claim 1 being applied to the migration of vitro inhibition prostate gland cancer cell PC-3 and LNCaP and 22RV1, or the purposes of androgen receptor mutant clone 22RV1 cell proliferation that vitro inhibition bicalutamide or MDV3100 fail to respond to any medical treatment.

Traditional Chinese medicine compound composition according to claim 1 is applied in body the transfer suppressing prostate gland cancer cell by 13., or suppresses growth and the transfer of castration-resistant prostate cancer in body, or in Mice Body, suppress growth or the transfer of prostate gland cancer cell 22RV1.

14. purposes as described in claim 4-13, it is characterized in that, described Traditional Chinese medicine compound composition and pharmaceutically acceptable carrier are made the medicine of injectable fluid, aerosol, emulsifiable paste, gel, pill, syrup, transdermal patch, capsule, microcapsule, tablet, granule or oral liquid formulation.

15. application according to claim 4-13, is characterized in that, described Traditional Chinese medicine compound composition is used alone or with other drug conbined usage.