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Fast detection method for dihydro flavanol 4-reductase/leucocyanidin reductase in tree plant

A technology of cyanidin reductase and dihydroflavan, applied in the field of rapid detection of dihydroflavanol 4-reductase/leucocyanidin reductase in tea tree, can solve the problem of high cost, long detection time, enzyme The active method is cumbersome and other problems, and the effect of low cost, high accuracy and simple method is achieved

Inactive Publication Date: 2009-07-15
ANHUI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of cumbersome methods, high cost and long detection time for detecting the enzyme activities of dihydroflavanol 4-reductase / leucocyanidin reductase (DFR / LAR) and anthocyanin reductase (ANR) in tea tree , the present invention provides a quick detection method of dihydroflavanol 4-reductase / leucocyanidin reductase and anthocyanin reductase in tea tree fresh leaves

Method used

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  • Fast detection method for dihydro flavanol 4-reductase/leucocyanidin reductase in tree plant
  • Fast detection method for dihydro flavanol 4-reductase/leucocyanidin reductase in tree plant
  • Fast detection method for dihydro flavanol 4-reductase/leucocyanidin reductase in tree plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Major equipment:

[0062] 1. UV-Vis spectrophotometer; 2. Constant temperature water bath; 3. Electronic scale; 4. Centrifuge; 5. pH meter.

[0063] Materials and Reagents:

[0064] 1. Materials:

[0065] Tea tree fresh leaves, tender stems, roots and other materials. Store at -80°C for later use.

[0066] 2. Main reagents

[0067] (1) Polyvinylpyrrolidone (PVPP): It is used to adsorb and precipitate polyphenols in the crude enzyme solution to maintain a high activity of the enzyme protein.

[0068] (2) Quartz sand: increase friction and improve the extraction rate of enzyme protein.

[0069] (3) Phosphate buffer solution (PBS) of 0.1mol / LpH7.0:

[0070] Solution A: Weigh sodium dihydrogen phosphate (NaH 2 PO 4 ) 27.8 grams, dissolved in 1000 milliliters of distilled water, set aside.

[0071] Solution B: Weigh disodium hydrogen phosphate (Na 2 HPO 4· 7H 2 (0) 53.62 grams, be dissolved in the distilled water of 1000 milliliters, standby.

[0072] Just befor...

Embodiment 2

[0096] Main equipment: same as embodiment 1

[0097] Materials and Reagents:

[0098] 1. Material: Same as Example 1

[0099] 2. Main reagents

[0100] (1) 0.1mol / L Phosphate buffer solution (PBS) of pH6.5:

[0101] Solution A: Weigh sodium dihydrogen phosphate (NaH 2 PO 4 ) 27.8 grams, dissolved in 1000 milliliters of distilled water, set aside.

[0102] Solution B: Weigh disodium hydrogen phosphate (Na 2 HPO 4 .7H 2 (0) 53.62 grams, be dissolved in the distilled water of 1000 milliliters, standby.

[0103] Just before use, take 68.5 ml of solution A and 31.5 ml of solution B, mix and dilute to 200 ml with water. Then under the pH meter, use 0.1mmol / L hydrochloric acid or sodium hydroxide solution to accurately adjust the pH value of the solution to 6.5, which is 0.1mol / L pH6.5 phosphate buffer solution (PBS).

[0104] (2) 3mmol / L pyridine hydrochloride (CYA) solution: Weigh 1 mg of CYA, add 1 ml of methanol and shake until completely dissolved, and store at 4°C in ...

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Abstract

The invention relates to a quick detection method of dihydrogen-flavanol4-reductase / leucocyanidin reductase and anthocyanin reductase in tea tree, which can solve the problems of the prior technique of complicated operation in enzymatic activity detection method, high cost, long detection time. The quick detection method comprises: preparing an enzyme extracting-solution, measuring the protein content in enzyme extracting-solution and enzymatic activity, wherein the enzymatic activity is detected by detecting the consumption of the reduction type nicotinamide adenine dinucleotide phosphate solution (NADPH) and the vitamine C is added into the reaction system to reduce the interference of the substrate DHM and CYA on enzymatic activity detection. The quick detection method using ultraviolet spectrophotometer has features of simple method, low cost, short detection time of 15-25 minutes; environmental protection, safety, no need of noxious organic solvent, higher detection accuracy due to continuous detection of the changed enzymatic activity.

Description

technical field [0001] The invention relates to a detection method of dihydroflavanol 4-reductase / leucocyanidin reductase (DFR / LAR) and anthocyanin reductase (ANR) in tea tree. Background technique [0002] Tea (Camellia sinensis) leaves are rich in flavonoids, alkaloids and other important secondary metabolites, which have important physiological and health effects on the human body, such as anti-allergic, anti-tumor and anti-aging, etc. [1] , is a popular health drink in the world. [0003] Dihydroflavanol 4-reductase / leucocyanidin reductase (DFR / LAR) and anthocyanin reductase (ANR) are important enzymes in the downstream pathway of flavonoid synthesis and are also non-ester-type catechins. It is a direct enzyme of catechin synthesis and plays an important regulatory role in the tea tree catechin pathway. [0004] At present, the determination methods of dihydroflavanol 4-reductase / leucocyanidin reductase (DFR / LAR) and anthocyanin reductase (ANR) enzymes at home and abro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33G01N33/68C12Q1/26
Inventor 张宪林高丽萍夏涛
Owner ANHUI AGRICULTURAL UNIVERSITY
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