Application of 4-(o-ascorbic acid)-(+)-catechin and detection method of 2,3-trans leucocyanidin

An ascorbic acid and detection method technology, applied in organic chemical methods, measuring devices, instruments, etc., can solve the problems of reduced detection speed, affected accuracy, and difficulty in distinguishing, and achieves the analysis of carbon flow distribution and improves reliability and accuracy. Effect

Active Publication Date: 2022-07-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] 2,3-trans-colorless anthocyanins are extremely unstable
However, the solvents used in this method can also cleave the condensed tannins in the sample, resulting in the release of additional catechin- or epicatechin-type carbocations, making 2,3-trans-leucocyanidin sources compatible with Carbocations from the cracking of condensed tannins are not easy to distinguish, thus affecting the accuracy of quantification
[0007] In addition, the reported extracts of catechin carbocations are not compatible with the extraction systems commonly used in the study of condensed tannins, which leads to the need to add an independent extraction step to extract catechin carbocations after obtaining soluble condensed tannins. For detection, the detection method is complicated and the detection speed is reduced

Method used

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  • Application of 4-(o-ascorbic acid)-(+)-catechin and detection method of 2,3-trans leucocyanidin
  • Application of 4-(o-ascorbic acid)-(+)-catechin and detection method of 2,3-trans leucocyanidin
  • Application of 4-(o-ascorbic acid)-(+)-catechin and detection method of 2,3-trans leucocyanidin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Screening of 2,3-trans leucocyanidin candidate endogenous indicators in plants

[0066] The design idea of ​​the endogenous indicator screening experiment in this example is that because 2,3-trans leucoanthocyanidin can spontaneously transform into an electrophilic (+)-catechin-type carbocation in plants , which can react with nucleophiles to form relatively stable adducts. Therefore, the content of 2,3-trans leucocyanidin can be obtained by detecting the content of the adduct, and the relationship between the two content is positively correlated.

[0067] As shown in formula (1), according to the molecular structure of 2,3-trans leucocyanidin, its C4 position is easy to form (+)-catechin or (-)-epicatechin type carbocation ; this position is attacked by a potential nucleophile, and finally a stable covalent bond is formed; therefore, the general structural formula of the adduct formed by the reaction of 2,3-trans leucocyanidin with a nucleophile is the formu...

Embodiment 2

[0082] Example 2 Confirms the specific structure of the substance whose parent ion information is m / z 463

[0083] In order to accurately infer the structure of the m / z 463 substance under low-resolution mass spectrometry, in this example, UHPLC-QToF was used to perform high-resolution scanning at m / z 100-1700 of tds4-4 mutant and wild-type Arabidopsis samples (MS1 scan ).

[0084] The UHPLC-QToF detection instrument was an Agilent 1290Infinity II UPLC liquid chromatography coupled with an Agilent6546Q-ToF mass spectrometry system, equipped with an Agilent Zorbax RRHD SB (3×150mm, 1.7μm, C18) chromatographic column. The elution program of liquid chromatography is: phase A (ultrapure water containing 0.1% formic acid), phase B (acetonitrile containing 0.1% formic acid), flow rate 0.4 mL / min; program: 0-1 min, 5% B; 1- 2 min, 5-10% B; 2-17 min, 10-31% B; 17-18 min, 31-95%. The parameters of the mass spectrometer detector are: sheath gas flow rate 10L / min, temperature 400°C; dr...

Embodiment 3

[0094] Example 3 Standard synthesis of 4-(O-ascorbic acid)-(+)-catechin

[0095] According to the results of Example 2, this example adopts 3,4-trans leucocyanidin and L-ascorbic acid to prepare 4-(O-ascorbic acid)-(+)-catechin; the specific preparation process is as follows:

[0096] (1) After mixing 500 μM 3,4-trans leucocyanidin with an equimolar L-ascorbic acid aqueous solution, adjust the pH of the solution system to 7.4 with NaOH; 3,4-trans leucocyanidin It can be obtained by chemical synthesis, or the finished product can be purchased.

[0097] (2) The mixed solution system was placed in the dark at 30°C for 1 hour.

[0098] (3) using ethyl acetate to extract the solution system after the reaction in step (2), and retaining the water phase after extraction; wherein, the volume of ethyl acetate used for extraction is 3 times that of the solution system.

[0099] (4) HPLC separation and preparation; the Agilent 1200 HPLC system was equipped with an Agilent Zorbax SB (9....

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Abstract

The invention relates to the technical field of plant condensed tannin detection, in particular to the application of 4-(O-ascorbic acid)-(+)-catechin and a method for detecting 2,3-trans leucocyanidin. The content of the endogenous indicator 4-(O-ascorbic acid)-(+)-catechin is proportional to the content of 2,3-trans leucocyanin. The detection method first extracts the soluble condensed tannins in the sample, and then conducts qualitative and quantitative analysis by UHPLC‑QToF and UHPLC‑QqQ. The detection method has the characteristics of accuracy and high efficiency, which is helpful to analyze the carbon flow distribution of plant flavonoid pathways and to elucidate the biosynthesis mechanism of condensed tannins.

Description

technical field [0001] The invention relates to the technical field of detection of plant condensed tannins, in particular to the application of 4-(O-ascorbic acid)-(+)-catechin and a detection method for 2,3-trans leucocyanidin. Background technique [0002] 2,3-trans leucocyanidin is the product of dihydroflavonol reductase (DFR) in plants, and is an important intermediate product of the flavonoid pathway, especially the biosynthetic branch of condensed tannins, and its chemical structural formula is: [0003] [0004] The compound can be further catalyzed by anthocyanin synthase (ANS) to form anthocyanin, and then catalyzed by UDP-flavonoid glycosidase (UFGT) or anthocyanin reductase (ANR) to form anthocyanin or condensed tannin (-) - Epicatechin-type units. This substance can also be converted to (+)-catechin-type starting units of condensed tannins as a substrate for leucoanthocyanidin reductase (LAR). In addition, free 2,3-trans leucocyanidins can directly partici...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/36G01N30/72C07D407/12
CPCG01N30/02G01N30/06G01N30/36G01N30/34G01N30/7266C07D407/12C07B2200/07
Inventor 段长青于可济兰义宾齐梦瑶
Owner CHINA AGRI UNIV
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