CN103690531A - Application of ethyl coumarin-3-carboxylyl L-theanine and the like in preparation of product used for preventing and treating disease such as cancers - Google Patents

Abstract

The invention relates to the field of medical technology. Ethyl coumarin-3-carboxylyl L-theanine, a novel synthetic compound, can inhibit the growth and invasion of cancer cells of human lung cancer, breast cancer, liver cancer, gastric cancer, colon cancer, prostatic cancer, pancreatic cancer, cervical cancer, lymphadenoma, leucocythemia, melanoma, and the like, and can remarkably inhibit the growth and metastasis of the tumors in a body, with an inhibiting effect exceeding the inhibiting effects of anti-cancer drugs. The mechanism of action involves the down regulation of receptors which are closely related to growth inhibition, invasion inhibition and metastasis inhibition of tumors, signal transduction regulation and control proteins, the kinase level and combination of a nuclear factor and DNA, and also upper regulation of factors such as cancer-inhibiting proteins and cell cycle inhibition proteins. The ethyl coumarin-3-carboxylyl L-theanine can directly inhibit the activity of histone deacetylase, the activity of histonemethylferase EZH2, and combination of a inflammatory factor NF-[Kabba]B and DNA. The activity of the ethyl coumarin-3-carboxylyl L-theanine exceeds the activity of the anti-cancer drugs. The invention provides new applications of the ethyl coumarin-3-carboxylyl L-theanine in prevention and treatment of disease such as tumors, inflammation, heart and cerebral vessels and immunodeficiency.

Description

The fragrant acid of tea second waits the application in the product in preparation with diseases such as prevention and treatment cancers

Technical field

The present invention relates to field of medical technology, be specifically related to the application in the diseases inhibitors such as preparation prevention and treatment tumor, inflammation and cardiovascular and cerebrovascular disease and immunodeficiency of the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) thereof and theanine ethyl ester (TE).

Background technology

Improper growth, invasion and attack and the transfer of the malignant tumor such as people's pulmonary carcinoma, breast carcinoma, hepatocarcinoma, colon cancer, carcinoma of prostate, cancer of pancreas, cervical cancer and lymphoma and melanoma is to cause annual number with the major reason of ten thousand note deaths, its morbidity also keeps certain level, has become the major disease of serious harm human health.Due to the toxic and side effects of clinical chemotherapy and radiotherapy, limited its effectively preventing.Theanine (having another name called Teaammonia acid), for a kind of characteristic aminoacid that shows its quality in Folium Camelliae sinensis, owing to being the food composition having no side effect, becomes the food additive of unrestricted consumption, is widely used in food industry; Research shows: theanine can increase the concentration of cancer therapy drug in tumor and Synergistic treatment human ovarian cancer (Sadzuki et al., Toxicol Lett.123:159-67,2001).The theanine that experimental results show that we are previous has effect (Liu, et al., Cytotechnology59:211-217,2009 that suppress hepatocarcinoma and pulmonary carcinoma; Zhang et al.Biosci Biotechnol Biochem.2002,66 (4): 711-6.).The present invention has formed the fragrant acid of a kind of new compound tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) and theanine ethyl ester (TE) by theanine by chemical method, and its anti-tumor activity surpasses cancer therapy drug and theanine.

Histone methylferase EZH2 inhibitor and deacetylase protein enzyme (HDAC) inhibitor are as SAHA (suberoylanilide hydroxamic acid), valproic acid, in the U.S., I phase or II phase blood and entity tumor clinical trial have been carried out with sodium butyrate etc., EZH2 and HDAC comprise breast carcinoma in the cancer of various human, pulmonary carcinoma, carcinoma of prostate, leukemia, cancer of pancreas, cervical cancer, overexpression in the malignant tumor such as intestinal cancer and hepatocarcinoma, the U.S. has carried out a large amount of research to its effect cancer therapy drug target spot, these EZH2 inhibitor and hdac inhibitor are widely used in the treatment of various tumor diseases, be considered to application prospect preferably potential new type anticancer medicine ( yamaguchi etal., Cancer Sci., 10:355-62,2010, Denis, et al., Clin Exp Metastasis 25:183-189,2008, Kelly et al., Nat Clin Pract Oncol2:150-157,2005, Mart í nez-Iglesias et al., clin Transl Oncol.10:395-8,2008).

Nucleoprotein factor NF-κ B is considered to promote the protein factor of the diseases such as kinds of tumors and inflammation and cardiovascular and cerebrovascular vessel, immunodeficiency, just becoming important drugs target spot (the Ishii et al. of this class disease of control, J Clin Biochem Nutr.50:91-105,2012).

The tumor signal of high-caliber receptor VEGFR, EGFR and c-Met, improper high expressed conducts relevant protein factor K-Ras, H-Ras, Akt, Cyclin D1, β-catenin occurs relevant with deterioration development with Bcl-2 to kinds of tumors, and rise tumor suppressor protein p53, p21, E-cadherin, Caspase3, Bax and endochylema cytochrome C level have shown the inhibitory action of multiple growth of cancer cells, invasion and attack and (or) transfer (Cengel, et al., Neoplasia9:341-8,2007; Huang et al., biochem Pharmacol.77:794-803,2009; Prasad et al., oncology.73:112-7,2007), therefore, these cancer correlation factors become the important drugs target spot of potential anti-curing cancers, can effectively affect the application prospect that these protein factor expressions and active compound have extensive anti-curing cancers.

The fragrant acid of tea second provided by the invention (TEC) and midbody compound 3-carboxylic acid coumarin (C) thereof and theanine ethyl ester (TE) have level and the activity of the above-mentioned a series of protein factors of significant impact, in the application of the diseases inhibitors such as preparation prevention and treatment tumor, inflammation, cardiovascular and cerebrovascular disease and immunodeficiency, hold out broad prospects.

Summary of the invention

1, the object of this invention is to provide the application in preparation prevention and treatment tumor and inflammation and cardio-cerebrovascular diseases inhibitor of the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) thereof and theanine ethyl ester (TE).

2, the invention provides the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) and the application of theanine ethyl ester (TE) in preparing histone methylferase EZH2 inhibitor.

3, the invention provides the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) and the application of theanine ethyl ester (TE) in preparing histone deacetylase (HDAC) inhibitor.

4, the invention provides the application in the factor NF-kB inhibitor of the disease associations such as preparation short tumor, inflammation and cardiovascular and cerebrovascular disease and immunodeficiency of the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) thereof and theanine ethyl ester (TE).

5, the invention provides the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin (C) thereof and theanine ethyl ester (TE) at the factor VEGFR of the disease associations such as preparation tumor, inflammation and cardiovascular and cerebrovascular disease and immunodeficiency, EGFR, c-Met, K-Ras, H-Ras, Akt, Cyclin D1, β-catenin, Bcl-2/Bax inhibitor and rise p53, p21, E-cadherin, Caspase3, the application in the ratio activator of endochylema and mitochondrial cytochrome C.

6, technical scheme provided by the invention is as follows:

The compound that is used for the treatment of tumor, its name is called the fragrant acid of tea second (TEC), full name is 5-ethylamino-5-oxo-2-(2-oxo-2H-.alpha.-5:6-benzopyran-3-formamido) ethyl valerate, and the chemical structural formula of its midbody compound 3-carboxylic acid coumarin (C) and theanine ethyl ester (TE) is as follows:

Suc as formula the compound shown in (I), physical behavior is white powder solid, and fusing point is in 180 ° of above decomposition of Celsius temperature.

Suc as formula the compound shown in (I), it is pharmaceutically acceptable carrier; Described pharmaceutically acceptable carrier comprises any material that is suitable for injection.Be preferably water for injection, or emulsifying agent and other pharmaceutical carriers.

Preparation method suc as formula compound shown in (I), mainly comprises the following steps:

1, the preparation of theanine ethyl ester, as shown in structural formula (II);

2, by the 3-carboxylic acid coumarin shown in structural formula (III) and the further fragrant acid of synthetic tea second of theanine ethyl ester, as shown in structural formula (I).

React total step as follows:

Step 1: prepare theanine ethyl ester

Theanine is dissolved in ethanol (referring to every liter of dissolve with ethanol 87-91g gram theanine) according to the ratio of 87-91g/L, with backward system, slowly adding thionyl chloride volume ratio is 17-19: 1 (volume ratio that refers to lysate and the thionyl chloride of theanine and ethanol), mixture stirs after 1-2 hour at ambient temperature, the mixture producing is concentrated under reduced pressure, obtain as the theanine ethyl ester of structural formula (II).

Step 2: prepare the fragrant acid of tea second (TEC)

20-22g theanine ethyl ester is dissolved in 2-2.2L anhydrous methylene chloride, adds 27--29g3-carboxylic acid coumarin, adds respectively 0.20-0.22L DIPEA (diisopropylethylamine), 76-78g EDCI.Mixture stirs 1-2 hour at ambient temperature, follow concentrating under reduced pressure, except desolventizing, by column chromatography, carry out purification, obtain as 5-ethylamino-5-oxo-2-of structural formula (I) (2-oxo-2H-.alpha.-5:6-benzopyran-3-formamido) ethyl valerate, being called for short the fragrant acid of tea second, is white powder solid.

7, the compound of the present invention as shown in structural formula (I), can be used for collaborative radiation and chemotherapy treated with combined medication tumor, in the application for the preparation of in Combined with Radiotherapy and chemotherapy tumour medicine.

8, the compound of the present invention as shown in structural formula (I), for 1%* is subcutaneous or muscle or vein or abdominal cavity (2%*) injection or oral liquid.

Described tumor comprises pulmonary carcinoma, breast carcinoma, hepatocarcinoma, rectal cancer, colon cancer, carcinoma of prostate, gastric cancer, esophageal carcinoma, laryngeal carcinoma, leukemia, lymphoma, melanoma, uterus carcinoma, ovarian cancer, skin carcinoma, bronchogenic carcinoma, bronchiolar carcinoma, carcinoma of urethra, renal carcinoma, oral cancer, cancer of vagina, cancer of biliary duct, cancer of pancreas, bladder cancer, nasopharyngeal carcinoma and other various tumors.

This compound at killing tumor cell effectively, can strengthen the curative effect of radiation and chemotherapy Drug therapy tumor, the drug effect that this compound suppresses tumor surpasses cancer therapy drug and theanine, and toxic and side effects reduces greatly, without obvious toxic-side effects.

The compound that the present invention proposes can be integrated the effect that chemotherapy and potentiation radiotherapy are integrated, be have application more extensively, better efficacy, toxic and side effects is less, indication is wider, potential using value and the larger new type antineoplastic medicine of market efficiency.

The 3-carboxylic acid coumarin of the fragrant acid of the resulting tea second of the present invention and theanine ethyl ester and use can pass through muscle, subcutaneous, vein and lumbar injection or oral, to various people's cancers and animal cancerous cell with to the treatment of animal people cancer transplanted tumor, is all better than theanine and more clinical cancer therapy drug rings as phosphamide and rhEndostatin etc.

The specific embodiment

Below provide the specific embodiment of the present invention, be used for that the present invention is further illustrated.

(in the application's book, all temperature that relate to are Celsius temperature).

Embodiment 1

A kind of compound for the treatment of tumor of the present embodiment, its name is called 5-ethylamino-5-oxo-2-(2-oxo-2H-.alpha.-5:6-benzopyran-3-formamido) ethyl valerate, is called for short the fragrant acid of tea second (TEC), has the chemical structural formula shown in formula (I):

Suc as formula the compound shown in (I), physical behavior is white powder solid, and fusing point is in 180 ° of above decomposition Celsius;

Suc as formula the compound shown in (I), it is pharmaceutically acceptable carrier; Described pharmaceutically acceptable carrier comprises any material that is suitable for injection, is preferably water for injection, or emulsifying agent, liposome, nanometer formulation and other pharmaceutical carriers.

Preparation method suc as formula the fragrant acid of the second of compound tea shown in (I), mainly comprises the following steps:

1, the preparation of intermediate theanine ethyl ester, as shown in structural formula (II);

2, by 3-carboxylic acid coumarin and the further fragrant acid of synthetic tea second of theanine ethyl ester, as shown in structural formula (I).

Concrete steps

Step 1: prepare theanine ethyl ester

Theanine is dissolved in ethanol (referring to every liter of dissolve with ethanol 87g gram theanine) according to the ratio of 87g/L, with backward system, slowly adding thionyl chloride volume ratio is 55ml, mixture stirs after 1 hour at ambient temperature, the mixture producing is concentrated under reduced pressure, obtain as the theanine ethyl ester of structural formula (III).

Step 2: prepare the fragrant acid of tea second (TEC)

20g theanine ethyl ester is dissolved in 2L anhydrous methylene chloride, adds 27g3-carboxylic acid coumarin, adds respectively 0.21L DIPEA (diisopropylethylamine), 76g EDCI.Mixture stirs one hour at ambient temperature, follow concentrating under reduced pressure, except desolventizing, by column chromatography, carry out purification, collect product and obtain the fragrant acid of tea second (TEC) as shown in structural formula (I), this product is pale yellow powder shape solid, fusing point 180 ° of above decomposition Celsius, and the following structural features of this compound molecule:

¹H?NMR(500MHz，CDCl ₃)δ1.11(t，3H，J＝7.2Hz)，1.28(t，3H，J＝7.1Hz)，2.17-2.28(m，3H)，2.31-2.40(m，1H)，3.23-3.28(m，2H)，4.10-4.13(m，1H)，4.18-4.22(q，2H，J＝7.1Hz)，5.61(br?s，1H)，6.90(dt，1H，J＝7.5，0.8Hz)，6.97(d，1H，J＝8.2Hz)，7.28(dd，1H，J＝7.7，1.6Hz)，7.34(dt，1H，J＝8.5，1.6Hz)，8.39(s，1H)，12.96(br?s，1H)； ¹³C?NMR(125MHz，CDCl ₃)δ14.2，14.8，29，32，34，61，70，117，118.5，118.9，131，132，160，167，170，171；ESI-MS?m/z375[M+1].

Embodiment 2

The difference of the present embodiment and embodiment 1 is

Step 1: prepare theanine ethyl ester

Theanine is dissolved in ethanol (referring to every liter of dissolve with ethanol 88g gram theanine) according to the ratio of 88g/L, with backward system, slowly adding thionyl chloride volume ratio is 56ml (volume ratio that refers to lysate and the thionyl chloride of theanine and ethanol), mixture stirs after 1.5 hours at ambient temperature, the mixture producing is concentrated under reduced pressure, obtain as the theanine ethyl ester of structural formula (II).

Step 2: prepare the fragrant acid of tea second (TEC)

21g theanine ethyl ester is dissolved in 2.1L anhydrous methylene chloride, adds 28g3-carboxylic acid coumarin, adds respectively 0.21L DIPEA (diisopropylethylamine), 77g EDCI.Mixture stirs 1.5 hours at ambient temperature, and then concentrating under reduced pressure, except desolventizing, carries out purification by column chromatography, obtain as the fragrant acid of the tea second of structural formula (I) (TEC), and be white powder solid.

Embodiment 3

The difference of the present embodiment and embodiment 2 is

Step 1: prepare theanine ethyl ester

Theanine is dissolved in ethanol (referring to every liter of dissolve with ethanol 91g gram theanine) according to the ratio of 91g/L, with backward system, slowly adding thionyl chloride volume ratio is 57ml (volume ratio that refers to lysate and the thionyl chloride of theanine and ethanol), mixture stirs after 2 hours at ambient temperature, the mixture producing is concentrated under reduced pressure, obtain as the theanine ethyl ester of structural formula (II).

Step 2: prepare the fragrant acid of tea second (TEC)

22g theanine ethyl ester is dissolved in 2.2L anhydrous methylene chloride, adds 32g3-carboxylic acid coumarin, adds respectively 0.22L DIPEA (diisopropylethylamine), 78g EDCI.Mixture stirs 2 hours at ambient temperature, and then concentrating under reduced pressure, except desolventizing, carries out purification by column chromatography, obtain as the fragrant acid of the compound tea second of structural formula (I) (TEC), and be white powder solid.

Embodiment 4

The preparation of the fragrant acid of tea second (TEC) sodium-salt parenteral solution

According to 1: 1 ratio, tea second is fragrant sour, add after 100% ethanol or other suitable organic solvent dissolution, with the NaOH of 0.2N and the HCL of 0.2N of water for injection preparation, regulate pH to 7-7.5, then, add normal saline dilution and be settled to 1000 times of cumulative volumes (being equivalent to concentration is the fragrant acid solution of tea second of 0.1%*), then bacteriological filtration pressurizes, gained filtrate is distributed in 1ml glass ampoule bottles, sealing by fusing by sampling pyrogenic test result negative and in efficient liquid phase chromatographic analysis result preparation the fragrant acid content of tea second consistent with sign, obtain the fragrant acid of 0.1% tea second (TEC) sodium-salt parenteral solution.

Embodiment 5

The fragrant acid of noval chemical compound tea second (TEC) and theanine ethyl ester (TE) etc. are to various human carcinoma cell line's deactivations

Reference literature method (Zhang Y, et al., Cytotechnology2009,59 (3): 191-200) measured the In vitro culture such as noval chemical compound (TEC) (the fragrant acid of tea second) and TE (theanine ethyl ester), C (3-carboxylic acid coumarin) and positive control cancer therapy drug to various human cancer cell deactivations, the results are shown in following table-1.

1, cell line and cell culture: people's pulmonary carcinoma A549, human breast carcinoma MCF-7, human prostata cancer PC-3, human lymphoma cell U937, people's hepatocarcinoma SMMC7721 and HepG2, human colon carcinoma HT29, human pancreas cancer PANC-1 and human cervical carcinoma Hela cell system, mouse melanoma B16 and Lewis lung cancer cell line are purchased from U.S. American Type Culture Collection.These cells are cultivated with DMEM and RPMI-1640 culture fluid respectively.

2, instrument and equipment:

CO2 gas incubator: 3111 types, U.S. Thermo company; Inverted fluorescence microscope: TE2000-U type, Japanese Nikon company.Inverted microscope: CKX31 type, Japanese Olympus company; Table-type high-speed refrigerated centrifuge: 5810R type, German Eppendorf company; Micro sample adding appliance: German Eppendorf company; Cell culture plastic board (96 hole): BD company; Microplate reader: SYNERGY HT type, U.S. BIO-TEK company; Ice machine: XB70 type, GRANT company.

Experiment adopts the mtt assay test tea fragrant acid of second (TEC), theanine ethyl ester (TE) and 3-carboxylic acid coumarin (C) and theanine (T) inhibitory action to growth of cancer cells under condition in vitro, and the blue staining of tongue sweet smell is verified it.Step is as follows:

1, main agents, cell line and instrument:

Cancerous cell line and instrument are as above described in 1 and 2.

RPMI1640 and DMEM culture fluid: Hyclone company; Inactivated fetal bovine serum: Hyclone company;

Trypsin trypsin): Amersco company; 0.4%* trypan blue: Sigma company;

Tetramethyl azo azoles salt (MTT): Sigma company;

2, experimental procedure:

(1) with the cancerous cell of 0.25% trypsinization exponential phase, make single cell suspension, adjusting cell concentration is 5 * 10 ⁴/ mL, is inoculated in 96 well culture plates with the 100 every holes of μ l;

(2) culture plate is moved into 37 ℃, in 5%CO2 saturated humidity incubator, cultivate 24h; Add the fragrant sour 1-1000 μ M/L of tea second, 3-carboxylic acid coumarin (1-1000 μ M/L), theanine ethyl ester (1-1000 μ M/L), theanine (1-1000 μ M/L), or positive drug to contrast its final concentration be 1-1500 μ M/L.If control wells (only adding cell suspension 200 μ l) and without medicine blank hole (containing solvent 0.01% ^*dMSO), all establish 8 multiple holes, put 37 ℃, 5% for every group ^*in CO2 saturated humidity incubator, cultivate;

(3) respectively at taking out 96 orifice plates after dosing 48h, careful suction abandoned former culture medium, and every hole adds DMEM culture medium and 10 μ lMTT (5mg/mL) solution of 100 μ l serum-frees, continues to cultivate after 4h, stops cultivating;

(4) careful suction abandoned supernatant in hole, and every hole adds 1501DMSO, and room temperature concussion 10-15min, fully dissolves crystallization.

(5) colorimetric: select 570nm wavelength, measure each hole light absorption value (A value) in microplate reader, record result;

(6) experiment repeats 3 times;

(7) experimental result is calculated as follows: relative survival rate=(each experimental group A value/cell control group A value) * 100%*

The calculating of middle effect concentration (IC50, drug level when suppression ratio is 50%* claim again half-inhibition concentration):

With regression equation, obtain the IC50 of the fragrant acid of tea second etc.

3, experimental result (in Table 1)

The fragrant acid of table 1 tea second (TCE) suppresses the growth of human cancer cell

Note: * p < 0.05 contrasts and compares with solvent; The method of relevant MTT test cell growth is shown in above-mentioned experimental technique part.The above results is used the fragrant blue staining of tongue to show that to its checking drug effect is identical errorless.

§ is that the fragrant acid of tea second (TCE), theanine ethyl ester (TE), 3 carboxylic acid coumarins (C) and theanine (T) philosophy are processed the drug level (IC50) that after 72 hours, 50% cancerous cell survives.

Embodiment 6

The fragrant acid of tea second waits the experiment that suppresses the interior various human cancer growth of xenografted of nude mouse and transfer

Reference literature method (George N.Naumov, et al.Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance Clin Cancer Res2009,15:3484-3494; Yang Zhenzhou etc.) measured the inhibitory action to various human cancer animal-transplanted tumor tumor growth such as the fragrant acid of noval chemical compound tea second (TEC) and theanine ethyl ester (TE), the results are shown in following table-2.

1, laboratory animal, cell line, main agents and instrument:

SPF level BALB/c nude mouse and C57/BL6J black rat, 4～5 week age, 18～22g, female, purchased from Beijing China Fukang Experimental Animal Center, animal credit number SCXK (capital) 2009-0004.Raise in SPF level Animal Lab.; IVC independent air-blowing-returning cage, Suzhou Su Hang science and technology equipment company limited; People's pulmonary carcinoma A549, people's hepatocarcinoma SMMC7721, Lewis lung cancer and Other Instruments are as mentioned above.RPMI1640 and DMEM culture fluid: Hyclone company; Inactivated fetal bovine serum: Hyclone company ,-20 ℃ of preservations; Trypsin trypsin): Amersco company; 0.4% trypan blue: Sigma company;

2, experimental procedure:

(1) foundation of cell culture and transplanted tumor animal model: human cancer cell strain is incubated at respectively containing 10% ^*in the DMEM of hyclone or RPMI-1640 culture fluid, at 37 ℃, 5%CO ₂condition under cultivate, collecting the cell of exponential phase and being prepared into concentration is 2 * 10 ⁷the single cell suspension of individual/ml, in superclean bench, every nude mouse, in the subcutaneous 0.1ml cell suspension of inoculating respectively of back of thighs, is observed each injection point every day and has or not red and swollen ulceration.About 2-5 is after week, and obvious skin mound appears in injection site, and the subcutaneous nodule of diameter 10-15mm all appears in all nude mices, and transplanted tumor model is set up; For setting up Mice Bearing Lewis Lung Cancer metastasis model, being prepared into concentration is 6 * 10 ⁶the single cell suspension of individual/ml, intravenous injection 0.1ml cell suspension divides into groups to start medication on the 2nd day after entering C57/BL6J black rat tail vein, and dosage and mode are same as nude mice and see following explanation.

(2) nude mice grouping and administration: in inoculation, about 2-5 is divided into 3 groups by the nude mouse of built vertical subcutaneous transplantation tumor model after week, 7 every group at random, negative control group (solvent DMDO0.05%, only, every 2-3 day is once for lumbar injection 0.2ml/, medicine and composition are lumbar injection (25mg/kg): cyclophosphamide positive controls, and rhEndostatin positive controls, fragrant sour group of tea second, theanine ethyl ester group, every 2-3 day, once 3-5 was all in medication treatment, impact for detection of drugs on in-vivo tumour EZH2H and HDAC enzyme enzymatic activity, in medication, after 6 hours, distinguish tumor resection, extract respectively total protein and extract nucleoprotein and detect and analyze for enzymatic activity, with negative control, it is DMSO (0.05%) solvent, positive control SAHA (suberoylanilide hydroxamic acid) 25mg/kg Mus) or the fragrant acid of compound tea second (TCLC/25mg/kg Mus) or theanine ethyl ester (TE/25mg/kg Mus) process mice and get tumor after 6 hours, total protein and nucleoprotein extract extract (total protein lysate by total protein lysate and nucleoprotein lysate, nucleoprotein lysate and PMSF are purchased from green skies biotechnology research institute), 1mg tumor tissues adds and in people 1mL lysate, adds 10 μ l PMSF, piping and druming mixes repeatedly, place 15min on ice, sample is transferred in 1.5mL EP pipe, 14000r/min, 4 ℃ of centrifugal 10min, transfer in aseptic EP pipe supernatant protein extract for detection of enzymatic activity.

(3) observation of nude mice and black rat tumor tumor bulk-growth: observe every day nude mice active situation (comprising feed, defecation character, the mental status etc.), transplanted tumor go out tumor time and growing state, the size of body weight of measurement in every 2-3 days, tumor body, major diameter a and minor axis b (mm) with vernier calliper dipstick metering tumor body, calculate its volume, volume V=π 1/2ab2 (mm3);

(4) execution of animal: treatment experiment finishes, anesthetized mice is taken a picture; Disconnected neck is put to death nude mice, under aseptic condition, peels off tumor tissue, takes tumor weight; Detection of drugs is to the pathology of each Organ and tissue and toxic effect situation.

(5) tumor heavy (or pulmonary metastases) of organizing by experiment transplanted tumor compare with blank group tumor weight (or pulmonary metastases) calculating tumour inhibiting rate, i.e. tumour inhibiting rate (%)=(the average tumor of 1-experimental group weigh/contrasts average tumor weight) * 100%.

3. experimental result (in Table 3)

The inhibitory action to animal-transplanted tumor tumor growth and transfer # such as the fragrant acid of table 2 tea second (TEC)

Note: * p < 0.05; Relevant method of testing is shown in above-mentioned experimental technique part.§ suppression ratio %* be compare with DMSO solvent matched group, the average suppression ratio (%) of medication group to tumor weight, # tumor lung metastasis inhibition rate %.

Animal pathology and toxicological analysis result show: at experimental session, we use the fragrant acid of tea second (TEC) and theanine ethyl ester (TE) to process people's cancer transplanted tumor nude mice and the C57/BL6J black rat of normal animal and treatment, have no generation toxic and side effects, body weight has no minimizing, and the heart, liver, spleen, lung, kidney, gastrointestinal tract, gonad, brain, skeletal muscle have no toxicity.The toxic reaction of positive control medicine cyclophosphamide group mice, performance weight loss, anorexia, medication occur after 2 weeks that abdominal part swelling and action keep away the phenomenons such as slow.The mice of rhEndostatin positive controls also occurs that weight loss, anorexia and action keep away the toxicity phenomenons such as slow.

Embodiment 7:

The fragrant acid of tea second is to EZH2 inhibition of enzyme activity effect experiment in animal body;

1, laboratory animal, cell line, instrument and main agents:

Laboratory animal nude mouse, cell line and instrument refer to above-mentioned (four) zoopery part.

EZH2Assay Kit, purchased from BPS Bioscience company.

Detect the inhibitory action of the fragrant acid of tea second to EZH2 enzymatic activity, experimental technique is in strict accordance with the subsidiary description operation of test kit, and step is as follows:

2, experimental procedure:

(1) to the TBST buffer that adds 150 μ l in each reacting hole in microwell plate, under room temperature, hatch 15min, get rid of buffer;

(2) according to explanation, preparation S-adenosylmethionine and EZH2 enzyme working solution, operation is carried out all the time on ice bath;

(3) dated ratio to specifications, preparation blank product, substrate reference substance, positive reference substance and inhibitor reference substance;

(4) each reference substance preparing and sample (respectively organize oncoprotein extract and replace EZH2 enzyme) to be tested are added respectively in reacting hole, 50 μ l/ holes, react 1h under room temperature, and each sample is all established 2 parallel holes; Each is organized oncoprotein extract preparation method and refers to as above (four) zoopery part.Wash plate sealing: each reacting hole adds 200 μ l TBST buffer to wash plate, repeats 3 times; To each reacting hole, add 100 μ l sealing buffer again, on shaking table, shake sealing 10min, discard liquid;

(5) the primary antibodie working solution having diluted is added in reacting hole, 100 μ l/ holes, shake bed reaction 1h;

(6) wash plate sealing, biconditional operation (5);

(7) two anti-working solutions of the HRP labelling having diluted are added in reacting hole, 100 μ l/ holes, shake bed reaction 30min;

(8) wash plate sealing, biconditional operation (5);

(9) by HRP chemical luminous substrate A and B the first-class volume mixture of ice bath evenly after, add in reacting hole 100 μ l/ holes;

(10) in microplate reader, read immediately fluorescence numerical value

3, experimental result (in Table 4)

The impact of the fragrant acid of table 4 tea second on closely related enzymatic activitys such as adjusting in body and tumor growth, invasion and attack and transfer, cardiovascular and cerebrovascular disease, immune deficiency disorder and inflammation

Note: ^*p < 0.05; Relevant method of testing is shown in above-mentioned experimental technique part.H3 and H4 Acetylation Level (%*) are analyzed and are obtained by Western Blotting, and method refers to following embodiment 8 partial contents.

Embodiment 8:

The experiment of the fragrant acid of tea second to the effect of histone deacetylase (HDAC) inhibition of enzyme activity;

1, laboratory animal, cell line, instrument and main agents

Laboratory animal nude mouse, cell line and instrument refer to above-mentioned (four) zoopery part.

EpiQuik HDAC Activity/Inhibition Assay Kit (Colorimetric), Epigentek company;

EpiQuik Nuclear Extraction Kit, Epigentek company;

2, experimental procedure:

(1) in strict accordance with the operation requirements of EpiQuik Nuclear Extraction Kit description, the tumor nuclear extract of preparation drug treating is shown in above-mentioned (four) zoopery part; Each sample is all established 2 parallel holes;

(2) in each reacting hole, add sample diluting liquid 50 μ ll, after shrouding film shrouding, under room temperature, react 30min;

(3) carefully take shrouding film off, discard liquid, dry, cleaning mixture 150 μ l are filled it up with in every hole, after standing 30 seconds, discard, and so repeat 2 times, pat dry;

(4) the HDAC enzyme of 2 μ l or tumor tissues nuclear extract are mixed with the sample diluting liquid of 28 μ l respectively, add in hand-hole, after shrouding film shrouding, at 37 ℃, react 60min;

(5) wash plate 3 times, operation is with (2); The capture antibodies working solution having diluted is joined in each reacting hole, and 50 μ l/ holes, are shaking bed reaction 60min under room temperature;

(6) wash plate 4 times, operation is with (2);

(7) the detection antibody working solution having diluted is joined in each reacting hole, 50 μ l/ holes, react 30min under room temperature;

(8) wash plate 5 times, operation is with (2);

Add developer, 100 μ l/ holes, lucifuge colour developing 2-10min under room temperature;

(9) when the color in standard substance hole becomes moderate strength blue, every hole adds the stop buffer cessation reaction of 50 μ l, now bluely vertically turns yellow;

(10) 450nm wavelength is sequentially measured the absorbance (OD value) in each hole.Mensuration should be carried out with interior for 15 minutes after adding stop buffer;

(12) inhibition of enzyme activity rate is calculated according to following formula:

Suppression ratio %=[1-(OD positive control-OD sample)/(OD positive control-OD blank)] 100% ^*

2, experimental result (in Table 4)

Embodiment 9:

The fragrant acid of tea second is to the horizontal regulating and controlling effect experiment of the closely related protein factor of kinds of tumors growth, invasion and attack and transfer, cardiovascular and cerebrovascular disease and immune deficiency disorder and inflammation etc.

Application Western Blotting method detects the fragrant acid of tea second to following tumor correlated albumen factor level regulating and controlling effect, and step is as follows:

1, main agents and instrument:

Antibody: VEGFR, EGFR, c-Met, K-Ras, H-Ras, Akt, Cyclin D1, β-catenin, Bcl-2, Bax, p53, p21, E-cadherin, Caspase3 albumen primary antibodie is purchased from U.S. Cell Signaling Technology company and Santa Cruz Technology company; H3 acetylation, H4 acetylation and HDAC (HDAC3 and HDAC4 etc.) Antibody Sample Kit antibody is purchased from U.S. Cell Signaling Technology company.

RPMI-1640, DMEM culture fluid and inactivated fetal bovine serum are purchased from Hyclone company; Trypsin trypsin) be purchased from Amersco company; Protein molecule Marker, 0.4% trypan blue: be purchased from U.S. Sigma company; Pvdf membrane is purchased from Millipore company;

Total protein lysate and nucleoprotein lysate and PMSF (Phenylmethanesulfonyl fluoride) solution are purchased from green skies biotechnology research institute; Two is anti-: horseradish peroxidase-labeled goat anti-mouse, horseradish peroxidase-labeled goat antirabbit, the anti-goat of horseradish peroxidase-labeled donkey, colour dye in advance molecular weight of albumen standard, ECL Plus luminescence reagent box, fixing powder, developing powder and be purchased from green skies biotechnology research institute; Film for medical X-ray radiography is purchased from Kodak.

CO2 gas incubator: 3111 types, U.S. Thermo company; Inverted microscope: CKX31 type, Japanese Olympus company; Table-type high-speed refrigerated centrifuge: 5810R type, German Eppendorf company; Micro sample adding appliance: German Eppendorf company; Cell culture plastic board (6 hole): Nunclon company; Small-sized Vertial electrophorestic tank: U.S. BIO-RAD company; Small-sized wet-type electrotransfer groove: U.S. BIO-RAD company; Decolorization swinging table: TS-1 type, Haimen City, Jiangsu kellin Bel instrument manufacturing company limited; Ice machine: XB70 type, GRANT company; OMEGA10 gel image analyser: U.S. μ lTRA LUM company; Sealing machine: SF-B type, the industrial Machinery Co., Ltd. in Wenzhou City; Analysis of protein lamp box: Shanghai Jing Ke Industrial Co., Ltd..

2, experimental procedure:

(1) cell is processed: the cell of the trophophase of taking the logarithm is inoculated in 6 orifice plates, treat that cell density grows to 70%～80% left and right, in cell, add the fragrant acid of tea second and cancer therapy drug 5-fluorouracil respectively, carmofur or daphnetin etc., its final concentration is respectively respectively organize the IC50 concentration (table 1 in foundation) of corresponding anticancer, separately establish the matched group containing solvent (0.01%DMSO) equal-volume cell culture fluid without medicine, continue to cultivate after 48h collecting cell;

(2) cell protein extracts: with cold PBS, wash after 2 times, with total protein or nucleoprotein cell pyrolysis liquid cell lysis, add 10 μ l PMSF in 1mL lysate, piping and druming mixes repeatedly, places 15min on ice; Sample is transferred in 1.5mL EP pipe, 14000r/min, 4 ℃ of centrifugal 10min, transfer to supernatant in aseptic EP pipe-80 ℃ of preservations;

(3) polyacrylamide gel electrophoresis (SDS-PAGE): use equivalent protein lysate sample separation albumen, transferring film, sealing, successively use respectively suitable primary antibodie and two anti-processing, wash film after, with the colour developing of ECL test kit, X-ray exposure tests trace protein band; With Gel-Pro Analyzer, carry out gray scale quantitative analysis, contrast is not compared with the optical density value of internal reference separately with each density component of drug treating, and the ratio of gained is compared with matched group respectively, sxemiquantitative protein expression level.

4, experimental result (in Table 5 and table 6)

The fragrant acid of table 5 tea second suppresses the closely-related protein factors such as tumor growth, invasion and attack and transfer, cardiovascular and cerebrovascular disease immune deficiency disorder and inflammation

Note: ^*p < 0.05; Relevant method of testing is shown in above-mentioned experimental technique part.

In table, about protein factor comes from the protein lysate by the cancerous cell lines such as the human breast carcinoma of the fragrant acid of tea second or positive control cancer therapy drug or DMSO vehicle treated, pulmonary carcinoma, hepatocarcinoma, Lewis lung cancer and these tumor tissues respectively, with Westem blotting, detect the result of analyzing.

The fragrant acid of table 6 tea second is on the impact with the closely related protein factor level such as tumor growth, invasion and attack and transfer, cardiovascular and cerebrovascular disease, immune deficiency disorder and inflammation and enzymatic activity

Note: ^*p < 0.05; Relevant method of testing is shown in above-mentioned experimental technique part.

#: about the ratio of tumor suppressor protein p53, expression of cyclin kinase inhibitor p21, apoptosis hydrolytic enzyme Caspase-3, cell cytosol and mitochondrial cytochrome C and cell adhesion protein E-cadherin come from the protein lysate by the cancerous cell lines such as the pulmonary carcinoma of the fragrant acid of tea second or positive control cancer therapy drug or DMSO vehicle treated, hepatocarcinoma Lewis lung cancer and these tumor tissues respectively, detect the result of analyzing with Western blotting.

Embodiment 10:

The fragrant acid of tea second is to nucleoprotein factor NF-κ B (p65)-DNA binding activity inhibition experiment in vitro (EMSA)

1, main agents and instrument:

Chemoluminescence method EMSA test kit: U.S. Pierce Biotechnology company;

Biotin labeling EMSA probe NF-κ B: U.S. Pierce Biotechnology company;

Protein molecule Marker and 0.4% ^*trypan blue: U.S. Sigma company; Pvdf membrane: Millipore company;

Nucleoprotein lysate, PMSF (Phenylmethanesulfonyl fluoride) solution: green skies biotechnology research institute

Colour dyes molecular weight of albumen standard, ECL Plus luminescence reagent box, fixing powder, developing powder in advance: green skies biotechnology research institute.Film for medical X-ray radiography: Kodak.

2, experimental procedure:

Use respectively corresponding IC50 concentration (in Table 1) and the DMSO (0.01% separately of the fragrant acid of tea second, positive control cancer therapy drug ^*) process the cancerous cell Nuclear extract extract that after 48 hours, the cancerous cell line such as pulmonary carcinoma, hepatocarcinoma Lewis lung cancer and these tumor tissues (seeing above-mentioned interior animal experiment) obtain, method refers to embodiment 6 experimental section contents.The chemoluminescence method EMSA test kit of NF-κ B (p65)-DNA binding activity inhibition experiment in vitro application U.S. Pierce Biotechnology company, experimental technique is in strict accordance with the description operation of test kit, and concrete steps are as follows:

(1) preparation of EMSA glue

Being formulated as follows shown in table of EMSA glue.The nucleoprotein extract 10 micrograms/pipe of the cell of drug treating after nucleoprotein extracting solution (green skies biotechnology research provides) extracts is respectively used to example reaction as follows and electrophoresis detection analysis.

Table 7-1 is about the preparation of 4%* polyacrylamide gel

(2) prerunning: glue is fixed in electrophoresis tank, fills it up with 0.5 * TBE electrophoretic buffer, according to the voltage electrophoresis of 10V/ centimetre 90 minutes.Probe association reaction is as follows:

The reaction of table 7-2 negative control

Table 7-3 example reaction

According to upper table, be added in successively in EP pipe, mix room temperature standing 15 minutes, eliminate the non-specific binding of contingent probe and albumen.Add respectively biotin labeled probe NF-κ B1 μ l, standing 20 minutes of room temperature.

(3) electrophoresis: renew fresh 0.5 * TBE electrophoretic buffer, according to the voltage electrophoresis of 10V/ centimetre 1.5 hours, guarantee that the temperature of glue is no more than 30 ℃.

(4) transferring film: it is that electricity turns 40 minutes in 380mA ice bath that the probe of EMSA glue coker protein factor and combination forwards nylon membrane process to.

(5) UV-crosslinked: nylon membrane to be placed under the uviol lamp of 254nm and to irradiate 15 minutes.

(6) chemoluminescence method detection of biological element label probe: get nylon membrane, the exposure of film X-ray, development and photographic fixing by test kit requirement to processing that confining liquid sealing was cross-linked, analysis result.

3, experimental result

The fragrant acid of table 8 tea second to tumor growth, invasion and attack and transfer, cardiovascular and cerebrovascular disease, immune deficiency disorder and the closely related nucleoprotein factor NF-κ B (p65) such as the scorching inhibitory action (cell and tumor tissues nucleoprotein) with DNA activity

When § is used in-vivo tumour tissue core protein lysate to detect, the fragrant acid of tea second and cyclophosphamide lumbar injection are 25mg/kg consumption.

Supplementary notes: present patent application is subsidized by National 863 problem " research and development (2012AA020206) of oncoprotein matter molecular marker " and Shandong Province's development in science and technology planning item " research (2009GG10002087) of ester catechin EGCG isoreactivity constituent structure transformation and optimization and anticancer candidate's new drug " with the research work that wherein achievement is relevant.

Claims (5)

1. the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin thereof and the theanine ethyl ester application in the medicine of the tumors such as preparation prevention and treatment people pulmonary carcinoma, breast carcinoma, hepatocarcinoma, colon cancer, carcinoma of prostate, cancer of pancreas, cervical cancer and lymphoma and melanoma.

2. the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin and the application of theanine ethyl ester in preparing histone methylferase EZH2 inhibitor.

3. the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin and the application of theanine ethyl ester in preparing histone deacetylase inhibitor.

4. the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin thereof and the theanine ethyl ester application in the factor NF-kB inhibitor of the disease associations such as preparation short tumor, inflammation and cardiovascular and cerebrovascular disease and immunodeficiency.

5. the fragrant acid of tea second (TEC) and midbody compound 3-carboxylic acid coumarin thereof and theanine ethyl ester are lowered the factor VEGFR of the disease associations such as short tumor, inflammation and cardiovascular and cerebrovascular disease and immunodeficiency in preparation, EGFR, c-Met, K-Ras, H-Ras, Akt, Cyclin D1, the protein levels such as β-catenin and Bcl-2/Bax and rise p53, p21, E-cadherin, the application in Caspase3 and endochylema Cyt C protein level.