CN103293149A - Novel screening method for medicines for treating liver diseases and gout - Google Patents
Novel screening method for medicines for treating liver diseases and gout Download PDFInfo
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- CN103293149A CN103293149A CN2012100450292A CN201210045029A CN103293149A CN 103293149 A CN103293149 A CN 103293149A CN 2012100450292 A CN2012100450292 A CN 2012100450292A CN 201210045029 A CN201210045029 A CN 201210045029A CN 103293149 A CN103293149 A CN 103293149A
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- xanthine oxidase
- xanthine
- solution
- gout
- treatment
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Abstract
The invention discloses a method for screening treatment medicines for treating liver diseases, gout and the like by virtue of xanthine oxidase inhibiting capability. The method in particular comprises the following steps: composition, extraction process and the like: firstly, establishment of a primary determination method by xanthine oxidase; preparing reagents for test, wherein the special steps are as follows: the total volume of each test tube is 8ml: an enzymatic reaction system contains 2ml of determination matrix liquid, 1ml of xanthine oxidase liquor, 2ml of reaction color developing agent, 1ml of xanthine liquor, 1ml of reaction stop buffer and the balance of blank matrix liquid; stopping reaction after water bath at 37 DEG C for 15 minutes. The method established by the steps screens and extracts medicinal materials by using xanthine oxidase inhibiting capability as an index.
Description
Technical field
The present invention relates to the screening technique of a kind of novel therapeutic hepatopathy and gout medicine.
Background technology
The xanthine oxidase brief introduction: xanthine oxidase (XOD) belongs to the aerobic dehydrogenation enzyme, is important enzyme in the nucleic acid in vivo metabolism.It is made up of by 140000 subunit two molecular weight, and a molybdenum atom, two Fe are arranged in each subunit
2S
2(SR)
4, cluster and a flavin adenine dinucleotide (FAD) (FAD).
Xanthine oxidase effect brief introduction:
Following reaction takes place in the hypoxanthine in the xanthine human body and xanthine in the presence of xanthine oxidase (XOD):
Reacted product is uric acid and hydrogen peroxide, and uric acid is accumulated in the blood, can cause goat, the superoxide radical O that forms in the reaction when superfluous
2 -Relate to pathologic processes such as inflammation, sclerosis, cancer.
Simultaneously because xanthine oxidase mainly is present in liver, when hepatocellular damage, serum XOD activity is rising in various degree, therefore the XOD inhibitor is favourable to hepatitis treatment, also can reduce the injury (E.J.Oliveira, et al.In vitro glucuronidation of kaempferol and quercetin by human UGT-1A9 microsomes.) that oxidation causes.
Based on above principle, we have designed a kind of triage techniques that act as evaluation means with the xanthine oxidase preparation, be that example is estimated with the Chinese crude drug rhizoma alismatis, the result shows that rhizoma alismatis has good xanthine oxidase inhibiting effect, this also can be used for hepatopathy (combination of Chinese tradiational and Western medicine magazine " rhizoma alismatis and hyperlipidemia with the rhizoma alismatis of bibliographical information, atherosclerotic and fatty liver ") and the treatment of gout (Liaoning traditional Chinese medical science is mixed " reusing dialectical treatment acute gouty arthritis 120 examples of rhizoma alismatis ") consistent, so this method also can extend to other to hepatopathy, in the screening of the effective medicinal material of gout.
Have two kinds of methods can reduce the formation of uric acid, superoxide radical and hydrogen peroxide in the human body: the one, reduce hypoxanthine and xanthic content in the human body; The 2nd, the activity of xanthine oxidase in the reduction human body, this can realize aspect medical science and the body metabolism research important meaning being arranged with the inhibiting effect of studying xanthine oxidase so seek the inhibitor of xanthine oxidase by the inhibiting effect to enzyme.
The assay method of xanthine oxidase XOD is a lot, mainly contains spectrophotometric method, electrochemical process, fluorescence method and radiochemical method etc., and instrument and equipment requires high, and what the applicant selected for use is the XOD colorimetric method that is applicable to common lab.
Summary of the invention
At problems of the prior art, the invention provides a kind of new method (suppressing ability with xanthine oxidase) and screen treatment of diseases medicines such as treatment hepatopathy, gout, specifically screen content and comprise prescription composition, extraction process etc.
A kind of screening technique for the treatment of hepatopathy and gout medicine is characterized in that:
(1) foundation of xanthine oxidase Preliminary Determination method: but the hypoxanthine in the XOD catalytic matrix (or xanthine) generates xanthine (or uric acid), the meanwhile superoxide ion (O of Chan Shenging
2 -) the nitro tetrazolium blue is reduced into mauve Jia Za.Growing amount according to the Jia Za can be calculated the XOD vigor.
(2) instrument: T6-new century UV, visible light spectrophotometer (Shanghai general rare general responsibility company limited), water-bath (going up the grand experimental facilities of Nereid company limited), PH counts (Nereid section on the thunder magnetic).
(3) test is with the preparation of each reagent:
1. blank matrix liquid: the 0.1MK of PH8.3
2HPO
4-KH
2PO
4Damping fluid.Include 0.2%TritonX-100.
2. measure matrix liquid: the blank matrix liquid of 0.034mg/ml xanthine.
3. reflect developer: 0.15mg/ml nitro tetrazolium blue distilled water solution.
4. reaction terminating liquid: 0.35M HCl.
5. xanthine oxidase solution: the blank matrix solution of 0.2% xanthine oxidase (50U).
6. test liquid.
7. allopurinol solution: 0.007mg/ml allopurinol solution.
(4) operation steps: cumulative volume is 8ml in each test tube: contains in the enzymatic reaction system and measures matrix liquid 2ml, and xanthine oxidase solution 1ml, reaction solution agent 2ml, xanthine solution 1ml, reaction terminating liquid 1ml, all the other are supplied with blank matrix liquid; 37 ℃ of water-bath 15min cessation reactions; According to the bibliographical information wavelength obtained the maximum absorption is arranged when the 560nm.Absorbance and XOD vigor are done typical curve: y=0.0163x+0.0044r=0.999096.
(5) screening:
The method of utilizing above-mentioned steps to set up is index screening medicinal material and extraction process with the xanthine oxidase inhibiting effect.
Beneficial effect of the present invention: provide a kind of new method (suppressing ability with xanthine oxidase) to screen treatment of diseases medicines such as treatment hepatopathy, gout, specifically screened content and comprise prescription composition, extraction process etc.
Embodiment
The foundation of xanthine oxidase Preliminary Determination method:
(1) method principle: but the hypoxanthine in the XOD catalytic matrix (or xanthine) generates xanthine (or uric acid), the meanwhile superoxide ion (O of Chan Shenging
2 -) the nitro tetrazolium blue is reduced into mauve Jia Za.Growing amount according to the Jia Za can be calculated the XOD vigor.
(2) instrument: T6-new century UV, visible light spectrophotometer (Shanghai general rare general responsibility company limited), water-bath (going up the grand experimental facilities of Nereid company limited), PH counts (Nereid section on the thunder magnetic).
(3) test is with the preparation of each reagent:
1. blank matrix liquid: the 0.1MK of PH8.3
2HPO
4-KH
2PO
4Damping fluid.Include 0.2%TritonX-100.
2. measure matrix liquid: the blank matrix liquid of 0.034mg/ml xanthine.
3. reflect developer: 0.15mg/ml nitro tetrazolium blue distilled water solution (be stored in the brown bottle that is surrounded by black paper, refrigeration can be stablized two months).
4. reaction terminating liquid: 0.35M HCl.
5. xanthine oxidase solution: the blank matrix solution of 0.2% xanthine oxidase (50U).
6. test liquid.
7. allopurinol solution: 0.007mg/ml allopurinol solution.
(4) operation steps:
Method: cumulative volume is 8ml in each test tube: contains in the enzymatic reaction system and measures matrix liquid 2ml, and xanthine oxidase solution 1ml, reaction solution agent 2ml, xanthine solution 1ml, reaction terminating liquid 1ml, all the other are supplied with blank matrix liquid.37 ℃ of water-bath 15min cessation reactions.According to the bibliographical information wavelength obtained the maximum absorption is arranged when the 560nm.Absorbance and XOD vigor are done typical curve: y=0.0163x+0.0044r=0.999096.
(5) screening:
The method of utilizing above-mentioned steps to set up is index screening medicinal material and extraction process with the xanthine oxidase inhibiting effect.
Embodiment rhizoma alismatis extract is investigated xanthine oxidase inhibiting effect dose-effect relationship.
Verify the effect that it suppresses xanthine oxidase with rhizoma alismatis medicinal material ethanol extract.
According to bibliographical information, find that rhizoma alismatis is comparatively obvious to the xanthine oxidase inhibiting effect, so the applicant carries out enzymatic determination to rhizoma alismatis medicinal material in the gout prescription, method is as follows: get rhizoma alismatis medicinal material 25g meal to be broken into, with 8 times of amount 80% alcohol extracts twice, each 2 hours, merge extract, standby.
Getting rhizoma alismatis extract (c=14.286ug/ml) xanthine oxidase inhibiting effect dose-effect relationship investigates:
Get from test findings, linear good when not adding rhizoma alismatis extract (c=14.286ug/ml) and add rhizoma alismatis extract 0.2ml-0.4ml, but adding 0.6ml when (being equivalent to rhizoma alismatis medicinal material 8.5716ug), absorbance minimum (inhibiting effect is the strongest).Calculate the 1mg allopurinol according to the absorbance of each test tube and be equivalent to 76.53mg rhizoma alismatis medicinal material.From experimental result as can be known: the rhizoma alismatis extract has good inhibition effect to xanthine oxidase, can be used as rhizoma alismatis extraction process selection indicators.
Embodiment is with xanthine oxidase inhibiting effect screening medicinal material and extraction process.
1, the screening of extracting method:
A decocting cooking method: get rhizoma alismatis medicinal material 25g, add 8 times of water gagings and decoct 2 times, each 2 hours, merge decoction liquor, standby.
B 50% ethanol refluxing process: get rhizoma alismatis medicinal material 25g, add 8 times of amount 50% alcohol refluxs 2 times, each 2 hours, merge phegma, standby.
C 80% ethanol refluxing process: get rhizoma alismatis medicinal material 25g, add 8 times of amount 80% alcohol refluxs 2 times, each 2 hours, merge phegma, standby.
D 95% ethanol refluxing process: get rhizoma alismatis medicinal material 25g, add 8 times of amount 95% alcohol refluxs 2 times, each 2 hours, merge phegma, standby.
Table A, B, C, D rhizoma alismatis Different Extraction Method are as follows to the experiment of the activity inhibition of xanthine oxidase:
Unit (ml)
This experimental result is as can be known: it is good that alcohol reflux technology is boiled technological effect than decocting, and optimised process is 95% ethanol refluxing process.
Though the present invention with preferred embodiment openly as above, they limit the present invention, anyly are familiar with this skill person, without departing from the spirit and scope of the invention, when doing various variations or retouching, belong to the present invention's protection domain equally certainly.Therefore protection scope of the present invention should with the application claim was defined is as the criterion.
Claims (5)
1. screening technique for the treatment of hepatopathy and gout medicine is characterized in that:
Be that the screening means are screened treatment of diseases medicines such as treatment gout, hepatopathy with the xanthine oxidase inhibiting effect.
2. the screening technique for the treatment of hepatopathy according to claim 1 and gout medicine is characterized in that:
The foundation of xanthine oxidase Preliminary Determination method: but the hypoxanthine in the XOD catalytic matrix (or xanthine) generates xanthine (or uric acid), the meanwhile superoxide ion (O of Chan Shenging
2 -) the nitro tetrazolium blue is reduced into mauve Jia Za, can calculate the XOD vigor according to the growing amount of Jia Za.
3. the screening technique for the treatment of hepatopathy according to claim 2 and gout medicine is characterized in that:
Test the preparation with each reagent:
Blank matrix liquid: the 0.1M K of PH8.3
2HPO
4-KH
2PO
4Damping fluid includes 0.2%TritonX-100;
Measure matrix liquid: the blank matrix liquid of 0.034mg/ml xanthine;
Reflection developer: 0.15mg/ml nitro tetrazolium blue distilled water solution;
Reaction terminating liquid: 0.35M HCl;
Xanthine oxidase solution: the blank matrix solution of 0.2% xanthine oxidase (50U);
Test liquid;
Allopurinol solution: 0.007mg/ml allopurinol solution.
4. according to the screening technique of claim 2 or 3 described treatment hepatopathys and gout medicine, it is characterized in that:
Operation steps: cumulative volume is 8ml in each test tube, contains in the enzymatic reaction system to measure matrix liquid 2ml, and xanthine oxidase solution 1ml, reaction solution agent 2ml, xanthine solution 1ml, reaction terminating liquid 1 ml, all the other are supplied with blank matrix liquid; 37 ℃ of water-bath 15min cessation reactions;
Screening: the method for utilizing described operation steps to set up is index screening medicinal material and extraction process with the xanthine oxidase inhibiting effect.
5. the screening technique for the treatment of gout according to claim 4 and liver disease drug is characterized in that:
In the described operation steps, absorbance and XOD vigor are done typical curve: y=0.0163x+0.0044 r=0.999096.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006050012A2 (en) * | 2004-11-01 | 2006-05-11 | Morinda, Inc. | Morinda citrifolia based compositions and methods for inhibiting xanthine oxidase |
| CN101092336A (en) * | 2006-06-23 | 2007-12-26 | 赵昱 | Compound in cinnamic alcohol category preparation method and usage |
| JP2008088107A (en) * | 2006-10-02 | 2008-04-17 | Fujiyakuhin Co Ltd | New pyridazine derivative |
| CN101508693A (en) * | 2009-02-02 | 2009-08-19 | 温州医学院 | Xylogen like flavonoid compounds, method of preparing the same and pharmaceutical use |
| CN102106516A (en) * | 2011-01-25 | 2011-06-29 | 天津科技大学 | Use of vegetable extract in inhibiting xanthine oxidase (XO) and application in preparing food for preventing and treating hyperuricemia or gout |
-
2012
- 2012-02-25 CN CN2012100450292A patent/CN103293149A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006050012A2 (en) * | 2004-11-01 | 2006-05-11 | Morinda, Inc. | Morinda citrifolia based compositions and methods for inhibiting xanthine oxidase |
| CN101092336A (en) * | 2006-06-23 | 2007-12-26 | 赵昱 | Compound in cinnamic alcohol category preparation method and usage |
| JP2008088107A (en) * | 2006-10-02 | 2008-04-17 | Fujiyakuhin Co Ltd | New pyridazine derivative |
| CN101508693A (en) * | 2009-02-02 | 2009-08-19 | 温州医学院 | Xylogen like flavonoid compounds, method of preparing the same and pharmaceutical use |
| CN102106516A (en) * | 2011-01-25 | 2011-06-29 | 天津科技大学 | Use of vegetable extract in inhibiting xanthine oxidase (XO) and application in preparing food for preventing and treating hyperuricemia or gout |
Non-Patent Citations (2)
| Title |
|---|
| 姜彤伟等: "玉米须中抑制黄嘌呤氧化酶活性组分的筛选及其作用", 《吉林大学学报》 * |
| 黎莉等: "中药提取物中黄嘌呤氧化酶抑制剂的筛选", 《武汉工程大学学报》 * |
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Application publication date: 20130911 |