A preparation method of dihydroxycinnamic acid and its application

Abstract

The present invention provides a method for preparing dihydrochlorogenic acid. Using chlorogenic acid (CQA) and various catechins as substrates, the chlorogenic acid and various catechins are subjected to an enzymatic oxidation reaction under the catalysis of pear homogenate or tea polyphenol oxidase to generate a reaction solution of a new type of tea pigment compound. After subjecting the reaction solution to vacuum concentration, freeze-drying, extraction, and column chromatography separation and purification, dihydrochlorogenic acid is obtained. The present invention synthesizes and separates four new dihydrochlorogenic acid pigment substances through in vitro enzymatic oxidation. The synthesis method and purification technology are simple to operate, easy to implement, and have a relatively low cost. Moreover, the four new natural pigments are orange-yellow amorphous powders with good color-toning ability and can be applied to the color-toning of tea beverages and new-style tea drinks. At the same time, the discovery of this type of compound helps to promote the research on pigment substances in black tea, improve the color quality of black tea soup, and develop and apply it in the field of food coloring.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation, separation and purification of natural products, and particularly relates to a method for synthesizing and preparing dihydrochlorogenic acid Background Art

[0002] According to different processing technologies, tea can be divided into six major categories: black tea, green tea, white tea, dark tea, oolong tea, and yellow tea. During the tea processing process, various pigment substances will be formed, which play an important role in the formation of the quality characteristics of tea. As early as the 1950s, experiments by Roberts E.A.H et al. confirmed that during the primary processing of black tea, polyphenolic substances mainly composed of catechins are catalyzed by polyphenol oxidase and peroxidase to generate two main characteristic pigments of black tea: thearubigins and theaflavins. Theaflavins are a general term for a class of compounds that can be dissolved in ethyl acetate and have a benzoylchromone structure formed by the oxidation of catechin substances. They are the main components responsible for the "brightness" of the black tea soup color and also the main substances that form the "golden ring" in the tea soup. Thearubigins are a class of complex reddish-brown phenolic compounds, including a variety of heterologous substances with extremely large differences in relative molecular mass, and are the main substances that make up the color of the black tea soup.

[0003] Previously, many scholars have focused on the research of pigment substances in black tea and carried out separation and purification. In 2001, Tanaka et al. used banana homogenate to catalyze the oxidation of catechins, and two theaflavin oxidation products with reddish-brown color, bis-theaflavin A and bis-theaflavin B, were isolated and purified (Tanaka T, Inoue K, Betsumiya Y, et al. Two Types of Oxidative Dimerization of the Black Tea Polyphenol Theaflavin [J]. Journal of Agricultural and Food Chemistry, 2001, 49(12): 5785-5789.). In 2002, Tanaka et al. oxidized the mixture of EC and EGCG with tea polyphenol oxidase and isolated a brown EGCG dimer from it (Tanaka T, Mine C, Kouno I. Structures of two new oxidation products of green tea polyphenols generated by model tea fermentation [J]. Tetrahedron, 2002, 58: 8851.). In addition, theacitrin is a class of unstable yellow pigments, which do not have a typical benzoyl tropolone skeleton, but also play an important role in the diversity of the formation of black tea polyphenols.Powell et al. isolated three theacitrin isomers when studying the auto-oxidation hydrolysis products of black tea polyphenols. They are a class of compounds in black tea that are insoluble in ethyl acetate and can form a precipitate with caffeine (Powell C. The polyphenolic pigments of black tea[M]. Guildford: University of Surrey, 1994; Matsuo Y, Keita O, Hitomi M, et al. Stereochemistry of the black tea pigments theacitrins A and C[J]. Journal of Natural Products, 2016, 79:189; Kuhernet N, Clifford M N, Mueller A. Oxidative cascade reactions yielding polyhydroxy-theaflavins and theacitrins in the formation of black tea thearubigins: evidence by tandem LC-MS[J]. Food&Function, 2010(1):180; Bailey RG, Nursten H E, McDowell I. Isolation and analysis of a polymeric thearubigin fraction from tea[J]. Journal of the Science of Food and Agriculture, 1992, 59:365.).

[0004] In addition, phenolic acids are an important component of polyphenols in tea. Research has shown that phenolic acids can also participate in the enzymatic oxidation reaction of catechins in tea to form pigment substances in black tea. In 2018, Zhang et al. first isolated and purified two red-colored catechin-chlorogenic acid adducts by catalyzing the oxidation of a mixed solution of chlorogenic acid and EGC, EGCG with horseradish peroxidase, and both of them contain a typical benzoyl tropolone skeleton (Zhang, Shuwei, Yang, et al. Novel Theaflavin-Type Chlorogenic Acid Derivatives Identified in Black Tea[J]. Journal of Agricultural and Food Chemistry, 2018, 66(13): 3402-3407.). The formation of the color of black tea soup is the result of the combined action of various pigment substances. The discovery of catechin-chlorogenic acid conjugates provides a reference for the study of potential pigment substances in black tea. However, the pigment substances in black tea have not been fully analyzed yet, and how such pigments are adducted and their contributions to tea soup or other tea beverages remain to be studied. Summary of the Invention

[0005] In view of the above deficiencies, the present invention provides a preparation and application of digalloyl chlorogenic acid with a simple synthesis method and purification technique, which is easy to implement, has a low cost, and has good color adjustment ability. The present invention is achieved by the following technical means:

[0006] A preparation method of digalloyl chlorogenic acid, comprising:

[0007] (1) Preparation of reaction solution: Mix catechin, chlorogenic acid, catalytic oxidizing substance and water, and perform magnetic stirring under open and appropriate temperature conditions to make the mixture react fully to obtain a crude reaction solution;

[0008] (2) Concentration, extraction and rough separation: Concentrate the crude reaction solution under reduced pressure and freeze-dry it to obtain a solid reactant. Redissolve the obtained solid reactant in water, then extract it with ethyl acetate, and remove the extraction phase to obtain an aqueous phase;

[0009] (3) Separation by macroporous adsorption resin: Filter the aqueous phase, pass the filtrate through macroporous adsorption resin, perform gradient elution with a methanol aqueous solution, collect the eluate, and dry it under reduced pressure to obtain a crude product of digalloyl chlorogenic acid;

[0010] (4) Medium and low pressure preparative column separation: The crude product of di-caffeoylquinic acid is separated by a medium and low pressure preparative column, followed by gradient elution. The eluate containing di-caffeoylquinic acid is detected and collected according to the ultraviolet absorption signal. The collected eluate is dried under reduced pressure to obtain four compounds with a purity > 80%, and their structures are shown in Formula I, Formula II, Formula III and Formula IV respectively:

[0011]

[0012] Furthermore, the catechin in step (1) is selected from any one or more of EGC, GC, EGCG, and GCG; the catalytic oxidizing substance is selected from any one of pear homogenate or tea polyphenol oxidase.

[0013] Furthermore, the mass ratio of the catechin to chlorogenic acid in step (1) is (1 - 15):(15 - 1); the mass of water is 10 - 500 times the total mass of the solid mixture; the mass of the catalytic oxidizing substance is 10 - 500 times the total mass of the solid mixture; the solid mixture includes catechin and chlorogenic acid.

[0014] Furthermore, the temperature of the magnetic stirring reaction in step (1) is 10 - 40 °C; the time of the magnetic stirring reaction is 10 - 300 min.

[0015] Furthermore, the macroporous adsorption resin in step (3) is HPD-300 resin; the gradient elution includes: pre-eluting with a methanol aqueous solution with a volume percentage of 0 - 10%, and then eluting with a methanol aqueous solution with a volume percentage of 15 - 60%.

[0016] Furthermore, the packing material of the medium pressure preparative column in step (4) is ODS C 18 particles, and the particle size range is 10 - 100 μm.

[0017] Furthermore, the gradient elution in step (4) includes: eluting with a methanol aqueous solution with a volume percentage of 0 - 60% (v / v, water containing 0.1% acetic acid).

[0018] Furthermore, the ultraviolet signal detection conditions in step (4) are Agilent C 18 chromatographic column (2.1×50 mm, 1.5 μm), the column temperature is 20 - 40 °C, the detection wavelength is 300 nm, the mobile phase A is 0.1% formic acid aqueous solution (v / v), B is acetonitrile, the chromatographic conditions are 0 - 26 min, 90% B, the injection volume is 0.1 - 1 mL, and the flow rate is 0.1 - 0.4 mL / min.

[0019] The present invention also discloses a di-caffeoylquinic acid prepared by any of the above preparation methods.

[0020] The present invention also discloses an application of the above-mentioned di-caffeoylquinic acid in beverage preparation, wherein: the addition amount of the di-caffeoylquinic acid is 20 mg / L to 1 g / L.

[0021] The beneficial effects of the present invention are as follows:

[0022] (1) The present invention has low cost and high product purity

[0023] The solvents used in extraction, the solvents used in separation by macroporous adsorption resin column and reverse-phase chromatography column can all be recycled; the materials used in reverse-phase chromatography separation can all be reused. The recycled solvents and the reused separation materials ensure a relatively low average separation cost.

[0024] (2) The preparation method of the present invention can meet the requirements of large-scale production

[0025] The raw material requirements are not high and the cost is low. Generally, analytically pure catechin and chlorogenic acid purchased can be used, and it is easy to prepare materials in batches; the concentration and extraction operations are simple; for separation, macroporous adsorption resin column is used for rough separation. This macroporous adsorption resin separation material can be installed in a medium-pressure column chromatography system, can be reused repeatedly, has a large separation amount, and is easy to realize large-scale production; after rough separation, reverse-phase chromatography column is used for further separation, which is also very suitable for large-scale production. Description of the Drawings

[0026] Figure 1 It is the preparation separation chromatogram of EGC-CQA-CQA in Example 1;

[0027] Figure 2 It is the preparation separation chromatogram of GC-CQA-CQA in Example 2;

[0028] Figure 3 It is the preparation separation chromatogram of EGCG-CQA-CQA in Example 3;

[0029] Figure 4 It is the preparation separation chromatogram of GCG-CQA-CQA in Example 4;

[0030] Figure 5 It is the structural schematic diagram of four kinds of di-caffeoylquinic acids in Test Example 2;

[0031] Figure 6 It is the comparison chart of the tea soup color before and after adding four kinds of di-caffeoylquinic acids in Application Example 1. Detailed Embodiments

[0032] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1

[0034] Separation, purification and identification of di-green procyanidin (EGC-CQA-CQA)

[0035] (1) Weigh 12.24 mg of EGC and 14.28 mg of CQA and dissolve them in 6 mL of water. After complete dissolution, add 4 mL of pear homogenate (or 4 mL of polyphenol oxidase). Then stir at room temperature for 90 min. Prepare multiple reaction solutions repeatedly so that the final amounts of EGC and CQA are 5.38 g and 6.22 g respectively. Combine all the reaction solutions, and after concentration under reduced pressure and freeze-drying, obtain a solid reactant. Dissolve the solid reactant in 100 mL of pure water, then add 100 mL of ethyl acetate, shake well, let stand for 5 min, and perform extraction. Remove the ethyl acetate layer obtained each time and retain the aqueous layer; repeat the above operation to perform multiple extractions on the aqueous layer, a total of 5 extractions, to obtain the extracted aqueous phase.

[0036] (2) Filter the extracted aqueous phase, pass the filtrate through macroporous adsorption resin, and perform gradient elution with a methanol aqueous solution. First, perform pre-elution with a methanol aqueous solution with a volume percentage concentration of 0-10%, and then elute with a methanol aqueous solution with a volume percentage of 15-60%. Detect and collect the eluate at a UV wavelength of 300 nm, and obtain a crude product rich in di-green procyanidin after drying under reduced pressure.

[0037] (3) Take 20 g of the solid reactant and dissolve it in 100 mL of 5% methanol water (v / v, water containing 0.1% acetic acid) solution. After mixing the obtained mixed solution (200 mg / mL) evenly, pass it through an ODS-C 18 reverse column for separation. The separation uses gradient elution, and elute with a methanol aqueous solution with a volume percentage of 0-60% (v / v, water containing 0.1% acetic acid). A total of 5 components are obtained after combination.

[0038] (4) Use high performance liquid chromatography to perform LC-DAD-MS detection on the components obtained by gradient elution to obtain the component where di-green procyanidin (EGC-CQA-CQA) is located, and its chromatogram is as Figure 1 shown. Finally, after concentration under reduced pressure of this component, obtain an orange-yellow amorphous powder with a purity > 80%.

[0039] Example 2

[0040] Separation, Purification and Identification of Di-catechin gallate (GC-CQA-CQA)

[0041] (1) Weigh 12.24 mg of GC and 14.28 mg of CQA, dissolve them in 6 mL of water. After complete dissolution, add 4 mL of pear homogenate (or 4 mL of polyphenol oxidase). Then stir at room temperature for 90 min. Prepare multiple reaction solutions repeatedly so that the final amounts of GC and CQA are 5.38 g and 6.22 g respectively. Combine all reaction solutions, and after concentration under reduced pressure and freeze-drying, obtain solid reactants. Redissolve the solid reactants in 100 mL of pure water, then add 100 mL of ethyl acetate, shake well, let stand for 5 min, and perform extraction. Discard the ethyl acetate layer obtained each time and retain the aqueous layer; repeat the above operation and perform multiple extractions on the aqueous layer, for a total of 5 extractions, to obtain the extracted aqueous phase.

[0042] (2) Filter the extracted aqueous phase, enrich it with macroporous adsorption resin, elute, detect, collect, and perform ODS-C 18 The steps of reverse column separation and LC-DAD-MS detection are the same as those in (2), (3), and (4) of Example 1. Finally, obtain the fraction containing di-catechin gallate (GC-CQA-CQA), and its chromatogram is as Figure 2 shown. Finally, after concentration under reduced pressure of this fraction, obtain an orange-yellow amorphous powder with a purity > 80%.

[0043] Example 3

[0044] Separation, Purification and Identification of Epigallocatechin gallate (EGCG-CQA-CQA)

[0045] (1) Weigh 18.32 mg of EGCG and 14.28 mg of CQA, dissolve them in 6 mL of water. After complete dissolution, add 4 mL of pear homogenate (or 4 mL of polyphenol oxidase). Then stir at room temperature for 90 min. Prepare multiple reaction solutions repeatedly so that the final amounts of EGCG and CQA are 8.05 g and 6.22 g respectively. Combine all reaction solutions, and after concentration under reduced pressure and freeze-drying, obtain solid reactants. Redissolve the solid reactants in 100 mL of pure water, then add 100 mL of ethyl acetate, shake well, let stand for 5 min, and perform extraction. Discard the ethyl acetate layer obtained each time and retain the aqueous layer; repeat the above operation and perform multiple extractions on the aqueous layer, for a total of 5 extractions, to obtain the extracted aqueous phase.

[0046] (2) Filter the extracted aqueous phase, enrich it with macroporous adsorption resin, elute, detect, collect, and perform ODS-C 18The steps of reverse column separation, LC-DAD-MS detection, etc. are the same as those in (2), (3), and (4) of Example 1. Finally, the fraction containing digalloyl chlorogenic acid (EGCG-CQA-CQA) is obtained, and its chromatogram is as shown in Figure 3 shown. Finally, after concentrating the fraction under reduced pressure, an orange-yellow amorphous powder with a purity > 80% is obtained.

[0047] Example 4

[0048] Separation, purification and identification of digalloyl chlorogenic acid (GCG-CQA-CQA)

[0049] (1) Weigh 18.32 mg of GCG and 14.28 mg of CQA and dissolve them in 6 mL of water. After complete dissolution, add 4 mL of pear homogenate (or 4 mL of polyphenol oxidase). Then stir at room temperature for 90 min. Prepare multiple reaction solutions repeatedly so that the final amounts of GCG and CQA are 8.05 g and 6.22 g respectively. Combine all the reaction solutions, and after concentration under reduced pressure and freeze-drying, obtain a solid reactant. Redissolve the solid reactant in 100 mL of pure water, then add 100 mL of ethyl acetate, shake well, let stand for 5 min, and perform extraction. Discard the ethyl acetate layer obtained each time and retain the aqueous layer; repeat the above operation and perform multiple extractions on the aqueous layer, a total of 5 extractions, to obtain the extracted aqueous phase.

[0050] (2) Filter the extracted aqueous phase, enrich it with macroporous adsorption resin, elute, detect, collect, and perform ODS-C 18 The steps of reverse column separation, LC-DAD-MS detection, etc. are the same as those in (2), (3), and (4) of Example 1. Finally, the fraction containing digalloyl chlorogenic acid (GCG-CQA-CQA) is obtained, and its chromatogram is as shown in Figure 4 shown. Finally, after concentrating the fraction under reduced pressure, an orange-yellow amorphous powder with a purity > 80% is obtained.

[0051] Test Example 1

[0052] Separation and purification of a mixture of four digalloyl chlorogenic acids

[0053] (1) Weigh 25 mg of a mixture of four catechins (EGC, GC, EGCG, GCG) mixed in any proportion and 14.28 mg of CQA, dissolve them in 6 mL of water. After complete dissolution, add 4 mL of pear homogenate (or 4 mL of polyphenol oxidase). Then stir at room temperature for 90 min. Prepare multiple reaction solutions repeatedly so that the final amounts of the four-catechin mixture and CQA are 11 g and 6.22 g respectively. Combine all the reaction solutions, and after concentration under reduced pressure and freeze-drying, obtain a solid reactant. Redissolve the solid reactant in 100 mL of pure water, then add 100 mL of ethyl acetate, shake well, let stand for 5 min, perform extraction, remove the ethyl acetate layer obtained each time, and retain the aqueous layer; repeat the above operation to perform multiple extractions on the aqueous layer, a total of 5 extractions, to obtain the extracted aqueous phase.

[0054] (2) Filter the extracted aqueous phase, enrich it with macroporous adsorption resin, elute, detect, collect, and perform ODS-C 18 Reverse column separation, LC-DAD-MS detection and other steps are the same as (2), (3), (4) in Example 1. Finally, obtain the components where the four di-caffeoyl chlorogenic acids are located respectively, and the chromatograms are as shown in Figures 1 to 4 the figure. Finally, after concentration under reduced pressure for each component, obtain an orange-yellow amorphous powder with a purity > 80%.

[0055] Test Example 2

[0056] Verification of the properties of the four di-caffeoyl chlorogenic acids prepared in Examples 1 - 4

[0057] Use UPLC-MS and NMR techniques to perform mass spectrometry identification and NMR analysis on the four monomeric compounds obtained by separation; for mass spectrometry detection, use a C 18 chromatographic column, mobile phase A is 0.1% formic acid in water (v / v), mobile phase B is acetonitrile, the flow rate is 0.25 mL / min, the column temperature is 40 °C, and the elution gradient is 0 - 26 min, 0 - 90% B. The structures of the four di-caffeoyl chlorogenic acids and their C positions are as shown in Figure 5 the figure:

[0058] The characteristics of the four di-caffeoyl chlorogenic acids detected are as follows:

[0059] 1), The four di-caffeoyl chlorogenic acids are orange-yellow amorphous powders;

[0060] 2), The ultraviolet detection wavelength UV of the four di-caffeoyl chlorogenic acids λmax (nm): 300;

[0061] 3), LC-MS (negative ion mode): EGC-CQA-CQA (m / z = 979.2123), GC-CQA-CQA (m / z = 979.2123), EGCG-CQA-CQA (m / z = 1131.2332), GCG-CQA-CQA (m / z = 1131.2332).

[0062] 4), The nuclear magnetic resonance spectroscopy data are as follows:

[0063] Compound I: 13 C-NMR δ: 79.78 (C-2), 69.03 (C-3), 29.4 (C-4), 98.83 (C-4a), 156.43 (C-5), 96.73 (C-6), 158.1 (C-7), 96.93 (C-8), 158.07 (C-8a), 197.84 (C-a), 85.48 (C-b), 133.11 (C-c), 149.51 (C-d), 42.94 (C-e), 124.4 (C-f), 119.89 (C-g), 147.0 (C-h), 156.82 (C-i), 114.15 (C-j), 138.21 (C-k), 123.72 (C-b1), 113.18 (C-b2), 145.7 (C-b3), 148.03 (C-b4), 122.9 (C-b5), 134.47 (C-b6), 76.93 (C-1’), 39.81 (C-2’), 72.6 (C-3’), 74.14 (C-4’), 71.69 (C-5’), 38.2 (C-6’), 177.43 (C-7’), 146.31 (C-8’), 121.39 (C-9’), 168.53 (C-10’), 76.39 (C-1”), 39.04 (C-2”), 72.41 (C-3”), 73.87 (C-4”), 72.32 (C-5”), 38.3 (C-6”), 177.67 (C-7”), 144.52 (C-8”), 117.42 (C-9”), 169.32 (C-10”); 11H-NMR δ: 4.72 (H-2), 4.0 (H-3), 2.81 (H-4a), 2.87 (H-4b), 5.83 (H-6), 6.04 (H-8), 6.39 (H-e), 6.37 (H-c), 7.07 (H-g), 6.96 (H-b2), 1.79 (H-2’a), 2.41 (H-2’b), 5.23 (H-3’), 2.50 (H-4’), 4.14 (H-5’), 2.04 (H-6’), 8.94 (H-8’), 6.02 (H-9’), 2.26 (H-2”a), 2.00 (H-2”b), 5.32 (H-3”), 3.78 (H-4”), 3.92 (H-5”), 2.15 (H-6”), 8.11 (H-8”), 6.15 (H-9”).

[0064] Compound II: 13 13C-NMR δ: 81.88 (C-2), 66.93 (C-3), 29.3 (C-4), 98.83 (C-4a), 156.13 (C-5), 96.53 (C-6), 158.0 (C-7), 96.73 (C-8), 158.37 (C-8a), 198.14 (C-a), 85.58 (C-b), 134.11 (C-c), 149.71 (C-d), 42.74 (C-e), 123.4 (C-f), 119.77 (C-g), 146.7 (C-h), 156.80 (C-i), 114.05 (C-j), 138.11 (C-k), 123.56 (C-b1), 113.38 (C-b2), 145.5 (C-b3), 148.3 (C-b4), 123.3 (C-b5), 134.47 (C-b6), 76.73 (C-1’), 39.91 (C-2’), 73.1 (C-3’), 74.54 (C-4’), 71.9 (C-5’), 37.8 (C-6’), 176.93 (C-7’), 145.71 (C-8’), 120.69 (C-9’), 167.93 (C-10’), 75.59 (C-1”), 38.04 (C-2”), 73.01 (C-3”), 73.67 (C-4”), 72.21 (C-5”), 38.2 (C-6”), 177.8 (C-7”), 144.5 (C-8”), 117.6 (C-9”), 169.32 (C-10”); 11H-NMR δ: 4.58 (H-2), 3.7 (H-3), 2.61 (H-4a), 2.47 (H-4b), 5.79 (H-6), 6.14 (H-8), 6.37 (H-e), 6.36 (H-c), 7.17 (H-g), 6.86 (H-b2), 1.87 (H-2’a), 2.43 (H-2’b), 5.33 (H-3’), 2.46 (H-4’), 4.14 (H-5’), 2.09 (H-6’), 8.74 (H-8’), 6.12 (H-9’), 2.3 (H-2”a), 2.10 (H-2”b), 5.42 (H-3”), 3.78 (H-4”), 3.9 (H-5”), 2.17 (H-6”), 8.10 (H-8”), 6.17 (H-9”).

[0065] Compound III: 13 13C-NMR δ: 79.78 (C-2), 67.03 (C-3), 28.4 (C-4), 98.83 (C-4a), 156.43 (C-5), 96.73 (C-6), 158.1 (C-7), 96.93 (C-8), 158.07 (C-8a), 197.84 (C-a), 85.48 (C-b), 133.11 (C-c), 149.51 (C-d), 42.94 (C-e), 124.4 (C-f), 119.89 (C-g), 147.0 (C-h), 156.82 (C-i), 114.15 (C-j), 138.21 (C-k), 123.72 (C-b1), 113.18 (C-b2), 145.7 (C-b3), 148.03 (C-b4), 122.9 (C-b5), 134.47 (C-b6), 76.93 (C-1’), 39.81 (C-2’), 72.6 (C-3’), 74.14 (C-4’), 71.69 (C-5’), 38.2 (C-6’), 177.43 (C-7’), 146.31 (C-8’), 121.39 (C-9’), 168.53 (C-10’), 76.39 (C-1”), 39.04 (C-2”), 72.41 (C-3”), 73.87 (C-4”), 72.32 (C-5”), 38.3 (C-6”), 177.67 (C-7”), 144.52 (C-8”), 117.42 (C-9”), 169.32 (C-10”), 121.6 (C-1”’), 109.8 (C-2”’), 146.1 (C-3”’), 138.7 (C-4”’), 146.1 (C-5”’), 109.8 (C-6”’), 166.1 (C-7”’); 11H-NMR δ: 4.72 (H-2), 4.0 (H-3), 2.81 (H-4a), 2.87 (H-4b), 5.83 (H-6), 6.04 (H-8), 6.39 (H-e), 6.37 (H-c), 7.07 (H-g), 6.96 (H-b2), 1.79 (H-2’a), 2.41 (H-2’b), 5.23 (H-3’), 2.50 (H-4’), 4.14 (H-5’), 2.04 (H-6’), 8.94 (H-8’), 6.02 (H-9’), 2.26 (H-2”a), 2.00 (H-2”b), 5.32 (H-3”), 3.78 (H-4”), 3.92 (H-5”), 2.15 (H-6”), 8.11 (H-8”), 6.15 (H-9”), 7.03 (2H, H-2”’, 6”’).

[0066] Compound IV: 13C-NMR δ: 81.78 (C-2), 66.83 (C-3), 28.4 (C-4), 98.83 (C-4a), 156.43 (C-5), 96.73 (C-6), 158.1 (C-7), 96.93 (C-8), 158.07 (C-8a), 197.84 (C-a), 85.48 (C-b), 133.25 (C-c), 149.51 (C-d), 42.16 (C-e), 124.4 (C-f), 119.89 (C-g), 147.0 (C-h), 156.82 (C-i), 114.15 (C-j), 138.21 (C-k), 123.72 (C-b1), 113.18 (C-b2), 145.7 (C-b3), 148.03 (C-b4), 122.9 (C-b5), 134.47 (C-b6), 76.93 (C-1’), 39.81 (C-2’), 72.6 (C-3’), 74.14 (C-4’), 71.69 (C-5’), 38.2 (C-6’), 177.43 (C-7’), 146.31 (C-8’), 121.39 (C-9’), 168.53 (C-10’), 76.39 (C-1”), 39.04 (C-2”), 72.41 (C-3”), 73.87 (C-4”), 72.32 (C-5”), 38.3 (C-6”), 177.67 (C-7”), 144.52 (C-8”), 117.42 (C-9”), 169.32 (C-10”), 121.6 (C-1”’), 109.8 (C-2”’), 146.1 (C-3”’), 138.7 (C-4”’), 146.1 (C-5”’), 109.8 (C-6”’), 166.1 (C-7”’); 1 H-NMR δ: 4.48 (H-2), 3.82 (H-3), 2.65 (H-4a), 2.35 (H-4b), 5.79 (H-6), 5.71 (H-8), 6.39 (H-e), 6.37 (H-c), 7.07 (H-g), 6.96 (H-b2), 1.79 (H-2’a), 2.41 (H-2’b), 5.23 (H-3’), 2.50 (H-4’), 4.14 (H-5’), 2.04 (H-6’), 8.94 (H-8’), 6.02 (H-9’), 2.26 (H-2”a), 2.00 (H-2”b), 5.32 (H-3”), 3.78 (H-4”), 3.92 (H-5”), 2.15 (H-6”), 8.11 (H-8”), 6.15 (H-9”), 7.03 (2H, H-2”’, 6”’).

[0067] Note:1 1H NMR and 13 13C NMR were measured at 600 MHz, with δ in ppm and deuterated methanol as the solvent.

[0068] It can be seen from Figures 1 to 5 that four di-catechin gallates were successfully synthesized, isolated and purified in this invention, namely EGC-CQA-CQA, GC-CQA-CQA, EGCG-CQA-CQA and GCG-CQA-CQA.

[0069] Application Example 1

[0070] Applications of Four Di-catechin Gallates

[0071] (1) Weigh 1 g of black tea dry tea, brew it with 50 mL of boiling water for 5 min, then pour the tea soup into a review bowl and cool it to room temperature. Repeat this process to prepare 8 portions of tea soup.

[0072] (2) Weigh 1 mg of each of the four di-catechin gallates obtained by separation and preparation, and add them to four portions of the tea soup respectively, then stir well. Then weigh 50 mg of each of the four di-catechin gallates obtained by separation and preparation, and add them to the remaining four portions of the tea soup respectively, and stir well. The color comparison of the tea soup before and after the addition is as Figure 6 shown. In addition, the color parameter values of each tea soup were measured, mainly including L*, a* and b* values. Among them, the L* value represents the illuminance, equivalent to the brightness, and its value range is [0, 100]. The larger the value, the higher the brightness of the tea soup; the a* value represents the range from red to green, and its value range is [-128, 127]. The larger the value, the more the tea soup color tends to red; the b* value represents the range from blue to yellow, and its value range is [-128, 127]. The larger the value, the more the tea soup color tends to yellow. The measurement results of the color parameter values of each tea soup are shown in the following table:

[0073] Table 1 Color Parameter Values of Each Tea Soup before and after the Addition of Di-catechin Gallates

[0074] tea soup L* a* b* stock solution 74.69±0.29 9.88±0.01 56.09±0.59 Add 1 mg EGC-CQA-CQA 76.74±0.39 13.71±0.79 67.32±0.09 Add 50 mg EGC-CQA-CQA 78.43±0.26 21.11±0.11 78.85±0.55 Add 1 mg GC-CQA-CQA 75.28±0.48 12.68±0.79 66.56±0.18 Add 50 mg GC-CQA-CQA 77.39±0.17 22.59±0.18 77.66±0.58 Add 1 mg EGCG-CQA-CQA 76.11±0.36 14.03±0.79 65.11±0.09 Add 50 mg EGCG-CQA-CQA 78.33±0.26 22.09±0.37 75.34±0.55 Add 1 mg GCG-CQA-CQA 76.02±0.62 13.22±0.79 68.13±0.44 Add 50 mg GCG-CQA-CQA 79.66±0.38 23.08±0.82 76.29±0.38

[0075] According to Figure 6 the results in Table 1, when four novel natural pigments (di-catechin gallates) are added to the black tea soup, the L*, a* and b* values of the tea soup all increase, indicating that the addition of this type of pigment can significantly change the tea soup color, making the red brightness of the drink increase, and the addition amount is 20 mg / L to 1 g / L.

[0076] The above are only the preferred embodiments of the present invention. After reading the above content of the present invention, for those of ordinary skill in the art, without departing from the concept, spirit and scope of the present invention, several improvements and refinements can be made to obtain the same or similar results. Specifically, some reagents and their ratios and other related contents can replace the reagents disclosed in this article to obtain the same or similar results. It should be noted that all similar improvements and refinements are obviously considered to be within the spirit, scope, concept and protection scope of the claims of the present invention.

Claims

1. A preparation method of di-caffeoylquinic acid, comprising: (1) Preparation of reaction solution: Catechin, chlorogenic acid, catalytic oxidizing substance and water are mixed, and magnetically stirred under open and appropriate temperature conditions to allow the mixture to react fully to obtain a crude reaction solution; (2) Concentration, extraction and rough separation: The crude reaction solution is concentrated under reduced pressure and freeze-dried to obtain a solid reactant. The obtained solid reactant is redissolved in water, and then extracted with ethyl acetate, and the extraction phase is removed to obtain an aqueous phase; (3) Separation by macroporous adsorption resin: The aqueous phase is filtered, and the filtrate passes through a macroporous adsorption resin, and gradient elution is carried out with an aqueous methanol solution, and the eluate is collected and dried under reduced pressure to obtain a crude product of di-caffeoylquinic acid; (4) Medium and low pressure preparative column separation: The crude product of di-caffeoylquinic acid is separated by a medium and low pressure preparative column, and gradient elution is carried out. According to the ultraviolet absorption signal, the eluate containing di-caffeoylquinic acid is detected and collected. The collected eluate is dried under reduced pressure to obtain four compounds with a purity > 80%, and their structures are shown in Formula I, Formula II, Formula III and Formula IV respectively: Formula I; Formula II; Formula III; Formula IV; Wherein: The catechin described in step (1) is selected from any one or more of EGC, GC, EGCG, GCG; The catalytic oxidizing substance is selected from any one of pear homogenate or tea polyphenol oxidase.

2. The preparation method according to claim 1, wherein: The mass ratio of the catechin and chlorogenic acid described in step (1) is (1~15):(15~1); The mass of the water is 10~500 times the total mass of the solid mixture; The mass of the catalytic oxidizing substance is 10~500 times the total mass of the solid mixture; The solid mixture includes: catechin, chlorogenic acid.

3. The preparation method according to claim 1, wherein: The temperature of the magnetic stirring reaction in step (1) is 10~40°C; the time of the magnetic stirring reaction is 10~300 min.

4. The preparation method according to claim 1, wherein: The macroporous adsorption resin described in step (3) is HPD-300 resin; The gradient elution includes: first pre-eluting with an aqueous methanol solution with a volume percentage concentration of 0~10%, and then eluting with an aqueous methanol solution with a volume percentage of 15~60%.

5. The preparation method according to claim 1, wherein: The packing material of the medium and low pressure preparation column described in step (4) is ODS C 18 particles, and the particle size range is 10~100 µm.

6. The preparation method according to claim 1, wherein: The gradient elution described in step (4) includes: eluting with an aqueous methanol solution with a volume percentage of 0~60%, and the water contains 0.1% acetic acid.

7. The preparation method according to claim 1, wherein: The ultraviolet absorption signal detection conditions described in step (4) are Agilent C 18 chromatographic column, 2.1×50 mm, 1.5 μm; the column temperature is 20~40 °C, the detection wavelength is 300 nm, mobile phase A is 0.1% formic acid aqueous solution, B is acetonitrile, the chromatographic conditions are 0~26 min, 90% B, the injection volume is 0.1~1 mL, and the flow rate is 0.1~0.4 mL / min.

8. A di-caffeoylquinic acid prepared by the preparation method according to any one of claims 1~7.

9. An application of the di-caffeoylquinic acid according to claim 8 in the preparation of beverages, wherein: The addition amount of the di-caffeoylquinic acid is 20 mg / L~1 g / L.

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