CN101851220A - Isoprene flavonoid compound and application thereof - Google Patents
Isoprene flavonoid compound and application thereof Download PDFInfo
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- CN101851220A CN101851220A CN200910133449A CN200910133449A CN101851220A CN 101851220 A CN101851220 A CN 101851220A CN 200910133449 A CN200910133449 A CN 200910133449A CN 200910133449 A CN200910133449 A CN 200910133449A CN 101851220 A CN101851220 A CN 101851220A
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Abstract
The invention relates to a novel isoprene flavonoid compound represented by a structural formula selected from the following group and a medicinal composition containing at least one such compound.
Description
Technical field
The present invention is about isoprene flavonoid compound of novelty and uses thereof.
Background technology
Propolis (Propolis) is that honeybee is collected from the resin compound of setting bud, fruit, myron stream or other plant origin.Report the biological activity that propolis contains various activeconstituentss and tool broad range, comprised antitumor, anti-oxidant, antibacterium, antiviral, antimycotic and antiphlogistic activity.Propolis from different areas can contain different activeconstituentss.Pertinent literature comprises: Cancer Lett.2007,252,235-243, Brain Res.2008,1201,135-142, J Appl Microbiol.2007,103,1914-1921, Antivir Chem Chemother.2008,19,7-13, J Pharmacol Sci.2005,99,39-44 and Experientia 1988,44,230-232.
We had before reported the Prenylflavonoid of 8 kinds of separation from the Taiwan propolis, and it is represented by following structural 1 to 8 respectively:
Formula 3 (the plain C of propolis)
Existing these propolis elements of report explanation have the biological activity of broad range, comprise anticancer, anti-oxidant and antimicrobial acivity.In addition, nearest studies confirm that activeconstituents and Taiwan propolis ingredient that Ryukyu's propolis (Okinawan propolis) is contained are similar to.Pertinent literature comprises: J.Nat.Prod.2003,66,503-506, Biochem.Pharmacol.2004,67,53-66, Evid.BasedComplement.Alternat.Med.2004,1,175-185, Cancer Lett.Cancer Lett.2007,245,218-231, J.Agric.Food.Chem.2007,55,7366-7376, J.Agric.Food.Chem.2007,55,5289-5298, J.Sci.Food Agric.2008,88,412-419, Biosci., Biotechnol.Biochem.2004,68,260-262 and J.Agric.Food Chem.2007,55,7722-7725.
Tissue protein deacetylase (HDAC, Histone deacetylase) is the deacetylation from the ε-amido of amino acid residue of catalysis tissue protein N terminal.Found and proved that some hdac inhibitor before clinical and clinical stage, has curative effect for the sufferer of suffering from dissimilar cancers.Model according to the anticancer mechanism of present hdac inhibitor; this inhibitor is the proteic acetylize excessively of initiated core heart tissue, therefore excites Chromatin Remodeling and activation silent gene (silent gene), such as; tumor suppressor gene, and cause the inhibition of growth of tumour cell.Pertinent literature comprises: Expert OpinInvestig Drugs.2007,16,1111-1120, Blood.2007,109,2781-2790, Adv ExpMed Biol.2008,615,261-298 and Cell Mol Immunol.2007,4,337-343.
Still have at present and need seek novel composition and assess its useful physiologically active from multi-functional propolis.
Summary of the invention
In an aspect, the invention provides a kind of isoprene flavonoid compound of novelty, it is to be selected from the following group's who is formed structural formula representative:
In another aspect, the invention provides a kind of medical composition, it comprises at least a above-mentioned isoprene flavonoid compound.
Description of drawings
Described and the embodiment of preamble can reach better description effect by accompanying drawing.In order to strengthen explanation of the present invention, the graphic of suitable embodiment is recited in this.Be noted that the present invention is not limited to be recited in this explanation.
Fig. 1 represents the important HMBC relation between the H and C among the plain I of propolis.
Fig. 2 represents the important HMBC relation between the H and C among the plain J of propolis.
Fig. 3 shows the cutting edge of a knife or a sword value that oppositely prepares HPLC among the embodiment 2.
Fig. 4 (A) shows that wherein (a) means the control group processing through the plain coloration result of handling 24 hours MCF-7 cell of the different propolis of various concentration; (b), (c) reaches and (d) means the plain F processing of propolis that is respectively 5,10 and 15 μ g/mL with concentration; (e), (f) reaches and (g) means the plain I processing of propolis that is respectively 2.5,5.0 and 7.5 μ g/mL with concentration; And (h), (i) and (j) mean that the plain J of propolis that is respectively 5,10 and 15 μ g/mL with concentration handles.
Fig. 4 (B) shows that wherein (a) means control treatment through the plain coloration result of handling 24 hours MDA-MB-231 cell of the different propolis of various concentration; (b), (c) reaches and (d) means the plain F processing of propolis that is respectively 5,10 and 15 μ g/mL with concentration; (e), (f) reaches and (g) means the plain I processing of propolis that is respectively 2.5,5.0 and 7.5 μ g/mL with concentration; And (h), (i) and (j) mean that the plain J of propolis that is respectively 5,10 and 15 μ g/mL with concentration handles.
Fig. 4 (C) shows that wherein (a) means control treatment through the plain coloration result of handling 72 hours MCF-7 cell of the different propolis of various concentration; (b), (c) reaches and (d) means the plain F processing of propolis that is respectively 5,10 and 15 μ g/mL with concentration; (e), (f) reaches and (g) means the plain I processing of propolis that is respectively 2.5,5.0 and 7.5 μ g/mL with concentration; And (h), (i) and (j) mean that the plain J of propolis that is respectively 5,10 and 15 μ g/mL with concentration handles.
Fig. 4 (D) shows that wherein (a) means control treatment through the plain coloration result of handling 72 hours MDA-MB-231 cell of the different propolis of various concentration; (b), (c) means the plain F processing of propolis that is respectively 10 and 15 μ g/mL with concentration; (d) and (e) mean that the plain I of propolis that is respectively 5.0 and 7.5 μ g/mL with concentration handles; And (f) and (g) mean that the plain J of propolis that is respectively 10 and 15 μ g/mL with concentration handles.
Fig. 4 (E) shows through the plain number of handling 72 hours MCF-7 cell of the different propolis of various concentration.
Fig. 5 (A) shows that wherein (a) means control treatment through the plain fluidic cell measurement result of handling 72 hours MCF-7 cell of the different propolis of various concentration; (b), (c) reaches and (d) means the plain F processing of propolis that is respectively 5,10 and 15 μ g/mL with concentration; (e), (f) reaches and (g) means the plain I processing of propolis that is respectively 2.5,5.0 and 7.5 μ g/mL with concentration; And (h), (i) and (j) mean that the plain J of propolis that is respectively 5,10 and 15 μ g/mL with concentration handles.
Fig. 5 (B) shows that wherein (a) means control treatment through the plain fluidic cell measurement result of handling 72 hours MDA-MB-231 cell of the different propolis of various concentration; (b), (c) reaches and (d) means the plain F processing of propolis that is respectively 5,10 and 15 μ g/mL with concentration; (e), (f) reaches and (g) means the plain I processing of propolis that is respectively 2.5,5.0 and 7.5 μ g/mL with concentration; And (h), (i) and (j) mean that the plain J of propolis that is respectively 5,10 and 15 μ g/mL with concentration handles.
Fig. 5 (C) shows that wherein (a) means control treatment through the plain fluidic cell measurement result of handling 72 hours MDA-MB-231 cell of the different propolis of various concentration; (b) and (c) mean with the concentration plain F processing of propolis of 10 and 15 μ g/mL respectively; And (d) and (e) mean that the plain I of propolis that is respectively 5.0 and 7.5 μ g/mL with concentration handles; And (f) and (g) mean that the plain J of propolis that is respectively 10 and 15 μ g/mL with concentration handles.
Fig. 6 (A) is presented at result's (handling the MCF-7 cell 6 hours) of immunocytochemical study among the embodiment 6.
Fig. 6 (B) is presented at embodiment 6 Chinese and westerns and inhales result's (handling the MCF-7 cell 48 hours) that stain is measured.
Embodiment
Unless otherwise defined, used herein all technology and science vocabulary have the affiliated skill person of this paper of haveing the knack of institute clear same meaning usually.
In this article, article " " means the grammatical object of this article of one or more (that is, at least one).For example, " assembly " means an assembly or the assembly more than.
In an aspect, the invention provides the isoprene flavonoid compound of a kind of separation from the Taiwan propolis.
A kind of specific embodiment of the present invention's isoprene flavonoid compound is 5,7,3 ', 4 '-tetrahydroxy-6-farnesyl flavones (be called " the plain I of propolis ", represent by following formula:
Formula I compound has molecular formula C30H36O6 and molecular weight is 492.25Da.
Another specific embodiment of isoprene flavonoid compound is 5,7, and 4 '-trihydroxy--6-geranyl flavones (being called " the plain J of propolis ") is represented by following formula:
Formula II compound has molecular formula C
25H
28O
5And molecular weight is 408.19Da.
The present invention's isoprene flavonoid compound is inhibited for the growth of HDAC enzymic activity and cancer cells, and preferable growth for breast cancer cell is inhibited.
Therefore, in another aspect, the invention provides a kind of medical composition as hdac inhibitor, it comprises at least a The compounds of this invention and medical acceptable vehicle or supporting agent.
In another aspect, the invention provides a kind of medical composition of anticancer growth, it comprises at least a The compounds of this invention and medical acceptable vehicle or supporting agent.
The present invention also provides the medical composition of a kind of treatment disease relevant with histone deacetylaseization, and it comprises at least a The compounds of this invention and medical acceptable vehicle or supporting agent.
In addition, many known hdac inhibitors have proved to have neuroprotective, and the hint hdac inhibitor can be used for treating neurodegenerative disorders.The example of these neurodegenerative disorders comprises, but be not limited to, multiple sclerosis (MS, Multiple sclerosis), Heng Dingdunshi disease (HD, Huntington ' s disease), spinal cord muscular dystrophy (SMA, Spinal muscular atrophy), spinal and bulbar muscular atrophy (SBMA, Spinal and bullar muscular atrophy) and amyotrophic lateral sclerosis (ALS, Amyotrophic lateral sclerosis).Pertinent literature comprises: F.Epigenetics.2006 Apr-Jun; 1 (2): 67-75.Epub 2006 Mar 5, Hum MolGenet.2007 Jun 1; 16 (11): 1293-306.Epub 2007, J Neurochem.2006Jul; 98 (1): 193-202, Hum Genet.2006 Aug; 120 (1): 101-10.Epub 2006 May, Hum Mol Genet.2005 May 1; 14 (9): 1171-82.Epub 2005 Mar, Hum Mol Genet.2004 Jun 1; 13 (11): 1183-92.Epub 2004 Apr, Curr Biol.2004 Mar23; 14 (6): 488-92, Proc Natl Acad Sci U S be Feb 18 A.2003; 100 (4): 2041-6.Epub2003 and Hum Mol Genet.2007 Jun 1; 16 (11): 1293-306.Epub 2007 Apr.
Therefore, the invention provides a kind of medical composition with neuroprotective, it comprises at least a The compounds of this invention and medical acceptable vehicle or supporting agent.
The present invention also provides a kind of medical composition for the treatment of neurodegenerative disorders, and it comprises at least a The compounds of this invention and medical acceptable vehicle or supporting agent.Neurodegenerative disorders is preferably multiple sclerosis (MS), Heng Dingdunshi disease (HD), spinal cord muscular dystrophy (SMA), spinal and bulbar muscular atrophy (SBMA) or amyotrophic lateral sclerosis (ALS).
When making the present invention's composition, to be diluted with vehicle usually or be encapsulated in the supporting agent as the The compounds of this invention of activeconstituents, its form can be capsule, anther sac (sachet), paper or other container.Vehicle is generally and is suitable for dispensing to human or other mammiferous vehicle.The example of appropriate excipients comprises, but be not limited to lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, gum arabic, calcium phosphate, alginates, tragcanth, gelatin, Calucium Silicate powder, crystallite shape Mierocrystalline cellulose, polyethylene Pyrrolizidine ketone, Mierocrystalline cellulose, water, syrup and methylcellulose gum.In addition, the present invention's composition can be the form of lozenge, pill, powder, suspension, emulsion, solution, syrup, aerosol (solid form or in fluid matrix), ointment, capsule or aseptic packaging powder.
Composition can be offerd medicine to the patient via oral route (for example), comprises the mankind, monkey, dog etc., perhaps offers medicine via the outer approach of enteron aisle with the injection formulations form.Typically, the present invention's composition contains the 125mg that has an appointment to the The compounds of this invention of about 250mg (propolis element I or J, or the two).Adult's dosage is preferable between every day about 500mg and 1000mg, its can single dose or the form of separate doses offer medicine.
The specification specified of each specific examples of the present invention as after.The present invention's further feature will be known via the detailed description in following each specific examples and presents.This field tool knows that usually the knowledgeable can understand the present invention and have various aspects, and following specific examples is only in order to explanation but not as the present invention's restriction.
Embodiment
Embodiment 1: preliminary extraction
Make 200g TW-I level Taiwan propolis (collecting) from the honeycomb of Taiwan Tai Nan
(29)Stir and homogenize at low temperature.Then the sample that will homogenize with 1.0L deionized water rinsing 3 times and with resistates with 95% alcohol extraction 3 times.The ethanolic extract that filters out is evaporated to dried under decompression, obtains brown powder (135.6g), it is stored in-20 ℃, to be further purified.
Embodiment 2: the separation of compound and purifying
Methyl alcohol will be dissolved in and put on Sephadex LH-20 tubing string that (Amersham Pharmacia Biotech AB, Uppsala Sweden), wherein use 95% ethanol as the dissolved solvent from the brown powder that ethanolic extract obtains.Detect the effect of all leachables (comprising the differentiation that follow-up chromatography obtains) for human breast cancer propagation, and by chromatography on Sephadex LH-20 tubing string and use 95% ethanol as the dissolved agent, and isolate active part once more.Then, (Darmstadt l Germany), uses normal hexane/EtOAc solvent systems for Kiesel gel 60, E.Merck to make active part carry out the silicone tube column chromatography.
With reverse preparation HPLC (normal hexane/EtOAc, 30: 70) the highest district's part of activity is carried out purifying.Experiment condition is as follows: tubing string is Luna Phenomenex (C18,250 x 4.6mm); Solvent systems is methanol (8: 2); Flow velocity is 1mL/min; And under UV280nm, detect.Collecting the residence time is district's part of 19.0 minutes (the plain I of propolis) and 10.5 minutes (the plain J of propolis), isolates plain I (pale yellow powder) of propolis and the plain J (weak yellow liquid) of propolis by this respectively.Fig. 3 shows the peak value of reverse preparation HPLC.
Embodiment 3: the evaluation of plain I of propolis and the plain J of propolis
Use each structure of plain I of following instrumental analysis propolis and the plain J of propolis: Perkin Elmer1760-X IR-FT spectrophotometer; Hitachi 150-20UV; Jasco J-710 spectropolarimeter; Finnigan MAT-95XL mass spectrograph (EI); And Bruker AV-500 spectrophotometer (using solvent spike (MeOH-d3) conduct) with reference to standard.
The plain I of propolis
The materialization data of the plain I of propolis are as follows:
[]
20 D+ 3.8 (c 0.26, CH
3OH); IR vmax (film) 3747,3390,2923,1636cm
-1UV (EtOH) λ max nm (log ε) 207.0 (1.65), 291 (0.73); CD (MeOH) 347nm (Δ ε+3.19), 294nm (Δ ε-3.98); HREIMS m/z 492.2512 (C
30H
36O
6Calculated value: 492.2512);
1H reaches
13C NMR.
From
1H NMR spectrum truly has 4 alkene methyl (δ as can be known
H1.52,1.56,1.63,1.74), 8 methene proton (δ
H1.87,1.95,2.05), 2 benzyl methene proton (δ
H3.20) and 2 vinyl proton (δ
H5.04,5.18), expression has the existence of farnesyl.In addition,
13C NMR spectrum shows 4 methyl (δ
c16.1,16.2,17.8,25.9), 5 mesomethylene carbon (δ
c21.8,27.3,27.8,40.8,40.9), 3 alkatrienes carbon (δ
c124.1,125.3,125.6) and 3 level Four alkene carbon (δ
c132.0,134.9,135.8), this further supports the existence of farnesyl.Moreover, δ
H5.21 (1H, dd, J=3.0,13.0Hz), 2.65 (1H, dd, J=3.0,17.0Hz) and 3.02 (1H, dd, J=13.0, ABX system 17.0Hz) shows the feature mode of flavones skeleton in H-2 and H-3 position respectively.
In addition,
1H reaches
13C NMR ownership and association are foundation
1H-
1H COSY, HSQC and deriving of HMBC data and determine.The HMBC spectrum of the plain I of propolis shows δ
HThe methylene signals of (3.20 H-1 ") respectively with C-5 (δ
c162.5) and C-7 (δ
c165.9) relevant, this suggestion method Thessaloniki is linked to C-6 (Fig. 1).In addition, CD spectrum shows that the configuration of C-2 is the S-type.
Therefore, the plain I of propolis is accredited as 5,7, and 3 ', 4 '-tetrahydroxy-6-farnesyl flavones is with above-mentioned formula I representative.This series of compounds separates first and is not disclosed in the open document.
The plain J of propolis
The materialization data of the plain J of propolis are as follows:
[]
20 D+ 2.4 (c 0.41, CH
3OH); IR vmax (film) 3747,3393,2925,2361,1637cm
-1UV (EtOH) λ max nm (log ε) 209.0 (1.82), 293 (0.92); CD (MeOH) 328.9nm (Δ ε+2.37), 289nm (Δ ε-3.98); HREIMS m/z 408.1944 (C
25H
28O
5Calculated value: 408.1937);
1H reaches
13C NMR.
From
1H NMR spectrum truly has 3 alkene methyl (δ as can be known
H1.55,1.61,1.74), 4 methene proton (δ
H1.93,2.04), 2 benzyl methene proton (δ
H3.18) and 2 vinyl proton (δ
H5.05,5.18), this shows the existence of geranyl (Geranyl).In addition,
13C NMR spectrum shows 3 methyl (δ
c16.2,17.7,25.9), 3 mesomethylene carbon (δ
c21.8,27.7,40.9), 2 three grades alkene carbon (δ
c123.9,125.5) and 2 level Four alkene carbon (δ
c132.0,135.3), this further supports the existence of geranyl.Moreover, δ
H5.29 (1H, dd, J=2.7,13.0Hz), 2.67 (1H, dd, J=2.7,17.0Hz) and 3.08 (1H, dd, J=13.0, ABX system 17.0Hz) shows the feature mode of flavones skeleton in H-2 and H-3 position respectively.
In addition,
1H reaches
13C NMR ownership and association are foundation
1H-
1H COSY, HSQC and deriving of HMBC data and determine.The HMBC spectrum of the plain J of propolis shows δ
HThe methylene signals of (3.18 H-1 ") respectively with C-5 (δ
c162.5) and C-7 (δ
c166.0) relevant, this hint geranyl is linked to C-6 (Fig. 2).In addition, CD spectrum shows that the configuration of C-2 is the S-type.
Therefore, the plain J of propolis is accredited as 5,7, and 4 '-trihydroxy--6-geranyl flavones is with above-mentioned formula II representative.This compound is for separating first and not being disclosed in the open document.
Table 1 is listed the materialization data of plain I of propolis and J.
A, bData are commutative
Embodiment 4: cell cultures and cytotoxic assay
Human breast cancer MCF-7 and MDA-MB-231 cell are available from foodstuffs industry institute (Hsin-chu).With cell cultures in following substratum: Dulbecco ' s modified Eagle ' s medium, contain 1% diluent and the 2mM Vetsin of 10% N of tire serum (FBS, Fetal boving serum), penicillin-Streptomycin sulphate.Cell is maintained at 37 ℃, 95% air and 5%CO
2Wet air in.Wherein, the MDA-MB-231 cell cultures in L-15 substratum (Gibco), is contained 1% diluent and the 2mM Vetsin of 10% N of tire serum (FBS), penicillin-Streptomycin sulphate.Cell is maintained at 37 ℃, 100% air and 0%CO
2Wet air.
Propolis plain F, I and J are dissolved in dimethyl Asia (DMSO, Dimethyl sulfoxide) and are prepared into fixed concentration is 10mg/mL.With cell (1.5 x 10
6/ dish) is incubated at the 100-mm dish, cultivated 14 hours, handled 48 hours with DMSO then or handled 48 hours with the propolis element (2.5,5.0,10 and 20.0 μ g/mL) of different concns.The propolis element is a micromolecular compound, and its molecular weight is 408 to 492Da.Change these numerical value into discrete not ear concentration.
Calculate cell number and measure its viability by trypan blue look eliminating assay method (Trypan blue exclusionassay).The inhibition of compound is active in IC
50Value representation (bringing out the required concentration of 50% cytotoxicity), mean value are to obtain from three repeating datas.
Table 2 is presented at the IC of plain I of propolis of the present invention resultant in the cytotoxic assay and J
50The IC of the plain F of value (μ M) and control group propolis
50Value.
Table 2
aIC
50Value (μ M) is from the representative experiment of one of three independent experiments.
Therefore, the present invention's compound (plain I of propolis and J) confirms to have the cellular cytoxicity activity that resists human breast cancer cell.
Embodiment 5: plain I of propolis and J are for the restraining effect of the growth of cancer cells
Cell dyeing
The breast cancer cell line MCF-7 (ER receptor positive) and the MDA-MB-231 cell (ER receptor negative) of two types are incubated at embodiment 5 described conditions.These cells were handled 24 or 72 hours so that the different propolis of various concentration are plain.With treated cell with trypan blue dyeing and assess its viability.
Fig. 4 A shows through the plain coloration result of handling 24 hours MCF-7 cell of the propolis of various concentration.Fig. 4 B shows through the plain coloration result of handling 24 hours MDA-MB-231 cell of the propolis of various concentration.Fig. 4 C shows through the plain coloration result of handling 72 hours MCF-7 cell of the propolis of various concentration.Fig. 4 D shows through the plain coloration result of handling 72 hours MDA-MB-231 cell of the propolis of various concentration.Fig. 4 E shows through the plain cell number of handling 72 hours MCF-7 cell of the propolis of various concentration.
Flow cytometry
(1.5x 10 for human breast cancer MCF-7 that will be in the 100-mm culture plate and MDA-MB-231 cell
6) handled 24 or 72 hours with the propolis of various concentration plain F, I and J.Cell is collected with trypsin treatment and with ice-cold PBS.These cell resuspending are in 200 μ L PBS and add ice-cold 100% ethanol of 800 μ L and fixed, overnight in-20 ℃ of cultivations then.With centrifugal collecting cell, with low the open damping fluid (0.5%Triton X-100 be dissolved in PBS and 1 μ g/mLRNase A) of its resuspending in 1mL, and in 37 ℃ of cultivations 30 minutes.Add the PI solution (50 μ g/mL) of 1mL then, and allow this mixture leave standstill 30 minutes in 4 ℃.Then with FACScan cell instrument (BectonDickinson) analysis of cells dna content.
Fig. 5 A shows through the plain result who handles 72 hours MCF-7 cell of the propolis of various concentration.Fig. 5 B shows through the plain result who handles 24 hours MDA-MB-231 cell of the propolis of various concentration.Fig. 5 C shows through the plain result who handles 72 hours MDA-MB-231 cell of the propolis of various concentration.
As Fig. 4 and shown in Figure 5, the present invention's compound confirms inhibited for the growth of cancer cells.
The west ink blok is analyzed
Human breast cancer MCF-7 cell (1.5 x 10 that will be on the 100-mm culture plate
6) handled 24 hours with the propolis of various concentration plain F, I and J.After the processing, collecting cell and with its resuspending in the molten damping fluid that splits of 100 μ L.The protein (30 μ g) of equivalent mixed with the 2x sample buffer and detect beta-actin, Bid, p21, Ac-tissue protein 3, CTPS and gelsolin (gelsolin) and resolve with 12.5%SDS-PAGE.The protein electromigration is gone to transfer film (immobilonmembrane) (PVDF; Millipore Corp.) and with reversible dyestuff amido black (Sigma ChemicalCo.) this film is dyeed, identical to confirm the protein heap(ed) capacity.Blockade overnight with 20mM Tris-HCl (pH 7.4), 125mM NaCl, 0.2%Tween 20 and solution that 3%BSA was formed subsequently.Used specific antibody is anti-human Bid (1: 500 rabbit polyclonal antibody; CellSignaling Technology, Inc.), anti--Ac-tissue protein (1: 1000 rabbit polyclonal antibody; Cell Signaling Technology, Inc.), anti--p21 (1: 1000 mouse monoclonal antibody; BDPharmingen Technology, Inc.), anti--CTPS (1: 1000 mouse monoclonal antibody; Taiwan ABNOVA company), anti--gelsolin (mouse monoclonal antibody of 1: 1000; Sigma ChemicalCo.) reaches anti--beta-actin (mouse monoclonal antibody of 1: 5000; Cell Signaling Technology, Inc.).(ECL Amersham) detects protein with chemoluminescence.
Embodiment 6:HDAC enzyme assay
Immunocytochemical study
The MCF-7 cell cultures was handled 6 hours on 6 holes cultivation slide and with propolis plain F, I and J.Use is known as the SAHA of hdac inhibitor as positive controls.Slide is fixed 30 minutes with 80% methanol solution under room temperature, then with PBS flushing 3 times.With cell in room temperature with 0.3%Triton X-100 infiltration processing 30 minutes; blockaded 1 hour in room temperature with the 10% N of tire serum (FBS) that is dissolved in PBS then, cultivate overnight in 4 ℃ with anti-ethanoyl tissue protein H3 (Cell Signaling) afterwards through dilution in 1: 500.After PBS flushing 3 times, use secondary antibodies to dye 1.0 hours slide in conjunction with anti-rabbit igg, then with PBS flushing 3 times, and (Sigma) give sealing with sealing substratum (mounting medium).In parallel control experiment, observe and do not add a then dye-free result generation of antibody.(Melville NY) analyzes slide with Olympus PM30 photographic camera under fluorescent microscope.Fig. 6 A shows the result of immunocytochemical study.
The west ink blok is measured
Carrying out the west ink blok according to embodiment 6 described methods for Ac-tissue protein 3, p21, gelsolin, CTPS and beta-actin measures.Fig. 6 B shows the result of west ink blok mensuration.
Shown in Fig. 6 A and 6B, the present invention's compound confirms inhibited for the HDAC enzymic activity.
Claims (8)
1. compound, it is by the structural formula representative that is selected from the following group who forms:
2. medical composition as tissue protein deacetylase (HDAC) inhibitor, it comprises at least a compound and medical acceptable vehicle or supporting agent according to claim 1.
3. medical composition that is used for the anticancer growth, it comprises at least a compound and medical acceptable vehicle or supporting agent according to claim 1.
4. as the medical composition according to claim 3, it is effective in the growth that suppresses breast cancer cell.
5. medical composition that is used for the treatment of the disease relevant with histone deacetylaseization, it comprises at least a compound and medical acceptable vehicle or supporting agent according to claim 1.
6. medical composition with neuroprotective, it comprises at least a compound and medical acceptable vehicle or supporting agent according to claim 1.
7. medical composition that is used for the treatment of neurodegenerative disorders, it comprises at least a compound and medical acceptable vehicle or supporting agent according to claim 1.
8. as the medical composition according to claim 7, wherein this neurodegenerative disorders system is selected from the group that multiple sclerosis (MS), Heng Dingdunshi disease (HD), spinal cord muscular dystrophy (SMA), spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are formed.
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| CN104470909A (en) * | 2012-03-23 | 2015-03-25 | Ptc医疗公司 | Compounds for treating spinal muscular atrophy |
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| CN104470909A (en) * | 2012-03-23 | 2015-03-25 | Ptc医疗公司 | Compounds for treating spinal muscular atrophy |
| CN104127442A (en) * | 2013-05-02 | 2014-11-05 | 彦臣生技药品股份有限公司 | Liver protecting composition |
| CN107987047A (en) * | 2017-12-22 | 2018-05-04 | 成都普思生物科技股份有限公司 | A kind of Mang ox base chromocor compound and its methods and applications extracted from the root bark of white mulberry |
| CN107987047B (en) * | 2017-12-22 | 2020-03-20 | 成都普思生物科技股份有限公司 | Geranylflavone compound extracted from cortex Mori, and its preparation method and application |
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