Rapid and efficient detection method of brassinosteroid

Brassinolide and detection method technology, applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problem of not involving the determination method of brassinolide, save reagent consumption and manpower, increase extraction rate, shorten extraction effect of time

Inactive Publication Date: 2015-12-02
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But none of the above-mentioned methods and patents involves the determination method of brassinosteroids in the grape fruit

Method used

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  • Rapid and efficient detection method of brassinosteroid
  • Rapid and efficient detection method of brassinosteroid
  • Rapid and efficient detection method of brassinosteroid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Accurately weigh 10.00g of grape fruit, quickly grind it into powder with liquid nitrogen, add 40ml of 80% methanol and add isotope internal standard 2 H3 Brassinolide 40ng and 2 H3 brassinosterone 40ng, ultrasonically extract at low temperature for 60min, then put it in a refrigerator at 4°C for 2h, add 4ml of methanol (80%) extract to the filtered residue for 1h, filter, and combine the extracts. Use nitrogen blowing to remove the moisture in the extract solution, put the column solution through the C18 column, and carry out sample adsorption, use 40mmol / L ammonium acetate buffer solution (pH value 6.5) as the eluent to elute to obtain the adsorption solution, and pass the adsorption solution through sephadexLH -20 column for impurity removal, use methanol as the eluent to obtain the eluted product, then pass through the silica column, elute with 10ml methanol to obtain the enriched sample solution, add 5mg of 9-phenanthrene boric acid, derivatize in a 70°C water bath...

Embodiment 2

[0025] Accurately weigh 10.00g grape fruit, quickly grind it into powder with liquid nitrogen, add 40ml methanol (95%) and add isotope internal standard 2 H3 Brassinolide 40ng and 2 H3 brassinosterone 40ng, and ultrasound at low temperature for 30min, then put it in a refrigerator at 4°C for 5h, filter, add 10ml of methanol (95%) to the filter residue, put it in a refrigerator at 4°C for 1h, filter, and combine the extracts. Use nitrogen blowing to remove the moisture in the extract solution, put the column solution through the C18 column, and carry out sample adsorption, use 40mmol / L ammonium acetate buffer solution (pH value 6.5) as the eluent to elute to obtain the adsorption solution, and pass the adsorption solution through sephadexLH -20 column for impurity removal, use methanol as the eluent to obtain the eluted product, then pass through the silica column, elute with 10ml methanol to obtain the enriched sample solution, add 5mg of 9-phenanthrene boric acid, derivatize ...

Embodiment 3-6

[0027] Carry out HPLC detection to the sample to be tested of embodiment 1, concrete test condition is as follows:

[0028] Instrument and chromatographic conditions:

[0029] Instrument: Waters high performance liquid chromatography, equipped with 1525 binary HPLC pump, 717 + Autosampler, 474 fluorescence detector, Empower chromatography workstation;

[0030] Chromatographic column: C18 column (150mm×4.6mm.i.d);

[0031] Mobile phase A: double distilled water;

[0032] Mobile phase B: acetonitrile;

[0033] Flow rate: 1m / min;

[0034] Detection wavelength: the excitation wavelength is 280nm, the emission wavelength is 700nm or 310nm and 375nm are selected as the excitation wavelength and emission wavelength respectively;

[0035] Column temperature: 37°C.

[0036] Standard solution: Accurately weigh 0.0250g brassinosteroid standard substance and dilute it to 25ml with acetonitrile to obtain a standard stock solution of brassinosteroid with a concentration of 100μg / ml an...

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Abstract

The present invention provides a rapid and efficient detection method of brassinosteroid. The rapid and efficient detection method comprises: adopting fresh grape fruits as a material, rapidly grinding into powder with liquid nitrogen, adding 80-95% methanol, isotope internal standard 2H3 brassinosteroid and 2H3 brassin sterone, carrying out low temperature ultrasonic extraction, standing for 2-5h, filtering, taking the filtration residue, adding 80-95% methanol, leaching for 1-2 h, filtering, merging the two filtrates, carrying out physical adsorption to remove the water, sequentially loading onto a C18 column, a sephadex LH-20 column and a silica column, eluting with methanol to obtain an enriched sample liquid, adding phenylboronic acid, carrying out a derivatization reaction, cooling, concentrating to achieve a dry state, redissolving with acetonitrile so as to be used for liquid chromatography determination, selecting acetonitrile and water as the mobile phase, and collecting the target peak, wherein a volume ratio of the acetonitrile to water is (6:4)-(9:1), and the elution way is isocratic elution. The method of the present invention has characteristics of rapidness, efficiency, stable baseline and good brassinosteroid separation.

Description

technical field [0001] The invention relates to the technical field of detection of plant effective components, in particular to a rapid and efficient detection method for brassinosteroids in grape fruits. Background technique [0002] Brassinosteroids (BRs) are also known as brassinosteroids and brassinosteroid hormones. Natural brassinolide is the latest plant endogenous hormone. It was first isolated from rapeseed pollen by American scholar J.W. Mitchell in 1970. The main active ingredient is BR with a steroid structure. Its physiological activity greatly exceeds that of the five existing hormones, and it is internationally recognized as the most active, broad-spectrum, and non-toxic plant growth regulator, and has been honored as the sixth hormone. Brassinolide widely exists in angiosperms, gymnosperms and some lower plants. From the perspective of distribution in plants, it exists in roots, stems, leaves, flowers, powder, pistils, fruits and seeds. Among them, pollen ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 吴月燕刘蓉饶慧云杨国伟
Owner ZHEJIANG WANLI UNIV
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