CN101627983A - Medicinal composition for inhibiting angiogenesis and application thereof - Google Patents
Medicinal composition for inhibiting angiogenesis and application thereof Download PDFInfo
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Abstract
The invention relates to a medicinal composition for inhibiting angiogenesis and application thereof. The medicinal composition comprises any one composition of 2-methoxy-6-methyl-P-benzoquinone(2-methoxy-6-methyl-p-benzoquinone), 2,3-dimethoxy-5-methyl-P-benzoquinone(2,3-dimethoxy-5-methyl-p-benzoquinone) or 2-hydroxy-5-methoxy-3-methyl-P-benzoquinone(2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) and appropriate pharmaceutically acceptable excipients or supporting agents; and the medicinal composition is used for inhibiting the angiogenesis and further treats or prevents angiogenesis-related diseases.
Description
Technical field
The invention relates to a kind of medical composition and application thereof of angiogenesis inhibiting, be meant especially a kind of can be in order to the medical composition and the application thereof of angiogenesis inhibiting relevant disease.
Background technology
Angiogenesis (angiogenesis) changes and is common in the process of period of development and wound healing for normal physiological, discovered in recent years angiogenesis and tumor be formed with confidential relation.When cancerous cell forms tumor, cancerous cell itself or connective tissue on every side, the secretion angiogenesis factor, this factor can promote vascular endothelial cell (endothelial cells) that following variation takes place: the decomposition and the destruction of connective tissue arround (1) tumor; (2) endotheli ocytosis; (3) endotheliocyte moves towards the position of the secretion angiogenesis factor; (4) endotheliocyte reconfigures, and generates neonatal blood vessels.Angiogenesis forms quite important for tumor, after tumor development is to a certain size, must generate new blood vessel effectively to obtain nutrient, oxygen and to get rid of refuse.Angiogenesis is also quite important for neoplasm metastasis, and tumor cell is essential to generate new blood vessel, entering blood circulation, and then is transferred to other organ-tissue; After treating that tumor cell arrives other organ-tissue, the new blood vessel of essential generation could be in this organ-tissue continued growth.Confirmed at present the growth of nearly all entity tumor and vascular tumor or shifted all to rely on angiogenesis.In theory, if can just can suppress the formation or the transfer of above-mentioned tumor by angiogenesis inhibiting.
The intravital blood vessel of people is woven into net just as highway, it is network system maximum in our health, be responsible for the transportation of oxygen and nutrition, it is one of mankind's major organ systems of depending on for existence, embryo period, article one blood vessel in the life promptly forms, and it is to be subjected to angiogenesis by blood cell elder generation guided cell to promote the influence of the factor, VEGF etc. to finish with growth.And regulate and control the normal and unusual of those factors, can cause all kinds of physiology and pathological phenomenon.Angiogenesis is too much, may cause nethike embrane pathological changes, endometriosis and the arthritis etc. of cancer, obesity, psoriasis, premature labor, diabetes all to think also too much relevant, then think very few relevant with angiogenesis as for Alzheimer disease, apoplexy, hypertension, diabetes, arteriosclerosis, myocardial infarction, ischemic heart desease, alopecia, emphysema and osteoporosis with angiogenesis.
In recent years, angiogenesis inhibitors is that treatment of cancer brings another kind of new the selection, the treatment that angiogenesis suppresses is different with the mode of traditional chemical treatment or radiotherapy direct aggression cancerous cell, the effect machine that replaces changes and uses is to be cancerous cell microenvironment (Microenvironment) on every side, as vascular endothelial cell (Endothelial Cells), angiogenesis growth factor (Angiogenic Growth Factors) and cell adhesion factor (Cell Adhesion Molecule) etc.It mainly is the new life who suppresses new vessels that the effect machine commentaries on classics of most anti-angiogenic class medicines is considered to, and can't cause disappearing of already present maturation (mature) blood vessel, only can suppress tumor growth or disappear slowly (regression) so be considered to those medicines; And unlikely cause tumor fast, significantly reduce.Therefore, anti-angiogenic drugs should belong to cell stability (cytostatic) medicine, differs from known anticancer chemotherapy medicine (belonging to cytotoxicity (cytotoxic) medicine).Existing in recent years evidence shows the anti-angiogenic rebirth therapy, be not as in the past the cognitive drug-fast problem that do not have produce.The heterogeneity of tumor cell (heterogeneity) is except the ability of known anti-chemicals, also comprise the heterogeneity of the interdependent degree of blood vessel or the heterogeneity (heterogeneity in vascular-dependence andangiogenic pathway) of bringing out the commentaries on classics of angiogenesis machine, so the use of anti-angiogenic drugs also will be towards compound recipe anti-angiogenic drugs therapy (combination anti-angiogenic agents therapy) and in conjunction with other Therapeutic Method in future, as operation, radiation cure or chemotherapy merge to be used, avoiding or to delay the generation of the cancerous cell of tool anti-angiogenic drugs, and preferable curative effect is arranged.
The angiogenesis of cancerous cell involves the secretion of various kinds of cell hormone, and wherein (vascular endothelial growth factor VEGF) is topmost regulatory factor with vascular endothelial cell growth factor.Known vascular endothelial cell growth factor (VEGF) all has the situation of excessive performance in kinds of tumors as cerebroma, pulmonary carcinoma, breast carcinoma, digestive tract tumor and urinary tract tumor etc.Studies confirm that in breadboard, to the antibody of anti-vascular endothelial cell growth factor (VEGF), the phenomenon of the angiogenesis that can cause in order to anticancer.To squeeze on one's body the infull mouse of the innate immunity that is implanted with JEG-3 the antibody of anti-vascular endothelial cell growth factor (VEGF), can effectively suppress growth of tumor and transfer.
The anti-angiogenic drugs that Avastin (Bevacizumab) is developed for Genentech company.This medicine is a kind of monoclonal antibody to anti-vascular endothelial cell growth factor (VEGF), acts on the blood vessel of cancer cell group, allows the blood vessel atrophy, makes cancerous cell dead gradually under the nutrient dificiency situation.Though Avastin is not the medicine of chemical classes, still has side effect.Those side effect are common blood vessel embolism, hypertension, epistaxis, apoplexy, cardiac infarction and slight albuminuria.In the case of colorectal cancer, a sufferer that imposes high dose Avastin is died from pulmonary infarction.In the case of pulmonary carcinoma, impose the sufferer of Avastin, there are four to cause death because of a large amount of hemoptysis.Avastin has obtained U.S. FDA at present and has checked and approved, and can merge with chemotherapy to use the cancer of treatment Metastatic Colorectal Cancer and other class.
This shows that the above-mentioned article of commonly using still have many disappearances, real non-one kindhearted designer, and demand urgently being improved.
The development of medicine now is the composition that will obtain in the natural plants, carries out active component analysis, cell toxicity test, antioxidation test, reaches different types of cell or animal experiment, and then inquire into this active component in order to treat different types of disease or purposes.The material of known a kind of angiogenesis inhibiting is resveratrol (Resveratrol), and this material belongs to a kind of natural polyphenol class material, can obtain from multiple natural edible-plant.Resveratrol can be in order to the angiogenesis inhibiting effect, and then reduces cancer cell metastasis, and also has abilities such as anti-inflammatory, oxidations.
Antrodia salmonea (Antrodia salmonea) genus polyporus Cordycepps (Polyporaceae) fungus, being grown in rotten fragrant Cunninghamia lanceolata (Lamb.) Hook. in Taiwan does, constant error is thought Antrodia camphorata, is accredited as the novel species that Antrodia belongs to (Antrodia) in the beginning in 2004 by the forestry test Zhang Dongzhu researcher of institute.Antrodia salmonea and Antrodia camphorata (Antrodia camphorate) are among the people commonly used with diseases such as treatment dysentery, stomachache, hypertension, scabies and hepatopathys in Taiwan.In view of Antrodia camphorata costs an arm and a leg, often replace, and effect is also good with the similar Antrodia salmonea of kenel, so the physiologically active of Antrodia salmonea and the very worth research of effective active composition, have the value of exploitation.Sporophore such as Antrodia salmonea, Antrodia camphorata can't obtain in tame mode at present, but can utilize suitable liquid culture medium culturing Antrodia salmonea mycelium, with mass production Antrodia salmonea active substance, and then remove to isolate the different activities composition, to inquire into its different purposes.At present, Antrodia salmonea mycelium is existing extensively to be sold, and for example can buy at Hsinchu City, Taiwan Province food industry institute.
Summary of the invention
Purpose of the present invention promptly is to provide a kind of application of compound of angiogenesis inhibiting.
An of the present invention purpose is to provide a kind of medical composition of angiogenesis inhibiting, in order to angiogenesis inhibiting, and then suppresses growth of tumor.
A time purpose of the present invention is the application that is to provide a kind of medical composition of angiogenesis inhibiting, is to throw the active component that gives the zooblast pharmacy effective dose, in order to suppress the angiogenesis of this cell.
Can reach the application of compound of a kind of angiogenesis inhibiting of foregoing invention purpose, include: 2-methoxyl group-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone) or 2,3-dimethoxy-5-methyl-p-benzoquinone (2,3-dimethoxy-5-methyl-p-benzoquinone) or 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) any, be used to prepare the medicine of the relevant disease of angiogenesis inhibiting.These three kinds of chemical compounds have common construction unit, referring to Fig. 3 or following formula (I).This chemical compound can be got by purification in the Antrodia salmonea mycelium in the present invention;
A kind of 2-methoxyl group-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone) or 2 that includes, 3-dimethoxy-5-methyl-p-benzoquinone (2,3-dimethoxy-5-methyl-p-benzoquinone) or any one or more the application process of medical composition of (have chemical compound-see also Fig. 3) of 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) suc as formula (I) structure, it is the active component that gives the zooblast pharmacy effective dose, this active component is for having the chemical compound suc as formula the structure of (I), in order to angiogenesis inhibiting, and then treatment is because the unusual disease that causes of angiogenesis.
The medical composition of a kind of angiogenesis inhibiting provided by the present invention and application thereof with aforementioned case and other located by prior art quoted as proof mutually relatively the time, have more following advantage:
The medical composition of a kind of angiogenesis inhibiting provided by the invention and application thereof, wherein this medical composition comprises the chemical compound that has suc as formula (I) structure, wherein R
1And R
2As defining in the literary composition,, and then suppress growth of tumor in order to angiogenesis inhibiting.
The medical composition of a kind of angiogenesis inhibiting provided by the invention, this medical composition includes the structure suc as formula (I)) chemical compound, this chemical compound is to obtain by purification from Antrodia salmonea mycelium, also can prepare and get by the chemosynthesis mode, so be obtainable angiogenesis inhibitors in the natural plants with Bai Li Lo alcohol, and its angiogenesis inhibiting effect is better than Bai Li Lo alcohol.
The medical composition of a kind of angiogenesis inhibiting provided by the invention, also can directly use through liquid culture and Antrodia salmonea mycelium, without extraction step, directly cooperate suitable dilution agent, excipient or carrier, to make various forms of preparations, in order to angiogenesis inhibiting.
(I) is as follows for the structural formula of above-mentioned three kinds of chemical compounds:
Wherein, R
1=H, R
2=H or R
1=OCH
3, R
2=H; Or R
1=H, R
2=OH.
Description of drawings
See also the detailed description and the accompanying drawing thereof of following relevant preferred embodiment of the present invention, can further understand technology contents of the present invention and purpose effect thereof; The accompanying drawing of relevant this embodiment is:
Fig. 1 is an Antrodia salmonea fermentation liquid lyophilized powder active component separating step;
Fig. 2 is the analysis of active component (As-4, As-5, As-6) in order to angiogenesis inhibiting;
Fig. 3 is the representative chemical structural formula of active component.
The specific embodiment
Used Antrodia salmonea (Antrodia salmonea) mycelium of embodiments of the invention is to derive to be deposited at the Antrodia salmonea mycelium BCRC36937 that Republic of China's Hsinchu City, Taiwan Province of China food industry institute strain is preserved the center, but Antrodia salmonea mycelium active substance of the present invention (has the chemical compound suc as formula (I) structure, see also Fig. 3) be not limited to strain gained thus, also can prepare by the chemosynthesis mode.
By methanol (MeOH) purification Antrodia salmonea mycelium active substance, and analysis composition wherein, this active component has suc as formula the chemical compound of (I) structure (seeing also Fig. 3), wherein R1 is that hydrogen and R2 are hydrogen, and then this chemical compound is that 2-methoxyl group-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone) (As-4); Wherein R1 is (OCH
3) and R2 be hydrogen, then this chemical compound is 2,3-dimethoxy-5-methyl-p-benzoquinone (2,3-dimethoxy-5-methyl-p-benzoquinone) (As-5); Wherein R1 is that hydrogen and R2 are that (OH), then this chemical compound is that 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) (As-6).
The medical composition of a kind of angiogenesis inhibiting provided by the invention and application thereof, this medical composition comprises and at least aly has suc as formula the chemical compound (seeing also Fig. 3) of (I) structure and suitable medical acceptable excipient or supporting agent, to make various forms of preparations, as lozenge, capsule, granule, solution, syrup etc.;
The acceptable excipient of this medicine can be diluent, filler, bonding agent, disintegrating agent, lubricant etc.;
The acceptable excipient of this medicine can be microcrystalline Cellulose (microcrystalline cellulose), polyvinylpyrrolidone (polyvinylpyrrolidone, PVP), corn starch, modify starch (modified starches) carboxymethylstach sodium (sodium starch glycolate), resin, gelatinized starch (gelatinized starches), saccharide, Polyethylene Glycol (polyethylene glycol, PEG), polyvinyl alcohol (polyvinyl alcohol), hyprolose (hydroxypropyl cellulose), methylcellulose (methylcellulose), hydrogen-oxygen methylcellulose (hydroxymethyl cellulose), hydroxypropyl emthylcellulose (hydroxypropyl methylcellulose) etc.;
This application is to throw the 2-methoxyl group-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone) or 2 that gives the zooblast pharmacy effective dose, 3-dimethoxy-5-methyl-p-benzoquinone (2,3-dimethoxy-5-methyl-p-benzoquinone) or arbitrary composition among 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) and suitable medical acceptable excipient or supporting agent;
The acceptable excipient of this medicine can be diluent, filler, bonding agent, disintegrating agent, lubricant etc.;
The acceptable excipient of this medicine can be microcrystalline Cellulose (microcrystalline cellulose), polyvinylpyrrolidone (polyvinylpyrrolidone, PVP), corn starch, modify starch (modified starches) carboxymethylstach sodium (sodium starch glycolate), resin, gelatinized starch (gelatinized starches), saccharide, Polyethylene Glycol (polyethylene glycol, PEG), polyvinyl alcohol (polyvinyl alcohol), hyprolose (hydroxypropyl cellulose), methylcellulose (methylcellulose), hydrogen-oxygen methylcellulose (hydroxymethyl cellulose), hydroxypropyl emthylcellulose (hydroxypropyl methylcellulose) etc.;
This zooblast is human umbilical cord endotheliocyte (HUVEC, human umbilical vein endothelial cells) including but not limited to the cell with angiogenesis ability in a preferred embodiment;
In a preferred embodiment, this pharmacy minimal effective dose is 2-methoxyl group-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone) of 0.08 μ g/ml, or be 2 of 0.02 μ g/ml, 3-dimethoxy-5-methyl-p-benzoquinone (2,3-dimethoxy-5-methyl-p-benzoquinone), or be 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) of 0.02 μ g/ml.This application is with the repeatedly sub-dosage dispenser of every day single dose or every day.
In addition, the medical composition of a kind of angiogenesis inhibiting provided by the invention, except available chemical compound through extraction cooperates suitable medical acceptable excipient or supporting agent to make various forms of preparations, also can directly use through liquid culture and Antrodia salmonea mycelium, without extraction step, directly cooperate suitable medical acceptable excipient or supporting agent, to make various forms of preparations;
This medical composition is that this relevant disease is including but not limited to nethike embrane pathological changes, endometriosis and the arthritis etc. of cancer, obesity, psoriasis, premature labor, diabetes in order to the relevant disease of treatment angiogenesis.
The present invention is demonstrated with the following examples to illustrating, but the present invention is not limited by following embodiment.Embodiment one carries out mycelial cultivation with Antrodia salmonea mycelium (BCRC 36937)
The present invention is with Republic of China letters patent I287991, and Antrodia salmonea mycelium active substance its preparation method and contain Antrodia salmonea mycelium and cultural method in those constituents to obtain enough Antrodia salmonea fermentation liquid lyophilized powders, is sketched wherein cultural method.
The mycelium bacterial strain:
For being deposited at the bacterial strain BCRC 36937 of food industry institute.
The dull and stereotyped cultivation:
Mycelium is inoculated on the flat board, and (Potato Dextrose Agar PDA), cultivates about 2 weeks down in 25 ℃ to use the detrine culture medium.
Flask is cultivated:
Scrape mycelium inoculation on the plate of making even in flask, use following culture medium, at about 25 ℃, pH 4.5 times, on shakeout machine with oscillation rate 10-250rpm shaken cultivation a couple of days;
Culture medium prescription:
Composition
Content (weight %)
Grains class (as the flour class) or beans (as analysis for soybean powder, Semen phaseoli radiati powder, Semen sojae atricolor powder etc.) 2
Peptone (peptone) 0.1
Magnesium sulfate 0.05
Dipotassium hydrogen phosphate 0.05
Iron sulfate 0.05
Sucrose 2
Yeast extract, powder, cream 0.5
The tank culture of Fermentation ferment:
Culture medium is the same, in flask culture Jie Zhong Yu Fermentation ferment tank culture base, at 25 ℃, groove is pressed 0.5-1.0 kilogram/square centimeter, about 4.5 times of 10-150rpm mixing speed and pH with 0.5-1.0VVM Ventilation Rate bubbling air, cultivated about 21 days, promptly get Antrodia salmonea mycelium liquid culture suspension, comprise mycelium and clear liquor.
The result:
100 Sheng Fermentation Jiao Ye Fermentation ferment finish and can get the filtrate of 1.2 kilograms of mycelium (dry weight) and 90 liters.
Embodiment two Antrodia salmonea fermentation liquid lyophilized powder angiogenesis inhibiting active component separate
Antrodia salmonea mycelium liquid culture suspension with embodiment one gained, comprise mycelium and clear liquor, carry out lyophilization to obtain Antrodia salmonea mycelium fermentation liquid freeze-dried powder, get this dry powder and carry out as shown in Figure 1 purification step, get 1 kilogram fragrant China fir fermentation liquid freeze-dried powder, can obtain methanol extraction liquid with after four liters methanol (MeOH) extraction three times, after concentrating the methanol extract.(60～230mesh, 5 * 60cm) do initial gross separation to the methanol extract, are that 10: 1 to 0: 1 ratio is dashed and carried with normal hexane (n-hexane)/ethyl acetate (EtOAc) in regular turn, obtain 7 graduation with the silica gel column chromatography tubing string.Belong to n-hexane/ethyl acetate (1: 1 to 1: 4) and dash and to propose the graduation 4 that obtains, use Sephadex LH-20 chromatography tubing string (3 * 60cm), dash to carry with methanol (MeOH) and can obtain 6 graduation (F4-1 to F4-6); Wherein graduation 4-1 use the silica gel column chromatography tubing string (60～230mesh, 2 * 60cm), dash to carry with n-hexane/ethyl acetate (1: 1) and can obtain compd A s-4 (630mg); Wherein graduation 4-3 uses silica gel column chromatography tubing string (60～230mesh, 2 * 60cm), with n-hexane/ethyl acetate (4: 1) dash carry after, re-use preparation type thin layer chromatography (preparative TLC), with n-hexane/ethyl acetate (3: 1) dash carry after, the separable compd A s-5 (30mg) that is purified into.Belong to n-hexane/ethyl acetate (1: 5 to 1: 10) towards proposing the graduation 6 that obtains, use Sephadex LH-20 chromatography tubing string (3 * 60cm), can obtain three graduation (F6-1 to F6-3) with methanol towards carrying, wherein graduation 6-3 can obtain compd A s-6 (130mg) with the methanol recrystallize after solvent evaporates.
Embodiment three Antrodia salmonea fermentation liquid lyophilized powder active component are identified
Be that three chemical compounds with gained among the embodiment two carry out chemical constitution and identify, form with mass spectrometer and high chemical constitution of resolving gas chromatography spectrometer analysis above-claimed cpd.
1. the chemical constitution of compd A s-4 is identified
The buff solid, it is 152 that MS (mass spectrometer, Finnigan GCQ) records molecular weight, by 1H-NMR and 13C-NMR (d-solvent:CDCl
3) (nuclear magnetic resonance analyser, Varian Unity Inova-500Spectrometer, 500MHz) measure comparison, δ H 2.07 (3H, d, J=1.5Hz) be at the locational methyl signal of C-7 (δ C15.7), (3H s) is the locational methoxyl group signal of C-2 (δ C 56.5), δ H 5.88 (1H to δ H 3.82, d, J=2.5Hz) and δ H 6.53 (1H, d are at C-3 (δ C107.5) and the locational fragrant hydrogen signal of C-5 (δ C 134.1) J=1.5Hz), learn that through HRMS (high gas chromatography mass spectrograph, the Shimadzu QP2010 of resolving) molecular formula is C again
8H
8O
3With list of references (A.A.LeslieGunatilaka, John M.Berger, Randy Evens, James S.Miller, Jan H.Wisse, Kim M.Neddermann, Isia Bursuker, and David G.I.Kingston, Isolation, synthesis, and structure-activity relationships of bioactivebenzoquinones from Miconia lepidota from the suriname rainforest.J.Nat.Prod., 64,2-5,2001), (Waldemar Adam and Masao Shimizu, Theacid-catalyzed oxidation of methoxybenzenes to p-benzoquinones bydimethyldioxirane, Synthesis, 560-562,1994) after the comparison, determine that this chemical compound is the 2-methoxyl group-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone) (hereinafter to be referred as As-4) with formula (I) structure as Fig. 3.
2. the chemical constitution of compd A s-5 is identified
The brownish red crystallization, it is 182 that MS (mass spectrometer, Finnigan GCQ) records molecular weight, through 1H-NMR and 13C-NMR (d-solvent:CDCl
3) (nuclear magnetic resonance analyser, Varian Unity Inova-500Spectrometer, 500MHz) measure comparison, δ H 2.01 (3H, d, J=1.0Hz) be at the locational methyl signal of C-7 (δ C15.7), δ H 3.97 (3H, s) and δ H 3.99 (3H s) is C-2 (δ C 61.4) and the locational methoxyl group signal of C-3 (δ C 61.5), δ H 6.41 (1H, d, J=2.0Hz) be at the locational fragrant hydrogen signal of C-6 (δ C 131.5), learn molecular formula C through HRMS (high gas chromatography mass spectrograph, the Shimadzu QP2010 of resolving) again
9H
10O
4With list of references (Steven T.Perri andHarold W.Moore, Rearrangements of Cyclobutenones.Synthesis ofbenzoquinones from 4-alkenyl-4-hydroxycyclobutenones, J.Am.Chem.Soc., 112,1897-1905,1990), (Waldemar Adam and Masao Shimizu, Theacid-catalyzed oxidation of methoxybenzenes to p-benzoquinones bydimethyldioxirane, Synthesis, 560-562,1994) after the comparison, determine that this chemical compound is to have 2 of formula (I) structure as Fig. 3, and 3-dimethoxy-5-methyl-p-benzoquinone (2,3-dimethoxy-5-methyl-p-benzoquinone) (hereinafter to be referred as As-5).
3. the chemical constitution of compd A s-6 is identified
Orange crystallization, it is 168 that MS (mass spectrometer, Finnigan GCQ) records molecular weight, through 1H-NMR and 13C-NMR (d-solvent:CDCl
3) (nuclear magnetic resonance analyser, Varian Unity Inova-500Spectrometer, 500MHz) measure comparison, (3H is at the locational methyl signal of C-7 (δ C 13.7) s) to δ H 1.89, δ H 3.85 (3H, s) be the locational methoxyl group signal of C-5 (δ C 56.6), (1H is at the locational fragrant hydrogen signal of C-6 (δ C 107.6) s) to δ H5.96, learn molecular formula C through HRMS (high gas chromatography mass spectrograph, the Shimadzu QP2010 of resolving) again
8H
8O
4, with list of references (FumiyukiKiuchi, Hiromi Takashima, and Yoshisuke Tsuda, Dimerization of2,5-dihydroxybenzoquinones in water, Chem.Pharm.Bull., 46 (8), 1129-1234,1998; Waldemar Adam and Masao Shimizu, The acid-catalyzedoxidation of methoxybenzenes to p-benzoquinones by dimethyldioxirane, Synthesis, 560-562,1994) after the comparison, determine that this chemical compound is the 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone) (hereinafter to be referred as As-6) with formula (I) structure as Fig. 3.
Above-mentioned three chemical compounds (As-4, As-5 and As-6) are through identifying its chemical constitution, be defined as the derivant of p-benzoquinone (p-benzoquinone), known different p-quinone derivatives has different purposes, for example improves circulation defective (circulatory disturbance), reduces different purposes such as allergy and inhibition insecticide appetite.Three p-quinone derivatives of gained of the present invention by following functional analysis, are tested the novel use whether these three p-quinone derivatives have angiogenesis inhibiting.
Embodiment four Antrodia salmonea fermentation liquid lyophilized powder active component angiogenesis inhibiting cell in vitro activity tests
(HUVEC?Tube?Formation?Test)
Be three chemical compounds that embodiment three is analyzed, according to list of references (the anti-angiogenic rebirth effect of Yang Pure person of outstanding talent Shui Fly Thistle Drug Wu Zai Body Wai With Huo Body pattern Right Da Intestinal cancer Fine born of the same parents strain, Country founds Yang Ming Da Learn Pro Chuan Medical Learn institute doctor Theory literary composition, the method of assessment angiogenesis 2005) is assessed the effect whether those chemical compounds have angiogenesis inhibiting.Be to be model organism with human umbilical cord endotheliocyte (HUVEC, human umbilicalvein endothelial cells) in the present embodiment, in order to the effect of assessment tester at angiogenesis, this appraisal procedure is as follows.
1. human umbilical cord endotheliocyte is cultivated
People Class cord vessels endotheliocyte is inoculated in the 6 hole culture plates, wherein 100 μ l complete mediums (the M200 culture medium (Invitrogen) that contains 20%FBS is contained in each hole, 30 μ g/ml ECGS, 5%EGM, 4mM L-glutamine, 100U/ml penicillin and 100 μ g/ml streptomycin), and place 37 ℃, 5%CO
2Constant automatic control incubator cultivated 24 hours, adjusting cell Number is 3 * 10
5Cells/well is to carry out following experiment.
2. human umbilical cord endotheliocyte angiogenesis test
Earlier not coalescent as yet basement membrane glue (Matrigel) (50 μ L/well) is inserted in 96 porose discs, reacted 30 minutes down, implant 100 μ l about 3 * 10 then in 37 ℃
5The human umbilical cord endotheliocyte (HUVEC) of cells/ml cell concentration, in the vascular endothelial cell growth factor that contains 10ng/ml (vascular endothelialgrowth factor, VEGF) in the M200 culture medium (Invitrogen) of (positive matched group), in addition, basic matched group (basal) is the M199 culture medium (Invitrogen) of no any additive, and negative matched group is that VEGF adds Bai Li Lo alcohol (Resveratrol) M200 culture medium (1.6mM).As described in the sample treatment group is divided into groups as following experiment.Need to add earlier low serum growth supplement (Low Serum GrowthSupplement before wherein the M200 culture medium is used, LSGS) (Invitrogen), human vascular endothelial cell (human vessel endothelialcells) is divided, otherwise do not add low serum growth supplement if having, then human vascular endothelial cell can't divide.In condition of culture is 6 hours 37 ℃, 5%CO
2After the cultivation, can under phase contrast microscope (phase-contrast photomicroscope), see two dimension (2D) vascularization, carry out the computational analysis of length of vessel with the Image-Pro.Plus.V4.5 program after taking pictures, with basic matched group income value is that basic value (1 or 100%) is done the numerical value analysis, again with the difference of basic matched group and positive matched group as 100% angiogenesis (tube formation), so that the ED50 of assessment medicine.This ED50 is defined as the effective dose that suppresses 50% angiogenesis degree.
The experiment grouping:
1. basic matched group (Basal): the M199 culture fluid of serum-free;
2. positive matched group (Positive control): the M200 culture fluid that contains 10ng/ml VEGF;
3. negative matched group (Negative control): contain 10ng/ml VEGF and Bai Li Lo alcohol (Resveratrol) M200 culture fluid (1.6mM);
4. the chemical compound among sample treatment group: the embodiment three (As-4, As-5 and As-6) adds the M200 culture fluid that contains 10ng/ml VEGF with variable concentrations respectively.
The result:
Table one angiogenesis degree
Shown in Fig. 2 and table one, the angiogenesis degree of positive matched group has 131.33% approximately, and the angiogenesis degree of basic matched group is good, shows that VEGF can promote angiogenesis really.The human umbilical cord endotheliocyte that negative matched group is handled with VEGF and Bai Li Lo alcohol (Resveratrol), its angiogenesis degree about 70.26%, the angiogenesis degree of basic matched group and positive matched group is low, shows that white Herba chenopodii Lo alcohol really can be in order to suppress the angiogenesis that VEGF promoted.Each is organized in the test group, and concentration of treatment is the As-4 chemical compound of 0.08 μ g/ml or higher concentration, and the angiogenesis degree of this test group (As-4) is 0%; Concentration of treatment is the As-5 chemical compound of 0.02 μ g/ml or higher concentration, and the angiogenesis degree of this test group (As-5) is 2.13% to 0%; Concentration of treatment is the As-6 chemical compound of 0.02 μ g/ml or higher concentration, and the angiogenesis degree of this test group (As-6) is 1.57% to 0%.Confirm that those chemical compounds can be in order to suppress the angiogenesis that VEGF promoted, it is better with respect to white Herba chenopodii Lo alcohol that it suppresses effect.
Above-listed detailed description is specifying at a possible embodiments of the present invention, only this embodiment is not in order to limit claim of the present invention, allly do not break away from the equivalence that skill spirit of the present invention does and implement or change, for example: the equivalence embodiment of pharmacy effective dose all should be contained in the claim of this case.
Claims (13)
1.2-methoxyl group-6-methyl-p-benzoquinone or 2, the application in the medicine of the relevant disease of preparation angiogenesis inhibiting of 3-dimethoxy-5-methyl-p-benzoquinone or 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone.
2. application according to claim 1 is characterized in that: the relevant disease of described angiogenesis is nethike embrane pathological changes, endometriosis or the arthritis of cancer, obesity, psoriasis, premature labor, diabetes.
3. the medical composition of an angiogenesis inhibiting, 2-methoxyl group-6-methyl-p-the benzoquinone or 2 that comprises pharmacy effective dose, in 3-dimethoxy-5-methyl-p-benzoquinone or the 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone any one or more, and suitable medical acceptable excipient or supporting agent.
4. medical composition according to claim 3 is characterized in that, described excipient is diluent, filler, bonding agent, disintegrating agent or lubricant.
5. medical composition according to claim 3, it is characterized in that described excipient is microcrystalline Cellulose, polyvinylpyrrolidone, corn starch, modification starch carboxymethylstach sodium, resin, gelatinized starch, saccharide, Polyethylene Glycol, polyvinyl alcohol, hyprolose, methylcellulose, hydrogen-oxygen methylcellulose or hydroxypropyl emthylcellulose.
6. medical composition according to claim 3 is characterized in that, described 2-methoxyl group-6-methyl-p-benzoquinone or 2, and the concentration of 3-dimethoxy-5-methyl-p-benzoquinone or 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone is for being equal to or greater than 0.02 μ g/ml.
7. medical composition according to claim 6 is characterized in that, the concentration of described 2-methoxyl group-6-methyl-p-benzoquinone is for being equal to or greater than 0.08 μ g/ml.
8. the medical composition of an angiogenesis inhibiting comprises Antrodia salmonea (Antrodiasalmonea) mycelium of pharmacy effective dose and suitable medical acceptable excipient or supporting agent.
9. the application of the medical composition of an angiogenesis inhibiting, be 2-methoxyl group-6-methyl-p-benzoquinone or 2 of throwing the pharmacy effective dose give zooblast, 3-dimethoxy-5-methyl-p-benzoquinone or 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone) any one or more composition and suitable medical acceptable excipient or supporting agent.
10. the medical composition of angiogenesis inhibiting according to claim 9 is used, and it is characterized in that described zooblast is human umbilical cord endotheliocyte.
11. the medical composition of angiogenesis inhibiting according to claim 9 is used, it is characterized in that, described 2-methoxyl group-6-methyl-p-benzoquinone or 2, the concentration of 3-dimethoxy-5-methyl-p-benzoquinone or 2-hydroxy-5-methyl oxygen base-3-methyl-p-benzoquinone is for being equal to or greater than 0.02 μ g/ml.
12. the medical composition of angiogenesis inhibiting according to claim 11 is used, and it is characterized in that the concentration of described 2-methoxyl group-6-methyl-p-benzoquinone is for being equal to or greater than 0.08 μ g/ml.
13. the application of the medical composition of angiogenesis inhibiting according to claim 9, wherein this application is with the repeatedly sub-dosage dispenser of every day single dose or every day.
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| JP2014058497A (en) * | 2012-09-18 | 2014-04-03 | China Medical Univ | Benzoquinone-type compound for inflammation suppression, and pharmaceutical composition and supplement using the same |
| JP2014058496A (en) * | 2012-09-18 | 2014-04-03 | China Medical Univ | BENZOQUINONE-TYPE COMPOUND FOR Wnt/β-CATENIN SIGNAL PATH SUPPRESSION, AND PHARMACEUTICAL COMPOSITION AND SUPPLEMENT FOR SKIN CARCINOMA SYMPTOM IMPROVEMENT USING THE SAME |
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| CN86105772A (en) * | 1986-07-13 | 1988-02-03 | 姚桂荣 | Reform method of cresol soap solution |
| CN1261401C (en) * | 2003-11-27 | 2006-06-28 | 复旦大学 | 2,3-dimethoxy-5-methyl-1,4-benzoquinone (I)preparation method |
| CN100389190C (en) * | 2005-08-15 | 2008-05-21 | 葡萄王生技股份有限公司 | Antrodia mycelium active substance preparation method and its components |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2014058497A (en) * | 2012-09-18 | 2014-04-03 | China Medical Univ | Benzoquinone-type compound for inflammation suppression, and pharmaceutical composition and supplement using the same |
| JP2014058496A (en) * | 2012-09-18 | 2014-04-03 | China Medical Univ | BENZOQUINONE-TYPE COMPOUND FOR Wnt/β-CATENIN SIGNAL PATH SUPPRESSION, AND PHARMACEUTICAL COMPOSITION AND SUPPLEMENT FOR SKIN CARCINOMA SYMPTOM IMPROVEMENT USING THE SAME |
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