CN104666306A - Application of triterpenoid compound in preparation of antitumor medicines - Google Patents
Application of triterpenoid compound in preparation of antitumor medicines Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to an application of a triterpenoid compound in preparation of antitumor medicines. A structural formula of the triterpenoid compound (tormentic acid) is shown in a formula I; and the tormentic acid is capable of obviously inhibiting proliferation of tumor cells, and significantly promoting apoptosis, so as to cause tumor cell death. The dosage of the tormentic acid is low; the toxic and side effects are lower than those of clinical common chemotherapeutic medicines; and the triterpenoid compound can be used as a novel anti-tumor medicine, is applied to treatment of tumors, and is simple in medication way and easy to operate.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of triterpenoid compound and preparing the application in antitumor drug.
Background technology
Tumor mortality rate is in and is beaten the world or second, and great threat the existence of the mankind with healthy.Oncotherapy is the difficult problem that medical circle faces always, and operation, chemotherapy and radiation therapy are common Therapeutic Method.And most of existing Therapeutic Method has very serious side effect, as hemorrhage, cognitive disorder, multiple etc., this is main relevant with its nonselective cytotoxicity.For chemotherapy, the drug resistance of emerging medicine is another serious problem.Find toxic and side effects new type anticancer medicine that is little, that have selective killing is one of main goal in research of whole world scientist always.Natural prodcuts, comprise plant, marine organisms and microorganism etc. and have attracted the concern of many scientists always.
Tormentic acid is that (molecular formula is C to a kind of triterpenoid compound that separation obtains from Fructus Rubi (Rubus chingii Hu)
30-H
48-O
5, molecular weight is 488.7).1986, the people such as Villar are separated from the aerial parts of a kind of plant Poterium ancistroides Deaf in Spain area first and obtain Tormentic acid (Villar A, Paya M, Hortiguela MD, Cortes D.Tormentic acid, a new hypoglycemic agent from Poterium ancistroides.Planta medica, 1986,1:43-45).Research reports that Tormentic acid has reduction blood glucose, antiinflammatory isoreactivity (Wu JB, Kuo YH, Lin CH, Ho HY, Shih CC.Tormentic Acid, a Major Component of Suspension Cells of Eriobotrya japonica, Suppresses High-Fat Diet-Induced Diabetes and Hyperlipidemia by Glucose Transporter 4and AMP-Activated Protein Kinase Phosphorylation.Journal of Agricultural and Food Chemistry, 2014, 62:10717-10726, Chang CT, Huang SS, Lin SS, Amagaya S, Ho HY, Hou WC, Shie PH, Wu JB, Huang GJ.Anti-inflammatory activities of tormentic acid from suspension cells of Eriobotrya Japonica ex vivo and in vivo.Food Chemistry, 2011,127:1131-1137.), but the research of anticancer aspect is not but reported.
Summary of the invention
The dosage that the object of the invention is to overcome antitumor drug clinically in prior art is high, drug effect is undesirable, lack selectivity, produce drug resistance and suppress the shortcoming and defect such as toxic and side effects of hemopoietic system, digestive system, immune system, nerve and hormonal system, provides a kind of triterpenoid compound preparing the application in antitumor drug.
Object of the present invention is realized by following proposal:
Triterpenoid compound is preparing the application in antitumor drug, and described triterpenoid compound is Tormentic acid, its structural formula as shown in Equation 1:
Described antitumor drug is preferably anti-breast cancer medicines, medicines resistant to liver cancer or anti-lung-cancer medicament;
Described triterpenoid compound Tormentic acid can be used as novel antitumor drug, is applied to the treatment of tumor.
A kind of antitumor drug, containing above-mentioned triterpenoid compound;
Principle of the present invention: the intrinsic propesties of tumor cell is pernicious increment, inhibition tumor cell propagation, arresting cell cycle and cell death inducing are the key links of containment tumor growth.The present invention finds and confirms the propagation of Tormentic acid energy inhibition tumor cell, and significantly promote apoptosis of tumor cells, and the using dosage of Tormentic acid is low, the toxic and side effects under same dosage is also lower, has cell selective.
The present invention has following advantage and effect relative to prior art:
(1) Late Cambrian triterpenoid compound Tormentic acid of the present invention significantly can suppress the propagation of tumor cell in vitro.
(2) Tormentic acid makes death of neoplastic cells mainly through the expression of inducing apoptosis of tumour cell, blocking-up cell cycle, interfering line mitochondrial membrane potential, change associated protein.
(3) dosage of Tormentic acid is lower, and endotoxic lower in effective pharmaceutical quantities scope.
Accompanying drawing explanation
Fig. 1 is the positive ion mass spectrum figure of Tormentic acid.
Fig. 2 is the interpretation of result figure that Tormentic acid affects HepG-2, Bel-7402, A549 and MCF-7 growth of tumour cell suppression ratio.
Fig. 3 is the interpretation of result figure that Tormentic acid affects MCF-7 tumor cell and LO2 normal liver cell growth inhibition ratio.
Fig. 4 is the electron microscope picture that Tormentic acid affects MCF-7 growth of tumour cell suppression ratio.
Fig. 5 is the streaming figure that Tormentic acid induces MCF-7 apoptosis of tumor cells, wherein, and A:control matched group; B:12.5 μ g/mL Tormentic acid treatment group; C:25 μ g/mL Tormentic acid treatment group; D:40 μ g/mL Tormentic acid treatment group.
Fig. 6 is the cycle streaming figure contrasting the MCF-7 tumor cell that (control) organizes in embodiment 7.
Fig. 7 is the cycle streaming figure of the MCF-7 tumor cell of 12.5 μ g/mL Tormentic acid treatment groups in embodiment 7.
Fig. 8 is the cycle streaming figure of the MCF-7 tumor cell of 25 μ g/mL Tormentic acid treatment groups in embodiment 7.
Fig. 9 is the cycle streaming figure of the MCF-7 tumor cell of 40 μ g/mL Tormentic acid treatment groups in embodiment 7.
Figure 10 is the interpretation of result figure of Tormentic acid on ROS impact in MCF-7 tumor cell.
Figure 11 is the interpretation of result figure that Tormentic acid affects MCF-7 tumor cell mitochondrial transmembrane potential, wherein, and A:control matched group; B:12.5 μ g/mL Tormentic acid treatment group; C:25 μ g/mL Tormentic acid treatment group; D:40 μ g/mL Tormentic acid treatment group.
The interpretation of result figure of Figure 12 to be Tormentic acid on MCF-7 tumor cell be correlated with mRNA level in-site impact, wherein, A:Cyclin D1; B:CDK4; *: compare with matched group, P<0.05; *: compare with matched group, P<0.01.
Figure 13 is the interpretation of result figure that Tormentic acid affects MCF-7 tumor cell associated protein level.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
In embodiment, people's HepG-2 cell line, Human hepatocarcinoma Bel-7402 cell, typeⅡ pneumocyte, MCF-7 Human Breast Cancer Cells and Human normal hepatocyte LO2 are all purchased from life science institute cellular resources center, Chinese Academy of Sciences Shanghai; Fructus Rubi is purchased from peaceful medical material market, Guangzhou.
Compound Tormentic acid prepares from Fructus Rubi;
Embodiment 1 prepares Tormentic acid from Fructus Rubi
(1) the Fructus Rubi 5Kg grinding and sieving will bought, use in batches after mix homogeneously the volume fraction of 10 times amount (quality) be 95% alcohol heating reflux extract three times, merge extractive liquid, filter, with Rotary Evaporators concentrating under reduced pressure evaporate to dryness at 40 DEG C, reclaim ethanol, obtain Fructus Rubi alcohol extracts extractum;
(2) dissolve with methanol of Fructus Rubi alcohol extracts extractum 1.5L step (1) prepared, then isopyknic petroleum ether solvent (namely adding the volume of petroleum ether volume=crude extract+methanol) is added, abundant vibrations mixing, leave standstill 2h, after the complete layering of two-phase solvent, the petroleum ether phase on sucking-off upper strata; Re-extract three times, merges petroleum ether extraction liquid, concentrating under reduced pressure evaporate to dryness, reclaims petroleum ether, obtains petroleum ether phase extract;
(3) in the methanol solution after the ether extraction of step (2) PetroChina Company Limited., isopyknic ethyl acetate solvent is added, abundant vibrations mixing, leave standstill 2h, by the two layers of solution concentrating under reduced pressure be respectively divided into, re-extract three times, obtain ethyl acetate extract (be extracted with ethyl acetate the extractum obtained, ethyl acetate phase extractum can be called); Get Fructus Rubi ethyl acetate extract, through silica gel column chromatography (Φ 8 × 120cm), chloroform-methanol gradient elution, 100:0 (chloroform and methanol volume ratio), 99:1,98:2,95:5,90:10,85:15,80:20,70:30,50:50 and 0:100 ten gradients are set, each gradient 20L, every 4L is gathered into portion (starting most to be respectively gathered into two parts respectively with these two gradients last), obtain 44 sub-fractions altogether with Rotary Evaporators concentrating under reduced pressure at 40 DEG C, be labeled as EA1-EA44 successively.EA-14 is through ODS column chromatography, methanol-water gradient elution, 30%, 60%, 80%, 90% 4 gradient is set, each gradient elution five column volumes, every half column volume is collected once, obtains 40 sub-fractions altogether with Rotary Evaporators concentrating under reduced pressure at 40 DEG C, wherein 28th ~ 32 fractions are placed a period of time and are separated out white powder material, obtain compound 1 through recrystallization, this compound is insoluble to methanol, is soluble in pyridine.
The structure of embodiment 2 deterministic compound 1
After compound 1 (trace) the chromatograph dissolve with methanol that embodiment 1 prepares, the ESI-MS of Esquire HCTplus type Large Copacity ion trap LC/MS is utilized to analyze, mass-to-charge ratio m/z sweep limits: 200 ~ 2000, obtains sample positive ion mass spectrum figure (Fig. 1).Compound 1 deuterated reagent pyridinium dissolution is placed in nuclear magnetic tube, utilize Bruker DRX-400 nuclear magnetic resonance analyser, using tetramethylsilane (TMS) as internal standard substance, measure its hydrogen spectrum (1H-NMR), full decoupled carbon spectrum (13C-NMR).
Compound 1 is white amorphous powder.ESI-MS m/z:511 [M+Na]
+(release molecular formula is C30-H48-O5).
1H-NMR(C
5D
5N)δ:1.02(3H,s),1.11(3H,s),1.13(3H,s),1.15(3H，d,J＝6.5Hz),1.30(3H,s),1.44(3H,s),1.74(3H,s),3.08(1H,s,18-H),3.41(1H,d,J＝9.3Hz,3-H),4.13(1H,m,2-H),5.59(1H,brs,12-H)。
13C-NMR(C
5D
5N)δ(C-1～30):48.3,69.1,84.3,40.4,56.4,19.5,34.0,40.9,48.4,39.0,24.6,128.4,140.5,42.6,29.9,26.9,48.8,55.1,73.1,42.9,27.4,39.0,29.8,18.2,17.4,17.7,25.2,181.3,27.6,17.3。Above spectroscopic data and document (Taniguchia S, Imayoshia Y, Kobayashi E, et al., Production of bioactive triterpenes by Eriobotrya japonica calli.Phytochemistry, 2002,59 (3): 315-323.) data consistent reported, therefore authenticating compound is Tormentic acid.
Embodiment 3 Tormentic acid suppresses different tissues tumor cell type propagation
Get eugonic people's HepG-2 cell line, Human hepatocarcinoma Bel-7402 cell, typeⅡ pneumocyte and MCF-7 Human Breast Cancer Cells respectively, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 3 × 10
4/ mL, 100 μ L are inoculated in 96 well culture plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, each group of DMEM culture medium (Tormentic acid final concentration is respectively 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/mL) added respectively containing Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, abandoning supernatant, PBS cleaning twice, every hole adds 200 μ L not containing DMEM culture medium and the 20 μ L tetrazolium bromides (MTT) of serum, puts into incubator and hatches 4h, abandoning supernatant, every hole adds 150 μ L dimethyl sulfoxide (DMSO), lucifuge is positioned over micro oscillator and evenly shakes 10min, the OD value measured under 490nm wavelength by microplate reader, calculates cell proliferation inhibition rate.
The suppression ratio of Tormentic acid to the growth of tumour cell of different tissues type increases along with the increase of concentration; The inhibition strength of different tissues tumor cell type is had nothing in common with each other, wherein the highest to the suppression ratio of MCF-7 Human Breast Cancer Cells, when Tormentic acid concentration is 100 μ g/mL, cell proliferation inhibition rate can reach 91.84%, and minimum to the suppression ratio of Bel-7402 hepatocarcinoma cells, be about 80% (Fig. 2).
Embodiment 4 Tormentic acid suppresses breast cancer tumor cells propagation
Get eugonic MCF-7 Human Breast Cancer Cells and the centrifugal 5min of Human normal hepatocyte LO2,1000rpm respectively, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 3 × 10
4/ mL, 100 μ L are inoculated in 96 well culture plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, each group of DMEM culture medium (Tormentic acid final concentration is respectively 6.25 μ g/mL, 12.5 μ g/mL, 25 μ g/mL, 30 μ g/mL, 40 μ g/mL and 50 μ g/mL) added respectively containing Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, abandoning supernatant, PBS cleaning twice, every hole adds 200 μ L not containing DMEM culture medium and the 20 μ L tetrazolium bromides (MTT) of serum, puts into incubator and hatches 4h, abandoning supernatant, every hole adds 150 μ L dimethyl sulfoxide (DMSO), lucifuge is positioned over micro oscillator and evenly shakes 10min, the OD value measured under 490nm wavelength by microplate reader, calculates cell proliferation inhibition rate.
Tormentic acid can the growth of inhibition tumor cell MCF-7 significantly, and has concentration dependent.When the concentration of Tormentic acid is increased to 25 μ g/mL from 12.5 μ g/mL, from 23.81%, 51.73% is increased to the suppression ratio of tumor cell MCF-7, and for normal liver cell LO2, when the concentration of Tormentic acid is 40 μ g/mL, 30.53% (Fig. 3) is only had to the suppression ratio of normal liver cell LO2.
The electron microscope observation that embodiment 5 Tormentic acid affects MCF-7 growth of tumour cell suppression ratio
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 3 × 10
4/ mL, 100 μ L are inoculated in 96 well culture plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, the each group of DMEM culture medium (Tormentic acid final concentration is respectively 12.5 μ g/mL, 25 μ g/mL and 40 μ g/mL) added respectively containing Tormentic acid, matched group adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
2cultivate 24h in incubator, then take out cell and take pictures under an electron microscope.
Compared with matched group, Tormentic acid can the growth of inhibition tumor cell MCF-7 significantly, and has concentration dependent.Along with the growth of Tormentic acid concentration, cell number tails off gradually, and cellular morphology also has significant change (Fig. 4).
Embodiment 6 Tormentic acid inducing tumor cell MCF-7 apoptosis
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 1 × 10
6/ mL, 2mL are inoculated in 6 orifice plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, the each group of DMEM culture medium (Tormentic acid final concentration is respectively 12.5 μ g/mL, 25 μ g/mL and 40 μ g/mL) added respectively containing Tormentic acid, matched group adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, collecting cell is in streaming pipe, 2 (2000rpm are cleaned with the PBS of pre-cooling, 5min), operate by apoptosis kit (Hangzhou Lian Ke Bioisystech Co., Ltd) description: 500 μ L Binding Buffer re-suspended cells, after adding 5 μ L Annexin V-FITC mixings, add 10 μ L propidium iodide (PI) working solution mixings, room temperature lucifuge hatches 5min, by the apoptosis situation of flow cytometer (FACscalibur, BD Biosciences) analysis of cells.
Compare with matched group, Tormentic acid can promote MCF-7 apoptosis, and apoptosis rate increases along with the increase of Tormentic acid concentration.When Tormentic acid concentration is 40 μ g/mL, MCF-7 apoptosis rate can reach 78.2% (Fig. 5).
Embodiment 7 Tormentic acid is to the cycle influences of MCF-7 tumor cell
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 1 × 10
6/ mL, 2mL are inoculated in 6 orifice plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, the each group of DMEM culture medium (Tormentic acid final concentration is respectively 12.5 μ g/mL, 25 μ g/mL and 40 μ g/mL) added respectively containing Tormentic acid, matched group adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, collecting cell is in streaming pipe, 2 (2000rpm are cleaned with the PBS of pre-cooling, 5min), operate by cell cycle test kit (Hangzhou Lian Ke Bioisystech Co., Ltd) description: 500 μ L Reagent A re-suspended cells, add 5 μ L Reagent B working solution mixings, room temperature lucifuge hatches 30min, with the cycle stage of flow cytometer (FACscalibur, BD Biosciences) analysis of cells.
The cell proportion that Tormentic acid can make MCF-7 tumor cell be in the Sub-G1 phase increases, and has concentration dependent (Fig. 6 ~ 9).
The impact that embodiment 8 Tormentic acid produces ROS in MCF-7 tumor cell
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 1 × 10
6/ mL, 2mL are inoculated in 6 orifice plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, the each group of DMEM culture medium (Tormentic acid final concentration is respectively 12.5 μ g/mL, 25 μ g/mL and 40 μ g/mL) added respectively containing Tormentic acid, matched group adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated, collecting cell and with the PBS of pre-cooling cleaning twice, then with the PBS re-suspended cell containing 10 μMs of DCFH-DA, hatch 20min, measure photon absorbing intensity by microplate reader for 37 DEG C, excitation wavelength and emission wavelength are respectively 488nm and 525nm in incubator.
Compared with matched group, Tormentic acid can significantly improve ROS level (P<0.01) (Figure 10) in MCF-7 tumor cell, and the result shows that intracellular ROS level improves is the key factor that Tormentic acid induces MCF-7 apoptosis of tumor cells.
Embodiment 9 Tormentic acid is on the impact of MCF-7 tumor cell mitochondrial transmembrane potential
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 1 × 10
6/ mL, 2mL are inoculated in 6 orifice plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, each group of DMEM culture medium (Tormentic acid final concentration is respectively 12.5 μ g/mL, 25 μ g/mL and the 40 μ g/mL) matched group added respectively containing Tormentic acid adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, collecting cell and with the PBS of pre-cooling cleaning twice, operate by mitochondrial membrane potential in anoxic detection kit (Beijing Mei Kemei biotechnology development corporation, Ltd.) description: draw 500 μ L 1 × Incubation Buffer, add 1 μ L JC-1, vortex mixing is made into JC-1 working solution, get 500 μ L JC-1 working solutions by cell even suspension, 37 DEG C, 5%CO
220min is hatched in incubator.Centrifugal (the 2000rpm of room temperature, 5min) collecting cell, wash twice with 1 × Incubation Buffer, then 500 μ L 1 × Incubation Buffer Eddy diffusion cells are drawn, by the mitochondrial membrane potential situation of flow cytometer (FACscalibur, BD Biosciences) analysis of cells.
Compared with matched group, MCF-7 tumor cell is after Tormentic acid process, and red fluorescence intensity significantly reduces and green fluorescence intensity significantly increases, and along with the increase of Tormentic acid concentration, this trend is more obvious.This illustrates that Tormentic acid can reduce the mitochondrial membrane potential (Figure 11) of tumor cell MCF-7, and then the apoptosis of inducing tumor cell.
Embodiment 10 Tormentic acid is correlated with on MCF-7 tumor cell the impact of mRNA level in-site
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 1 × 10
6/ mL, 2mL are inoculated in 6 orifice plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, the each group of DMEM culture medium (Tormentic acid final concentration is respectively 12.5 μ g/mL, 25 μ g/mL and 40 μ g/mL) added respectively containing Tormentic acid, matched group adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, collecting cell and with the PBS of pre-cooling cleaning twice, utilize Trizol reagent to extract RNA, and with RevertAid First Strand cnthesis Kit (Thermo company) Reverse Transcription box, 20 μ L reaction systems, carry out reverse transcription to RNA.Adopt DyNAmo Flash SYRB Green qPCR Kit (Thermo company) test kit, ABI real-time fluorescence quantitative PCR instrument (Rrism7500, Applied Biosystems, Foster City, CA, USA) mRNA is increased, after amplification, carry out automatic analysis to obtain the relative expression quantity of genes of interest by Relative Quantification (ddCt) Study method in ABI PRISM 7500SDS software.Related gene mRNA primer sequence is CDK4 (forward, 5 '-TCT GGT ACC GAG CTC CCG AA-3 ', reverse, 5 '-GAT TTG CCC AAC TGG TCG G-3 '), Cyclin D1 (forward, 5 '-ATG CCA ACC TCC TCA ACG AC-3 ', reverse, 5 '-CGC AGA CCT CCA GCA TCC-3 '), β-actin (forward, 5 '-TCA CCC ACA CTG TGC CCA TCT-3 ', reverse, 5 '-GTG AGG ATC TTC ATG AGG TAG TCA GTC-3 ').
Cyclin D1 and CDK4 gene expression dose significantly reduce (P<0.01) compared with matched group, and this illustrates that Tormentic acid carrys out induction of cell cycle arrest (Figure 12) by change cell Cyclin D1 and CDK4 gene expression dose.
Embodiment 11 Tormentic acid is on the impact of MCF-7 tumor cell associated protein level
Get eugonic MCF-7 tumor cell, the centrifugal 5min of 1000rpm, abandoning supernatant, adjusting cell number by the DMEM culture medium containing mass fraction being 10% hyclone is 1 × 10
6/ mL, 2mL are inoculated in 6 orifice plates, are placed in 5%CO
2in incubator, after 37 DEG C of cultivation 24h, abandoning supernatant, each group adds DMEM culture medium containing Tormentic acid (final concentration be 12.5 μ g/mL, 25 μ g/mL and 40 μ g/mL) matched group respectively and adds not containing the DMEM culture medium of Tormentic acid.At 37 DEG C, 5%CO
224h is cultivated in incubator, collecting cell and with the PBS of pre-cooling cleaning twice, collecting cell is in 1.5mL centrifuge tube, add the PIPA lysate (phosphoric acid enzyme inhibitor (PhosSTOP of 80 μ L, and protease inhibitor (cOmplete ULTRA Tablets Roche), Mini, EDTA-free, EASYpack, Roche)), drawing lysate with the syringe of 1mL and repeatedly blow and beat cell to fully mixing, in placing 30min on ice, to vibrate on turbula shaker 30s every 10min.With the centrifugal 15min of 12000rpm, supernatant is transferred to new 1.5mLEP pipe, is placed in protein concentration to be determined on ice.BCA method is adopted to detect protein concentration (carrying out according to BCA Protein Assay Kit description): to be first that the BSA solution of 2mg/mL gives the standard solution that ultra-pure deionized water dilution is series mass concentration (0 μ g/mL, 25 μ g/mL, 125 μ g/mL, 250 μ g/mL, 500 μ g/mL, 1000 μ g/mL) by concentration, then to configure a certain amount of working solution (BCA Solution:4% (volume fraction) Cupric Sulfate=200:4) as required.Get one piece of 96 orifice plate without substrate, every hole adds the serial standards solution 25 μ L diluted respectively, and needs the protein solution 25 μ L (having diluted 5 times) of detection, often organizes and all arranges multiple hole.Add 200 μ L/ hole working solutions simultaneously, shake mixing gently, be placed in 37 DEG C hatch 30min after, take out, in multiple labeling microwell plate detector 570nm place detection OD value.Survey OD value drawing standard curve according to standard substance protein solution, calculate testing protein solution concentration.According to the protein concentration that BCA method records, calculate 40 μ g total protein desirable proteins volumes, add 5 μ L 5 × SDS-PAGE sample-loading buffers simultaneously, ultra-pure deionized water to the cumulative volume supplementing respective volume is again 25 μ L, and mixing is fully centrifugal, make liquid accumulation in bottom, 100 DEG C of heat denatured 10min, centrifugal, be placed in for subsequent use on ice.Select 12% (volume fraction) separation gel and the concentrated glue of 5% (volume fraction), protein sample good for degeneration is added each swimming lane successively, and the swimming lane of sample both sides adds 5 μ L Marker respectively.Add the TGS buffer of q.s in electrophoresis tank, deposition condition is: 100V, about 150min, until bromophenol blue indicator runs to when being about 0.5cm apart from glue lower edge, stops electrophoresis.Then in ice bath with the voltage transferring film 100min of 100V.Close with the TBST containing 5% (mass fraction) defatted milk powder, slowly sway 1h.One anti antibody is respectively: GAPDH (14c10) Rabbit mAb, caspase-3, caspase-9, NF-κ B and p-ERK1/2 (all purchased from CST company), two anti antibodys are goat antirabbit (HRP labelling), purchased from Ju Yan bio tech ltd.The dilution ratio (1:1000) of recommending to specifications, preparation primary antibodie, under room temperature, jog hatches 4h or 4 DEG C hold over night.After primary antibodie hatches end, change two and resist, two anti-corresponding proportions of pressing of HRP labelling dilute (1:2000), room temperature jog 1h.After two resistive connection bundles, add luminescent solution (purchased from BioFuture company) and utilize gel imaging instrument protein band to develop.
Tormentic acid process MCF-7 tumor cell can make caspase-3 and caspase-9 of non-activity cut, caspase-3 and caspase-9 take part in the apoptosis process that Tormentic acid induces.Tormentic acid process can lower the ERK1/2 protein expression (Figure 13) of NF-κ B and phosphorylation.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (3)
1. triterpenoid compound is preparing the application in antitumor drug, it is characterized in that: described triterpenoid compound is Tormentic acid, its structural formula as shown in Equation 1:
2. triterpenoid compound according to claim 1 is preparing the application in antitumor drug, it is characterized in that:
Described antitumor drug is anti-breast cancer medicines, anti-lung-cancer medicament or medicines resistant to liver cancer.
3. an antitumor drug, is characterized in that: containing triterpenoid compound according to claim 1.
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| CN106176764A (en) * | 2016-07-12 | 2016-12-07 | 河南省医药科学研究院 | 2a in Isodon excisoides, the application in preparing antitumor drug of 3 β, 19a trihydroxy 12 alkene 28 ursolic acid |
| CN111297820A (en) * | 2020-03-25 | 2020-06-19 | 哈高科白天鹅药业集团有限公司 | Thymosin enteric-coated tablet and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106176764A (en) * | 2016-07-12 | 2016-12-07 | 河南省医药科学研究院 | 2a in Isodon excisoides, the application in preparing antitumor drug of 3 β, 19a trihydroxy 12 alkene 28 ursolic acid |
| CN106176764B (en) * | 2016-07-12 | 2018-10-09 | 河南省医药科学研究院 | 2a in Isodon excisoides, 3 β, -12 alkene -28- ursolic acid application in preparations of anti-tumor drugs of 19a- trihydroxies |
| CN111297820A (en) * | 2020-03-25 | 2020-06-19 | 哈高科白天鹅药业集团有限公司 | Thymosin enteric-coated tablet and preparation method thereof |
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