Methods for biologically synthesizing and detecting catechin intermediates

Abstract

The invention discloses methods for synthesizing and detecting catechin intermediates. The methods include acquiring CsANRa genes from tea trees by means of cloning and then constructing binary expression vectors; transforming the binary expression vectors into plant bodies to obtain transgenic plants; extracting polyphenols substances such as flavane-3-alcohol intermediates in the transgenic plants by the aid of extracting solution which is 80% methanol and hydrochloric acid mixed solution so as to obtain the catechin intermediates; qualitatively and quantitatively authenticating catechin intermediate products by means of UPLC-QQQ-MS / MS (ultra-performance liquid chromatography-triple quadrupole-mass spectrometry / mass spectrometry). Compared with the prior art, the methods have the advantages that the methods are easy to operate, and organic solvents are inexpensive and are low in cost; the catechin intermediates acquired by the aid of the corresponding method do not contain catechin and are relatively simple, accordingly, the corresponding method is suitable to be used for preparing catechin intermediates with high purity, and the methods for synthesizing and detecting the catechin intermediates are reported for the first time.

Description

technical field [0001] The invention relates to the technical field of natural product chemistry, in particular to a biosynthesis and detection method of a catechin intermediate. Background technique [0002] Catechin intermediates are intermediate products in the formation of catechins, which generally exist in the form of carbocations, so they are extremely unstable. Catechins are widely distributed in different plants and have been continuously detected and reported by researchers, but intermediate products in the formation of catechins have been reported. The existence of flavan-3-ol intermediates in plants is of great significance. On the one hand, it is the precursor for the formation of catechins. On the other hand, it is also an indispensable intermediate in the formation of proanthocyanidins. unit. At the same time, because the flavan-3-ol intermediate itself has certain electrophilic properties, it will also form conjugates with a series of substances with nucleo...

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Problems solved by technology

[0003] However, the source of the intermediate has not been reported. On the one hand, it is because it is not sure which enzymes are involved in the synthesis of the intermediate. On the other hand, the method for extracting and detecting the intermediate is not perfect. The conventional reagents for extracting phenolic compounds There are 70% acetone / 0.05% acetic acid, 80% methanol, etc., but in the extracts using these chemical reagents as extraction solvents, no intermediates have been reported to be detected (Pang Y, Peel GJ, Wright E et al.Early Stepsin Proanthocyanidin Biosynthesis in the Model Legume Medicago truncatula.[J].Plant Physiol.2007,145(3):601-615.)

Therefore, flavan-3-ol intermediates have not been discovered and reported largely because our extraction and detection methods are not perfect

Although people such as Xie Deyu proposed that such intermediates are more stable and more easily detected under acidic conditions, and the structures of intermediates were predicted (such as figure 1 structures of four predicted flavan-3-ol intermediates), but until now there have been no reports on the synthesis and detection of intermediates

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