Method for preparing delphinium acylated anthocyanin

Patent metadata

Field	Value
Publication number	US-11981697-B2
Application number	US-202217674860-A
Country	US
Kind code	B2
Filing date	Feb 18, 2022
Priority date	Sep 14, 2020
Publication date	May 14, 2024
Grant date	May 14, 2024

Abstract

Disclosed is a method for separating and preparing delphinidin-3-O-(6″-O-p-coumaroyl) glucoside. By means of extraction, macroporous resin purification, extraction, preparative liquid chromatography and high-speed countercurrent chromatography and the like, a high-purity delphinidin-3-O-(6″-O-p-coumaroyl) glucoside monomer can be obtained from grapes by separation and purification. By means of this method, at least 80 mg of delphinidin-3-O-(6″-O-p-coumaroyl) glucoside can be obtained from 10 kg of grape skins, with the purity can be no less than 98%. The method has the advantages of simple operation, large handling capacity, good repeatability, etc., which provides a new idea for the development and utilization of grape resources.

First claim

What is claimed is:

1. A method for separating and preparing delphinidin-3-O-(6″-O-p-coumaroyl) glucoside, comprising:
using grapes as a raw material, and extracting by an acid ethanol solution, filtering and concentrating to obtain a crude grape skin anthocyanin extract;
macroporous resin purification: injecting the crude grape skin anthocyanin extract into macroporous resin, and eluting and concentrating to obtain an anthocyanin eluent;
extraction: extracting the anthocyanin eluent with an organic solvent, concentrating under reduced pressure, and freeze-drying to obtain anthocyanin lyophilized powder;
purification by preparative liquid chromatography: dissolving the lyophilized anthocyanin powder, injecting the solution into a preparative liquid chromatography system for purification, and detecting with an ultraviolet detector, wherein the specific conditions are as follow:
mobile phase: phase A is pure acetonitrile, and phase B is a formic acid aqueous solution with a volume percentage of 1%-2% formic acid;
the gradient elution program is: 0 min-4 min, 5%-20% phase A; 4 min-18 min, 20%-25% phase A; 18 min-21 min, 25%-35% phase A; 21 min-24 min, 35%-60% phase A; 24 min-27 min, 60%-5% phase A; 27 min-30 min, 5% phase A;
the flow rate is 8 mL/min-10 mL/min, the column temperature is 30° C., and the detection wavelength is 520 nm; and
collecting the components with retention time of 16.5 min-18.0 min according to the liquid chromatogram, and concentrating under reduced pressure and freeze-drying to obtain the crude product of anthocyanin monomer,

separation by high-speed countercurrent chromatography: mixing methyl tert-butyl ether, methanol, water and trifluoroacetic acid at a volume ratio of 2:2:3:0.001 to act as a two-phase solvent system, using the upper phase as a stationary phase, and using the lower phase as a mobile phase, pumping the stationary phase and the mobile phase into a high-speed countercurrent chromatographic instrument in turn, after the two phases reach balance in a pipeline, dissolving the crude product of anthocyanin monomer in the mobile phase, injecting the sample, and detecting by the ultraviolet detector, wherein the detection wavelength is 280 nm, collecting the fractions with a retention time of 105 min-115 min, concentrating under reduced pressure, and freeze-drying to obtain a target compound delphinidin-3-O-(6″-O-p-coumaroyl) glucoside.

2. The method according to claim 1 , wherein extracting by an acid alcohol solution, filtering and concentrating to obtain the crude anthocyanin extract of grape skins comprises: washing grapes and peeling them, mixing the grape skins with an acid ethanol solution and pulping, ultrasonic extracting below 50° C. and filtrating, and concentrating the filtrate under reduced pressure at 40° C.-50° C. for removing ethanol, so as to obtain the crude anthocyanin extract of grape skins; wherein the material-to-liquid ratio of the grape skins to the acid ethanol solution is 1 g:4 mL-8 mL; in the acid ethanol solution, the volume concentration of ethanol is 50%-80%, the volume concentration of the acid is 0.1%-1%; and the time of the ultrasonic extraction is 40 min-120 min.

3. The method according to claim 2 , wherein the acid is selected from at least one of hydrochloric acid, formic acid, or acetic acid in the acid ethanol solution.

4. The method according to claim 1 , wherein the method of the macroporous resin purification comprising:
injecting the crude grape skin anthocyanin extract into the macroporous resin, eluting by sequentially using acid ethanol solutions in which the volume concentration of ethanol is 0, 5%, 20% and 40% each at a dose of 4 times the bed volume (4 BV), collecting acid ethanol eluent in which the volume concentration of ethanol is 40%, and evaporating under reduced pressure at 40° C.-50° C. for removing ethanol to obtain the anthocyanin eluate, wherein the macroporous resin is selected from AB-8, D101, XAD-7, HPD-100 or DM-130, with a specific surface area of 450 m 2 /g-550 m 2 /g, an average pore diameter of 10 nm-50 nm, and a particle size range of 0.3 mm-1.25 mm; and the acid ethanol solution is selected from ethanol solutions in which the volume percent concentration of the acid is 0.1%-1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid, or acetic acid.

5. The method according to claim 1 , wherein the organic solvent used in the step “extration” is ethyl acetate.

6. The method according to claim 1 , wherein the liquid chromatographic column is a C18 column, a single injection is 10 mg-40 mg based on the lyophilized anthocyanin powder, and the volume after concentration under reduced pressure is 40%-70% of the volume before concentration.

7. The method according to claim 1 , wherein the temperature of the high-speed countercurrent chromatographic instrument is stabilized at 20° C.-30° C., it is at the forward-inlet elution mode, pumping the stationary phase, and the rotating speed is adjusted to 800 r/min-950 r/min, the mobile phase is introduced at a flow rate of 2 mL/min and allowed to be balanced, and the amount of each injection is 30 mg-200 mg based on the crude product of anthocyanin monomer.

Assignees

• Univ Zhejiang

Inventors

• Chen Wei
• Xie Jiahong
• Xu Yang
• Cui Haoxin

Classifications

• C07H17/065 Primary

  Benzo[b]pyrans · CPC title

• B01D11/0265

  Applying ultrasound · CPC title

• B01D15/426

  Specific type of solvent · CPC title

• C07H1/08 Primary

  from natural products · CPC title