Detection method of polyamine substances in tobacco roots, stems and leaves
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A detection method, technology of tobacco roots, applied in the direction of measuring devices, instruments, scientific instruments, etc.
Active Publication Date: 2016-03-23
GUIZHOU TOBACCO SCI RES INST
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[0004] At present, there is no simple, rapid, highly sensitive and environmentally friendly method for the detection of polyamines in tobacco roots, stems and leaves
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Embodiment 1
[0027] The detection method of polyamine substances in tobacco roots, stems and leaves of the present invention comprises the following steps:
[0028] (1) Weigh 100mg of freeze-dried samples (roots, stems or leaves) in a 10mL centrifuge tube, then add 1mgmL of 5uL 0.1M hydrochloric acid -1 Hexamethylenediamine hydrochloride, then add 6mL of pre-cooled 0.3M perchloric acid aqueous solution to extract by vortexing at 2000rpm for 30min, centrifuge at 4000rpm for 5min, and pass the supernatant through a 0.45um water filter membrane;
[0029] (2) Take 2mL of the filtrate in a 10mL centrifuge tube, put it in an ice bath to cool for 5min, then add 400uL of 4M sodium hydroxide solution, vortex and mix well, then add 50mM 3,5-dinitrate prepared in pre-cooled 1050uL of acetonitrile Base benzoyl chloride, vortex mix, react in ice bath for 2min, add 500uL 2M hydrochloric acid aqueous solution to stop the reaction;
[0030] (3) Add 1mL ultrapure water to the derivatized product, adjust i...
Embodiment 2
[0048] The polyamines in different parts of the same tobacco were detected by the method as in Example 1, and the measurement results are shown in Table 6. From the detection results, it can be known that the content of polyamines in leaf buds is the highest, especially the content of tyramine. The composition of polyamine content in leaves was leaf bud>upper leaf>middle leaf>lower leaf. Generally speaking, the content of polyamine in young leaves was higher than that in old leaves. The root has relatively high tyramine content, but other polyamine content is low, which is similar to the polyamine composition of the lower leaves, and the polyamine content in the stem is higher than that of the root, which is similar to the polyamine composition of the middle leaves.
[0049] Table 6 Polyamine content composition between different parts of the same tobacco
[0050]
[0051] a Data are mean ± SD of 5 determinations; ND, not detected.
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Abstract
The invention discloses a detection method of polyamine substances in tobacco roots, stems and leaves. The method comprises the steps that vortex extraction is conducted on tobaccos to obtain polyamines through a pre-cooled perchloric acid solution, derivatization is conducted through 3,5-dinitro benzoyl chloride, extraction and concentration are conducted on a derivative product through vortex auxiliary-liquid-liquid microextraction, and detection is conducted through an ultra-efficient liquid chromatography-photodiode array detector. In addition, in order to obtain the best enrichment factors and the recovery rate in vortex auxiliary-liquid-liquid microextraction, method optimization is conducted through fractional factorial design and Doehlert design. Compared with other methods of analyzing and detecting the polyamines, the detection method of the polyamine substances in the tobacco roots, stems and leaves has the advantages of being rapid in derivative reaction, stable in product, high in sensitivity, good in reproducibility, green and friendly to environment and the like, and accurate qualitativeness and quantification can be conducted on the polyamines in the tobacco roots, stems and leaves. In addition, other four kinds of polyamines, namely, tyramine, 1,3-propylene diamine, high spermidine and canavalmine are identified through high-resolution mass spectrometry, wherein the canavalmine is found in the tobaccos for the first time.
Description
technical field [0001] The invention belongs to the technical field of measuring methods for chemical components of tobacco, and in particular relates to a method for detecting polyamines in tobacco roots, stems and leaves. Background technique [0002] Polyamines are biologically active low molecular weight fatty nitrogenous bases produced during the metabolism of organisms. Common polyamines are putrescine, cadaverine, spermidine, and spermine. In higher plants, it mainly exists in free form, and there are differences among different tissues and organs. In plants, polyamines can stimulate growth and delay aging. It is generally believed that polyamines can be used as plant hormones in a broad sense, and some people think that polyamines may be "second messengers" like cAMP to regulate plant growth and development. In addition, polyamines are also closely related to the stress resistance of plants. Studies have found that when plants independently resist the adversity st...
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