Method for screening 1-deoxidation-D-xylulose5-phosphoric acid reduction isomerization enzyme inhibitor from plant extract

A plant extract and isomerase technology, which is applied in the field of antibiotic screening, can solve the problems of large and expensive, high detection cost, etc., and achieve the effect of strong feasibility and simple operation

Inactive Publication Date: 2010-07-07
NORTHWEST UNIV
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a simple and fast screening method for DXR inhibitors, which is suitable for using common paper chromatography (PC), thin layer chromatography (TLC), or reversed-phase ion pairing in the laboratory. Detection methods such as liquid chromatography screen DXR inhibitors from plant extracts, which solves the technical problem that the method for screening DXR inhibitors in the background technology needs large and expensive instruments for detection, such as nuclear magnetic resonance, etc., and the detection cost is high.

Method used

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  • Method for screening 1-deoxidation-D-xylulose5-phosphoric acid reduction isomerization enzyme inhibitor from plant extract
  • Method for screening 1-deoxidation-D-xylulose5-phosphoric acid reduction isomerization enzyme inhibitor from plant extract
  • Method for screening 1-deoxidation-D-xylulose5-phosphoric acid reduction isomerization enzyme inhibitor from plant extract

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Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: PC method screening DXR inhibitor

[0056] 1) Preparation of samples for detection of DXR inhibitors

[0057] In this design, a reaction system with a volume of 50-200 μl is used. The reaction system is shown in Table 1, and the reaction is carried out in a water bath at 25-40° C. for 2-16 hours to prepare samples for detecting DXR inhibitors.

[0058] Table 1 Reaction system

[0059] Reactant

Final concentration

DXP

5-20mM

NADPH

5-20mM

Mg 2+

5-50mM

DXR

1-10μg / μl

Plant extracts

1-100μg / μl

Tris-HCl (pH=6-8)

50-500mM

[0060] The optimal conditions are: 100 μl reaction system, the final concentration of DXP, NADPH, and Mg2+ is 10 mM, the final concentration of DXR is 1.0 μg / μl, and the rest can be adjusted according to the experimental design. Tris-HCl with a pH of about 7.4 is used as the buffer because it is closest to the physiological environment and can maximize th...

Embodiment 2

[0068] Embodiment 2: TLC method screening DXR inhibitor

[0069] 1) Repeat step 1) of the above PC method to prepare samples for detection of DXR inhibitors, and then pretreat the samples: add 5-50U phosphatase to the reaction system, and continue the reaction in a water bath at 30-40°C for 2 -8h, evaporate the water in the reaction system under reduced pressure, and redissolve the residue with 50-300 μl of organic solvent. The preferred conditions are: for a 100 μl reaction system, after adding 10 U alkaline phosphatase, continue the reaction in a 37° C. water bath for 4 hours, evaporate the water in the reaction system under reduced pressure, and redissolve the residue in 100 μl methanol.

[0070] 2) Repeat step 2) of the above PC method to prepare a control sample, which is pretreated in the same way.

[0071] 3) Take the reaction solution without plant extract as a control, take 0.5-2 μl of the above methanol extract for spotting, and select the expansion system as chloro...

Embodiment 3

[0074] Embodiment 3: HPLC method screens the inhibitor of DXR

[0075] 1) Repeat step 1) of the PC method to prepare samples for detecting DXR inhibitors.

[0076] 2) Repeat step 2) of the above PC method to prepare a control sample.

[0077] 3) Take 2-10 μl of sample, dilute it at 1:10, filter it into the HPLC system, and treat the control sample in the same way. The optimal injection volume is 20 μl;

[0078] Chromatographic conditions: mobile phase A: 5-20% methanol aqueous solution containing 10-30mM TBAS (tetra-n-butyl ammonium bisulfate); mobile phase B: 40-70% methanol aqueous solution containing 10-30mM TBAS; flow rate: 0.3- 3mL / min; injection volume: 10-100μl; column temperature: 20-40°C; detector: DAD ultraviolet detector (Agilent); detection wavelength: 210-280nm. The preferred conditions are: mobile phase A: 5% aqueous methanol containing 10mM TBAS; mobile phase B: 70% aqueous methanol containing 10mM TBAS, flow rate: 0.75mL / min; injection volume: 50μl; column t...

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Abstract

The invention relates to a method for screening 1-deoxidation-D-xylulose5-phosphoric acid reduction isomerization enzyme (DXR) inhibitor from plant extract. In the method, paper chromatography, thin layer chromatography or reversal phase ion pairing HPLC method are employed to detect decline of substrate transformation quantity to evaluate inhibiting effect of the plant extract on DXR. In addition, as far as developing solvents employed in the process of paper chromatography are concerned, ethanol with a concentration of 95%: 1M ammonium acetate: 1M EDTA is 70:29:1(v / v / v), colour-developing agent is ethylenediamine sulfate water solution, in the developing system employed in the process of thin layer chromatography, chloroform: methanol is 3:1 (v / v), the colour-developing agent is sulfuric acid ethanol solution; in the reversal phase ion pairing HPLC method, the double phase solution gradient elution method is employed for detection. In the process of screening inhibiting agent in the method, the substrate 1-deoxidation-D-xylulose5-phosphoric acid and coenzyme NADPH do not contain isotope labeling. In addition, the screening process features simpleness, speediness, good feasibility and no detection by expensive large instruments.

Description

technical field [0001] The invention belongs to the field of antibiotic screening, and relates to a simple method for screening 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) inhibitors from plant extracts. Background technique [0002] Terpenoids exist in almost all forms of life, and they play an important role in organisms, such as the regulation of dolichol diphosphates on cell wall and glycoprotein biosynthesis; quinones (such as dolichol diphosphates) Body quinone and coenzyme Q) can be used as carriers for electron transfer and redox reactions; side chains of carotenoids and chlorophyll participate in photosynthesis; steroids can help form cell membranes (such as cholesterol); estrogen participates in intercellular signal transmission and can Control the growth and development of individuals; capsidiol, etc. can be used as antibiotics and phytoalexins for mutual defense between species; gibberellin and abscisic acid can be used as plant hormones; the presence o...

Claims

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Application Information

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IPC IPC(8): C12Q1/533G01N30/90G01N30/89G01N30/02
Inventor 高文运李恒田洁王辉杨少青
Owner NORTHWEST UNIV
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