CN108714149A

CN108714149A - Purposes of the garden burnet active constituent in preparing anti-tumor drug

Info

Publication number: CN108714149A
Application number: CN201810456594.5A
Authority: CN
Inventors: 吴建明; 秦大莲; 柏冲飞; 杨靖; 孙悦珊
Original assignee: Southwest Medical University
Current assignee: Southwest Medical University
Priority date: 2018-05-14
Filing date: 2018-05-14
Publication date: 2018-10-30

Abstract

The invention belongs to field of medicaments, specifically disclose garden burnet active constituent 3,3 ', purposes of the 4 '-trimethoxy benzoaric acids in preparing anti-tumor drug, disclose 3,3 ', 4 '-trimethoxy benzoaric acids are by lowering vegf expression, inhibit the inhibition of phosphorylation angiogenesis of PI3K/AKT/mTOR, simultaneously by activating Bax/Bcl-2/Caspase-3 access inducing apoptosis of tumour cell, have the economic benefits and social benefits antitumor efficacy for inhibiting neonate tumour blood vessel and cytotoxicity, is used to prepare anti-cancer drug preparation in use, therapeutic effect is good.

Description

Purposes of the garden burnet active constituent in preparing anti-tumor drug

Technical field

The invention belongs to field of medicaments, and in particular to 3,3 ', 4 '-trimethoxy benzoaric acid of garden burnet active constituent is being made Standby angiogenesis inhibiting and the purposes in cell toxicant economic benefits and social benefits anti-tumor drug.

Background technology

Cancer, i.e. malignant tumour are the high diseases of a kind of lethality, seriously threaten the life and health of people.According to WHO It delivers《Global cancer report 2014》It points out, whole world cancer patient morbidity and mortality were gradually passed every year from 2012 Increase, considerable part increases cases of cancer newly and is found in Chinese [1].In four big malignant tumours (liver, lung, stomach and cancer of the esophagus), China New cases and Died Patients rank the first in the world.According to statistics, Cancer in China number of patients in 2012 accounts for about global illness people Number 1/5, about 306.5 ten thousand；Cancer mortality patient accounts for about global cancer mortality sum 1/4, and about 220.5 ten thousand, cancer patient is every The medical expense in year is up to nearly hundred billion yuan, it is seen then that cancer is a heavy social burden.Currently, clinically still with surgery hand Art, radiotherapy and chemotherapy are main treating cancer, but there are apparent adverse reactions for above-mentioned therapy, seriously affect the life of patient Quality, therefore be badly in need of finding less toxic anticancer drug.

The mankind have been deep into molecular level to cellular biology of tumor and science of heredity, and new treatment concept and method are constantly gushed Existing, on the basis of traditional operation, radiotherapy, chemotherapy, the molecular targeted therapy of tumour has become the popular development side of oncology To using the focus treated as numerous focus of attention that Tumor Angiongesis is target spot.Harvard University Folkman is carried for the first time Going out the growth of tumour and transfer has blood vessel dependence theoretical, i.e., angiogenesis makes tumour obtain enough nutriments, promotes Tumour continued propagation and transfer, thus tumors of nutrients supply is cut off by blocking tumor angiogenesis, inhibition can be reached With the purpose for the treatment of tumour.Therefore, the targeting preparation of " angiogenesis inhibiting " clinically uses extensive and significant in efficacy, no Good reaction is few, is not likely to produce drug resistance, such as Sorafenib, but such targeting preparation mainly passes through suppression in angiogenesis inhibiting The secretion of VEGF processed.And VEGF has different physiological roles, it will necessarily be with hypertension, heart failure to its long-term extra-inhibitory Equal adverse reactions (referring to non-patent literature 1-2).

From FARBER in 1948, S report for the first time cell toxicity medicament for oncotherapy so far up to 66 years, main mechanism DNA of tumor cell damage and cell inhibitory effect effect can be generated for high dose cell toxicity medicament, is directly acted on to reach Tumour cell and the purpose for killing tumour cell.But bone marrow suppression and normal cell is easily caused to be damaged with the administration of this conventional method Wound, while also easy ting produce tolerance and outer row of the tumour cell to cell toxicity medicament.Therefore, for reduce tumour cell tolerance and The adverse reaction of cytotoxic drug promotes different conditions and the sensitivity of tumor cells in period, and clinic generally use is more at present Medicine joins application method, improves the therapeutic effect to tumour to a certain extent.Especially with neovascularization inhibitor and cell The use in conjunction of malicious class drug makes treatment income be obviously improved (referring to non-patent literature 3-4).However the combination of drug is very big Degree is influenced by patient's Liver and kidney function, causes the ADME processes of drug to significantly change, while improving Operative risk (referring to non-patent literature 5).

It can be seen that a kind of drug molecule having vascular study and cell toxicant double effects concurrently of research and development, in current anticarcinogen Have tremendous expansion advantage and significance in object research field.

Garden burnet (Sanguisorba OfficinalisL.) is rose family garden burnet platymiscium, and there is cooling blood and hemostasis, removing toxic substances to hold back The effect of sore, be conventionally used to treatment have blood in stool, scald carbuncle sore tumefacting virus, modern pharmacology research show garden burnet also have antibacterial, It is anti-inflammatory, promote hematopoiesis and it is anti-oxidant the effects that.Modern pharmacology research show garden burnet tannin have antibacterial, antitumor, immunological regulation, Promote hematopoiesis, it is antiviral and anti-oxidant the effects that, there is good rise with the Chinese patent drug kind " Diyushengbai Tablet " that single garden burnet is developed High leukocytic and rush thrombocytopoiesis effect, are widely used in tumor aid treatment.

But there has been no the active constituents with vascular study and cell toxicant double effects in document report garden burnet at present, also not See and be based on the double effects active constituent, can play simultaneously and inhibit HUVEC cells to VEGF, PI3K, mTOR mRNA and albumen Expression, to inhibit tumor vascular growth, and promotes in tumour cell to Bax, Bcl-2 and Caspase-3mRNA and albumen table It reaches, plays a dual role of cytotoxicity mechanism, to play safety, notable anticancer function for common cancers tumour cell Report.

Non-patent literature

1、HAYMAN S R,LEUNG N,GRANDE J P,et al.VEGF Inhibition,Hypertension, and Renal Toxicity[J].Current Oncology Reports,2012,14(4):285-294.

2、KIERAN M W,KALLURI R,CHO Y J.The VEGF Pathway in Cancer and Disease:Responses,Resistance,and the Path Forward[J].Cold Spring Harbor Perspectives in Medicine,2012,2(12):a6593.

3, Tu Zhewei, Tao Yifei, Dong's red silk wait cell toxicity medicaments and Tumor Angiongesis [J] Chinese Journal of Modern Applied Pharmacy, 2011,28(7):618-622.

4、YOKOYAMA Y,XIN B,SHIGETO T,et al.Combination of ciglitazone,a peroxisome proliferator-activated receptor gamma ligand,and cisplatin enhances the inhibition of growth of human ovarian cancers[J].Journal of Cancer Research&Clinical Oncology,2011,137(8):1219-1228.

5, Jin Chaohui, Xu Ting, Ma Yin, wait Positive and Negative Aspects [J] China Dispensaries of tumor patient drug combinations, and 2009,20 (2):152-154.

Invention content

The purpose of the present invention is to provide garden burnet 3,3 ', 4 '-trimethoxy of active constituent benzoaric acid, (english abbreviation is TMEA) the application in preparing anti-tumor drug.

Use of compound 3,3 ', the 4 '-trimethoxy benzoaric acid in preparing anti-tumor drug as shown in formula (I) On the way.

Further, the compound of the formula (I) is extracted from garden burnet.

Further, the compound of the formula (I) prepare it is antitumor with angiogenesis inhibiting and/or cytotoxicity Drug in purposes.Preferably, the compound of the formula (I) is preparing inhibition HUVEC cells to VEGF, PI3K, mTOR MRNA and protein expression, to the purposes in the anti-tumor drug of angiogenesis inhibiting.Preferably, the chemical combination of the formula (I) Object promotes apoptotic protein Bax, Caspase-3 to express and inhibit Bcl-2 protein expressions in preparation, to play cytotoxicity Purposes in anti-tumor drug.

Further, the compound of the formula (I) is in the medicine for preparing anti-liver cancer and anti-tumour, inhibitor against colon carcinoma cells tumour or anti-lung cancer tumour Purposes in object.

Further, the anti-tumor drug further includes pharmaceutically acceptable carrier or auxiliary material.Preferably, described The drug of tumour is the targeted drug preparation for including 3,3 ', 4 '-trimethoxy benzoaric acids.It is highly preferred that the targeting Pharmaceutical preparation is capsule, injection or oral agents.

Experimental study proves that garden burnet ellagic acid constituents TMEA has both simultaneously and significantly inhibits neonate tumour blood vessel and to tumour The cytotoxicity double effects of cell, and especially by vegf expression is lowered, inhibit the phosphorylation of PI3K/AKT/mTOR, to Angiogenesis inhibiting；By promoting apoptotic protein Bax, Caspase-3 to express and Bcl-2 protein expressions being inhibited to induce tumour cell Apoptosis plays cytotoxicity, and the purposes that can be used for preparing the drug of economic benefits and social benefits antitumor action uses.Wherein,

RT-PCR is tested and Western blotting (protein immunoblotting experiment) result is shown：

(1) TMEA can obviously inhibit the expression of VEGF, PI3K, mTORmRNA, and obviously lower the table of HUVEC cell VEGEs It reaches, promotes p-PI3Kp85 (Tyr458)/PI3K, p-AKT (Ser473)/AKT, p-mTOR (Ser2448)/mTOR protein expressions Decline (P<0.01 or P<0.05) phosphorylation for, inhibiting PI3K/AKT/mTOR, plays the role of angiogenesis inhibiting；

(2) TMEA can significantly inhibit in SW620 cells inhibit Apoptosis Bcl-2mRNA and Bcl-2 protein expressions, Promote Bax, Caspase-3mRNA and its protein expression (P of Apoptosis<0.01 or P<0.05), to induce tumour thin Born of the same parents' apoptosis, plays cytotoxicity.

Experiment in vitro result of study is shown：Compared with blank group, TMEA (5,10,20,40 μ g/mL) can obviously inhibit Proliferation, migration and its formation of cell tubule of HUVEC cells, to play the role of significantly inhibiting angiogenesis.

Experiment in vivo result of study is shown：Compared with model group, high, middle dosage (200,100mg/kg) TMEA can obviously press down The growth of SW620 transplantable tumors mouse tumor volume processed, knurl quality are decreased obviously (P<0.01 or P<0.05), TMEA refers to liver Number has no significant effect, but can significantly reduce index and spleen index.And it is shown through Immunohistochemical Method testing result：High, middle dose group (200, 100mg/kg) TMEA can obviously inhibit CD31 in SW620 transplantable tumor Mice Bodies to express (P<0.01 or P<0.05), and promote thin The expression of Caspase-3, Bax of born of the same parents' apoptosis inhibit the expression of the Bcl-2 of suppression Apoptosis, can be generated to tumour cell aobvious The cytotoxicity of work.

In addition, experiment in vitro research further confirm TMEA (5,10,20,40 μ g/mL) to HepG2 cell lines, Acellular lung cell A549, colon carcinoma cell line SW620 proliferation there is obvious inhibiting effect, and its inhibited proliferation M- concentration effect (P when being presented apparent<0.01 or P<0.05), to be directed to human liver cancer tumour, lung cancer tumor and colon cancerous swelling Tumor has notable anticancer therapy effect.

Description of the drawings：

Fig. 1 show 3,3 ', 4 '-trimethoxy benzoaric acids in embodiment 1 to HUVEC cell Proliferations influence (N=6)；

Fig. 2, Fig. 3 show that 3,3 ', 4 '-trimethoxy benzoaric acids are to HUVEC cell migration abilities in example 2 Influence (N=3)；

Fig. 4 shows that 3,3 ', 4 '-trimethoxy benzoaric acids form HUVEC cells in vitro tubules in embodiment 3 Influence；

Fig. 5 show 3,3 ', 4 '-trimethoxy benzoaric acids in example 4 to HepG2 cell Proliferations influence (N=6)；

Fig. 6 show 3,3 ', 4 '-trimethoxy benzoaric acids in embodiment 5 to A549 cell Proliferations influence (N=6)；

Fig. 7 show 3,3 ', 4 '-trimethoxy benzoaric acids in embodiment 6 to SW620 cell Proliferations influence (N=6)；

Fig. 8 shows that 3,3 ', 4 '-trimethoxy benzoaric acids live to normal liver cell's LO-2 cells in embodiment 7 Property influence (N=6)；

Fig. 9 shows that 3,3 ', 4 '-trimethoxy benzoaric acids are thin to normal human embryonic kidney cells HEK-293 in embodiment 8 Cytoactive influence (N=6)；

Figure 10 shows that 3,3 ', 4 '-trimethoxy benzoaric acids live to bone marrow cells in mice BMC cells in embodiment 9 Property influence (N=6)；

Figure 11 shows that 3,3 ', 4 '-trimethoxy benzoaric acids are to SW620 transplantable tumor nude mouse tumor bodies in embodiment 10 Long-pending influence；

Figure 12 shows that immunohistochemistry microexamination 3,3 ', 4 '-trimethoxy benzoaric acid is to SW620 in embodiment 10 The influence (200 ×) of new vessels in transplantable tumor mouse tumor tissue；

Figure 13 shows that immunohistochemistry microexamination 3,3 ', 4 '-trimethoxy benzoaric acid is to SW620 in embodiment 10 The influence (200 ×) of transplantable tumor mouse Bax expression；

Figure 14 shows that immunohistochemistry microexamination 3,3 ', 4 '-trimethoxy benzoaric acid is to SW620 in embodiment 10 The influence (200 ×) of transplantable tumor mouse Bcl-2 expression；

Figure 15 shows that immunohistochemistry microexamination 3,3 ', 4 '-trimethoxy benzoaric acid is to SW620 in embodiment 10 The influence (200 ×) of transplantable tumor mouse Caspase-3 expression；

Figure 16 shows that Immunohistochemical Method detects TMEA to SW620 transplantable tumor mouse CD31, Bax, Bcl-2 in embodiment 10 With the influence of Caspase expression；

Figure 17 shows 3,3 ', 4 '-trimethoxy benzoaric acids in embodiment 1101 to HUVEC cell VEGEs, PI3K, MTORmRNA expression influence (N=6)；

Figure 18,19 show in embodiment 1,102 3,3 ', 4 '-trimethoxy benzoaric acids to HUVEC cell VEGEs, The influence of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR protein expression；

Figure 20 shows that 3,3 ', 4 '-trimethoxy benzoaric acids are to colon cancer cell SW620 cells in embodiment 1201 Bax, Bcl-2, Caspase-3mRNA expression influence (N=6)；

Figure 21,22 show that 3,3 ', 4 '-trimethoxy benzoaric acids are in SW620 cells in embodiment 1202 The influence of Caspase-3, Bax, Bcl-2 expression of apoptosis protein；

Figure 23 is 3,3 ', 4 '-trimethoxy benzoaric acid angiogenesis inhibitings and the signal of cytotoxicity economic benefits and social benefits mechanism Figure.

Specific implementation mode

With reference to test example and specific implementation mode, the present invention is described in further detail.But this should not be understood It is only limitted to embodiment below for the range of the above-mentioned theme of the present invention, it is all that this is belonged to based on the technology that the content of present invention is realized The range of invention.

Main agents and instrument in embodiment：Matrigel matrigels (Coring, article No.：354248) HUVEC cells：People Huve cell system is purchased from Sciencell companies；HepG2 cells：Bel7402 derives from Chinese Typical Representative culture The Wuhan object collection (CCTCC) cell bank；A549 cells：Lines derive from the Wuhan CCTCC cell Library；SW620 cells：Human colon cancer cell line derives from the Wuhan CCTCC cell bank.Refrigerated centrifuge：Sigma, USA, 3-18KS Type；LO-2 cells：Normal liver cell derives from the Wuhan CCTCC cell bank；HEK-293 cells：Human embryonic kidney cells derive from The Wuhan CCTCC cell bank；SPF Kunming mouses (16-20g), half male and half female are purchased from Chengdu and reach large experimental animal company, qualified Card number：SCXK (river) 2013-24.Each cell English breviary vocabulary is as shown in table 1 in specification：

The English breviary vocabulary of table 1

1 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment inhibits Human umbilical vein endothelial cells HUVEC cell Proliferations Effect

With containing 5%FBS (fetal calf serum, Fetal bovine serum, Gibco, specification：500mL/ bottles, lot number： 42Q9462K), 1% Pen .- Strep mixture (Beyotime, specification：100mL/ bottles, lot number：C0222), 1% endothelium is thin ECM complete mediums (Sciencell, the specification of intracellular growth factor ECGS：500mL/ bottles, lot number：1001), at 37 DEG C, 5% CO₂Incubator in cultivate HUVEC cells, change liquid every other day, cell fusion degree about 90% waited for, by 1：2 carry out passage processing.

After taking the good HUVEC of growth conditions cells trypsinised, take 10 μ L cell suspensions in being counted on tally, Cell density is adjusted to 3 × 10⁴A/mL is inoculated in 3000/hole in 96 orifice plates, per 100 μ L of hole.After cell is adherent, go Except culture medium, it is added the fresh culture of drug containing, experimental setup blank group, TMEA various concentration groups, totally 6 groups.The end of drug Concentration is as follows, TMEA final concentration of 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, and 5 μ g/mL, 1 μ g/mL, blank group give equivalent DMSO, 6 multiple holes of every group of setting, in 37 DEG C, 5%CO₂Routine culture in incubator.It cultivates for 24 hours, 36h, 48h, after 60h, adopts respectively Cell proliferation rate is detected with MTS methods, in triplicate.MTS presses 1:9 with 96 orifice plates are added after culture medium mixing, per 100 μ L of hole, incubate After case is incubated 2h, its absorbance OD values are detected at 490nm, calculate opposite proliferation rate, and formula is as follows：

Opposite proliferation rate (%)=(medicine group OD/ blank group OD) × 100%

Experimental result is as shown in Figure 1.As can be seen from FIG. 1：Compared with blank group, after TMEA intervenes HUVEC cells for 24 hours, 5, 10,20,40 μ g/mL can significantly inhibit the proliferation (P of HUVEC cells<0.01 or P<0.05)；Drug TMEA intervenes HUVEC cells After 36,48,60h；The drug of 1,5,10,20,40 μ g/mL can obviously inhibit the proliferation (P of HUVEC cells<0.01), and right The dependence of time and concentration is presented in the inhibited proliferation of HUVEC cells.

2 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment inhibits Human umbilical vein endothelial cells HUVEC cell migrations Effect

Influences of the TMEA to HUVEC cell migration abilities is investigated using scratch experiment.Used in experimentation Marker, ruler, the experiment equipments such as 200 sterile pipette tips of μ L be put into ultraviolet irradiation 30min in super-clean bench, specific experiment in advance Steps are as follows, in triplicate：

Before cell inoculation, 3 horizontal lines are uniformly drawn at the 6 orifice plates back side with Maker, wait for that ink marks dries.By growth conditions After the good cells trypsinised liquid digestion of HUVEC, tally counts and adjusts cell density to 1 × 10⁶A/mL connects Kind, per hole 1mL, makes cell uniformly be laid on 6 orifice plates bottom, is cultivated for 24 hours in incubator after 6 orifice plates.

Cell fusion degree about 80% or so removes culture medium, and 3mL PBS rinses cell is added twice.Ruler compares, with nothing The 200 μ L pipette tips of bacterium do vertical cut in every hole cell growth single layer same position, and PBS cleanings wash away the thin of floating afterwards twice The culture medium of 0.5%FBS, Sorafenib a concentration of 5 μm of ol/mL, TMEA final concentration of 40,20,10,5,1 μ is then added in born of the same parents Blank group is arranged in g/mL.Using the intersection of straight line and cut as reference point detection scratch width, 40 × take pictures, it is opened per hole 3.Add Photograph to record the scratch width of 0h after medicine under 40 × microscope immediately；Pharmaceutical intervention is taken pictures after for 24 hours under microscope, and method is same On.Image Pro Plus (IPP) software is used to calculate the variation of cell scratch width, and every group of scratch width mean value is used as should The average scratches width of group, is repeated 3 times, scratch width is calculated by following formula：

Migration distance=scratch width_0hScratch width_24h

2 3,3 ', 4 '-trimethoxy benzoaric acid of table inhibits the influence of HUVEC cell migrations

(N=3)

Note：Compared with blank group, * P<0.05, * * P<0.01.

For experimental result as shown in Fig. 2, Fig. 3 and table 2, positive drug Sorafenib can inhibit the migration (P of HUVEC cells< 0.01).TMEA intervenes HUVEC cells for 24 hours, and compared with blank group, 40,20,10,5 μ g/mL concentration groups can obviously inhibit HUVEC Migration (the P of cell<0.01 or P<0.05), and with the increase of drug concentration, the migration distance of HUVEC cells significantly reduces, It can be seen that TMEA inhibits HUVEC cell migrations, there are apparent concentration effect relationships.

3 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment inhibits Human umbilical vein endothelial cells HUVEC cells in vitro small The effect that pipe is formed

Martrigel matrigels are liquefied in 4 DEG C of refrigerator overnights in advance, while by the pipette tips used in experimentation It is used etc. the liquefaction after 4 DEG C of refrigerator overnights is both needed to, avoids the occurrence of glue surface out-of-flatness.Matrigel bases are remained in experimentation Matter glue is placed in ice face, its solidification is avoided to influence to use.

Liquefied Martrigel matrigels are laid in per hole in 96 orifice plates with 50 μ L, in slightly concussion makes it in ice face 96 orifice plate bottoms are uniformly distributed in, being then transferred to 37 DEG C of incubator 30min makes its solidification.After matrigel fully solidifies, 2 × 10 are inoculated with per hole⁵The drug containing cell suspension of a/mL, 40 × microscopically observation tubule is formed after incubator is incubated.Experiment is set altogether Blank group is set, the TMEA of Sorafenib group (10 μm of ol/mL) and five various concentrations (40,20,10,5,1 μ g/mL), every group sets Set 3 multiple holes.40 after culture 6h × under the microscope and take pictures, it takes pictures at every group of random selection 3, observation TMEA is to external small tubular At influence.

Laboratory test results are as shown in figure 4, note：A blank groups；B Sorafenib groups；C 1μg/mL；D 5μg/mL；E 10μ g/mL；F 20μg/mL；G 40μg/mL

As shown in figure 4, compared with blank group, Sorafenib can obviously inhibit HUVEC tubules to form (P<0.01 or P< 0.05)；TMEA (1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL) can obviously inhibit HUVEC tubules to be formed.

Integrated embodiment 1-3 is it is found that TMEA can be by obviously inhibiting Human umbilical vein endothelial cells HUVEC cell Proliferations, moving It moves and cell is at pipe ability, to effectively inhibit Human umbilical vein endothelial cells HUVEC cell Proliferations, show that TMEA has significantly suppression Angiogenesis effect processed.

Specific indication research

4 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment inhibits the effect of HepG 2 cell proliferation

Experimental setup blank group, TMEA various concentration groups, totally 6 groups.The final concentration of drug is respectively：TMEA is final concentration of 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 1 μ g/mL, blank group give the DMSO of equivalent, 6 multiple holes of every group of setting, in 37 DEG C, 5%CO₂Routine culture in incubator.It cultivates respectively for 24 hours, 36h, 48h, after 60h, cell proliferation rate is detected using MTS methods, In triplicate.The concrete operation methods such as cell inoculation are the same as referring to embodiment 1.

Experimental result as shown in figure 5, TMEA intervene cell for 24 hours after, it is thin that 5,10,20,40 μ g/mL can significantly inhibit HepG2 Proliferation (the P of born of the same parents<0.01 or P<0.05)；The drug of 1,5,10,20,40 μ g/mL after HepG2 cells 36,48,60h is intervened in dosing Proliferation (the P of HepG2 cells can obviously be inhibited<0.01 or P<0.05) time, and to the inhibited proliferation of HepG2 cells is presented With the dependence of concentration.

5 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment inhibits non-small cell lung cancer cell A549 cell Proliferations Effect

Experimental setup blank group, TMEA various concentration groups, totally 6 groups.The final concentration of drug is as follows, and TMEA is final concentration of 40,20,10,5,1 μ g/mL, blank group give the DMSO of equivalent, 6 multiple holes of every group of setting, in 37 DEG C, 5%CO₂In incubator often Rule culture.After cultivating 24,36,48,60h respectively, cell proliferation rate is detected using MTS methods, in triplicate.Cell inoculation etc. is specific Operating method is the same as referring to embodiment 1.

Experimental result as shown in fig. 6, TMEA intervene cell for 24 hours after, 5,10,20,40 μ g/mL can significantly inhibit A549 cells Proliferation (P<0.01 or P<0.05)；The drug of 5,10,20,40 μ g/mL can be apparent after dosing intervention A549 cells 36,48,60h Inhibit the proliferation (P of A549 cells<0.01 or P<0.05) time and concentration, and to the inhibited proliferation of A549 cells is presented Dependence.

6 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment inhibits the effect of colon carcinoma cell line SW620 cell Proliferations

Experimental result as shown in fig. 7, TMEA intervene cell for 24 hours after, 1,5,10,20,40 μ g/mL can significantly inhibit SW620 Proliferation (the P of cell<0.01)；The drug that 36,48,60h, 1,5,10,20,40 μ g/mL of SW620 cells are intervened in dosing can obviously press down Proliferation (the P of SW620 cells processed<0.01) dependence of concentration, and to the inhibited proliferation of SW620 cells is presented.

Drug safety Journal of Sex Research

The influence of 7 3,3 ', 4 '-trimethoxy benzoaric acid normal liver cell's LO-2 cell activity of embodiment

Using RPIM1640 (10%FBS, 1% Pen .- Strep) medium culture Human normal hepatocyte (LO-2), often It changes the liquid once within 2 days, after passing on for 3 generations, after well-grown normal liver cell's trypsin digestion, in being counted on tally Number, adjustment cell density to 3 × 10⁴A/mL is inoculated in 3000/hole in 96 orifice plates, per 100 μ L of hole.Wait for that cell is adherent Afterwards, culture medium is removed, be added the fresh culture (40,20,10,5,1 μ g/mL) containing TMEA, while blank group and solvent are set Control group, using its OD value of MTS methods detection cell proliferation rate after incubator culture 48h.

Experimental results are shown in figure 8, after TMEA intervenes normal liver cell 48h, MTS the experimental results showed that high concentration 20, LO-2 cells are presented weaker cytotoxicity in 40 μ g/mL, and the inhibiting rate to the proliferation of LO-2 is 15% (P<0.01 or P< 0.05).It is small to the mankind's normal liver cell toxic side effect when showing TMEA as anticancer medication.

Shadow of 8 3,3 ', the 4 '-trimethoxy benzoaric acid of embodiment to normal human embryonic kidney cells HEK-293 cell activity It rings

With containing 10%FBS, the DMEM medium culture HEK-293 of 1% Pen .- Strep, cell is used after 3 passages In experimental study.To be in the good HEK-293 of growth conditions it is cells trypsinised after, take 10 μ L in being counted on tally Number, with 5 × 10⁴A/hole is inoculated in 96 orifice plates, per 100 μ L of hole.After cell is adherent, culture medium is removed, is added containing TMEA's Fresh culture (40,20,10,5,1 μ g/mL), while blank group and solvent control group are set, incubator culture 48h uses MTS methods Detect OD values.

Experimental result is as shown in Figure 9：After TMEA intervenes normal human embryonic kidney cells HEK-29348h, MTS the experimental results showed that Apparent cytotoxic effect (P is presented to HEK293 cells in 20,40 μ g/mL of high concentration<0.01).

Influence of 9 3,3 ', the 4 '-trimethoxy benzoaric acid of embodiment to bone marrow cells in mice BMC cell activity

Kunming mouse 8, half male and half female, cervical dislocation is taken to put to death, the soaking disinfection 10min in 75% ethyl alcohol, separation Mouse both sides femur.Femur one end is cut off, is inserted into from the other end with 1mL syringes, is rushed repeatedly with sterile RPIM1640 culture mediums It washes marrow to femur to whiten, then goes to bone marrow suspension in 15mL centrifuge tubes, medium supernatant is taken after its natural subsidence. Cell suspension is settled to 4mL, 3mL monocyte separation mediums (being slowly added to along tube wall) are added, are centrifuged with 400 × g at 4 DEG C 30min, acquires intersection milky-white layer bone marrow nucleated cell (BMC) cell, and 1640 culture mediums of serum-free RPIM wash 3 repeatedly It is secondary, 1640 culture mediums containing 10%FBS are then added and are resuspended to get mouse BMC suspensions.After routine culture cell, wait for that cell passes For after 2-3 times, the BMC cells of logarithmic growth phase are used for experimental study.The good BMC of growth conditions is taken, in being counted on tally Cell density is adjusted after number to 5 × 10⁴A/mL, is inoculated with 180 μ L per hole, it is rear various concentration is added pastille culture medium (40,20, 10,5,1 μ g/mL), while blank group and solvent control group are set, its OD value is detected using MTS after incubator culture 48h.

Experimental result is as shown in Figure 10：After TMEA intervenes mouse bone marrow cells karyocyte 48h, MTS is the experimental results showed that highly concentrated It spends 10,20,40 μ g/mL and the apparent small (P of cytotoxic effect is presented to BMC cells<0.01 or P<0.05), this shows TMEA high Weaker toxic effect is presented when dosage to bone marrow cells in mice.

Internal pharmacodynamic study

10 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment is to colon carcinoma cell line SW620 transplantable tumor nude mice models Internal pharmacodynamic study

Test agents useful for same：Fluorouracil Injection (5-Fu, specification:0.25g, 10mL) to be purchased from Tianjin gold credit medicine company limited Company；Sodium carboxymethylcellulose (CMC, specification：250g, CAS：9004-32-4) it is purchased from the good rich limited public affairs of biotechnology that are full of in Xi'an Department；Polyoxyethylene sorbitan monoleate (Tween 80, specification：500mL, CAS：500-019-9) it is purchased from Tianjin Zhi Yuan chemical reagents corporations；Poly- second Glycol 400 (PEG-400, specification：500mL/ bottles, CAS：112-60-7) it is purchased from Tianjin Zhi Yuan chemical reagents corporations.

Test instrument：Vernier caliper：Upper marine site Jin Lanyou e-commerce Co., Ltd, 0-150mm types；Electronic scale： Hauswirt, HE-51 type.

Experimental animal：SPF grades of Balb/c nude mices are purchased from Chengdu and reach large experimental animal company, quality certification number：SCXK (river) 2013-24。

Specific experiment method and step is：

1, the foundation of Human colon cancer SW620 transplantable tumors nude mice model：SPF grades of Balb/c nude mices, 50 males (4-6 weeks Age, 16-20g) adaptability culture one week.By SW620 cells after 2-3 passage, the SW620 cells for taking growth conditions vigorous disappear It is resuspended after change, adjustment cell density to 1 × 10⁷A/mL, oxter is inoculated with 0.1mL on the right side of nude mice, and posterior tuberosity body is formed within 7 days, establishes Human colon carcinoma SW620 Transplanted tumor models.

2, grouping administration：For SW620 cell inoculations nude mice after 14 days, mouse tumor volume reaches 120mm3, shows SW620 nude mices Mouse is then divided into 5 groups, i.e. model group by Transplanted tumor model success according to mouse tumor volume, 5-FU groups, and TMEA is high, medium and low Five groups of dosage.Isometric Vehicle controls are administered in model group mouse, and the 5- of 25mg/kg is injected intraperitoneally in fluorouracil group every other day The TMEA of the high, medium and low dosage group difference gastric infusion 200mg/kg, 100mg/kg and 50mg/kg of FU, TMEA, by 0.1mL/10g Administration, once a day, successive administration 21 days.Every group of administrations statistics is as shown in table 3.

Table 3 is grouped administrations

3, Testing index：Especially by following experimental method test experience result.

3.1 general status are observed

Observe modeling before, administration after mouse the state of mind, diet, changes of weight and death condition.

It is observed that after 50 mouse inoculation SW620 cells, mouse oxter tumour starts to grow after 7 days, measures within the 14th day small Mouse gross tumor volume is up to 120mm³, modeling success 47, tumor formation rate is up to 94%.And Transplanted tumor model is successfully established rear mouse essence God is dispirited, and activity is reduced, weight loss.

The measurement of 3.2 gross tumor volumes

The 3d after administration, 7d, 10d, 14d, after 17d, 21d (day), with the volume of vernier caliper measurement tumour knurl Size (V=1/2ab², a indicates that tumour longest diameter, b indicate tumour shortest diameter), draw tumour knurl growth inhibition figure, and by with Lower formula calculates tumor control rate：

Tumor control rate (%)=medicine group gross tumor volume/model group gross tumor volume × 100%.

3.3 tumour knurl weights and tumor control rate

After last time is administered, cervical dislocation puts to death mouse, detaches knurl, weighs, and detects the difference of mouse tumor weight.It is real It is as shown in table 4 to test statistical result.

4 TMEA of table is to SW620 transplantable tumors nude mice to the inhibiting rate (x ± s, n=6) of tumour

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

According to known to Figure 11 and 4 statistical result of table：After SW620 Transplanted tumor model mouse are administered in TMEA, it can obviously inhibit small The growth of mouse gross tumor volume.Compared with model group, 5-FU groups, inhibiting rate of the high, medium and low dosage groups of TMEA to mouse tumor weight Respectively 45%, 41%, 37% and 27%.

3.4 organ index

After administration, the liver of separating mouse, spleen are simultaneously weighed, and calculate organ index by following formula：

Organ index=organ weights/mouse weight

The results are shown in Table 5 for experiment statistics：

Influences of 5 TMEA of table to SW620 transplantable tumors mouse liver, index and spleen index

Note：Compared with model group, * P<0.05, * * P<0.01.

According to known to 5 statistical data of table：Compared with model group, TMEA administration mouse have no significant effect liver index, and And TMEA can inhibit mouse spleen index (P<0.05 or P<0.01).

3.5 detecting TMEA to tumor tissues CD31, Bax, Bcl-2, the protein expression of Caspase-3 acts on

TMEA is observed to tumor tissues CD31, Bax, Bcl-2, the egg of Caspase-3 using immunohistochemistrySABC SABC method White expression, concrete operation step are as follows：

(1) paraffin section makes：Take about 1cm in the middle part of the tumour tumor mass under being cut in 3.3³Fritter it is solid in neutral formalin It is 24 hours fixed, after being transferred to 70% alcohol then overnight；Again with the dehydration of alcohol of low concentration to high concentration, dehydration is as follows： Twice with 75% alcohol of alcohol 1h → 80% alcohol of 1h → 90% alcohol of 30min → 95% alcohol of 30min → 100% 30min, by Step sloughs moisture in tissue, then will be organized in dimethylbenzene it is transparent twice, each 10min is advisable so that tissue block is just transparent.It connects It above-mentioned transparent tissue block being put into the liquid wax melted and impregnates 1h, be put into then in embedding basket, then pour into and melt in advance Embedding basket is gently transferred to accelerated solidification in cold water by the embedding wax of change after surface solidification.The wax that will finally be formed after solidification Block is fixed on slicer, and the thin slice for being cut into 5 μ m-thicks is attached on the glass slide that neutral gum has been added dropwise, and clean coverslip is tilted It puts down, completes mounting, slice is put into 42 DEG C of baking ovens after labelled and is dried overnight to get to the corresponding paraffin of each group mouse Slice.

(2) related solution is prepared：

PBS liquid：PBS powder is dissolved in the phosphate buffer (pH 7.3) that 1000mL distilled waters are made into 0.01mol/L；

Citrate buffer：0.4g citric acids .H₂O, 3g trisodium citrates .H₂O₂1000mL distilled waters are dissolved in be made into 0.01mol/L(pH 6.0)；

3% hydrogen peroxide：A concentration of 30% hydrogen peroxide is according to 1：10 (i.e. 1 part 30% of hydrogen peroxide, 9 parts of distilled waters) (now with the current)；

CD31 primary antibodies：According to 1：25 dilutions are prepared；Caspase-3 primary antibodies：1：100 dilutions are prepared；Bax primary antibodies：1：250 is dilute Release preparation；Bcl-2 primary antibodies：1:250 dilution configurations；

Biotin labeling goat anti-rabbit igg secondary antibody：With 1：100 dilutions are prepared；

DAB working solutions：Seminal plasma fructose detection kit 1 and reagent 2 press 1：20 are configured to (now with the current).

(3) immunohistochemical staining detects

A, paraffin section de-waxing：The paraffin section for taking first part 3.5.4 to make, constant temperature toasts 2h in 65 DEG C of baking ovens, Each dewaxing 10min of dimethylbenzene I, dimethylbenzene II, dimethylbenzene III；

B, alcohol gradient aquation：Successively through each 2min in 100%, 95%, 90%, 80% alcohol, finally distillation washing 1min.After aquation, PBS buffer solution rinse 5min × 3；

C, antigen retrieval：Water-bath heats 0.01M sodium citrate buffer solutions (PH 6.0) to 95 DEG C, is put into histotomy and boils 20min after liquid cooled to room temperature to be buffered, takes out slice.PBS buffer solution rinse 5min × 3；

D, the 3%H that 50 μ L deionized waters are prepared is added dropwise in every slice₂O₂It is incubated 10min, to block Endogenous peroxide Enzyme.PBS buffer solution rinse 5min × 3；

E, it closes：Every slice is added dropwise 50 μ L and closes serum working solution, and room temperature closes 30min；

F, primary antibody is incubated：Filter paper sucks remaining closing serum, and the primary antibody (Caspase-3 of 50 μ L is added dropwise in every slice：1: 25；Bax:1:100；CD31:1:250；Bcl-2：1:250) it is incubated overnight in 4 DEG C of refrigerators.

G, secondary antibody is incubated：Slice is warmed to room temperature again in setting in wet box, PBS buffer solution rinse 5min × 3, filter paper blots residual 50 μ L biotin secondary antibody working solutions are added dropwise in the PBS stayed, in incubation at room temperature 45min, PBS buffer solution rinse, 5min × 3；

H, SABC compounds are added dropwise, in incubation at room temperature 30min, PBS buffer solution rinse 5min × 3；

I, it develops the color：DAB develops the color 2-9 minutes, in microscopically observation, grasps dye levels；

J, it redyes：Haematoxylin carries out nuclear targeting 2min, 5min is rinsed with tap water, under the microscope, if dyed It is shallow, then carry out a haematoxylin dyeing and broken up with hydrochloric acid if it is hyperchromasia, again nuclear staining is carried out with haematoxylin；

K, serial dehydration：80% ethyl alcohol 5min, 85% ethyl alcohol 5min, 95% ethyl alcohol 5min, 100% ethyl alcohol 5min；

L, mounting：The transparent 10min of dimethylbenzene, neutral gum mounting；

M, Image Acquisition and semi-quantitative analysis：Using CCD (DP73；Olympus, Tokyo, Japan) in 200 times of times magnifications Under several, every slice randomly chooses 5 visuals field (marginal zone of non-necrosis and bleeding, unspecific staining) acquisition high-quality Piece.Integral optical density (IOD) is measured with Olympus Cellsense, half is carried out with average optical density (MOD, i.e. IOD/Area) Quantitative analysis；

(4) data analysis：Using SPASS21.0 softwares, P<0.05, which is considered as difference, has statistical significance, and data are using equal Value ± standard deviation, group is interior, comparison among groups use one-way analysis of variance.

Laboratory test results are as shown in Figure 12,13,14,15,16, it was found from experimental result data analysis：With model group phase Than 5-FU groups can obviously inhibit the expression (P of CD31, Bcl-2 in SW620 transplantable tumor mouse tumor tissues<0.01), TMEA (200mg/kg, 100mg/kg) group can obviously inhibit CD31, Bcl-2 to express (P<0.05 or P<0.01)；5-FU groups can obviously promote Into the expression (P of Bax, Caspase-3<0.01), and TMEA can be obviously promoted Bax in SW620 transplantable tumor tumor tissues, Expression (the P of Caspase-3<0.05 or P<0.01).

Known to above-mentioned In vivo study experiment：For model group, high, middle dosage (200,100mg/kg) TMEA can be bright The aobvious growth for inhibiting SW620 transplantable tumor mouse tumor volumes, knurl quality are decreased obviously (P<0.01 or P<0.05), TMEA is to liver Dirty index has no significant effect, but can significantly reduce index and spleen index.In addition, compared with model group, high, middle dose group can obviously inhibit CD31 expresses (P<0.01 or P<0.05) expression, high, middle dose group TMEA can promote Caspase-3, Bax, inhibits Bcl-2's Expression.

Study on mechanism

11 3,3 ', 4 '-trimethoxy benzoaric acid angiogenesis inhibiting Study on mechanism of embodiment

3,3 ', 4 '-trimethoxy benzoaric acids of experimental example 11-01 inhibit HUVEC cell VEGEs, PI3K, mTOR MRNA expressional functions

By HUVEC cells with 1 × 10⁵After a/mL is inoculated in 6 orifice plates, after cell is adherent, cell culture medium is removed, point Not Jia Ru 10 μm of ol/mL Sorafenibs, 1 μ g/mLTMEA, 5 μ g/mL TMEA, 10 μ g/mL TMEA, 20 μ g/mL TMEA, 40 μ G/mL TMEA intervened HUVEC cells after 36 hours, using Trizol method traditional extraction HUVEC cell total rnas, and with reference to RT- PCR kit specification amplifying target genes.Wherein, Trizol methods extraction RNA concrete operation steps are as follows：

(1) twice with the PBS rinses cell of precooling, 1mL RZ lysate lytic cells are added into 6 orifice plates, use liquid-transfering gun It blows and beats repeatedly transparent to solution several times；

(2) RZ lysates are transferred in no enzyme EP pipes, 200 μ L of chloroform is added, cover EP pipe lids, after acutely shaking 15s, It is placed at room temperature for 3min；

12000rpm centrifuges 10min under the conditions of (3) 4 DEG C, and sample can be divided into three layers, the i.e. colourless water phase in upper layer after centrifugation, Middle layer and lower layer's yellow organic phase, mainly the volume in water phase is about 0.5mL to RNA.Water intaking 400 μ L of phase are transferred to new EP pipes In, carry out subsequent experimental operation；

(4) 200 μ L of absolute ethyl alcohol are slowly added into EP pipes, pipette tips blow and beat mixing.Subsequent mixture is transferred to adsorption column CR3 In, 12000rpm centrifuges 30s under cryogenic conditions；

(5) 500 μ L protein liquid removals are added into RD adsorption columns CR3,12000rpm centrifuges 30s, waste liquid in reject collecting pipe Afterwards, adsorption column is put back in collecting pipe；

(6) 500 μ L rinsing liquids RW are added to adsorption column, are stored at room temperature 2min, 4 DEG C of 12000rpm centrifuge 30s, abandon waste liquid；

(7) repetitive operation (6)；

(8) adsorption column is put into 2mL collecting pipes, 12000rpm centrifuges 2min under the conditions of 4 DEG C, removes remaining in collecting pipe Waste liquid.Adsorption column CR3 is placed on superclean bench the 2min that divulges information after centrifugation, fully dries moisture；

(9) adsorption column CR3 is transferred in the EP pipes of a 1.5mL without enzyme, the RNase-Free ddH2O of 30 μ L is added Dissolving, is placed at room temperature for 2min, and 4 DEG C of 12000rpm centrifuge 2min；

(10) 2 μ LRNA samples is taken to detect its purity, A260/ with NanoDrop One/Onec ultramicrospectrophotometers Sample of the A280 values between 1.8-2.0 then can be used for subsequent experimental.

Trans Script Reverse Transcriptase kits are reused by total serum IgE reverse transcription into cDNA, using GAPDH as internal reference, are carried out Fluorescence quantitative polymerase chain reaction (qRT-PCR) detects.Specially：

(1) synthesis of cDNA

Reverse transcription reaction system is as shown in the table：

(2) real-time fluorescence quantitative PCR amplification system and program

One step RT-PCR reaction system is as shown in the table：

(3) VEGF, PI3K, mTOR and GAPDH reaction condition are shown in Table 6：

6 qRT-PCR parameters of table

At the end of experiment, with 2^-ΔΔCTIt indicates experimental result, is calculated as the relative value after being compared with blank group, blank group It is set as 1.Primer information is shown in Table 7.

7 gene primer sequence of table and product clip size

Experimental result is as shown in figure 17, and A indicates the relative expression levels of VEGF mRNA in figure；B indicates PI3K mRNA's Relative expression levels；C indicates mTOR mRNA relative expression levels.According to figure：Compared with blank group, Sorafenib group can obviously press down Expression (the P of mTOR processed, PI3K, VEGF mRNA<0.01 or P<0.05)；TMEA (10 μ g/mL, 20 μ g/mL, 40 μ g/mL) can be bright It is aobvious to inhibit HUVEC cell VEGEs, the expression (P of PI3K, mTOR mRNA<0.01 or P<, but 1, the TMEA of 5 μ g/mL 0.05) To its gene expression indifference.

It follows that high, middle dose group (10 μ g/mL, 20 μ g/mL, 40 μ g/mL) TMEA can obviously inhibit HUVEC Cell VEGE, the expression (P of PI3K, mTORmRNA<0.01 or P<0.05), angiogenesis inhibiting.

3,3 ', 4 '-trimethoxy benzoaric acids of embodiment 11-02 are to HUVEC cell VEGEs, PI3K, p-PI3K, The influence of AKT, p-AKT, mTOR, p-mTOR protein expression

By HUVEC cells with 1 × 10⁵After/mL is inoculated in 6 orifice plates, after cell is adherent, cell culture medium is removed, is added Fresh pastille culture medium (Sorafenib 10 μm of ol/mL, the TMEA of VEGF 40ng/mL, VEGF 40ng/mL and 10 μ g/mL, 1, The TMEA of 5,10,20,40 μ g/mL) intervene HUVEC cells extract total protein after 48 hours and carry out subsequent experimental.

Steps are as follows for specific experiment：

(1) preparation of protein sample：After the cell removal supernatant in 6 orifice plates, after the PBS rinses 3 times of precooling, often (specific ratio is lysate to the cell pyrolysis liquid of hole addition certain volume：Protease inhibitors：Inhibitors of phosphatases=8：1： 1), lytic cell 20min on ice, is scraped attached cell with cell scraper, after cracking 5min in liquid nitrogen, 4 DEG C, 12000rpm/min centrifuges 20min, takes supernatant up to sample protein.

(2) total protein concentration is measured and is denaturalized：It is dense in strict accordance with kit specification detection sample protein using BCA methods Degree.Boiling 5min in 95 DEG C after addition sample-loading buffer makes protein denaturation, and it is spare to be stored in -20 DEG C of refrigerators.

(3) electrophoresis.It is dried after glass plate is cleaned first spare.After glue-pouring device is fixed, separation gel and slowly is configured Separation gel is injected to suitable height, and ethyl alcohol is then added and flattens glue surface, being placed at room temperature for makes it fully solidify.By the ethyl alcohol of moulding It pours out rear and is sufficiently absorbed through extra moisture with filter paper.It is injected after configuration concentration glue, is inserted into 10 hole combs.Be placed at room temperature for wait for it is dense Contracting gelling is solid, and both hands gently extract comb upwards.Electrophoretic buffer is simultaneously added in fixed electrophoretic apparatus, with the albumen of 20 μ g of every hole into Row loading.60V electrophoresis 30min, 100V electrophoresis to destination protein band separates.

(4) transferring film.Ready PDVF films, which are put into immersion 5min in methanol, makes it fully activate.Sponge and filter paper are put Enter and be soaked for a period of time in transferring film liquid, is sponge-filter paper-PVDF- filter paper-spongy " sandwich " knot successively from anode to cathode Structure, and it is put into transferring film in transferring film slot, 250mA constant current transferring films 90min.

(5) it closes:5% skimmed milk power floods pvdf membrane and shaking table closing 1h, PBST are rinsed 3 times at room temperature.

(6) primary antibody is incubated：Antibody is pressed into proper proportion mTOR (1：1000), (1 p-mTOR：1000), (1 PI3K：1000), p-PI3K(1：1000), (1 AKT：1000), (1 p-AKT：1000), (1 VEGF：400), (1 Caspase-3：300), (1 Bax： 600), (1 Bcl-2：500), β-actin (1：1000) it dilutes, 4 DEG C of refrigerator overnights are incubated, and next day is incubated at room temperature 20min, PBST Wash film 3 times, each 3min.

(7) secondary antibody is incubated：After secondary antibody is incubated at room temperature 1h, PBST washes film 5 times, each 4min.

(8) develop：By luminescent solution A, B component is with 1：After 1 ratio mixing, PDVF films are placed in luminescent solution and impregnate 5min Afterwards, develop, ImageJ softwares is used in combination to analyze gray value.

Testing result is analyzed as shown in Figure 18,19, Tu19Zhong, A indicate shadows of the TMEA to VEGF/ β-actin protein expressions It rings, B indicates that the influence that TMEA expresses p-PI3K/PI3K albumen and its phosphorylated protein, C indicate TMEA to p-AKT/AKT eggs The influence of bletilla its phosphorylated protein expression, D indicate the shadow that TMEA expresses p-mTOR/mTOR albumen and its phosphorylated protein It rings.

According to the graph：1, compared with blank group, the differential expression unobvious of Sorafenib group total protein, VEGF, p-PI3K P85 (Tyr458)/PI3K, p-AKT (Ser473)/AKT, p-mTOR (Ser2448)/mTOR protein expressions reduce (P<0.01 or P <0.05)；2, compared with blank group, the expression no significant difference of VEGF group total proteins, VEGF, p-PI3Kp85 (Tyr458)/ PI3K, p-AKT (Ser473)/AKT, p-mTOR (Ser2448)/mTOR protein expressions increase (P<0.01 or P<0.05)；3, The expression no significant difference of TMEA+VEGF group total proteins, VEGF, p-PI3K p85 (Tyr458)/PI3K, p-AKT (Ser473)/ AKT, p-mTOR (Ser2448)/mTOR protein expression no significant differences；4, compared with blank group, the expression of TMEA group total proteins Difference unobvious, VEGF, p-PI3Kp85 (Tyr458)/PI3K, p-AKT (Ser473)/AKT, p-mTOR (Ser2448)/mTOR Protein expression declines (P<0.01 or P<0.05).

It can be seen that TMEA can effectively inhibit HUVEC cell VEGEs, p-PI3K, p-AKT, p-mTOR protein expressions.

The cytotoxicity mechanism study of 12 3,3 ', 4 '-trimethoxy benzoaric acid of embodiment

3,3 ', 4 '-trimethoxy benzoaric acids of embodiment 12-01 are to SW620 cells Bax, Bcl-2, Caspase- The expressional function of 3mRNA

By SW620 cells with 2 × 10⁶After a/mL is inoculated in 6 orifice plates, after cell is adherent, cell culture medium is removed, is added Enter various concentration fresh pastille culture medium (5-FU of 20 μ g/mL, the TMEA of 1 μ g/mL, the TMEA of 5 μ g/mL, 10 μ g/mL's TMEA, the TMEA of 20 μ g/mL, the TMEA of 40 μ g/mL) intervene SW620 cells after 12 hours, using Trizol method traditional extractions After SW620 cell total rnas, and with reference to RT-PCR kit specification amplifying target genes.Wherein, Trizol methods extraction RNA tools Body operating procedure and RT-PCR reaction systems and response procedures, primer information list are referring to embodiment 10.

Experimental result is as shown in figure 20, and part A indicates relative expression levels' statistical chart of Bax mRNA in figure；Part B table Show relative expression levels' statistical chart of Bcl-2mRNA；C portion indicates Caspase-3mRNA relative expression levels' statistical charts.

According to the graph：Compared with blank group, 5-FU can be obviously promoted Caspase-3, the expression (P of Bax mRNA<0.01 or P<0.05).TMEA (1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL) can be obviously promoted SW620 cells Expression (the P of Caspase-3, Bax mRNA<0.01 or P<0.05).Compared with blank group, 5-FU can obviously inhibit Bcl- Expression (the P of 2mRNA<0.01 or P<0.05)；TMEA (1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL) can be apparent Inhibit the expression of SW620 cells Bcl-2mRNA.

It follows that TMEA can significantly inhibit the expression of Bcl-2mRNA in SW620 cells, at the same promote Bax, Expression (the P of Caspase-3mRNA<0.01 or P<0.05).

3,3 ', 4 '-trimethoxy benzoaric acids of experimental example 12-02 promote SW620 apoptosis protein Caspase-3, The expressional function of Bax, Bcl-2

By SW620 cells with 1 × 10⁵After/mL is inoculated in 6 orifice plates, after cell is adherent, cell culture medium is removed, is added Various concentration fresh pastille culture medium (5-FU of 20 μ g/mL, the TMEA of 1 μ g/mL, the TMEA of 5 μ g/mL, 10 μ g/mL's TMEA, the TMEA of 20 μ g/mL, the TMEA of 40 μ g/mL) intervene after HUVEC cells extract total protein after 48 hours, and utilize The total protein extracted is detected analysis by western blot conventional analysis processing methods, and specific experiment step is referring to reality Apply example 1102.

Testing result is analyzed as shown in Figure 21,22, A indicates to influence statistics to Caspase-3/ β-actin expression in Figure 22 Figure；B indicates to influence statistical chart to Bax/ β-actin expression；C indicates to influence statistical chart to Bcl-2/ β-actin expression；D is indicated Expressing Bcl-2/Bax influences statistical chart；Wherein, * P<0.05, * * P<0.01.

According to the graph：5-FU and TMEA can be obviously promoted the expression (P of pro apoptotic protein Caspase-3<0.01 or P< 0.05)；Compared with blank group, 5-FU groups and TMEA (1,5,10,20,40 μ g/mL) can be obviously promoted the table of pro apoptotic protein Bax Up to (P<0.01 or P<0.05), while inhibiting the expression of apoptotic proteins Bcl-2 albumen, and Bcl-2/Bax ratios reduce (P< 0.01 or P<0.05).

In summary known to all experimental results：TMEA is by obviously inhibiting in HUVEC cells to promoting angiogenesis to rise VEGF mRNA and its protein expression and PI3K, mTOR mRNA and its protein expression to key effect, to inhibit blood vessel new It is raw, and then inhibit cancer tumor cells proliferation or growth.Also, TMEA can also play inhibition carefully by significantly inhibiting in cancer cell The expression of the Bcl-2mRNA and its protein of born of the same parents' apoptotic effect, at the same promote can induce and accelerate Apoptosis Bax, Expression (the P of Caspase-3mRNA and its protein<0.01 or P<0.05), and then the economic benefits and social benefits of induced cancer Apoptosis are played Therapeutic effect (as shown in figure 23).Fully confirm that TMEA can inhibit the phosphorylation of PI3K/AKT/mTOR by lowering vegf expression Angiogenesis inhibiting, and be to have inhibition blood vessel by activating Bax/Bcl-2/Caspase-3 access inducing apoptosis of tumour cell The antitumor medicinal active ingredient of newborn and cytotoxicity economic benefits and social benefits.Also, show the TMEA of high concentration by experimental study To normal liver cell LO-2 cell activity, mouse bone marrow cells karyocyte BMC cell activity and normal human embryonic kidney cells HEK-293 Cell activity has different degrees of toxic effect, and toxic side effect is relatively weak, meets human administration's safety, further demonstrates that TMEA It is used to prepare angiogenesis inhibiting to use with cell toxicant economic benefits and social benefits antineoplastic targeting pharmaceutical preparation, to including colon cancer, liver cancer, lung The kinds cancers oncotherapy effect such as cancer is safe efficient.

Claims

1. purposes of compound 3,3 ', the 4 '-trimethoxy benzoaric acid of formula (I) in preparing anti-tumor drug.

2. purposes according to claim 1, which is characterized in that the compound of the formula (I) is extracted from garden burnet.

3. purposes according to claim 1, which is characterized in that the compound of the formula (I) is being prepared with inhibition blood vessel Purposes in newborn and/or cytotoxicity anti-tumor drug.

4. purposes according to claim 3, which is characterized in that the compound of the formula (I) is preparing inhibition HUVEC cells To VEGF, PI3K, mTOR mRNA and protein expression, to the purposes in the anti-tumor drug of angiogenesis inhibiting.

5. purposes according to claim 3, which is characterized in that the compound of the formula (I) is preparing promotion apoptotic proteins Bax, Caspase-3 express and inhibit Bcl-2 protein expressions, the use in anti-tumor drug to play cytotoxicity On the way.

6. purposes according to claim 1, which is characterized in that the compound of the formula (I) is preparing anti-liver cancer and anti-tumour, resisting Purposes in colon cancer tumours or the drug of anti-lung cancer tumour.

7. purposes according to claim 1, which is characterized in that the anti-tumor drug further includes pharmaceutically acceptable Carrier or auxiliary material.

8. according to any purposes of claim 1-7, and be characterized in that, the anti-tumor drug be comprising 3,3 ', The targeted drug preparation of 4 '-trimethoxy benzoaric acids.

9. purposes according to claim 8, which is characterized in that the targeted drug preparation is capsule, injection or mouth Take agent.