阅读说明：本技术 一种原飞燕草素b9没食子酸酯的化学合成方法 (Chemical synthesis method of delphinidin B9 gallate ) 是由 陈士国 徐新雷 潘海波 叶兴乾 王艺 程焕 陈健乐 于 2021-05-21 设计创作，主要内容包括：本发明的目的是提供一种原飞燕草素B9没食子酸酯的化学合成方法,以杨梅叶原花色素为原料,利用EGCG作为亲核试剂,在盐酸催化作用下攻击杨梅叶原花色素的结构单元EGCG和EGC的C4位点,合成原飞燕草素B9没食子酸酯。相较于从茶叶、杨梅叶等材料中提取、分离原飞燕草素B9没食子酸酯,本发明提供的方法所制得的原飞燕草素B9没食子酸酯纯度和得率均较高,可直接应用于食品领域中作为营养增强剂和天然抗氧化剂。(The invention aims to provide a chemical synthesis method of prodelphinidins B9 gallate, which comprises the steps of taking myricetin as a raw material, utilizing EGCG as a nucleophilic reagent, attacking structural units EGCG of the myricetin proanthocyanidin and C4 sites of the EGC under the catalytic action of hydrochloric acid, and synthesizing the prodelphinidins B9 gallate. Compared with the method for extracting and separating the prodelphinidins B9 gallate from the materials such as tea leaves, waxberry leaves and the like, the prodelphinidins B9 gallate prepared by the method provided by the invention has higher purity and yield, and can be directly applied to the food field as a nutrition enhancer and a natural antioxidant.)

1. A chemical synthesis method of delphinidin B9 gallate is characterized by comprising the following steps:

respectively preparing a hydrochloric acid solution containing the proanthocyanidin of the waxberry leaves at the concentration of 10-400mg/mL and a hydrochloric acid solution containing the epigallocatechin gallate at the concentration of 10-400 mg/mL; wherein, the hydrochloric acid concentration of the two hydrochloric acid-containing solutions is 0.1-1.0 mol/L; mixing myricetin and EGCG-containing hydrochloric acid solution with the same hydrochloric acid concentration at a volume ratio of 1:2-2:1, reacting at 20-60 deg.C for 40min, drying, separating, and purifying to obtain delphinidin B9 gallate, wherein the delphinidin B9 gallate comprises delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate.

2. The chemical synthesis method according to claim 1, wherein the solvent used for preparing the hydrochloric acid solution containing myrica rubra leaf proanthocyanidin at a concentration of 10-400mg/mL and the hydrochloric acid solution containing epigallocatechin gallate at a concentration of 10-400mg/mL is methanol, ethanol, etc.

3. The chemical synthesis method according to claim 1, wherein the myrica rubra leaf proanthocyanidin purity is 86.4%; the purity of EGCG is more than or equal to 98 percent.

4. The chemical synthesis method according to claim 1, wherein the drying method is specifically:

and (3) rotationally evaporating the reaction liquid after reacting for 40min at 40 ℃, and removing methanol and a small amount of water to obtain reaction crude product dry powder.

5. The chemical synthesis method according to claim 1, wherein the separation and purification method specifically comprises:

a S series X5H column was used, mobile phase A was acetonitrile containing 0.5 vol% acetic acid and mobile phase B was ultrapure water. Dissolving the crude reaction product in a volume ratio of 88: 12, mobile phase A, B to prepare a 20mg/mL solution. After 2mL of sample injection, carrying out mobile phase elution purification, wherein the flow rate of the mobile phase is 5mL/min, and the elution gradient is as follows: 0-10min, 12-27 vol% B, 10-20min, 60 vol% B, 20-26min 12 vol% B. Setting detection wavelength at 280nm, collecting eluate of 5.5-6.0min according to liquid phase chromatogram, rotary evaporating the eluate at 40 deg.C to remove acetonitrile, and freeze drying to obtain delphinidin B9 gallate purified product.

6. The chemical synthesis method according to claim 5, wherein the separation and purification further comprises a second purification:

the second purification was carried out using Shim-pack GIST C18, mobile phase A being ultrapure water and mobile phase B being 98 vol% acetonitrile (containing 0.05 vol% phosphoric acid). Dissolving the delphinidin B9 gallate purified product in a solvent with the volume ratio of 80: 20 was mixed with A, B to prepare a 50mg/mL solution. After 2mL of sample injection, carrying out mobile phase elution purification, wherein the flow rate of the mobile phase is 15mL/min, and the elution gradient is as follows: 0-5min, 20-40 vol% B; 5-15min, 40 vol% B. Setting detection wavelength at 280nm, collecting effluent at different peak discharge time according to liquid phase chromatogram, rotary evaporating the effluent at 40 deg.C to remove acetonitrile, and freeze drying to obtain delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate respectively.

Technical Field

The invention relates to the field of plant functional components, in particular to a preparation method of delphinidin B9 gallate.

Background

Proanthocyanidins are a class of polymeric polyphenols with flavan-3-ols as building blocks, widely present in plants, and the second major class of dietary polyphenols behind lignin. The main structural units of the compound are (epicatechin), (epigallocatechin, (E) GC and gallate thereof. A large number of researches show that the proanthocyanidins have various beneficial biological activities of resisting oxidation, reducing blood sugar, losing weight and the like. These functions are closely related to their structure, especially the type of building blocks and the degree of polymerization. Firstly, the degree of polymerization of proanthocyanidins determines their bioavailability, which decreases with increasing degree of polymerization, proanthocyanidins with a degree of polymerization greater than 4 being completely unabsorbed. The density of phenolic hydroxyl of structural units of proanthocyanidins is closely related to the biological activity of the proanthocyanidins. It has been shown that proanthocyanidins containing galloyl groups in their building blocks exhibit greater biological activity due to their higher density of phenolic hydroxyl groups than proanthocyanidins without galloyl groups. Wherein the delphinidin having the structural unit (E) GC and the gallic acid ester (E) GCG thereof is a proanthocyanidin having a high activity.

At present, the more researched proanthocyanidins take (epicatechin), (epicatechin) and gallic acid ester thereof as structural units, and because the sources are rich, the structure-activity relationship research of taking dimers thereof as materials is clearer; the studies on prodelphinidins were based on proanthocyanidin mixtures. Proanthocyanidin derived from waxberry leaves has a typical delphinidin structure, main structural units are EGC and EGCG, and the in vitro antioxidant activity is extremely strong, but the polymerization degree is high, most polymerization degrees are distributed between 9.5 and 26.7, the bioavailability is extremely low, the in vivo activity is poor, and the action mechanism is fuzzy. The low-polymerization-degree protodelphinidin has extremely low content and is difficult to separate and purify, so that the research on the structure-activity relationship is stagnated.

Disclosure of Invention

The invention aims to provide a chemical synthesis method of delphinidin B9 gallate, which takes myrica rubra leaf proanthocyanidin as a raw material and EGCG as a nucleophilic substrate to synthesize two dimeric delphinidin gallate esters (the delphinidin B-3 '-gallate and the delphinidin B-3,3' -digallate) under the action of acid catalysis, so that the component content of the delphinidin B9 gallate is improved, and the further purification difficulty is reduced. The dimeric delphinidin gallate is a B-type proanthocyanidin dimer with EGCG as a structural unit, so that the bioavailability of the delphinidin-type proanthocyanidin is improved, and a high-purity material is provided for the structure-activity relationship research.

The technical scheme adopted by the invention is specifically as follows:

a chemical synthesis method of delphinidin B9 gallate comprises the following steps:

respectively preparing a hydrochloric acid solution containing the proanthocyanidin of the waxberry leaves at the concentration of 10-400mg/mL and a hydrochloric acid solution containing the epigallocatechin gallate at the concentration of 10-400 mg/mL; wherein, the hydrochloric acid concentration of the two hydrochloric acid-containing solutions is 0.1-1.0 mol/L; mixing myricetin and EGCG-containing hydrochloric acid solution with the same hydrochloric acid concentration at a volume ratio of 1:2-2:1, reacting at 20-60 deg.C for 40min, drying, separating, and purifying to obtain delphinidin B9 gallate, wherein the delphinidin B9 gallate comprises delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate.

Wherein, the delphinidin B-3 '-gallate and the delphinidin B-3,3' -digallate have the following structural formulas:

the extraction and purification method of the myrica rubra leaf proanthocyanidin refers to a patent of a method for separating and preparing the myrica rubra leaf proanthocyanidin, and the purity of the prepared myrica rubra leaf proanthocyanidin is 86.4%; EGCG is a commercial HPLC-grade reagent, and the purity is more than or equal to 98 percent.

As a preferred scheme, the drying method specifically comprises the following steps: carrying out rotary evaporation on the reaction liquid after reacting for 40min at 40 ℃, removing methanol and a small amount of water, and obtaining reaction crude product dry powder;

as a preferred scheme, the obtained reaction crude product is separated and purified by Shimadzu LC-20 semi-preparative liquid chromatography to obtain delphinidin B9 gallate.

Purification A series S X5H preparative column (10mm X250 mm, 5 μm) was used, mobile phase A being acetonitrile containing 0.5 vol% acetic acid and mobile phase B being ultrapure water. Dissolving the crude reaction product in a volume ratio of 88: 12, mobile phase A, B to prepare a 20mg/mL solution. After 2mL of sample injection, carrying out mobile phase elution purification, wherein the flow rate of the mobile phase is 5mL/min, and the elution gradient is as follows: 0-10min, 12-27 vol% B, 10-20min, 60 vol% B, 20-26min 12 vol% B. Setting detection wavelength at 280nm, collecting eluate of 5.5-6.0min according to liquid phase chromatogram, rotary evaporating the eluate at 40 deg.C to remove acetonitrile, and freeze drying to obtain delphinidin B9 gallate purified product.

Further, the separation and purification also comprises a second purification:

the second purification was performed using Shim-pack GIST C18(20 mm. times.250 mm, 5 μm), mobile phase A as ultrapure water and mobile phase B as 98 vol% acetonitrile (containing 0.05 vol% phosphoric acid). Dissolving the delphinidin B9 gallate purified product in a solvent with the volume ratio of 80: 20 was mixed with A, B to prepare a 50mg/mL solution. After 2mL of sample injection, carrying out mobile phase elution purification, wherein the flow rate of the mobile phase is 15mL/min, and the elution gradient is as follows: 0-5min, 20-40 vol% B; 5-15min, 40 vol% B. Setting detection wavelength at 280nm, collecting effluent at different peak discharge time according to liquid phase chromatogram, rotary evaporating the effluent at 40 deg.C to remove acetonitrile, and freeze drying to obtain delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate respectively.

The method takes myricetin as a raw material, EGCG is used as a nucleophilic reagent, and structural units EGCG and C4 sites of EGC of the myricetin are attacked under the catalytic action of hydrochloric acid to synthesize delphinidin B9 gallate. Compared with the method for extracting and separating the prodelphinidins B9 gallate from the materials such as tea leaves, waxberry leaves and the like, the prodelphinidins B9 gallate prepared by the method provided by the invention has higher purity and yield. Can be directly applied to the food field as a nutrition enhancer and a natural antioxidant.

Drawings

FIG. 1 is a synthetic HPLC analytical map of delphinidin B9 gallate in example 1;

FIG. 2 is the HPLC analysis chart of the synthesis of delphinidin B9 gallate in example 2;

FIG. 3 is the HPLC analysis chart of the synthesis of delphinidin B9 gallate in example 3;

FIG. 4 is the HPLC analysis chart of the synthesis of delphinidin B9 gallate in example 4;

FIG. 5 is the HPLC analysis chart of the synthesis of delphinidin B9 gallate in example 5;

FIG. 6 is the HPLC analysis chart of the synthesis of delphinidin B9 gallate in example 6;

FIG. 7 is the HPLC analysis of delphinidin B-3' -gallate prepared in example 1, with a purity of 93.2%

FIG. 8 is an HPLC analysis chart of delphinidin B-3,3' -digallate prepared in example 1, with a purity of 96.6%.

FIG. 9 shows the mass spectrum of delphinidin B-3' -gallate prepared in example 1, and the molecular weights are 762 respectively.

FIG. 10 is a mass spectrum of delphinidin B-3,3' -digallate prepared in example 1, with a molecular weight of 914.

FIG. 11 shows the preparation of delphinidin B-3' -gallate prepared in example 1¹³C NMR chart;

FIG. 12 shows the preparation of delphinidin B-3,3' -digallate prepared in example 1¹³C NMR chart;

in the figures 1-6, the time of the appearance of the delphinidin B-3 '-gallate (i) and the time of the delphinidin B-3,3' -digallate (ii) are respectively 9.9min and 11.7 min.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of making the objects and technical solutions of the present invention clearer, but the present invention is not limited thereto. The difference between the examples lies in the reaction conditions, which are drying, isolation and purification (two purifications), as follows:

and (3) drying: carrying out rotary evaporation on the reaction liquid after reacting for 40min at 40 ℃, removing methanol and a small amount of water, and obtaining reaction crude product dry powder;

separation and purification:

first purification an S series X5H preparative column (10mm X250 mm, 5 μm) was used, mobile phase A being acetonitrile containing 0.5 vol% acetic acid and mobile phase B being ultrapure water. Dissolving the crude reaction product in a volume ratio of 88: 12, mobile phase A, B to prepare a 20mg/mL solution. After 2mL of sample injection, carrying out mobile phase elution purification, wherein the flow rate of the mobile phase is 5mL/min, and the elution gradient is as follows: 0-10min, 12-27 vol% B, 10-20min, 60 vol% B, 20-26min 12 vol% B. Setting detection wavelength at 280nm, collecting eluate of 5.5-6.0min according to liquid phase chromatogram, rotary evaporating the eluate at 40 deg.C to remove acetonitrile, and freeze drying to obtain delphinidin B9 gallate purified product.

The second purification was performed using Shim-pack GIST C18(20 mm. times.250 mm, 5 μm), mobile phase A as ultrapure water and mobile phase B as 98 vol% acetonitrile (containing 0.05 vol% phosphoric acid). Dissolving the delphinidin B9 gallate purified product in a solvent with the volume ratio of 80: 20 was mixed with A, B to prepare a 50mg/mL solution. After 2mL of sample injection, carrying out mobile phase elution purification, wherein the flow rate of the mobile phase is 15mL/min, and the elution gradient is as follows: 0-5min, 20-40 vol% B; 5-15min, 40 vol% B. Setting detection wavelength at 280nm, collecting effluent at different peak discharge time according to liquid phase chromatogram, rotary evaporating the effluent at 40 deg.C to remove acetonitrile, and freeze drying to obtain delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate respectively.

In addition, the synthesis detection method of the prepared delphinidin B9 gallate comprises the following steps:

analyzing delphinidin B9 gallate by High Performance Liquid Chromatography (HPLC), wherein the specific analysis conditions are as follows: the high performance liquid chromatography is Waters 2695, the detector is a Waters 2489 ultraviolet-visible light detector, the detection wavelength is 280nm, and the type of the chromatographic column is Luna hilc (4.6mm x 250mm, 5 mu m, Phenomex Inc.); mobile phase A is acetonitrile (containing 0.5 vol% acetic acid), and mobile phase B is ultrapure water; gradient elution: 0-30min, 12-27 vol% B, 30-45min, 27-80 vol% B, 45-46min, 80-12 vol% B, 46-55min, 12 vol% B; flow rate: 0.8 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 30 ℃.

The purity and structure detection method of the prepared delphinidin B9 gallate comprises the following steps:

LC-MS analysis: the analysis was performed using an Agilent 1290/6460Triple Quad coupled mass spectrometer with the same chromatographic conditions as above. The mass spectrum conditions are as follows: the ionization mode is an ESI negative ion mode; the mass-to-charge ratio scanning range is 50-1500 m/z; the desolvation temperature is 400 ℃; the capillary voltage is 3.5 kV; the collision energy was 16 eV.

¹³C NMR analysis: the analysis is carried out by adopting an Agilent DD2-600 superconducting nuclear magnetic resonance spectrometer, and deuterated methanol is used as a solvent.

Example 1:

weighing 1g each of bayberry leaf proanthocyanidin and EGCG, dissolving in 50mL of 0.1mol/L HCl methanol solution respectively, stirring well to clarify, mixing 50mL of bayberry leaf proanthocyanidin solution and 50mL of LEGCG solution in a conical flask with a plug, heating in water bath at 40 deg.C for 40min, drying, separating, purifying, and detecting, as shown in figure 1, the yield of prodelphinidins B-3 '-gallate is 8.5%, and the yield of prodelphinidins B-3,3' -digallate is 20.0%.

FIGS. 7 and 8 show HPLC analysis spectra of the prepared delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate, with the purities of 93.2% and 96.6%, respectively.

FIGS. 9 and 10 are mass spectrograms of prodelphinidins B-3 '-gallate and prodelphinidins B-3,3' -digallate prepared according to the present invention, showing that the molecular weights are 762 and 914, respectively.

FIGS. 11 and 12 show the preparation of delphinidin B-3 '-gallate and delphinidin B-3,3' -digallate¹³C NMR chart; the carbonyl group (166ppm) and galloyl ring carbon signals (138ppm and 110ppm) of gallic acid are the characteristics of the gallic acid esterified flavan-3-ol, which indicates that the structural unit contains EGCG, and usually, the A-type proanthocyanidin has a broad peak signal at 102-104ppm, and the signal is not detected, which indicates that the A-type bonds are absent in the structure and are protoporphyrin B-3' -gallate and protoporphyrinAnd 4, the prairitin B-3,3' -digallate.

Example 2:

weighing 1g each of bayberry leaf proanthocyanidin and EGCG, dissolving in 100mL of 0.1mol/L HCl methanol solution respectively, stirring uniformly to clarify, mixing 50mL of bayberry leaf proanthocyanidin solution and 25mL of EGCG solution in a conical flask with a stopper, heating in water bath at 40 deg.C for 40min, drying, separating, purifying, and detecting, as shown in figure 2, the yield of prodelphinidins B-3 '-gallate is 6.2%, and the yield of prodelphinidins B-3,3' -digallate is 11.6%.

Example 3:

weighing 1g each of bayberry leaf proanthocyanidin and EGCG, dissolving in 50mL of 0.1mol/L HCl methanol solution respectively, stirring uniformly to clarify, mixing 50mL of bayberry leaf proanthocyanidin solution and 50mL of EGCG solution in a conical flask with a stopper, heating in water bath at 60 ℃ for 40min, drying, separating and purifying, and detecting, wherein the yield of prodelphinidins B-3 '-gallate is 4.0%, and the yield of prodelphinidins B-3,3' -digallate is 18.0%, as shown in figure 3.

Example 4:

weighing 1g each of myrica rubra leaf proanthocyanidin and EGCG, dissolving in 5.0mL of 0.1mol/L HCl methanol solution, stirring well to clarify, mixing 2.5mL of myrica rubra leaf proanthocyanidin solution and 5.0mL of EGCG solution in a conical flask with a stopper, heating in water bath at 20 deg.C for 40min, drying, separating, purifying, and detecting, as shown in FIG. 4, the yield of prodelphinidins B-3 '-gallate is 16.6%, and the yield of prodelphinidins B-3,3' -digallate is 19.5%.

Example 5:

weighing 2g each of bayberry leaf proanthocyanidin and EGCG, dissolving in 5.0mL of 0.1mol/L HCl methanol solution, stirring well to clarify, mixing 2.5mL of bayberry leaf proanthocyanidin solution and 5.0mL of GCG solution in a conical flask with a stopper, heating in water bath at 40 deg.C for 40min, drying, separating, purifying, and detecting, as shown in FIG. 5, the yield of prodelphinidins B-3 '-gallate is 27.9%, and the yield of prodelphinidins B-3,3' -digallate is 21.2%.

Example 6:

weighing 1g each of bayberry leaf proanthocyanidin and EGCG, dissolving in 50.0mL of 1mol/L HCl methanol solution respectively, stirring well to clarify, mixing 50mL of bayberry leaf proanthocyanidin solution and 50mL of EGCG solution in a conical flask with a plug, heating in water bath at 40 deg.C for 40min, drying, separating, purifying, and detecting, as shown in figure 4, the yield of prodelphinidins B-3 '-gallate is 6.8%, and the yield of prodelphinidins B-3,3' -digallate is 18.0%.

The above description is only a preferred embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, so that all equivalent structural changes made by using the content of the present specification should be included in the scope of the present invention.