CN101519395A

CN101519395A - Preparation method of fully substituted acetylate of epigallocatechin-gallate (EGCG)

Info

Publication number: CN101519395A
Application number: CN200810121636A
Authority: CN
Inventors: 江和源; 刘晓辉; 张建勇; 袁新跃
Original assignee: Tea Research Institute Chinese Academy of Agricultural Sciences
Current assignee: Tea Research Institute Chinese Academy of Agricultural Sciences
Priority date: 2008-10-16
Filing date: 2008-10-16
Publication date: 2009-09-02

Abstract

The invention discloses a preparation method of fully substituted acetylate of epigallocatechin-gallate (EGCG) by taking EGCG as materials. The acetylate of EGCG obtained by the existing method has different substituting degree until full substituted substance and the fully substituted acetylate has low purity, which is adverse for further research and application. The invention takes EGCG and acetylation reagent as substrates, an acetylate reaction happens in the presence of an organic solvent and a pyridine catalyst, the mole ratio between EGCG and the acetylate reagent is controlled within the range of 1:80-200, and the fully substituted acetylate of EGCG is generated by the reaction and the products are purified then. The invention can obtain fully substituted acetylate with high purity, which is convenient for further research and application.

Description

The preparation method of fully substituted acetylate of epigallocatechin-gallate (EGCG)

Technical field

The present invention relates to a kind of is the method for its fully substituted acetylate of feedstock production with the NVP-XAA 723.

Background technology

Natural catechin is a most important physiologically active substance in the tealeaves, mainly comprise l-Epicatechol (EC), epigallocatechin (EGC), L-Epicatechin gallate (ECG) and 4 kinds of materials of NVP-XAA 723 (EGCG), account for the 60%-80% of tea-polyphenol total amount.Wherein the highest with EGCG content again, active function is the strongest, and is the most noticeable with its research in recent years.Modern scientific research shows that EGCG has anti-oxidant significantly, anti-mutation, radioprotective, antibiotic and sterilizing effect, can the enhancing immunity system function, suppress the growth of fat and cholesterol, and the inhibition growth of tumor has prophylactic effect to cardiovascular diseases and acquired immune deficiency syndrome (AIDS) etc.

EGCG unique and widely physiologically active be the basis of its application, but still have following problem with regard to its application: 1, EGCG water-soluble limited its application in the lipid product, particularly as the antioxidant of lipid; Because fat-soluble difference is difficult for seeing through the double resin layer cytolemma, is difficult to reach targeting point and reduces its due active function greatly in the body.2, various metabolic reactions easily take place in EGCG in vivo, be exactly in addition in vivo under the physiological environment poor stability cause its bioavailability not high.Bioavailability at the EGCG of present existence is low, portioned product active low, product category is abundant inadequately, do not have problems such as the substitute products of renewal, carries out EGCG derivatize, the efficient focus that becomes research with product innovation that utilizes.

In recent years, the scientist of the U.S. and Japan discovers: EGCG can effectively improve its bioavailability, strengthen its biological action after acetylize, thereby improves utility value.This all has profound significance to the added value that makes full use of tea resources, raising tea resources, the product category of enriching the tealeaves functional ingredient etc.

But at present; the research of external EGCG acetylate also is in the laboratory study; the product that obtains is still based on breadboard activity and Study on mechanism; wherein more with the research of aspects such as its arrestin enzyme body activity, anticancer anti-tumor activity, cell death inducing, be proved to be the anticancer prodrug of a kind of potential.But related acetylate also rests on experimental stage and lacks the research of other physiologically active, does not see systematic research and report about it synthesizes or prepares.

Summary of the invention

Technical problem to be solved by this invention provides the preparation method of a kind of high purity, high yield EGCG fully substituted acetylate, and further research and application are provided convenience for it.

For this reason; the present invention adopts following technical scheme: the preparation method of fully substituted acetylate of epigallocatechin-gallate (EGCG); its step is as follows: with NVP-XAA 723 and acetylation reagent is reaction substrate; under the condition of organic solvent and the existence of catalyzer pyridine, carry out acetylization reaction; the mol ratio of control table nutgall catechin gallic acid ester and acetylation reagent is at 1:80-200; reaction obtains the fully substituted acetylate of NVP-XAA 723, and this product carries out purification process again.

Adopting chemical synthesis to prepare in the process of EGCG acetylate, except that the target product of full replacement, the EGCG that generates in also responding in its product partly replaces acetylate.

The structural formula of EGCG acetylate is as follows:

Wherein the structural formula of EGCG fully substituted acetylate is as follows:

Influencing all multifactor of reaction as time, acetylation reagent, solvent, catalyst levels and substrate mol ratio etc.; have the greatest impact with kind, composition and the content of substrate (EGCG and diacetyl oxide) mol ratio, mainly recently carry out different degree of substitution synthesizing until the EGCG of full replacement acetylate by the control substrate to reaction yield, product.The mol ratio of control EGCG and acetylation reagent is reacted the full replacement acetyl product that has only that obtains at 1:80-200; The mol ratio of EGCG and acetylation reagent is below 1:80, and full replacement and part that reaction obtains EGCG replace acetylate.

Above-mentioned preparation method, reaction raw materials is highly purified EGCG (purity is more than 90%), the organic solvent of reaction is ethyl acetate, dithiocarbonic anhydride, tetrahydrofuran (THF), halohydrocarbon equal solvent, preferred chloroform, ethylene dichloride or ethyl acetate; Acetylation reagent is the donor reagent of ethanoyl such as acetate, Acetyl Chloride 98Min., diacetyl oxide, preferred diacetyl oxide; Reaction times 3-20 hour, preferred 5 hours, mixing speed was 100-700rpm.

Above-mentioned preparation method, described purification process adopt that solvent wash is handled, the combination of any or two kinds in the column chromatography purification, and preferred purification process adopts that first solvent wash is handled, dry concentrating, back column chromatography purification processing.

Described solvent wash treating processes is as follows: reaction solution processing such as washes through washing, Diluted Acid Washing, alkali cleaning and salt, each volume of handling washings is 1/10～1 of a reaction solution volume, wherein be advisable with 1/2, repeat for 3-7 time, being washed till reaction solution does not have tangible diacetyl oxide and pyridine flavor, and used diluted acid is HCl or H ₂SO ₄Deng the aqueous solution, volumetric concentration is less than 20%, used alkali is saturated NaHCO ₃Or mass concentration is less than the aqueous solution of 20% NaOH, KOH etc., and used salt is saturated NaCl, Na ₂SO ₄Or K ₂SO ₄Deng solution.The anhydrous MgSO of the dry employing of reaction solution after the washing ₄, Na ₂SO ₄Deng water-removal agent, dried reaction solution can adopt the rotation vacuum concentration, and temperature is controlled at 30-80 ℃, vacuum tightness 〉=0.09M Pa.

Described column chromatography purification is handled: the chromatography column filler is 80-300 purpose silica gel; the dress post can adopt wet method and dry method dual mode; wet method can become homogenate dress post with sherwood oil, chloroform equal solvent; must be before the silica gel dress post through pre-treatment, organic solvents such as the available alcohols of dissolving, ketone or the ester class of the EGCG acetylate of last sample.Eluent should be selected the combination of polarity than big and the little solvent of polarity, the solvent that polarity is bigger can be selected methyl alcohol, ethanol, acetoneand ethyl acetate etc., the solvent that polarity is little can be normal hexane, benzene and sherwood oil etc., preferred washing composition is the mixed solution of normal hexane and ethyl acetate, its volume ratio is 1:1-1:10, preferred 1:1.The Balance Treatment flow velocity of chromatography column be the 1.5-2 column volume/hour, the flow velocity of eluent be the 1-1.5 column volume/hour, the hydraulic pressure of elutriant can change in certain scope, pressure boost is quickened elution process, passes through chromatographic column fast.After silica filler used, available organic solvent washed alone or in combination or adopts 1% sodium hydroxide of 5-10 volume to boil half an hour earlier, and washing is boiled half an hour with 1% hydrochloric acid of 3-6 volume again, is washed to neutral 110 ℃ of activated silica gels for reusing.

Above-mentioned preparation method, the EGCG acetylate that obtains is the white powder material, after the carrying out washing treatment purity of products therefrom more than 80%, again after column chromatography is handled purity in (high performance liquid chromatography (HPLC) mensuration) more than 90%.

Reaction conditions gentleness provided by the invention makes high yield, highly purified EGCG fully substituted acetylate (purity is more than 90%) by the control reaction conditions; When keeping the physiological property of natural catechin own, have higher stability and bioavailability, made things convenient for its further research and application.

The invention will be further described below in conjunction with specification drawings and specific embodiments.

Description of drawings

Fig. 1 is the liquid phase analysis collection of illustrative plates of the EGCG fully substituted acetylate ACEGCG that obtains among the embodiment 5.

Fig. 2 is the liquid phase analysis collection of illustrative plates of the EGCG fully substituted acetylate ACEGCG that obtains among the embodiment 6.

Embodiment

Embodiment 1

Take by weighing EGCG sample 0.46g in the 100ml chloroformic solution; add 8ml diacetyl oxide and 1ml pyridine (substrate EGCG is more than the 1:80 with the diacetyl oxide ratio); under the room temperature behind stirring reaction 5h on the magnetic stirring apparatus; rotating speed is 300rpm; when finishing, reaction adds 10mL distilled water termination reaction system; obtain the EGCG fully substituted acetylate, its content reaches more than 95.60%.

Embodiment 2

Take by weighing EGCG sample 0.46g in the 100ml chloroformic solution; add 15ml diacetyl oxide and 1ml pyridine (substrate EGCG is 1:159.0 with the diacetyl oxide ratio); under the room temperature behind stirring reaction 5h on the magnetic stirring apparatus; rotating speed is 400rpm; when finishing, reaction adds 10mL distilled water termination reaction system; obtain the EGCG fully substituted acetylate, wherein the transformation efficiency of EGCG reaches 98.20%.

Embodiment 3

Take by weighing EGCG sample 0.46g in the 100ml chloroformic solution; add 20ml diacetyl oxide and 1ml pyridine (substrate EGCG is more than the 1:200 with the diacetyl oxide ratio); under the room temperature behind stirring reaction 5h on the magnetic stirring apparatus; rotating speed is 300rpm; when finishing, reaction adds 10mL distilled water termination reaction system; prepare the EGCG fully substituted acetylate, its content reaches more than 98.50%.

Embodiment 4

Take by weighing EGCG4.6g in the 1000ml chloroformic solution; add 100ml diacetyl oxide and 10ml pyridine (substrate EGCG is 1:106.0 with the diacetyl oxide ratio); under the room temperature behind stirring reaction 5h on the magnetic stirring apparatus; rotating speed is 500rpm; add 100mL distilled water termination reaction system when reaction finishes and carry out the synthetic of EGCG fully substituted acetylate, wherein the transformation efficiency of EGCG can reach more than 98%.

Embodiment 5

Mixed solution after embodiment 4 reactions finish, at 40 ℃, under vacuum tightness 〉=0.09M Pa condition, be concentrated into about 200ml, then respectively with 100ml water washing reaction solution 7 times, again with the saturated sodium bicarbonate solution of 70ml washing 5 times, with the saturated NaCl solvent wash processing reaction liquid of 50ml 2 times, resulting organic phase adds anhydrous Na at last ₂SO ₄Leave standstill dewater after, at 40 ℃, rotation concentrates under vacuum tightness 〉=0.09M Pa condition, obtains EGCG fully substituted acetylate 6.15g, is white powdered material, yield is 77.21%, purity is 89.53%.

Embodiment 6

Silica gel 80-160 order is soaked (spending the night) with concentrated hydrochloric acid, use distilled water wash silica gel repeatedly then after, it is carried out decompress filter, back constantly adding distil water is washed to the water of suction filtration and is neutral, after drying, activation got final product in 2 hours in 110 ℃ of baking ovens.

It is 1.6cm that pretreated silica gel material is filled in diameter, and length is in the experimental scale post of 40cm, and packed height is 35.9cm, with 2-3 column volume of elutriant balance chromatography column of ethyl acetate/normal hexane=1:1.

Take by weighing the EGCG acetylate 1.0g that embodiment 5 obtains and be dissolved in the 3ml ethyl acetate solution, as the sample solution of column chromatography.Load sample solution in the chromatography column top; at room temperature with 1BV (column volume)/hour constant flow rate acetylate wash-out from chromatography column is come out; the main peak position of taking off curve according to Xian merges collection liquid; at 40 ℃; concentrate drying under vacuum tightness 〉=0.09M Pa condition; just can obtain highly purified EGCG fully substituted acetylate, reclaim altogether and obtain 0.85g powdered material, detecting purity is 96.04%.

Protection scope of the present invention has more than and is limited to above embodiment, and technical scheme all and of the present invention content identical or that be equal to all falls into protection scope of the present invention.

Claims

1, the preparation method of fully substituted acetylate of epigallocatechin-gallate (EGCG); its step is as follows: with NVP-XAA 723 and acetylation reagent is reaction substrate; under the condition of organic solvent and the existence of catalyzer pyridine, carry out acetylization reaction; the mol ratio of control table nutgall catechin gallic acid ester and acetylation reagent is at 1:80-200; reaction obtains the fully substituted acetylate of NVP-XAA 723, and this product carries out purification process again.

2, preparation method according to claim 1 is characterized in that described acetylation reagent is acetate, Acetyl Chloride 98Min. or diacetyl oxide.

3, preparation method according to claim 1 is characterized in that described organic solvent is any or the mixture more than two kinds in ethyl acetate, halohydrocarbon, dithiocarbonic anhydride, the tetrahydrofuran (THF).

4, preparation method according to claim 3 is characterized in that described organic solvent is chloroform, ethylene dichloride or ethyl acetate solution.

5, preparation method according to claim 1 is characterized in that described purification process adopts that solvent wash is handled, the combination of any or two kinds in the column chromatography purification.

6, preparation method according to claim 5 is characterized in that described purification process adopts first solvent wash processing, dry concentrating, and back column chromatography purification is handled.

7, preparation method according to claim 6, the reaction solution after it is characterized in that washing adopts anhydrous Na earlier ₂SO ₄The drying that dewaters then adopts the mode of rotation vacuum concentration, and condition is 30-80 ℃, vacuum tightness 〉=0.09M Pa.

8, according to claim 5 or 6 described preparation methods, it is characterized in that described solvent wash processing, its washings of selecting for use is water, diluted acid, alkali and saturated salt solution, described diluted acid is HCl or H ₂SO ₄The aqueous solution, its concentration is less than 20%, described alkali is saturated NaHCO ₃Or concentration is less than 20% NaOH, the KOH aqueous solution, and described salt is saturated NaCl, Na ₂SO ₄Or K ₂SO ₄Solution; Described column chromatography purification is handled, and its filler of selecting for use is a 80-300 purpose silica gel, and eluent is the mixed solution of normal hexane and ethyl acetate, and volume ratio is 1:1-1:10.

9, preparation method according to claim 1 is characterized in that stirring reaction at room temperature, and the reaction times is 3-20 hour.

10, preparation method according to claim 1 is characterized in that stirring reaction at room temperature, and the reaction times is 5 hours.