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Method for quickly analyzing anthocyanin in tobacco corolla

An anthocyanin and rapid analysis technology, which is applied to the analysis of materials, material separation, and measurement devices, can solve the problems of reduced practicability of analysis methods, long analysis operation time, and increased analysis costs, so as to achieve accurate identification and shorten analysis time , Analyzing the effect of improving efficiency

Inactive Publication Date: 2011-04-06
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0009] The method established by Nakatsuka et al. in 2008 using HPLC-ESI-MS technology to analyze anthocyanin components in tobacco corolla. The chromatographic column adopted in the glucoside process, the eluent composition and its proportion and the elution program of the eluent are different, the HPLC analysis time is 32min, and the retention time of cyanidin-3-O-rutinoside (Cy3R) It is about 14 minutes, which makes it take a long time to complete an analysis of anthocyanins, resulting in low analysis efficiency, increased analysis cost, and reduced practicability of the analysis method

Method used

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  • Method for quickly analyzing anthocyanin in tobacco corolla
  • Method for quickly analyzing anthocyanin in tobacco corolla
  • Method for quickly analyzing anthocyanin in tobacco corolla

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] 1. Extraction of anthocyanins

[0084] 1) collect the fresh corolla of tobacco in full bloom period, collect the sample of corolla coloring part;

[0085] 2) Freezing the colored corolla sample with liquid nitrogen, grinding it into powder, adding the extract to the corolla powder under light-shielding conditions, immersing the corolla powder in the extract, and extracting anthocyanins, the extraction temperature is maintained at 4 ℃, extraction time is 24h, and wherein, extracting solution is made up of concentrated hydrochloric acid and methanol, wherein the mass percent concentration of concentrated hydrochloric acid is 36.5%, and the volume ratio of concentrated hydrochloric acid and methanol is 1:999 (that is to prepare 1L of extracting solution, It consists of 1mL of concentrated hydrochloric acid with a mass percentage concentration of 36.5% mixed with 999mL of methanol); 4mL of extract solution is added to every 1g of corolla powder, and the extraction mixture i...

Embodiment 2

[0119] In the step of extracting anthocyanins, except that the volume ratio of concentrated hydrochloric acid to methanol in the extract is 1:199 (that is, to prepare 1 L of extract, it is composed of 5 mL of concentrated hydrochloric acid with a concentration of 36.5% by mass and 995 mL of methanol) ; Add 6mL extract solution for every 1g Corolla powder; Extraction time is 20h, all the other are identical with embodiment 1;

[0120] In the step of separating anthocyanins by HPLC, except for the elution gradient of the mobile phase, it is as follows:

[0121] 0min, 90% A solution and 10% B solution, that is, the volume ratio of A solution and B solution in the mobile phase is 9:1;

[0122] 20min, 70% A solution and 30% B solution, that is, the volume ratio of A solution and B solution in the mobile phase is 7:3;

[0123] 25 minutes, 90% solution A and 10% solution B, that is, the volume ratio of solution A and solution B in the mobile phase is 9:1.

[0124] That is, the elut...

Embodiment 3

[0130] In the step of extracting anthocyanins, except that the volume ratio of concentrated hydrochloric acid to methanol in the extract is 1:99 (that is, to prepare 1 L of extract, it is composed of 10 mL of concentrated hydrochloric acid with a concentration of 36.5% by mass and 990 mL of methanol) ; Extraction time is outside 72h, and all the other are identical with embodiment 1;

[0131] In the step of separating anthocyanins by HPLC, liquid A in the mobile phase is composed of formic acid and water with a volume ratio of 2:8, and liquid B is composed of acetonitrile and methanol with a volume ratio of 80:20;

[0132] The rest are the same as in Example 1, HPLC elution separation, tobacco anthocyanin HPLC elution analysis operation time is 15min, collect and separate anthocyanin, wherein the retention time of anthocyanin is 6.33min, and the degree of separation is ≥ 1.5, HPLC elution separation profile as Figure 5 .

[0133] The molecular structure of anthocyanins iden...

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Abstract

The invention discloses a method for quickly analyzing anthocyanin in tobacco corollas. The method comprises the steps of: separating an extracting solution of the tobacco corolla by high efficiency liquid chromatography; and identifying the molecular structure of the anthocyanin by using a mass spectrometry. By the method, anthocyanin samples in a large amount of tobacco corollas can be analyzed in a short time; the method has quick analysis speed and accurate analysis result, reduces the analysis cost and provides an effective, quick and practical analysis method for heterogenous expression research of anthocyanin-synthesized related genes in the tobacco without a high-tech analytical instrument, such as UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry).

Description

technical field [0001] The invention relates to a method for fast qualitative and quantitative analysis of natural substances, in particular to a fast analysis method for anthocyanins. Background technique [0002] Anthocyanins are an important material basis for the formation of plant flower color, so the analysis of anthocyanin composition and content is one of the important contents of flower color research. At present, tobacco (Nicotiana tabacum) is often used as the receptor material in the research on the expression of heterologous genes related to flower color in plants. Some studies have been carried out on the analysis methods of anthocyanin components in tobacco, mainly including the following four methods: ( 1) thin layer chromatography (thin layer chromatography, TLC), (2) high-performance liquid chromatography-photodiode array detection technology (high-performance liquid chromatography with a photodiodarray detector, HPLC-PAD / DAD), (3) high-efficiency Liquid c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/72G01N30/34G01N30/56
Inventor 戴思兰孙翊李慧王亮生
Owner BEIJING FORESTRY UNIVERSITY
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