CN101548969B - Use of ethyl malonyl silybin derivant in preparing antioxidant medicine - Google Patents
Use of ethyl malonyl silybin derivant in preparing antioxidant medicine Download PDFInfo
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Abstract
The invention relates to use of ethyl malonyl silybin derivant in preparing antioxidant medicine, particularly relates to uses of a silybin esters with a replacement of ethyl malonyl in the 23 position in preparing medicine for preventing and curing senile dementia or gout. Pharmacodynamic experiments showing that the compound has function of cleaning superoxide anion free-radical, repressing generation of lipoperoxide reduced by free-radical, and has antioxidation damage protection action to PC12 cell damage caused by free-radical namely PC12 cell for simulating brain nerve cell, for this reason, it is expected to be developed as medicine for protecting brain nerve and curing senile dementia caused by free-radical damage. The compound can also be used for repressing cativity of xanthine oxidase, so it is expected to be developed as medicine for curing gout.
Description
Technical field
The present invention relates to medical technical field; particularly; the silybin esters derivatives that the present invention relates to 23 ethyl malonyls replacement is chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2; 3-dihydro-3; 5; 7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt be used to prepare the medicinal usage of antioxidant.Pharmacology test to this chemical compound shows: this chemical compound has external removing ultra-oxygen anion free radical, suppresses the activity of the lipoperoxide generation of free yl induction; In addition; this chemical compound also shows the very strong rat adrenal chromaffin tumor PC12 cell injury effect that free radical resisting is caused; promptly the PC12 cell to the simulation cranial nerve cell has the anti-oxidative damage protective effect; illustrate its to protection brain cell tissue antioxidation and cranial nerve antioxidation, prevent and treat senile dementia positive role arranged, can expect the purposes that is used to prepare such medicine.Moreover this chemical compound also shows certain inhibitory action to xanthine oxidase, can expect the medicine that is used to prepare the goat that treatment causes by xanthine oxidase.
Background technology
In numerous free radical resisting natural products, Herba Silybi mariani is one of medicine of a few extensive use clinically.This medical instrument of the clinical trial certificate of three more than ten years has definite curative effect and hypotoxicity (to consult people such as Flora K., Am.J.Gastroenterol, 1998,93,139-143; People such as Saller R., Drugs, 2001,61 (14), 2035-2063).Silibinin content is maximum in the Herba Silybi mariani, and activity is also the highest.This medicine effect mainly contain following some: (one) free radical resisting activity: silymarin has protective effect for the hepatic injury that is caused by carbon tetrachloride, galactosamine, alcohols and other hepatotoxin.People such as nineteen ninety Lotteron have reported in the Mouse Liver microsome, silymarin can reduce external lipid peroxidation that is caused by the carbon tetrachloride metabolism and the peroxidation that is caused separately by reduced coenzyme, and these show that all silymarin is the chain interruption antioxidant or is free radical scavenger.(2) protection liver plasma membrane: keep flowability of cell membranes by the anti peroxidation of lipid reaction, the protection liver plasma membrane.Can also block combining of special receptor on mycotoxin phalloidine and α-amanitin etc. and the hepatocyte, suppress it, interrupt its liver sausage circulation, thereby the enhance hepatocyte film be for the resistance of multiple damage factor hepatocellular attack and transmembrane transport.(3) promote hepatocellular reparation and regeneration: silibinin can combine with estradiol receptor after entering cell, and make it to activate, activated receptors can enhance hepatocyte nuclear RNA polymerase 1 activity, rna transcription is strengthened, promote enzyme and proteinic synthetic, and promote the synthetic of DNA indirectly, help hepatocellular reparation and regeneration.(4) antitumor action: various active oxygens can form 8-hydroxyl guanine by the oxidation guanine, cause DNA damage, and then cause tumor, and silibinin has also shown the effect of prevention and treatment tumor as an effective free radical resisting material.
The silibinin compounds has definite curative effect, but because the market that the some shortcomings on its water solublity and the bioavailability have limited this medicine.So seek the new derivant of silibinin class, make it can have pharmacologically active higher or that upgrade, in the hope of obtaining the new drug of independent intellectual property right, real genus is necessary.
Numerous studies show that: free radical and nervous system disease such as senile dementia (A Zihaimo disease, AD), Parkinson's disease, apoplexy etc. be closely related.Its reason is main relevant with the brain tissue metabolism characteristics: the cerebral tissue oxygen consumption is big, the fat content height, and the content of various antioxidases is on the low side for other tissue, thereby be subject to free radical and attack and to cause damage (Gilgun-Sherki, Y. etc., Antioxidant therapy inacute central nervous system injury:current state, Pharmacol.Rev., 2002,54,271-284).Thereby, think that at present free radical has participated in AD patient's pathological process.In addition, free radical also can cause the amyloid beta deposition, produces reaction with cell membrane, causes oxidative damage in the cell.Therefore, research and development mesencephalic centre nervous system antioxidant is one of effective way of treatment alzheimer disease, fashionable catching a glimpse of (Green, K.N. etc., Melatonin from recent melatonin (Melatonin), aging, andage-related diseases:perspectives for prevention, intervention, and therapy.J.Physiol (Lond), 2002,541 (3): 1013-1023).Therefore, lead compound or the existing huge social benefit of medicine of seeking novel effective inhibition brain cell oxidative damage has remarkable economic efficiency again.
In recent years, international the world of medicine has dropped into great amount of manpower and material resources, financial resources continuously, strive to find antioxidation significantly, have an antioxidant lead compound of potent neurocyte protection effect.The cell in vitro model that senile dementia (mainly being cholinergic nerve system) is studied in more sure being used at present mainly contains 3 kinds: former generation cultured rat hippocampal, cortex, basal forebrain neurocyte, PC12 cell and Hippocampus HT22 cell strain.The PC12 cell that this test is adopted becomes important tool cell in the neuroscience vitro study with its easy to be acquired and cell homogeneity in incubation, and be used to study multiple nervous system disease, [heart-benefiting recipe is to H for Wang Jian, Chen Shawei as senile dementia, parkinson disease etc.
2O
2The protective effect research of oxidative damage PC12 cell, 2004 47 phases of volume of Traditional Chinese Medicine magazine hurricane; Cui Xu etc., the compound recipe Fructus Alpiniae Oxyphyllae is loose to increment of PC12 cell and anti-apoptotic effect, Chinese old cardiovascular and cerebrovascular vessel magazine, 2006,8 (3), 98-201; Xu Ying etc., daidzein is to the influence of PC12 cell toxic action due to the oxygen-derived free radicals, Pharmacology and Clinics of Chinese Materia Medica, 2007,5 phases].
In addition, one of paathogenic factor of modern medical theory confirmation goat is xanthine oxidase (EC1.2.3.2 abbreviates XO as).This enzyme is the metabolic important enzyme of nucleic acid in vivo, and energy catalysis hypoxanthine and xanthine generate uric acid and superoxide anion, if body can not in time be removed the uric acid of generation, uric acid crystal salt is deposited at joint, will cause gout; Produce too much superoxide anion and also can cause many tissue damageds, it is significant to finding new XO inhibitor and its antioxidation to the effect of XO therefore to measure chemical compound.From natural product with and derivant filter out the vital task that the xanthine oxidase inhibitor lead compound also is an exploitation treatment goat new drug.
In sum, it is active and be used to prepare the purposes of relevant disease medicine to the present invention relates to the multiple antioxidation mechanism pharmacodynamics of the silybin esters derivatives that ethyl malonyl replaces.Find that after tested this chemical compound has the activity of the lipid peroxide generation of removing the ultra-oxygen anion free radical activity, suppressing free yl induction and the PC12 cell injury effect that free radical resisting is caused, thereby can expect and develop into medicine or the pharmaceutical composition that in body, to remove free radical, especially can be used to prepare the medicine of the impaired senile dementia that causes of control cranial nerve oxidation.In addition, this chemical compound also shows certain inhibition xanthine oxidase activity, thereby it is also contemplated that and be developed further into medicine or the pharmaceutical composition that the disease that is caused by xanthine oxidase for treatment comprises goat.Finished the present invention thus.
Summary of the invention
The silybin esters derivatives that the purpose of this invention is to provide 23 ethyl malonyls replacements shown in a kind of following formula is chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2; 3-dihydro-3; 5; 7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2; 3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt be used to prepare the purposes of a kind of oxygen free radical scavenger and antioxidant medicine.
10,11 need be the R configuration simultaneously in the formula (I), or is the S configuration simultaneously.
Particularly, one of purpose of the present invention has provided malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt be used to prepare that control is caused by oxygen-derived free radicals or the purposes of the alzheimer disease disease drug relevant with oxygen-derived free radicals.
Another object of the present invention has provided malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt be used to prepare the purposes of the medicine of the goat that control causes by xanthine oxidase.
Another purpose of the present invention has provided a kind of medicine or pharmaceutical composition of the goat that is used to prepare senile dementia that the control free radical causes and is caused by xanthine oxidase, wherein contain the malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2 that treats effective dose, 3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt and pharmaceutic adjuvant.
Usefulness of the present invention is: drug actions such as PC12 cell injury that the silybin esters derivatives that 23 ethyl malonyls replace has potent removing free radical, suppresses lipid peroxidation, cause free radical resisting and xanthine oxidase inhibition; and its activity is better than silibinin; showing that this chemical compound can be expected is applied to prepare the new indication medicine of treatment; provide new material base for developing new anti-senile dementia and these two kinds of commonly encountered diseases of goat and the innovative medicine of frequently-occurring disease, had potential social benefit and economic benefit.Characteristics are again: this chemical compound derives from the semi-synthetic structure of modification of natural product, and preparation process is easy, and cost is low, pollutes for a short time, is beneficial to large-scale production.
The specific embodiment
In order to understand essence of the present invention better, at first use the process of the formal specification compound of embodiment below, embodiment has provided part physics and the chemistry and the Wave Spectrum data of chemical compound, and wherein OEt is meant ethyoxyl (OCH
2CH
3).Mandatory declaration, embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1: the preparation of chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester
In exsiccant reaction bulb, add 1 gram silibinin (available from Liaoning Panjin lucky chance pharmaceutcal corporation, Ltd, HPLC detects purity 98%), 0.56 gram monoethyl malonate and 1.6 gram triphenylphosphines are with 20 milliliters of anhydrous tetrahydro furan dissolvings, add 1 gram diethyl azodiformate, add the back in stirring at room 10 hours, distilling under reduced pressure removes and desolvates, and adds 5 milliliters of chloroforms, remove by filter white solid, mother solution gets buff powder 0.46 gram, yield 37% through column chromatography.
R
f(chloroform/ethyl acetate/acetic acid=50: 1: 0.25): 0.11; Proton nmr spectra
1H NMR (400MHz, deuterochloroform) δ: 1.28 (triplet, J=7.2Hz, 3H, CH
3), 3.41 (unimodal, 2H, COCH
2CO), 3.76 (unimodal, 3H, OCH
3), 4.08 (multiplet, 1H, H-23a), 4.22 (multiplet, 2H, H-3,10), 4.30 (multiplet, 2H, OCH
2CH
3), 4.36 (multiplet, 1H, H-23b), 4.90 (bimodal, J=7.6Hz, 1H, H-11), 4.98 (bimodal, J=10.8Hz, 1H, H-2), 5.99 (unimodal, 1H, H-6), 6.07 is (unimodal, 1H, H-8), 6.82～8.25 (multiplet, 9H, aromatic rings Ar-H), 11.19 (unimodal, 1H, 5-OH); Electrospray Mass Spectrometry ESI-MS:595 (M-1)
+
Chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester has multiple important biological, and the present invention tests this chemical compound antioxidant radical activity by multiple pharmacodynamics test model, finds that it has definite external removing ultra-oxygen anion free radical and suppresses the activity that lipoperoxide of free yl induction generates.In addition, this compounds also shows the very strong PC12 cell injury effect that free radical resisting is caused, promptly pheochromocytoma PC12 cell on the kidney of rats of simulation cranial nerve cell is had the anti-oxidative damage protective effect.Illustrate its to protection cranial nerve antioxidation, prevent and treat senile dementia positive role arranged, can be used to the purposes for preparing protection cranial nerve antioxidation, prevents and treats the alzheimer disease disease drug.In addition, this chemical compound also is found has the effect that suppresses xanthine oxidase activity, thereby can expect as the medicine for the treatment of gout class diseases.
Chemical compound malonic acid of the present invention [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt can combine with adjuvant or carrier pharmaceutically commonly used, and preparation is used for the treatment of the medicine or the pharmaceutical composition of the senile dementia that free radical causes.Said medicine or pharmaceutical composition can adopt dosage forms such as injection, tablet, capsule, aerosol, suppository, membrane, drop pill, externally-applied liniment.
Chemical compound malonic acid of the present invention [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester or its officinal salt can also unite use with the free radical resisting oxidant drug that has now gone on the market such as superoxide dismutase (SOD) etc., prepares to have the active antioxidant compositions of infringement that the defence free radical causes, is used for the treatment of above-mentioned disease.It also can be protected the cranial nerve antioxidation with other, prevent and treat the medication combined use of senile dementia, obtains anti-ageing year dementia pharmaceutical composition.Aforementioned pharmaceutical compositions can adopt dosage forms such as injection, tablet, capsule, aerosol, suppository, membrane, drop pill, externally-applied liniment.
Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof can also be united use with treatment goat medicine that has now gone on the market such as xanthine oxidase inhibitor allopurinol etc., prepares the pharmaceutical composition with treatment goat, is used for the clinical treatment of goat.Above-mentioned various kinds of drug compositions can adopt dosage forms such as injection, tablet, capsule, aerosol, suppository, membrane, drop pill, externally-applied liniment.
Dosage form described in the present invention such as tablet, capsule, injection, aerosol, suppository, membrane, drop pill and externally-applied liniment with and various slow release, controlled release form or nanometer formulation can get via the routine preparation according to the pharmaceutics general knowledge of now having generally acknowledged, the medicine of preparing on these knowledge and technical foundation that contains claim compound or pharmaceutically acceptable salt thereof of the present invention or other dosage forms of pharmaceutical composition are all in protection scope of the present invention.
In order to understand essence of the present invention better, enumerate chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2 with the form of pharmacology related embodiment respectively below, 3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester is to the test of ultra-oxygen anion free radical inhibitory action, to oxydol H
2O
2Due to the PC12 cell injury protective effect test, suppress the activity test that the lipid peroxide of free yl induction generates; and, its purposes in pharmaceutical field and the basis that is used to prepare relevant antioxidant medicine are described to the result of the inhibitory action test of xanthine oxidase XO.The pharmacology related embodiment has provided the part activity data of chemical compound.Same mandatory declaration, these embodiment that the present invention enumerates are used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 2The activity test of malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-external removing ultra-oxygen anion free radical of methyl ethyl ester
2.1 experiment material and sample
2.1.1 experiment reagent:
2.1.1.1 phenazine methosulfate (phenazine methosulfate, PMS), nitro tetrazole orchid (nitrobluetetrazolium, NBT), luxuriant and rich with fragrance alloxazine (ferrozine) is available from Sigma company;
2.1.1.2 Quercetin (quercetin) provides (purity is 99%) by pharmaceutical college of Zhejiang University Chinese medicine and natural drug research department laboratory; Silibinin (Silybin) is available from Liaoning Panjin lucky chance pharmaceutcal corporation, Ltd, and HPLC detects purity 98%.
2.1.1.3Tris alkali, the DMEM culture medium is available from Gibco company;
2.1.1.4 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and NADH (DPNH) are available from Amresco company for 3-;
2.1.1.5 other reagent is homemade analytical reagent, available from Hangzhou Huadong Medicine reagent company limited.
2.1.2 instrument:
2.1.2.1 microplate reader: Synergy-HT type, BIO-TEK company;
2.1.2.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
2.1.2.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
2.1.2.4 ultra-pure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
2.1.2.5 sterilizing filter: Sterifil500 type, Millipore company;
2.1.2.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory.
2.2 experimental technique:
2.2.1 malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-detection that methyl ethyl ester (abbreviation formula (I) chemical compound) is removed the ultra-oxygen anion free radical ability is to use azophenlyene-N metilsulfate-NADH (phenazinemethosulfate-NADH) system, with the calibrating of nitroblue tetrazolium (nitroblue tetrazolium) method of reducing.
2.2.2 in pH value is 8.0 16mM Tris-HCl buffer, produce ultra-oxygen anion free radical with 3 milliliters of NADH, 50 μ M nitroblue tetrazolium and 10 μ M azophenlyene-N metilsulfates that contain 78 μ M, to its activity of formula (I) compound test of variable concentrations.The color of ultra-oxygen anion free radical and nitroblue tetrazolium reaction product is monitored under the 560nm wavelength with spectrophotometer, and silibinin is product in contrast, and Quercetin is used as the positive control medicine.
2.3 experimental result: result of the test sees Table one.
Table one
2.4 conclusion: result of the test shows, formula (I) Compound I is malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester has certain ultra-oxygen anion free radical scavenging action, though under same concentration, its removing ability is less than the Quercetin height, but its removing ability is a little more than silibinin.Conclusion: the silybin esters derivatives that these 23 ethyl malonyls replace belongs to the antioxidant with certain removing ultra-oxygen anion free radical.
Embodiment 3Chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester is to oxydol H
2O
2Due to the protective effect activity test of PC12 cell injury
3.1 experiment material and sample
3.1.1 cell: rat adrenal chromaffin oncocyte (PC12) is available from Shanghai cell institute of the Chinese Academy of Sciences.
3.1.2 experiment reagent:
3.1.2.1 hydrogen peroxide (hydrogen peroxide, H
2O
2), nitro tetrazole orchid (nitroblue tetrazolium, NBT), luxuriant and rich with fragrance alloxazine (ferrozine) is available from Sigma company;
3.1.2.2 Quercetin (quercetin) provides (purity is 99%) by pharmaceutical college of Zhejiang University Chinese medicine and natural drug research department laboratory; Silibinin (Silybin) is available from Liaoning Panjin lucky chance pharmaceutcal corporation, Ltd, and HPLC detects purity 98%.
3.1.2.3Tris alkali, the DMEM culture medium is available from Gibco company;
3.1.2.4 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is available from Amresco company for 3-;
3.1.2.5 calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.;
3.1.2.6 penicillin and streptomycin are produced by Shijiazhuang Pharmaceutical Group Co Ltd;
3.1.2.7 other reagent is homemade analytical pure, available from Hangzhou Huadong Medicine reagent company limited.
3.1.3 instrument:
3.1.3.1 microplate reader: Synergy-HT type, BIO-TEK company;
3.1.3.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
3.1.3.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
3.1.3.4 ultra-pure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
3.1.3.5 sterilizing filter: Sterifil500 type, Millipore company;
3.1.3.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory;
3.1.3.7CO
2Cell culture incubator: MMM, German company;
3.1.3.8 inverted microscope: XD-2 type, Chongqing Optical ﹠ Electrical Instrument Co., Ltd..
3.2 experimental principle:
H
2O
2It is a kind of precursor of main living radical, it can cause the apoptosis of central nervous system cell, PC12 cell (pheochromocytoma on the kidney of rats) can be simulated cranial nerve cell, therefore use it always and be used as studying the model that concerns between medicine and the neurocyte, with the survival rate of MTT survey cell, if testing compound has removing by H
2O
2The effect of the free radical that causes, neuroprotective cell, its cell survival rate height then, the OD value is just high; Otherwise it is just low.The present invention adopt to Tang Xican institute reported method (Xiaoqiu Xiao etc., Neurosci Letter, 1999,275:73-76) and to improved, in order to measure the anti-oxidative damage protective effect of chemical compound to the PC12 cell.
3.3 cell culture: the PC12 cell is with containing 10% calf serum, and the DMEM culture medium culturing of 100U/ ml penicillin and 100 mcg/ml streptomycins is in 37 ℃, 5%CO
2Hatch the conventional method cultivation of going down to posterity in the cell culture incubator.
3.4 experimental technique:
3.4.1 Cytotoxic mensuration: at first measured the effect of testing compound to the PC12 cell proliferation with the mtt assay of improvement.The PC12 cell digests with the pancreas enzyme-EDTA Digestive system, collecting cell, counting, be diluted to 8000/milliliter density with the DMEM culture medium that contains 10% calf serum, be inoculated in then in the 96 porocyte plates, continue in the cell culture incubator to cultivate, after 24 hours, the dimethyl sulfoxide solution to be measured that to newly join respectively joins in each hole with Concentraton gradient, makes that the ultimate density of chemical compound is respectively 16,8 and 4 mcg/ml in the hole.After 72 hours, add the normal saline solution of 10 microlitre MTT (5 mg/ml), continue at 37 ℃ 5%CO
2Cultivated 3 hours in the incubator of humid air, discard stock solution, add 150 microlitre dimethyl sulfoxines in every hole, the MTT crystal Jia Za that the vibration dissolving generates, with microplate reader colorimetric under the 570nm wavelength, the inhibitory rate of cell growth computing formula is as follows:
% inhibitory rate of cell growth=(OD
Solvent control-OD
Sample)/OD
Solvent control* 100%.
3.4.2 formula (I) chemical compound is to H
2O
2Due to the mensuration of protective effect of PC12 cell injury
PC12 cell DMEM culture medium culturing contains 10% Ox blood serum in the culture medium, 100U/ ml penicillin and 100U/ milliliter streptomycin.Cell is added in 96 orifice plates with the density in 8000 in every hole, at 37 ℃, and 50%CO
2Cultivated 36 hours in the incubator of humid air.With the metamorphosis of inverted microscope observation of cell and the survival rate of measuring cell with mtt assay.Cell is after 36 hours hatch, and the dimethyl sulfoxide solution of the formula that will newly join (I) chemical compound joins in each hole with Concentraton gradient respectively.Act on the H that adding is newly joined after 2 hours
2O
2(final concentration is 600 μ M) effect 3 hours, the microscopic examination record discards original fluid, adds new culture fluid 100 microlitres, adds MTT 10 microlitres then, and after 3 hours, the careful suction removed culture fluid, adds 150 microlitre DMSO Rong Xie Jia Za, in 570nm place reading.Adopt silibinin product in contrast, the positive control drug of Quercetin.
3.4.3 result of the test: see Table two.
Table two formula (I) chemical compound and reference substance are to hydrogen peroxide damage PC12 cytoprotection
3.5 conclusion: result of the test shows; malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2; 3-dihydro-3; 5; 7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2; 3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester has the stronger PC12 cell injury effect that free radical resisting is caused, and promptly the PC12 cell to the simulation cranial nerve cell has clear and definite anti-oxidative damage protective effect.And under same concentration; its ability of removing free radical protection cell is close with the positive control Quercetin; and its protection PC12 cell is avoided the radical damage ability and is better than silibinin greatly, illustrates that the silybin esters derivatives of these 23 ethyl malonyls replacements belongs to the antioxidant of the PC12 cytosis with potent protection simulation cranial nerve cell.
Measure chemical compound to H
2O
2Due to the protective effect of PC12 cell injury can be used as the foundation of the mechanism of action of its protection maincenter cranial nerve cell of preliminary discussion.So show protective effect according to this chemical compound, can expect that its treatment to senile dementia has positive role to PC12 cell due to the hydrogen peroxide damage.
Embodiment 4Chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester suppresses the activity test that the lipoperoxide of free yl induction generates
4.1 experimental principle: lipid peroxidation is the product that free radical acts on polyunsaturated fatty acid, and the generation positive correlation of content and free radical because body has the protective effect of antioxidative enzyme system and non-enzyme system, constantly produces free radical and constantly is eliminated again.With advancing age, intravital antioxidant constantly descends, and the ability of removing free radical weakens gradually, and lipid peroxidation then strengthens, and lipid peroxidation product increases.Therefore, the working sample lipoid peroxidization resistant is one of important indicator of the anti-liver brain tissue oxidizing damage medicine of screening.This pharmacodynamic study vitamin C-Fe
2+The system induction rat liver homogenate produces lipid peroxidation product malonaldehyde (MDA), MDA can react with thiobarbituricacid (TBA), product has absorption maximum at the 532nm place, and the variation of measuring by MDA reflects the inhibitory action of chemical compound to the liver homogenate lipid peroxidation indirectly.
4.2 experiment material and sample
4.2.1 experimental animal: SD (sprague-dawley) rat is available from Zhejiang University's Experimental Animal Center.The quality certification number: SCXK (Zhejiang 2007-0029).
4.2.2 experiment reagent:
4.2.2.1 ferrous sulfate FeSO
4(4 μ M) solution: homemade FeSO
4Analyze pure reagent, available from East China, Hangzhou reagent company, autogamy as requested before the use;
4.2.2.2 vitamin C (50 μ M) Vc solution: Domestic V c analyzes pure reagent, available from East China, Hangzhou reagent company, and autogamy as requested before the use;
4.2.2.3 trichloroacetic acid CCl
3CO
2H (20%) solution: homemade CCl
3CO
2H analyzes pure reagent, available from Hangzhou Huadong Medicine reagent company limited, and autogamy as requested before the use;
4.2.2.4 potassium dihydrogen phosphate, dipotassium hydrogen phosphate, thiobarbituricacid and other reagent are homemade analytical pure (Hangzhou Huadong Medicine reagent company limited).
4.2.2.5 Quercetin (quercetin) provides (purity is 99%) by pharmaceutical college of Zhejiang University Chinese medicine and natural drug research department laboratory; Silibinin (Silybin) is available from Liaoning Panjin lucky chance pharmaceutcal corporation, Ltd, and HPLC detects purity 98%.
4.3 experiment material and sample
4.3.1 microplate reader: Synergy-HT type, BIO-TEK company;
4.3.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
4.3.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
4.3.4 ultra-pure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
4.3.5 constant water bath box: Shanghai gloomy letter test science instrument company;
4.3.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory;
4.3.7 desk type high speed refrigerated centrifuger: TGL-16G, last Hai'an booth instrument plant.
4.4 experimental technique:
4.4.1 the preparation of hepatomicrosome: the lipoperoxide generation that suppresses free yl induction is to detect by the Hepar Mus microsome.Get 5 sacrificed by decapitation of SD experimental rat, take out liver rapidly, prepare hepatomicrosome with supercentrifugation.
4.4.2 sample is to the inhibitory action of Hepar Mus lipid peroxidation: containing FeSO
4Add in the 200 mcg/ml microsomes in 1 milliliter of buffer of (4 μ M) and Vc (50 μ M), to induce the generation lipid peroxidation, the testing compound sample that adds variable concentrations simultaneously, hatch for 37 ℃ and add 1 milliliter of trichloroacetic acid (20%) stopped reaction adding, 1.5 milliliters of thiobarbituricacids (0.76%) after 30 minutes, 100 ℃ of boiling water baths 20 minutes, centrifugal removal albumen precipitation is measured the supernatant absorbance under the 532nm wavelength.Lipid peroxidation is represented with the malonaldehyde that generates in the reaction, tests with Quercetin as the positive control medicine.As the blank group, what add same volume DMSO is the solvent control group with buffer surrogate response liquid.
Sample is as follows to the computing formula computing formula of rat liver homogenate lipid peroxidation suppression ratio:
Suppression ratio (%)=(OD
Sample cell-OD
Blank pipe)/(OD
Standard pipe-OD
Blank pipe) * 100%.
4.5 result of the test: result of the test sees Table three.
Table three. sample formula (I) chemical compound is to inhibitory action (the half-inhibition concentration IC of rat liver microsomal lipid peroxidation
50)
Result of the test shows, chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester has the lipoperoxide generative capacity of stronger inhibition free yl induction, its half-inhibition concentration IC
50Be 19.89 mcg/ml, promptly pair cell has clear and definite anti-oxidative damage protective effect; Compare with silibinin, it suppresses the LPO activity and has improved 68%.
4.6 conclusion: this chemical compound belongs to and has potent protection cell antioxidant, points out it to have protection liver brain cell tissue and reduces oxidative damage and can expect and be used to prepare the effect that protection liver brain tissue oxidizing damage class disease comprises the alzheimer disease disease drug.
Embodiment 5. malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-methyl ethyl ester is to the inhibitory action test of xanthine oxidase XO
5.1 experiment material and sample
5.1.1 experimental animal: SD (sprague-dawley) rat is buied in Zhejiang University's Experimental Animal Center.The quality certification number: SCXK (Zhejiang 2007-0029).
5.1.2 experiment reagent:
5.1.2.1 phenazine methosulfate (phenazine methosulfate, PMS), nitro tetrazole orchid (nitrobluetetrazolium, NBT), xanthine, luxuriant and rich with fragrance alloxazine (ferrozine) be available from Sigma company;
5.1.2.2 Triton X-100 is available from Gibco company;
5.1.2.3 positive control medicine: allopurinol, Taizhou Mei Tong pharmaceutcal corporation, Ltd;
5.1.2.4 potassium dihydrogen phosphate, dipotassium hydrogen phosphate and other reagent are homemade analytical pure (East China, Hangzhou reagent company).
5.1.3 instrument:
5.1.3.1 microplate reader: Synergy-HT type, BIO-TEK company;
5.1.3.2 vertical automatic electric heating pressure steam sterilizer: LDZX-40BI type, Shen, Shanghai peace medical apparatus plant;
5.1.3.3 ultraviolet spectrophotometer: UV-1201 type, Beijing Rayleigh Analytical Instrument Co.,Ltd;
5.1.3.4 ultra-pure water system: UPWS-I-60D type, Hangzhou Yongjieda Cleaning Technology Co.,Ltd;
5.1.3.5 sterilizing filter: Sterifil500 type, Millipore company;
5.1.3.6 gas bath constant temperature oscillator: THZ-C, Jintan City, Jiangsu Province Medical Instruments factory;
5.1.3.7 glass homogenizer: homemade.
5.2 experimental technique:
5.2.1 get the SD rat, sacrificed by decapitation, take out the phosphate buffer (100mM of liver rapidly to pre-cooling, pH 8.75) in, remove blood vessel, shred the back and dilute the back in directly homogenate on ice with phosphate buffer 1: 1 (w/v), in-20 ℃ of preservations, with phosphate buffer (1: 7) dilution, centrifugal under 4 ℃ of conditions (8000rpm/10 minute) gets supernatant as proenzyme with preceding.
5.2.2 chemical compound is measured the enzyme linked immunological ELISA method that adopts to the inhibitory action of XO.Add substrate xanthine (0.1 mg/ml in the sample well, 600 microlitres), enzyme (30 microlitre), chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester dissolves with dimethyl sulfoxine, with phosphate buffer dilution, every hole 30 microlitres, making its final concentration is 40 mcg/ml, 20 mcg/ml and 10 mcg/ml, add blue tetrazole of nitro and phenazine-methionine ester (30 microlitre), add Triton X-100 (0.4%, 10 microlitre) at last, water bath heat preservation is 2 hours in 37 ℃, colorimetric determination under the 550nm wavelength.Chemical compound is calculated for blank and contrast OD value by sample OD value the xanthine oxidase suppression ratio.Xanthine oxidase suppression ratio (%)=[(OD
Standard pipe-OD
Blank pipe)-(OD
Sample cell-OD
The sample blank pipe)]/(OD
Standard pipe-OD
Blank pipe) * 100%.
5.3 experimental result:
Chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester is 32.32% to the xanthine oxidase suppression ratio when 40 mcg/ml, when 20 mcg/ml, be 13.52% to the xanthine oxidase suppression ratio, when 40 mcg/ml, be 9.47% to the xanthine oxidase suppression ratio, and suppression ratio is 89.2% during positive control allopurinol 40 mcg/ml, half-inhibition concentration IC
50=2.35 * 10
-4M.
5.4 conclusion: do not have positive drug allopurinol height though it suppresses activity, chemical compound malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester has certain inhibition activity to xanthine oxidase, and its inhibition activity is dose-effect relationship.Illustrating that this chemical compound has further develops the potential that becomes the xanthine of determined curative effect enzyme inhibitor.
When above-mentioned description elaboration was of the present invention, the purpose that the pharmacology related embodiment is provided was to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of in entering claim of the present invention and its equivalent scope, practical application of the present invention comprises all general variations, cooperates, or improves.
Claims (2)
1. the silibinin ester derivant of 23 ethyl malonyls replacements shown in the formula (I) or its officinal salt application in the pharmaceutical preparation of preparation antioxidant, the name of formula (I) is called: malonic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy-4-oxo-.alpha.-5:6-benzopyran-2)-2,3-dihydro-1,4-benzodioxane-2]-the methyl ethyl ester;
Formula (I)
Its condition is: prosposition is the R configuration in the formula (I); 10,11 is the R configuration simultaneously, or is the S configuration simultaneously.
2. the silibinin ester derivant that replaces according to 23 ethyl malonyls shown in claim 1 Chinese style (I) or its officinal salt are prevented and treated application in the alzheimer disease disease drug in preparation.
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