CN101091707A - Medicinal usage of kuhseng extractive - Google Patents
Medicinal usage of kuhseng extractive Download PDFInfo
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- CN101091707A CN101091707A CN 200610028072 CN200610028072A CN101091707A CN 101091707 A CN101091707 A CN 101091707A CN 200610028072 CN200610028072 CN 200610028072 CN 200610028072 A CN200610028072 A CN 200610028072A CN 101091707 A CN101091707 A CN 101091707A
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Abstract
The present invention belongs to the field of medicine, and discloses an application of flavescent sophora flavone compound or its mixture. It can be used for preparing inhibitor for inhibiting NF-kB, EGFR, Her-2 or KDR activity. Further, said flavescent sophora flavone compound or its mixture can be used for preparing medicine for curing the diseases of tumor or inflammation resulted from high-expression or high-activity of NF-kB, EGFR, Her-2 or KDR.
Description
Technical field
The invention belongs to field of medicaments, specifically, the present invention relates to the medical usage of a kind of Radix Sophorae Flavescentis flavone compound or its mixture.
Background technology
Chinese medicine Radix Sophorae Flavescentis (Sophora flavescens Ait) is a cassia leguminous plant, and its bitter in the mouth cold in nature has heat-clearing and toxic substances removing, the arresting lacrimation that makes eye bright, wind dispelling insecticide, settling five organs, an effect of turning round sedate.Therefrom isolated main chemical compositions can be divided into two big class, i.e. Radix Sophorae Flavescentis alkaloid and Radix Sophorae Flavescentis flavone.Someone has isolated 23 kinds of alkaloids and 32 kinds of flavone and isoflavonoid (Miao Kangli, Zhang Jianzhong etc., research and development of natural products, 2000,13 (2): 69～73) from radix sophorae, stem, leaf with spending.
Chinese patent " a kind of kuh-seng total flavone extract and its production and use " (CN1676520) discloses the purposes of the flavone extract (comprising kurarinone, 2-methoxyl group-kurarinone and Chinese scholartree flavanone) of a class Radix Sophorae Flavescentis as TNF-α and IL-1 beta inhibitor, and some monomers or the mixture that exist in the visible Radix Sophorae Flavescentis extract have medical value preferably.
Therefore, be necessary further the extract or the active ingredient of Radix Sophorae Flavescentis to be studied, find out its new action target spot, to develop valuable drug.
Summary of the invention
The object of the present invention is to provide a kind of Radix Sophorae Flavescentis flavone compound or its mixture purposes, it can be used for preparing the medicine that suppresses NF-κ B, EGFR, Her-2 or the active inhibitor of KDR or preparation treatment tumor or inflammation.
In a first aspect of the present invention, the Radix Sophorae Flavescentis flavone compound shown in a kind of formula (I) is provided or contains the purposes of the mixture of the Radix Sophorae Flavescentis flavone compound shown in the formula (I),
In the formula, R1 represents H or CH
3
R2 represents H or CH
3
Wherein, chemical compound shown in the formula (I) or the mixture that contains chemical compound shown in the formula (I) are used to preparation and suppress NF-κ B, EGFR, Her-2 or the active inhibitor of KDR.
In a preference of the present invention, the Radix Sophorae Flavescentis flavone compound shown in the formula (I) is selected from: kurarinone, 2 '-methoxyl group-kurarinone or Chinese scholartree flavanone G.
In another preference of the present invention, the mixture of described Radix Sophorae Flavescentis flavone compound is the mixture that contains two or more flavone compound that is selected from down group: kurarinone, 2 '-methoxyl group-kurarinone or Chinese scholartree flavanone G.
In another preference of the present invention, the mixture of described Radix Sophorae Flavescentis flavone compound contains following component:
Kurarinone: 20～60 weight portions;
2 '-methoxyl group-kurarinone: 1～5 weight portion; With
Chinese scholartree flavanone G:1～12 weight portions.
Preferred, the mixture of described Radix Sophorae Flavescentis flavone compound contains following component:
Kurarinone: 35～45 weight portions;
2 '-methoxyl group-kurarinone: 2～4 weight portions; With
Chinese scholartree flavanone G:5～8 weight portions.
Most preferred, the mixture of described Radix Sophorae Flavescentis flavone compound contains following component:
Kurarinone: 45 weight portions;
2 '-methoxyl group-kurarinone 2 weight portions; With
Chinese scholartree flavanone G 6 weight portions.
In another preference of the present invention, the mixture of described Radix Sophorae Flavescentis flavone compound is the extract of Radix Sophorae Flavescentis.
In another preference of the present invention, described inhibition NF-κ B, EGFR, Her-2 or the active inhibitor of KDR are used to prepare the medicine of prevention or treatment tumor or inflammation.
In another preference of the present invention, described tumor includes, but is not limited to: bladder cancer, cerebral glioma, gastric cancer, pulmonary carcinoma, carcinoma of prostate, tumor of head and neck, breast carcinoma, ovarian cancer, carcinoma of endometrium, carcinoma of fallopian tube, cervical cancer, carcinoma of prostate, hepatocarcinoma, colon cancer, human primary gastrointestinal cancers or renal cell carcinoma; Perhaps
Described inflammation includes, but is not limited to: rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, inflammatory bowel, systemic lupus erythematosus (sle), bacterial infection, acute pancreatitis, sepsis, Pyoderma gangrenosum, septicemia or septic shock.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, described pharmaceutical composition contains:
(a) the Radix Sophorae Flavescentis flavone compound of effective dose or its compositions; With
(b) the chemotherapy of tumors preparation of effective dose.
In another preference of the present invention, described chemotherapy of tumors preparation is selected from: 5-fluorouracil, paclitaxel, Docetaxel, cisplatin, methotrexate, amycin, vincristine, mitomycin, bleomycin, table podophyllin, Irinotecan, special willing or its combination of topology.
Preferred, described chemotherapy of tumors preparation is selected from: paclitaxel, Docetaxel or its combination.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
The specific embodiment
The inventor is through long term studies and test, find that first Radix Sophorae Flavescentis flavone compound or its mixture (as plant extract) can suppress the activity of NF-κ B, EGFR, Her-2 or KDR, and then can be used for preparing by the high expressed of NF-κ B, EGFR, Her-2 or KDR or the disease that high activity causes; Such as tumor and/or inflammation.Finished the present invention based on this.
Radix Sophorae Flavescentis flavone compound or its mixture
As used herein, described " Radix Sophorae Flavescentis flavone compound " is meant that a class generally is present in the flavone compound in the Radix Sophorae Flavescentis.Described " flavone compound " is meant the middle general name that is connected to form a compounds of C6-C3-C6 basic framework by three carbochains of two phenyl ring.
Preferred, it is the structure shown in (I) that Radix Sophorae Flavescentis flavone compound of the present invention has:
Wherein, R1 represents H or CH
3
R2 represents H or CH
3
Preferred, described Radix Sophorae Flavescentis flavone compound is selected from: kurarinone; 2 '-methoxyl group-kurarinone; Or Chinese scholartree flavanone G.
The main plant source of kurarinone (Kurarinone) is the Radix Gentianae Macrophyllae (Gentiana macrophylla Pall) of Radix Sophorae Flavescentis of pulse family (Sophora flavescens Ait) and Gentianaceae, and its chemical structural formula is suc as formula shown in (II):
Molecular formula C
26H
30O
6, molecular weight 438.52,121～123 ℃ of fusing points.
2 '-methoxyl group-kurarinone (2 '-methoxy-Kurarinone) has another name called isokurarinone (Isokurarinone), is yellow crystals, and its chemical structural formula is suc as formula shown in (III):
Molecular formula C
27H
32O
6, molecular weight 438, fusing point 102-104 ℃.
Chinese scholartree flavanone G (Sophoraflavanone G) has another name called Vexibinol (Norkurarinone), is colourless needle (benzene), and its chemical structural formula is suc as formula shown in (IV):
Molecular formula C
25H
28O
6, molecular weight 424.49, fusing point 173-175 ℃.
Described Radix Sophorae Flavescentis flavone compound can be the chemical compound that exists with respective pure form, also can be to contain 0.1-95wt%, and more preferably the form of the plant extract (as Radix Sophorae Flavescentis extract) of the Radix Sophorae Flavescentis flavone compound of 1-90wt% exists.
In the present invention, described " mixture of Radix Sophorae Flavescentis flavone compound " mixture that two or more described Radix Sophorae Flavescentis flavone compound mixes of serving as reasons.
Preferably, the mixture of described Radix Sophorae Flavescentis flavone compound is to contain the mixture that two or more is selected from down the chemical compound of group: kurarinone, 2 '-methoxyl group-kurarinone or Chinese scholartree flavanone G.
In a kind of optimal way of the present invention, the mixture of described Radix Sophorae Flavescentis flavone compound is a kind of plant extract that contains above-mentioned two or more described Radix Sophorae Flavescentis flavone compound.
Preferably, the mixture of described Radix Sophorae Flavescentis flavone compound is a kind of Radix Sophorae Flavescentis extract; Preferred, be the kuh-seng total flavone extract.The mixture that wherein contains kurarinone, 2 '-methoxyl group-kurarinone and Chinese scholartree flavanone G.Further preferred, the mixture of described Radix Sophorae Flavescentis flavone compound contains: kurarinone 20～60 weight portions; 2 '-methoxyl group-kurarinone 1～5 weight portion; With Chinese scholartree flavanone G1～12 weight portions.Most preferred, the mixture of described Radix Sophorae Flavescentis flavone compound contains: kurarinone 35～45 weight portions; 2 '-methoxyl group-kurarinone: 2～4 weight portions and Chinese scholartree flavanone G:5～8 weight portions.Such as, the mixture of described Radix Sophorae Flavescentis flavone compound contains: kurarinone 45 weight portions, 2 '-methoxyl group-kurarinone 2 weight portions and Chinese scholartree flavanone G6 weight portion.
The preparation process of mixture of above-mentioned Radix Sophorae Flavescentis flavone compound of the present invention or described chemical compound can be referring to Chinese patent CN1676520.
The purposes of Radix Sophorae Flavescentis flavone compound or its mixture
The inventor studies have shown that, described Radix Sophorae Flavescentis flavone compound or its mixture can suppress nuclear factor κ B (Nuclear factor-kappa b, NF-κ B), the activity of EGF-R ELISA (EGFR), proto-oncogene Her-2 or kinases insert structure threshold receptor (KDR), thereby can prevent or treat because the high expressed of the above-mentioned factor or the disease that high activity causes.
Nuclear factor κ B (Nuclear factor-kappa b, NF-κ B) is that a class extensively is present in the many dominance nuclear factor in the cell, belongs to the Rel gene family.It has the ability of regulation and control several genes transcriptional expression in tumor cell, close with growth, propagation and infiltration, the relations of metastasis of tumor cell, apoptosis and immunocompetence that can also the modulate tumor cell, it also be the pass key switch that body brings out inflammatory reaction simultaneously.Therefore, suppress the activity of NF-κ B, can stop growth, propagation and the infiltration of tumor cell or shift, and generation that can the inflammation-inhibiting reaction.
Radix Sophorae Flavescentis flavone compound of the present invention or its mixture can be used for preparing the inhibitor of NF-κ B, thereby are used to prevent or treat because the disease that NF-κ B high expressed or high activity cause; Preferred, described disease is inflammation or tumor.Wherein, described tumor includes but not limited to: bladder cancer, cerebral glioma or gastric cancer; Perhaps, described inflammation includes but not limited to: rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, inflammatory bowel, systemic lupus erythematosus (sle), bacterial infection, acute pancreatitis, sepsis, Pyoderma gangrenosum, septicemia, septic shock.
EGF-R ELISA (EGFR) signal transduction pathway plays an important role at the aspects such as propagation, injury repairing, invasion and attack and new vessels formation of tumor cell.Discover,, can suppress the growth of tumor, promote the death of tumor cell by suppressing EGFR.The expression of EGFR is all relevant with kinds of tumors.
Radix Sophorae Flavescentis flavone compound of the present invention or its mixture can be used for preparing the EGFR inhibitor, thereby can be used for preventing or treat because the disease that EGFR high expressed or high activity cause; Preferred, described disease is a tumor.Wherein, described tumor includes but not limited to: pulmonary carcinoma, carcinoma of prostate or tumor of head and neck.
Proto-oncogene Her-2 is also referred to as C-erB-2 or Her-2/neu, is the 2nd member of EGFR family, and it regulates cell growth, differentiation with other EGF-R ELISA by the signal network of complexity.Her-2 is high expressed in Placenta Hominis, embryo's epithelial tissue and many tumor cells, and in normal structure negative or trace expression.Existing studies show that, in the mankind tumor tissue more than 30%, all with the amplification of Her-2/neu gene, as (Hynes NE such as breast carcinoma, ovarian cancer, carcinoma of endometrium, carcinoma of fallopian tube, cervical cancer, carcinoma of prostate, gastric cancer, Stem DF.The biology of erbB-2/neu/Her-2 and its role incancer[J] .Biochim Biophys Acta Rev Cancer.1994,7798:165-184).
Radix Sophorae Flavescentis flavone compound of the present invention or its mixture can be used for preparing the Her-2 inhibitor, thereby can be used for preventing or treat because the disease that Her-2 high expressed or high activity cause; Preferred, described disease is a tumor.Wherein, described tumor includes but not limited to: breast carcinoma, ovarian cancer, carcinoma of endometrium, bladder cancer, carcinoma of fallopian tube, cervical cancer, carcinoma of prostate, hepatocarcinoma, pulmonary carcinoma, colon cancer or gastric cancer.
Kinases insert structure threshold receptor (KDR) is the specific receptor of VEGF (VEGF), and the two combines on the one hand can the induced tumor endothelial cell division, propagation, promotes vascularization; Can impel vascular permeability to increase on the other hand, cause the interior macromolecular substances of blood vessel to spill, thereby help the formation of blood vessel epimatrix, for the propagation of tumor vascular endothelial cell, the formation of blood vessel provide support, secondary vessel forms, and promotes growth of tumor.KDR not only is expressed in the vascular endothelial cell of tumor tissues, and tumor cell itself also has expression.Bibliographical information, the high expressed of KDR and WEGF is relevant with the formation of kinds of tumors, as (Shen Xiaoli, thunderclap, Wan Feng etc. such as pituitary adenoma, gastric cancer, colorectal cancer, pulmonary carcinoma, bladder cancer, breast carcinoma; The expression in pituitary adenoma of VEGF and receptor KDR thereof and the relation of tumor vessel formation; Treatment and prevention of tumour research, 2005,32 (1): 8-10).
Described Radix Sophorae Flavescentis flavone compound or its mixture can be used for preparing the KDR inhibitor, thereby can be used for preventing or treat because the disease that KDR high expressed or high activity cause; Preferred, described disease is a tumor.Wherein, described tumor includes but not limited to: human primary gastrointestinal cancers, renal cell carcinoma or nonsmall-cell lung cancer.
Pharmaceutical composition
The mixture of described Radix Sophorae Flavescentis flavone compound or described Radix Sophorae Flavescentis flavone compound can be prepared into pharmaceutical composition, prevent or treat being used to by the high expressed of NF-κ B, EGFR, Her-2 or KDR or the disease that high activity causes, as tumor or inflammation.
Described pharmaceutical composition can contain effective dose " Radix Sophorae Flavescentis flavone compound " or " mixture of Radix Sophorae Flavescentis flavone compound ", and pharmaceutically acceptable carrier.
As used herein, " effective dose " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.
The composition of " pharmaceutically acceptable " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and allergy), the material of rational benefit/risk ratio is promptly arranged." pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991) at Remington ' s Pharmaceutical Sciences.Acceptable carrier can contain liquid on combination of Chinese medicine is learned, as water, saline, glycerol and ethanol.In addition, also may there be complementary material in these carriers, as filler, disintegrating agent, lubricant, fluidizer, effervescent, wetting agent or emulsifying agent, correctives, pH buffer substance etc.
Dosage form for described pharmaceutical composition has no particular limits, and can be any dosage form that is applicable to that mammal is taken; Preferably, described dosage form can be selected from: granule, capsule, tablet, powder agent, oral liquid, suspension or Emulsion.
The effective dose of the used Radix Sophorae Flavescentis flavone compound or the mixture of described Radix Sophorae Flavescentis flavone compound can change with the order of severity of the pattern of administration and disease to be treated.Yet, when Radix Sophorae Flavescentis flavone compound of the present invention or its mixture every day give with the dosage of about 0.5-500mg/kg the weight of animals, can obtain gratifying effect usually, preferably give with the dosage that separates for 2-4 time every day, or with the slow release form administration.For most of large mammal, the accumulated dose of every day is about 0.1-100mg, preferably is about 0.2-80mg.This dosage of scalable is replied so that optimal treatment to be provided.For example, by the needs of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
In addition, the inventor also finds unexpectedly, when described Radix Sophorae Flavescentis flavone compound or its mixture are treated tumor with some other chemotheraping preparation drug combination, can significantly strengthen the antitumor action of chemotheraping preparation or alleviate the toxic and side effects of chemotheraping preparation.
Therefore, the present invention also provides a kind of pharmaceutical composition, and described pharmaceutical composition comprises: (a) the Radix Sophorae Flavescentis flavone compound of effective dose or its mixture; And (b) the chemotherapy of tumors preparation of effective dose.
Preferably, described chemotherapy of tumors preparation is selected from: 5-fluorouracil, paclitaxel, Docetaxel, cisplatin, methotrexate, amycin, vincristine, mitomycin, bleomycin, table podophyllin, Irinotecan, special willing or its combination of topology.
Preferred, described chemotherapy of tumors preparation is selected from: paclitaxel, Docetaxel or its combination.
In optimal way of the present invention, in the described pharmaceutical composition, the ratio of described Radix Sophorae Flavescentis flavone compound or its mixture and described chemotherapy of tumors preparation is 5-100: 1; Preferred, the ratio of described Radix Sophorae Flavescentis flavone compound or its mixture and described chemotherapy of tumors preparation is 10-50: 1; Most preferred, the ratio of described Radix Sophorae Flavescentis flavone compound or its mixture and described chemotherapy of tumors preparation is 20-40: 1; Such as described ratio can be 20: 1.
Certainly, for the ease of administration, pharmaceutical composition described here also can contain pharmaceutically acceptable carrier, as previously mentioned.
Major advantage of the present invention is:
The present invention has confirmed that first Radix Sophorae Flavescentis flavone compound or its mixture can suppress the activity of NF-κ B, EGFR, Her-2 or KDR, and then can be used for preparing by the high expressed of NF-κ B, EGFR, Her-2 or KDR or the disease that high activity causes; Such as tumor and/or inflammation.
The present invention confirms that first Radix Sophorae Flavescentis flavone compound or its mixture can strengthen the effectiveness of chemotherapy of tumors preparation, thereby has started new approach for tumor treatment.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1 Radix Sophorae Flavescentis extract is to the inhibition activity of NF-κ B
On human embryo kidney (HEK) 293 cell strains, mensuration kurarinone, 2 '-methoxyl group-kurarinone, Chinese scholartree flavanone G, paclitaxel (Taxol) and kurarinone and Taxol share the inhibition activity to NF-κ B.
1. material
(1) cell strain: human embryo kidney (HEK) 293 cells (293 HEK)
(2) test specimen: kurarinone, 2 '-methoxyl group-kurarinone, Chinese scholartree flavanone G, Taxol, kurarinone+Taxol (wherein, the preparation method of kurarinone, 2 '-methoxyl group-kurarinone, Chinese scholartree flavanone G can referring to Chinese patent CN1676520)
(3) positive control: Triptolide (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number is 1566-200201)
(4) negative control: blank cell
(5) reagent: pcDNA3.1 carrier (purchasing company) in California, USA Invitrogen; PNFkB-Luc carrier (purchasing company) in California, USA Clonetech; Lipofectamine
TM2000 transfection reagent boxes (purchasing company) in California, USA Invitrogen; Luciferase detection kit (purchasing company) in Wisconsin, USA Promega; Reorganization human TNF alpha albumen and dimethyl sulfoxide (DMSO) (purchasing company) in Sigma.
2. method
(1) human embryo kidney (HEK) 293 cell strains are available from U.S. ATCC DSMZ; Human embryo kidney (HEK) 293 cell attachments grow in and contain in 10% hyclone (FBS) the DMEM culture medium, in 37 ℃, and 5%CO
2The saturated humidity incubator in cultivate.PNFkB-Luc plasmid and pcDNA3.1 cultivate with the culture fluid that contains the 0.6mg/ml neomycin by liposome cotransfection 293 cells, set up the pNFkB-Luc-293 cell strain of stable transfection through the G418 screening.The pNFkB-Luc-293 cell is according to 1 * 10
5/ hole density is inoculated in 96 orifice plates, is used for follow-up pharmaceutically active evaluation.
(2) each test specimen carries out 3 times of gradient dilutions with the DMEM culture medium respectively, and each cell culture hole adding 10ul sample (the medicine final concentration is respectively 3,10,30,100ug/ml), in 37 ℃, 5%CO
2The saturated humidity incubator in hatched 15 minutes, then induced 6 hours with the human TNF alpha (final concentration 10ng/ml) of recombinating.Triptolide is as positive control drug, and its final concentration is 0.1ug/ml; Add 10 μ LDMEM culture medium in the blank cell hole, as negative control.
(3) adopt conventional method cell lysis, adopt luciferase detection kit (purchasing company) to experimentize, detect 96 orifice plate fluorescence intensities in Perkin-Elmer Victor 3 spectrophotometers in Wisconsin, USA Promega.
Calculate the test specimen suppression ratio with this formula:
IC
50Value is analyzed by XLfit2.0 software.
3. result
Experiment shows that on human embryo kidney (HEK) 293 cell strains, kurarinone, 2 '-methoxyl group-kurarinone and Chinese scholartree flavanone G are to the IC of NF-κ B
50Value has certain cytotoxicity to human embryo kidney (HEK) 293 cell strains, and has the dependent inhibitory action of drug level for 1-50 μ M.
The result shows that also kurarinone and Taxol share, and its suppression ratio is apparently higher than independent use Taxol, and visible kurarinone can strengthen the antitumor action of Taxol.
Embodiment 2 suppresses the analysis of Phosphorylation of EGFR and HER-2
1. experiment material and reagent
Cell strain: A431 (being used for the EGFR analysis of Phosphorylation), MDA-MB-453s (being used for the HER-2 analysis of Phosphorylation) be available from cell institute of the Chinese Academy of Sciences, is incubated to contain in 10% hyclone (Gibco) and the antibiotic DMEM culture medium.
Antibody: (Cat.AF231 is available from R﹠amp for polyclonal antibody anti-EGFR; D) and anti-HER 2 (Cat.E2777 is available from Sigma) and monoclonal antibody Eu
+-PY66 (Cat.AD0040 is available from Perkin Elmer) is respectively applied for bag by the detection of 96 orifice plates and phosphorylated tyrosine.
Other reagent: DELFIA detects buffer (Cat.1244-106), cleaning mixture (Cat.1244-114), reinforcing agent (enhance reagent) (Cat.4001-0010) reach low fluorescence background 96 orifice plates (Cat.AAAnd-0001) and all buy from PerkinElmer.
Cell pyrolysis liquid: 0.1%Triton X-100,50mM Tris.HCl/pH8.0,0.5M NaCl, 0.2mM EDTA, Gastric inhibitory polypeptide (pepstatin) 1 μ g/ml, leupetin (leupeptin) 0.75 μ g/ml, DTT 1mM, Na
3VO
4500uM and PMSF 1uM.
Sample diluting liquid: 20mM Tris HCl/pH7.3,150mM NaCl, 0.1%BSA, 0.05%Tween20.
Test specimen: kurarinone; 2 '-methoxyl group-kurarinone; Chinese scholartree flavanone G; With the Radix Sophorae Flavescentis crude extract (preparation method of this crude extract is seen Chinese patent CN1676520) that comprises kurarinone 45%, 2 '-methoxyl group-kurarinone 2% and Chinese scholartree flavanone G6%.
Above-mentioned sample is dissolved in DMSO respectively, and 10mg/ml is stored in-20 ℃.Be diluted to 10 * final concentration with serum-free medium before the dosing, add in 10 μ l to the 90 μ l cells.
Positive control: D-82041 DEISENHOFEN (Staurosporine), available from Sigma, S4400.
Detecting instrument: Victor
TM-3 (Perkin Elmer)
2. experimental technique
(1) exponential phase A431 (or MDA-MB-453s) cell inoculation is in 96 orifice plates, and 5 * 10
4Cells/well, overnight incubation.
(2) the careful suction gone culture medium, washes 2～3 times with 150 μ l PBS, adds the culture medium that 90 μ l do not contain FBS, and hunger is spent the night.(, then need not hunger for the MDA-MB-453s cell.)
(3) 10 μ l dilution test samples are added in the cell, in 37 ℃ of incubators, be incubated 15min.6 porocytes add 10 μ l culture medium in every plate, and difference is signal and 100% phosphorylation signal as a setting.
(4) remove the extracellular of signal as a setting, every hole adds 10 μ l 20ng/ml EGF (available from Biosource 55-130), places 37 ℃ of incubator moderate stimulation 30min.
(5) carefully absorb all culture medium, add 4 ℃ of cell pyrolysis liquids of 70 μ l, frozen in-80 ℃.
(6) carry out analysis of Phosphorylation before, with cell pyrolysis liquid in dissolving on ice, with pipettor mix homogeneously up and down.
(7) 96 orifice plates are used in advance 100 μ l 0.4ug/ml anti-EGFR antibody A F231 (or 1 μ g/mlanti-HER 2) bag spent the night, the PBS that contains 1.0%BSA with 200 μ l sealed 1 hour, and every hole adds the 80ul sample diluting liquid.
(8) add the 20ul cell pyrolysis liquid.The room temperature insulation is 1 hour on shaking table.The method Eu that provides according to manufacturer
2+-PY66 and DELFIA analytical system (PerkinElmer) are analyzed.
3. the result calculates and analyzes
The IC50 value is analyzed by XLfit2.0 software.
Wherein: A
DrugThe fluorescence value of reading in-dosing hole
A
Cell-background the value of reading, neither dosing, also not adding EGF stimulates
A
Phos.-not dosing only adds the value of reading that EGF stimulates
To HER 2 analysis of Phosphorylation:
A
DrugThe fluorescence value of reading in-dosing hole
A
Cell-background the value of reading replaces cell pyrolysis liquid with cell lysis buffer solution
A
Phos.-do not add the cell pyrolysis liquid (HER2 composition autophosphorylation signal) of medicine
IC
50Value is analyzed by XLfit2.0 software.
The result shows, kurarinone; 2 '-methoxyl group-kurarinone; Chinese scholartree flavanone G; Or the Radix Sophorae Flavescentis crude extract that comprises kurarinone 45%, 2 '-methoxyl group-kurarinone 2% and Chinese scholartree flavanone G 6% is inhibited to the phosphorylation of EGFR and HER-2, and its IC50 is 1-50 μ M.
The inhibitory action analysis of 3 couples of KDR of embodiment
1. experiment material
Reagent: KDR catalytic domain (Cat.PV3660), PDGFR-beta catalytic domain (Cat.P3082), Z-lyte Tyr 1 Peptide Assay Kit (Cat.PV3190) and Tyr 4 Peptide Assay Kit (Cat.PV3298) all buy from Invitrogen.
Specimen: kurarinone; 2 '-methoxyl group-kurarinone; Chinese scholartree flavanone G; With the Radix Sophorae Flavescentis crude extract that comprises kurarinone 45%, 2 '-methoxyl group-kurarinone 2% and Chinese scholartree flavanone G 6%.
Above-mentioned each sample is dissolved in DMSO respectively, and 10mg/ml is stored in-20 ℃.Being diluted to final concentration with 4%DMSO before the dosing is 100,33,11,3.7,1.2.
Positive control: D-82041 DEISENHOFEN (Staurosporine), available from Sigma, S4400.
2. method
(1) specimen (the control wells C1 of adding 2.5 μ l4 * final concentrations in 384 orifice plates, C2, add 2.5 μ l of 4%DMSO among the C3 and replace specimen), 5 μ l kinases/peptide (Kinase/peptide) mixture (0.2 μ g/ml KDR, 1.8 μ g/ml PDGFR-beta, Tyr 1/Tyr 4 Peptide of 4 μ M are dissolved in 50mM HEPES pH7.5,0.01%BRIJ-35,10mM MgCl
2, 1mM EGTA) and (add 5 μ l phosphor-peptides (phosphor-peptide) among the C3 and replace normal peptide substrate) and 2.5 μ l400 μ MATP (add 2.5 μ l reaction buffers among C1 and the C3 and replace ATP).
(2) mix homogeneously, room temperature reaction 1 hour.
(3) every hole adds 5 μ l reagent (development reagent), continues reaction 1 hour.
(4) add 5 μ l and stop reagent.Use Victor
TM-3 read fluorescent value, excitation wavelength 400nm/ emission wavelength 435nm and 520nm.
3. the result calculates and analyzes
Wherein:
Emissivity (Emission Ratio)=sample well coumarin (Coumarin)/fluorescein (Fluorescein) ratio
C
100%The average Coumarin signal value of=100%Phos.Control phosphorylation
C
0%The average Coumarin signal value of=0%Phos.Control phosphorylation
F
100%The mean F luorescein signal value of=100%Phos.Control phosphorylation
F
0%The mean F luorescein signal value of=0%Phos.Control phosphorylation
IC
50Value is analyzed by XLfit2.0 software.
The result shows, kurarinone; 2 '-methoxyl group-kurarinone; Chinese scholartree flavanone G; The Radix Sophorae Flavescentis crude extract that comprises kurarinone 35%, 2 '-methoxyl group-kurarinone 4% and Chinese scholartree flavanone G 8% is inhibited to the phosphorylation of KDR, and its IC50 is 10-50 μ M.
And the result shows that also kurarinone can strengthen the inhibitory action of Taxol.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (11)
1. the Radix Sophorae Flavescentis flavone compound shown in the formula (I) or contain the purposes of the mixture of the Radix Sophorae Flavescentis flavone compound shown in the formula (I),
In the formula, R1 represents H or CH
3
R2 represents H or CH
3
It is characterized in that,
Chemical compound shown in the formula (I) or the mixture that contains chemical compound shown in the formula (I) are used to preparation and suppress NF-κ B, EGFR, Her-2 or the active inhibitor of KDR.
2. purposes as claimed in claim 1 is characterized in that, the Radix Sophorae Flavescentis flavone compound shown in the formula (I) is selected from: kurarinone, 2 '-methoxyl group-kurarinone or Chinese scholartree flavanone G.
3. purposes as claimed in claim 1 is characterized in that, the mixture of described Radix Sophorae Flavescentis flavone compound is the mixture that contains two or more flavone compound that is selected from down group: kurarinone, 2 '-methoxyl group-kurarinone or Chinese scholartree flavanone G.
4. purposes as claimed in claim 3 is characterized in that, the mixture of described Radix Sophorae Flavescentis flavone compound contains following component:
Kurarinone: 20～60 weight portions;
2 '-methoxyl group-kurarinone: 1～5 weight portion; With
Chinese scholartree flavanone G:1～12 weight portions.
5. purposes as claimed in claim 4 is characterized in that, the mixture of described Radix Sophorae Flavescentis flavone compound contains following component:
Kurarinone: 35～45 weight portions;
2 '-methoxyl group-kurarinone: 2～4 weight portions; With
Chinese scholartree flavanone G:5～8 weight portions.
6. as the arbitrary described purposes of claim 3-5, it is characterized in that the mixture of described Radix Sophorae Flavescentis flavone compound is the extract of Radix Sophorae Flavescentis.
7. purposes as claimed in claim 1 is characterized in that, described inhibition NF-κ B, EGFR, Her-2 or the active inhibitor of KDR are used to prepare the medicine of prevention or treatment tumor or inflammation.
8. purposes as claimed in claim 7, it is characterized in that described tumor comprises: bladder cancer, cerebral glioma, gastric cancer, pulmonary carcinoma, carcinoma of prostate, tumor of head and neck, breast carcinoma, ovarian cancer, carcinoma of endometrium, carcinoma of fallopian tube, cervical cancer, carcinoma of prostate, hepatocarcinoma, colon cancer, human primary gastrointestinal cancers or renal cell carcinoma; Perhaps
Described inflammation comprises: rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, inflammatory bowel, systemic lupus erythematosus (sle), bacterial infection, acute pancreatitis, sepsis, Pyoderma gangrenosum, septicemia or septic shock.
9. a pharmaceutical composition is characterized in that, described pharmaceutical composition contains:
(a) the Radix Sophorae Flavescentis flavone compound of effective dose or its compositions; With
(b) the chemotherapy of tumors preparation of effective dose.
10. pharmaceutical composition as claimed in claim 9, it is characterized in that described chemotherapy of tumors preparation is selected from: 5-fluorouracil, paclitaxel, Docetaxel, cisplatin, methotrexate, amycin, vincristine, mitomycin, bleomycin, table podophyllin, Irinotecan, special willing or its combination of topology.
11. pharmaceutical composition as claimed in claim 10 is characterized in that, described chemotherapy of tumors preparation is selected from: paclitaxel, Docetaxel or its combination.
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| CN 200610028072 CN101091707A (en) | 2006-06-23 | 2006-06-23 | Medicinal usage of kuhseng extractive |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200610028072 CN101091707A (en) | 2006-06-23 | 2006-06-23 | Medicinal usage of kuhseng extractive |
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| CN 200610028072 Pending CN101091707A (en) | 2006-06-23 | 2006-06-23 | Medicinal usage of kuhseng extractive |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104961717A (en) * | 2015-07-24 | 2015-10-07 | 黑龙江中医药大学 | Enrichment method and application of Sophoraflavanone G |
| CN106176697A (en) * | 2015-05-25 | 2016-12-07 | 中国医学科学院药物研究所 | The application in prevention or treatment hepatocarcinoma of the one class flavanone compound |
| CN112535695A (en) * | 2019-09-21 | 2021-03-23 | 井冈山市红扁担科技有限公司 | Traditional Chinese medicine extract composition for treating intestinal cancer, preparation method and application thereof in preparing medicines |
| CN113304163A (en) * | 2021-06-09 | 2021-08-27 | 中国中医科学院中药研究所 | Application of trifolium pterocarpus santalin in treating arthritis |
| CN118370763A (en) * | 2024-06-27 | 2024-07-23 | 山东方舟生物科技有限公司 | Application of isopentenyl flavonoid compound in kuh-seng extract in preparation of product for preventing and treating pathogenic bacteria of dairy cows |
-
2006
- 2006-06-23 CN CN 200610028072 patent/CN101091707A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106176697A (en) * | 2015-05-25 | 2016-12-07 | 中国医学科学院药物研究所 | The application in prevention or treatment hepatocarcinoma of the one class flavanone compound |
| CN104961717A (en) * | 2015-07-24 | 2015-10-07 | 黑龙江中医药大学 | Enrichment method and application of Sophoraflavanone G |
| CN112535695A (en) * | 2019-09-21 | 2021-03-23 | 井冈山市红扁担科技有限公司 | Traditional Chinese medicine extract composition for treating intestinal cancer, preparation method and application thereof in preparing medicines |
| CN113304163A (en) * | 2021-06-09 | 2021-08-27 | 中国中医科学院中药研究所 | Application of trifolium pterocarpus santalin in treating arthritis |
| CN113304163B (en) * | 2021-06-09 | 2022-05-20 | 中国中医科学院中药研究所 | Application of trifolium pterocarpus santalin in treating arthritis |
| CN118370763A (en) * | 2024-06-27 | 2024-07-23 | 山东方舟生物科技有限公司 | Application of isopentenyl flavonoid compound in kuh-seng extract in preparation of product for preventing and treating pathogenic bacteria of dairy cows |
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Open date: 20071226 |