CN102787146A

CN102787146A - Method utilizing microwave assisted lipase catalysis to synthesize esterification modified EGCG

Info

Publication number: CN102787146A
Application number: CN2012102641186A
Authority: CN
Inventors: 王洪新; 朱松; 李哲; 马朝阳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-07-28
Filing date: 2012-07-28
Publication date: 2012-11-21
Anticipated expiration: 2032-07-28
Also published as: CN102787146B

Abstract

The invention relates to a method utilizing microwave assisted lipase catalysis to synthesize esterification modified EGCG (Epigallocatechin Gallate), and belongs to the biosynthesis filed of organic compounds. The method is characterized in that EGCG and fatty acid vinyl ester are taken as raw materials to synthesize the esterification modified EGCG through the co-action of microwave radiation and lipase catalysis in an organic solvent system, so that the product can be obtained. The method provided by the invention comprehensively utilizes the advantages of both the microwave radiation and the lipase catalysis, so as to achieve the effect which is not achieved by the lipase catalysis under a conventional heating condition. With the action of the microwave radiation, the reaction system can be uniformly affected by a microwave field, the heating rate of the system is high, the temperature is kept stable, the reaction is obviously accelerated, the reaction rate is improved, and the reaction time is greatly shortened. Therefore, the synthesis method provided by the invention is efficient and fast, and is consistent with the requirements of green chemistry.

Description

A kind of method with the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted

Technical field

The present invention relates to a kind of method with the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted; Be to be raw material specifically with EGCG and vinyl fatty ester; In organic solvent system; Through microwave radiation and lipase-catalyzed acting in conjunction synthesis modification EGCG, belong to the biosynthesizing field of organic cpds.

Background technology

Catechin is a most important physiologically active substance in the tealeaves, accounts for the 60%-80% of tea-polyphenol total amount, is the essential substance composition of tea health-care function.The highest in the catechin with NVP-XAA 723 (EGCG) content, account for about 50% of catechin total amount.Because anti-oxidant, the mutation that EGCG is excellent, radioprotective, antitumor, regulate immunity and physiological activity such as delay senility; The research of recently relevant EGCG is noticeable; But with regard to its application, exist fat-soluble poor, bioavailability is low, absorb problems such as slow in instability and the body under the physiological environment.

Based on these problems,, become one of focus of EGCG research in recent years through keeping the molecular modification that its reactive group makes its modification.Molecular modification is the important channel of transforming physical property at present both at home and abroad, is the important means that changes the material BA.The acidylate molecular modification of catechin utilizes biological or chemical synthetic method exactly, with the hydroxylic moiety at some position of catechin molecular structure or all by the acidylate modification, makes the new molecule that constitutes become the novel substance of fat-soluble or activity change.

In the process that adopts the chemical process acidylate, because the EGCG hydroxyl is more, and environmental facies of living in seemingly, and each hydroxyl all might form ester bond with acylating reagent, is to have much challenging problem in the organic synthesis so the regioselectivity of compounds is synthesized always.Utilize chemical method acidylate EGCG to have many shortcomings: regioselectivity is poor, is prone to produce a large amount of by products, and the product productive rate is low; Adopt measures such as radical protection, reactions step is many, length consuming time, and it is many to introduce solvent, the separation and purification difficulty; Use basic catalyst more, produce a large amount of alkali wastes etc.And enzymatic esterification EGCG has the reaction conditions gentleness, and regioselectivity is high, and side reaction is few, and reactions step is few, simple controllable, advantages of environment protection.

Since Gedye in 1986 etc. utilized microwave radiation to quicken organic reaction effectively, the research that microwave promotes organic chemical reactions gradually deeply.Microwave radiation can not only fast reaction speed, reduces catalyst levels, can also change reaction preference sometimes.Since nineteen ninety, microwave radiation is applied to biocatalytic reaction especially in the nonaqueous phase enzymic catalytic reaction.In a lot of concrete enzymatic reaction systems, suitable microwave radiation is compared with the routine heating and is shown remarkable advantages, and its can fast reaction speed, improve reaction yield and product selectivity, sometimes even can change the stereoisomerism structure of product.The report that does not also have at present microwave radiation and lipase-catalyzed acting in conjunction synthesis modification EGCG.

Summary of the invention

The objective of the invention is to overcome the weak point of prior art; A kind of method with the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted is provided; With EGCG and vinyl fatty ester is raw material, in the Virahol system, through microwave radiation and lipase-catalyzed acting in conjunction synthesis modification EGCG.

According to technical scheme provided by the invention; A kind of method with the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted; Step is following: with NVP-XAA 723 and vinyl fatty ester in molar ratio 1-2 ︰ 1 be dissolved in the Virahol of 400-500 times of quality, add the lypase of 80-120U/g EGCG, reactor drum is placed the microwave reaction device; Start microwave radiation; Microwave power is chosen in 30-60W, and temperature is set between 40-60 ℃, stopped reaction behind the reaction 30-60min; Get the gained reacting liquid filtering, the 50-60 ℃ of rotary evaporation of filtrating is vacuum-drying 2-4h under the 0.05-0.1MPa condition in vacuum tightness, obtains the NVP-XAA 723 of product esterification modification.

Said vinyl fatty ester is a vinyl-acetic ester, propionate, vinyl butyrate, valeric acid vinyl acetate, vinyl caproate, new n-nonanoic acid vinyl acetate, vinyl neodecanoate, vinyl laurate or palmitinic acid vinyl acetate.

Said lypase is lypase LIPOZYME TL IM (description of product is seen examination as to substances bibliography 1) and the LPOZYME RM IM (description of product is seen examination as to substances bibliography 2) that Novo Nordisk company produces, the lypase Lipase AYS " Amano " (description of product is seen examination as to substances bibliography 3) that Amano Pharmarceutial company produces.

The EGCG acetylate that the present invention prepares (EGCG and vinyl-acetic ester reaction) is through mass spectrum confirmation (Fig. 1), and product is that 1 hydroxyl is acetylation, 2 hydroxyls are acetylation, 3 EGCG modification derivants that hydroxyl is acetylation.

The present invention has following advantage: the method synthesis of synthetic esterification EGCG provided by the present invention has utilized the advantage of microwave heating and enzymic catalytic reaction, can reach the not available effect of enzymic catalytic reaction under the conventional heating condition.Microwave radiation can make reaction system evenly receive the effect of microwave field, and the system heat-up rate is fast, and it is stable that temperature keeps, and obviously fast reaction speed improves reaction yield, shortens the reaction times greatly.Thereby provide a kind of efficient, fast, meet the compound method of Green Chemistry.

Description of drawings

EGCG acetylate mass spectroscopy figure among Fig. 1 embodiment 1.A: the liquid chromatogram of acetylization reaction after product under 280nm; The be acetylation mass spectrum of product (Mw=585) of 3 hydroxyls is selected ion figure in the B:EGCG molecule; The be acetylation mass spectrum of product (Mw=543) of 2 hydroxyls is selected ion figure in the C:EGCG molecule; The be acetylation mass spectrum of product (Mw=501) of 1 hydroxyl is selected ion figure in the D:EGCG molecule; The mass spectrum of E:EGCG molecule (Mw=459) is selected ion figure).

Embodiment

Below provide embodiment, the present invention is described further.

Embodiment 1

In 50mL tool plug round-bottomed flask, add EGCG 50mg respectively, vinyl-acetic ester 9.5mL (EGCG and vinyl-acetic ester mol ratio are 1:1) adds Virahol 20mL again; Reactor drum is placed the microwave reaction device; The lypase LIPOZYME TL IM that adds 80U/g EGCG reacts 30min down at 40 ℃, 40W, behind the stopped reaction; Reaction solution obtains EGCG esterified prod 35.8mg through filtration, rotary evaporation, vacuum-drying, and the recovery is 71.6%.Transformation efficiency through efficient liquid phase chromatographic analysis EGCG is 65.4%.

The mass spectroscopy of EGCG acetylate is as shown in Figure 1.

A: the liquid chromatogram of acetylization reaction after product under 280nm; The be acetylation mass spectrum of product (Mw=585) of 3 hydroxyls is selected ion figure in the B:EGCG molecule; The be acetylation mass spectrum of product (Mw=543) of 2 hydroxyls is selected ion figure in the C:EGCG molecule; The be acetylation mass spectrum of product (Mw=501) of 1 hydroxyl is selected ion figure in the D:EGCG molecule; The mass spectrum of E:EGCG molecule (Mw=459) is selected ion figure)

The comparative example 1

In 50mL tool plug round-bottomed flask, add EGCG 50mg respectively; Vinyl-acetic ester 9.5mL (EGCG and vinyl-acetic ester mol ratio are 1:1) adds Virahol 20mL again, the lypase LIPOZYME TL IM of 80U/g EGCG; 40 ℃ of water-baths 12 hours; Behind the stopped reaction, reaction solution obtains EGCG esterified prod 36.4 mg through filtration, rotary evaporation, vacuum-drying, and the recovery is 72.8%.Transformation efficiency through efficient liquid phase chromatographic analysis EGCG is 67.9%.

Embodiment 2

In 50mL tool plug round-bottomed flask, add EGCG 50mg respectively, vinyl butyrate 16.1mL (EGCG and vinyl butyrate mol ratio are 1:1) adds Virahol 20mL again; Reactor drum is placed the microwave reaction device; The lypase Lipase AYS " Amano " that adds 100U/g EGCG reacts 40min down at 60 ℃, 50W, behind the stopped reaction; Reaction solution obtains EGCG esterified prod 30.8 mg through filtration, rotary evaporation, vacuum-drying, and the recovery is 61.6%.Transformation efficiency through efficient liquid phase chromatographic analysis EGCG is 73.4%.

The comparative example 2

In 50mL tool plug round-bottomed flask, add EGCG 50mg respectively; Vinyl butyrate 16.1mL (EGCG and vinyl butyrate mol ratio are 1:1) adds Virahol 20mL again, the lypase Lipase AYS " Amano " of 100U/g EGCG; 60 ℃ of water-baths 16 hours; Behind the stopped reaction, reaction solution obtains EGCG esterified prod 29.8 mg through filtration, rotary evaporation, vacuum-drying, and the recovery is 59.6%.Transformation efficiency through efficient liquid phase chromatographic analysis EGCG is 72.7%.

Embodiment 3

In 100mL tool plug round-bottomed flask, add EGCG 150mg respectively, vinyl caproate 15.6mL (EGCG and vinyl caproate mol ratio are 1.5:1) adds Virahol 40mL again; Reactor drum is placed the microwave reaction device; The lypase Lipase AYS " Amano " that adds 100U/g EGCG reacts 50min down at 50 ℃, 60W, behind the stopped reaction; Reaction solution obtains EGCG esterified prod 81.5mg through filtration, rotary evaporation, vacuum-drying, and the recovery is 81.5%.Transformation efficiency through efficient liquid phase chromatographic analysis EGCG is 76.3%.

Embodiment 4

In 100mL tool plug round-bottomed flask, add EGCG 50mg respectively, vinyl neodecanoate 22mL (EGCG and vinyl neodecanoate mol ratio are 1:1) adds Virahol 30mL again; Reactor drum is placed the microwave reaction device; The lypase LPOZYME RM IM that adds 60U/g EGCG reacts 50min down at 60 ℃, 40W, behind the stopped reaction; Reaction solution obtains EGCG esterified prod 37.9 mg through filtration, rotary evaporation, vacuum-drying, and the recovery is 75.8%.Transformation efficiency through efficient liquid phase chromatographic analysis EGCG is 76.9%.

Claims

1. method with the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted; It is characterized in that: step is following: with NVP-XAA 723 and vinyl fatty ester in molar ratio 1-2 ︰ 1 be dissolved in the Virahol of 400-500 times of quality, add the lypase of 80-120U/g EGCG, reactor drum is placed the microwave reaction device; Start microwave radiation; Microwave power is chosen in 30-60W, and temperature is set between 40-60 ℃, stopped reaction behind the reaction 30-60min; Get the gained reacting liquid filtering, the 50-60 ℃ of rotary evaporation of filtrating is vacuum-drying 2-4h under the 0.05-0.1MPa condition in vacuum tightness, obtains the NVP-XAA 723 of product esterification modification.

2. use the method for the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted according to claim 1, it is characterized in that: said vinyl fatty ester is a vinyl-acetic ester, propionate; Vinyl butyrate; The valeric acid vinyl acetate, vinyl caproate, new n-nonanoic acid vinyl acetate; Vinyl neodecanoate, vinyl laurate or palmitinic acid vinyl acetate.

3. use the method for the lipase-catalyzed synthetic esterification modification EGCG of microwave-assisted according to claim 1; It is characterized in that: said lypase is lypase LIPOZYME TL IM and the LPOZYME RM IM that Novo Nordisk company produces, the lypase Lipase AYS " Amano " that Amano Pharmarceutial company produces.