CN116969966A - A natural dimeric catechin pigment and its preparation method and application - Google Patents

Abstract

The invention provides a method for preparing new natural pigment by using phenotypic catechin synthesis, which takes various epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate as substrates, mixes the substrates and then carries out non-enzymatic oxidation reaction under the damp-heat condition to generate a new tea pigment compound; concentrating under reduced pressure, freeze drying, extracting, and separating and purifying by column chromatography to obtain dimeric catechin pigment. The six new dimeric catechin pigment substances prepared by the invention have simple synthesis method. Six new dimeric catechin pigments obtained by the preparation method are orange or orange red amorphous powder, have better color mixing capability, and can be applied to color mixing of tea beverages and novel tea beverages. The discovery of the natural pigment is helpful for promoting the research of the color-developing substances in the tea hot processing process, the improvement of the tea soup color quality and the development and application of the food coloring field.

Description

A natural dimerized catechin pigment and its preparation method and application

Technical Field

The invention relates to the technical field of chemical synthesis and separation and purification of natural products, and in particular to a natural dimerized catechin pigment and a preparation method and application thereof.

Background Art

Catechins are the main polyphenols in tea, and have obvious bitterness and astringency. They are one of the main substances that form the comprehensive taste of tea soup. In addition, catechins have strong biological activity and are the main health active substances in tea. Thermal processing (high temperature drying or baking) is a very important step in the tea production process. It can not only reduce the moisture in tea leaves, but also significantly improve the flavor of tea leaves. After high-temperature baking, the color of the tea soup deepens, and the color of the dry tea is brown or even dark brown. In addition to being related to the Maillard reaction and caramelization reaction, this may also be due to the non-enzymatic oxidation reaction of polyphenols to form a more stable polymeric pigment, which in turn promotes the formation of the comprehensive color of the tea soup. This type of natural pigment is more suitable for development and application in food.

In recent years, the results of studying the transformation products of catechins using in vitro models or real tea matrices have shown that during the thermal reaction, catechins mainly undergo isomerization, degradation and polymerization reactions. At present, the transformation products of EC, EGC, EGCG or other catechins in in vitro models of aqueous solution or solid powder are mainly studied, but the temperature of the research model is lower than 90°C. In 2003, Jack M. Miller's research group used catechins EC and C to study the transformation products of catechins under normal temperature aqueous solution (pH 6.5) conditions, and identified some dehydrodimers of catechins using LC-MS (Weixing Sun and Jack M. Miller. Tandem mass spectrometry of the B-type procyanidins in wine and B-type dehydrodicatechins in an autoxidation mixture of (+)-catechin and (-)-epicatechin [J]. Journal of Mass Spectrometry, 2003.). In 2008, Chang-Qing Duan's research group used catechins EC and C to react in aqueous solution (pH 6) at room temperature for 24 hours to study the transformation products of catechins, and used LC-MS to identify some dehydrogenated dimers of catechins (Fei He., Qiu-Hong Pan., Ying Shi., et al. Identification of autoxidation oligomers of flavan-3-ols in model solutions by HPLC-MS/MS[J]. Journal of Mass Spectrometry, 2008.). In 2012, Lisa J. Mauer's research group used EGCG in an in vitro aqueous solution model or solid model at 85°C to study its structural stability and color changes. They found that the color gradually deepened with time, and identified two main EGCG dimers (polyester catechin A and D) using LC-MS (Na Li., Lynne S. Taylor., Mario G. Ferruzzi., et al. Color and chemicalstability of tea polyphenol (-)-epigallocatechin-3-gallate in solution and solid states [J]. Food Research International, 2012.). In 2016, Liang Yuerong's research group used 8 catechins (EC, C, EGC, GC, ECG, CG, EGCG and GCG) to study the transformation products of catechins in an in vitro aqueous solution model at 90°C, and identified some dimers or isomerization and degradation products of catechins by using LC-MS (Fang-Yuan Fan., Meng Shi., Ying Nie., et al. Differential behaviors of tea catechins under thermal processing: Formation of non-enzymatic oligomers[J]. Food Chemistry, 2016.).

In 2019, Thomas Hofmann's research group used catechin EC or C to study the catechin conversion products in vitro under alkaline solution and 90°C heating conditions, and identified some catechin dimer compounds by separation and purification (Daniel German, Timo D. Stark, and Thomas Hofmann. Formation and characterization of polyphenol-derived red chromophores: Enhancing the color of processed cocoa powders: Part 1 [J]. Journal of Agricultural and Food Chemistry, 2019.). In 2020, Jean-Paul Vincken's group used EC and EGC aqueous solution models to study the main products and solution color changes at different reaction times at 80°C, and identified some catechin dimers or trimers by LC-MS (Junfeng Tan., Wouter J.C. de Bruijn., Annemiek van Zadelhoff., et al. Browning of Epicatechin (EC) and Epigallocatechin (EGC) by Auto-Oxidation [J]. Journal of Agricultural and Food Chemistry, 2020.).

Researchers have used catechins to study their structural stability, transformation products, and color changes under in vitro heating conditions. However, the main polymerization products were mainly identified through LC-MS. The reaction temperature was low, the time to produce coloring substances was long, and the content was low. No efficient synthesis and separation and purification methods were established; and no separation, purification, and identification of key coloring substance structures were conducted to study their color characteristics. This makes it difficult to separate the polymerized pigments after catechins are heated at high temperatures, greatly limiting the development and application of this type of natural polymerized pigments in the fields of tea beverages, new tea beverages, and food coloring.

Summary of the invention

In view of this, the present invention provides a natural dimer catechin pigment and a preparation method and application thereof. The present invention adopts in vitro synthesis, separation, purification and identification to obtain the natural dimer catechin pigment, which has the effect of promoting the formation of red and yellow colors of tea soup, and promotes the development and application of natural dimer catechin pigment in the field of color adjustment of tea beverages and new tea beverages and food coloring.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The present invention provides a method for preparing a natural dimerized catechin pigment, comprising the following steps:

Step 1, preparation of a crude reaction product: mixing phenotypic catechins, a pH regulator and water, and performing a non-enzymatic oxidation reaction under open and heated conditions to obtain a crude reaction product containing dimerized catechin pigments;

Step 2, extraction, concentration and crude separation: adding water to the crude reaction product for redissolution, extracting with ethyl acetate, removing the aqueous phase to obtain an ethyl acetate extraction phase, and evaporating and concentrating to obtain a concentrated and dried ethyl acetate extraction phase;

Step 3, separation by ODS-C ₁₈ reversed phase chromatography column: the concentrated and dried ethyl acetate extract phase is redissolved with 10% methanol water (v/v, water containing 0.2% formic acid), the redissolved methanol aqueous solution is separated by ODS-C ₁₈ reversed phase chromatography column, methanol aqueous solutions of different concentrations are sampled for gradient elution, and the eluate is collected to obtain a crude product rich in six dimerized catechin pigments or monomeric dimerized catechin pigments;

Step 4, separation by medium- and low-pressure preparative columns: Separate the crude product rich in six dimerized catechin pigments or monomeric dimerized catechin pigments using medium- and low-pressure preparative columns, perform gradient elution with a methanol aqueous solution containing 0.2% formic acid (v/v, water containing 0.2% formic acid), collect the eluate containing dimerized catechin pigments according to the peak position of the ultraviolet absorption signal, and dry the collected eluate under reduced pressure to obtain six compounds with a purity greater than 80%, whose structures are respectively shown in Formula I (EGCG), Formula II (EGC), Formula III (EC), Formula IV (ECG), Formula V (EGC-ECG) and Formula VI (EGCG-EC), and detect them;

Furthermore, the reaction substrate phenotypic catechin in step 1 is selected from any one of EGCG, ECG, EGC, EC or a mixture of four thereof.

Furthermore, the quality of the water in step 1 is 50 to 1,000,000% of the quality of the phenotypic catechins.

Furthermore, the pH adjuster in step 1 is an acid or a base, and the pH value of the non-enzymatic oxidation reaction is 3-12.

Furthermore, the heating temperature in step 1 is 20 to 200° C., and the non-enzymatic oxidation reaction time is 10 to 180 min.

Furthermore, the filler used for separation in step 3 is ODS-C ₁₈ particles, and the ODS-C ₁₈ particles have a particle size range of 5 to 200 μm.

Furthermore, the gradient elution in step 3 comprises:

The eluent used in elution gradient 1 was 0.2% formic acid in water (v/v, water containing 0.2% formic acid) and methanol, and the volume ratio of the 0.2% formic acid in water mixture to methanol was 90:10;

The eluent used in elution gradient 2 was 0.2% formic acid in water (v/v, water containing 0.2% formic acid) and methanol, and the volume ratio of the 0.2% formic acid in water mixture to methanol was 75:25;

The eluent used in elution gradient 3 was 0.2% formic acid in water (v/v, water containing 0.2% formic acid) and methanol, and the volume ratio of the 0.2% formic acid in water mixture to methanol was 50:50;

The eluent used in elution gradient 4 was pure methanol.

Furthermore, the filler used for the low-pressure preparative column separation in step 4 is ODS-C ₁₈ particles with a particle size range of 5 to 100 μm; gradient elution is adopted, and the eluent is 5% to 80% methanol aqueous solution (v/v, water containing 0.2% formic acid).

Furthermore, the chromatographic column used in the detection method of step 4 is a _C18 reverse phase chromatographic column, and the mobile phase includes phase A and phase B, wherein: phase A is a formic acid aqueous solution with a volume concentration of 0.1% (v/v, water containing 0.2% formic acid), and phase B is acetonitrile; the flow rate is 0.15-1 mL/min, the column temperature is 25-40°C, the detection wavelength is 278 nm, the injection volume is 1-50 μL, and the elution time is 0-25 min.

The invention also discloses a natural dimerized catechin pigment prepared according to any one of the above preparation methods.

The invention also discloses an application of the natural dimerized catechin pigment in the production of tea beverages.

Furthermore, the application includes:

The dimerized catechin pigment is added to the tea beverage in an amount of 20 mg/L to 1 g/L.

The beneficial effects of the present invention are:

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

The solvent used for extraction, the solvent used for ODS-C ₁₈ reverse chromatography column and the solvent used for reverse phase chromatography column separation can all be recycled; the materials used for reverse phase chromatography separation can all be reused, and the recycled solvent and reused separation materials ensure a relatively low average separation cost.

(2) The preparation method of the present invention can realize the needs of large-scale production

The raw material requirements are not high and the cost is low. Generally, analytically pure catechins purchased can be used, and batch preparation is easy. The concentration and extraction operations are simple. The separation adopts an ODS-C ₁₈ reverse chromatography column for coarse separation. The ODS-C ₁₈ reverse chromatography column filler can be installed in a medium-pressure column chromatography system and can be repeatedly used. The separation volume is large and it is easy to realize large-scale production. After the coarse separation, a reverse phase chromatography column is used for further separation, which is also very suitable for large-scale production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a separation step of dimerized catechin pigment in Example 1;

FIG2 is a mass spectrum of the preparation and separation of the dimerized catechin pigment of EGCG in Example 1;

FIG3 is a mass spectrum of the preparation and separation of dimerized catechin pigment of EGC in Example 2;

FIG4 is a mass spectrum of the preparation and separation of dimerized catechin pigments of EC in Example 3;

FIG5 is a mass spectrum of the preparation and separation of dimerized catechin pigment of ECG in Example 4;

FIG6 is a mass spectrum of the preparation and separation of dimerized catechin pigment of EGC-ECG in Example 5;

FIG7 is a mass spectrum of the preparation and separation of dimerized catechin pigment of EGCG-EC in Example 6;

FIG8 is a schematic diagram of the structures of six dimerized catechin pigments in Experimental Example 2;

FIG9 is a comparison of the tea soup colors before and after the six dimerized catechin pigments are added to the tea soup in Application Example 1;

FIG. 10 is a color difference diagram of the tea soup color before and after the six dimerized catechin pigments are added to the tea soup in Application Example 1; a, b, c, and d marked in the figure indicate significance (P<0.05).

DETAILED DESCRIPTION

In the present invention, unless otherwise specified, the required raw materials for preparation are all commercially available products well known to those skilled in the art.

The invention mixes epicatechins, a pH regulator and water, and performs non-enzymatic oxidation reaction under open and heating conditions to obtain a polymer product.

In the present invention, the purity of epicatechins is preferably independently ≥ 95%.

In the present invention, during the non-enzymatic oxidation reaction, the mass of water is 50 to 1,000,000%, preferably 100 to 100,000%, of the mass of phenotypic catechins.

In the present invention, the pH regulator is an acid or a base, the acid is preferably a 0.02 mol/L hydrochloric acid solution, the base is preferably a 0.02 mol/L sodium hydroxide solution, and the pH value of the non-enzymatic oxidation reaction is 3-12.

In the present invention, the method of mixing the phenotypic catechin mixture or the monomeric catechin and water comprises: vortex mixing and ultrasonic mixing performed in sequence; the vortex mixing rate is 500r/min, the time is 1 to 3min, preferably 2.5min; the ultrasonic mixing power is 200W, the frequency is 40kHz, the time is 1 to 3min, preferably 2.5min.

The present invention has no special limitation on the opening method, and any method that can achieve the opening effect is acceptable; in the embodiment of the present invention, the open reaction device is a test tube, a beaker or a glass bottle. In the present invention, the temperature of the non-enzymatic oxidation reaction is 20-200°C, preferably 120-150°C; the reaction time is 10-180min, preferably 30-120min, and more preferably 80-100min. After high-temperature heating of the phenotypic catechins, a crude reaction product containing dimerized catechin pigments is obtained, which is redissolved in water, extracted three times with an equal volume of ethyl acetate, and the aqueous phase is removed to obtain an ethyl acetate extraction phase, and the dimerized catechin pigment is detected by high performance liquid chromatography to determine the presence of dimerized catechin pigments in the ethyl acetate extraction phase, and then concentrated by rotary evaporation.

In the present invention, the chromatographic column used for the high performance liquid chromatography detection is preferably a _C18 reverse phase chromatographic column, the mobile phase includes phase A and phase B, phase A is 0.2% formic acid aqueous solution (v/v, water containing 0.2% formic acid), and phase B is methanol; the flow rate is 1 mL/min, the detection wavelength is 278 nm, the detector is DAD, the column temperature is 30°C, and the elution conditions are: 0-5 min, 25% methanol aqueous solution to 42% methanol aqueous solution; 5-11 min, 42% methanol aqueous solution to 50% methanol aqueous solution; 11-13 min, 50% methanol aqueous solution to 42% methanol aqueous solution; 13-14 min, 42% methanol aqueous solution to 25% methanol aqueous solution; 14-17 min, 25% methanol aqueous solution.

After obtaining the ethyl acetate extract containing the dimerized catechin pigment, column chromatography purification and rotary evaporation concentration are continued to obtain the dimerized catechin pigment having the structure shown in Formula I to VI. The present invention has no special limitation on the rotary evaporation concentration process, which can be carried out according to the process known in the art.

In the present invention, the column chromatography purification process comprises:

The concentrated and dried ethyl acetate extract was redissolved in 10% methanol water (v/v, water containing 0.2% formic acid) to obtain an isolated product, which was then separated using an ODS-C ₁₈ reverse phase column chromatography. The reverse phase column chromatography separation included 4 elution gradients to obtain 4 elution components.

In the present invention, the filler used for the column chromatography separation is preferably an ODS-C ₁₈ reverse column, and the glass column specification is 8×40 cm; the chromatographic separation preferably includes four elution gradients, the eluent used in elution gradient 1 is a formic acid water mixture with a volume concentration of 0.2% and methanol, the volume ratio of the formic acid water mixture to methanol is 90:10, and the dosage is 250-500 mL, preferably 350 mL; the eluent used in elution gradient 2 is a formic acid water mixture with a volume concentration of 0.2% and methanol, the volume ratio of the formic acid aqueous solution to methanol is 75:25, and the dosage is 2 50～500mL, preferably 350mL; the eluent used in elution gradient 3 is a formic acid water mixture with a volume concentration of 0.2% and methanol, the volume ratio of the formic acid water mixture to methanol is 50:50, and the dosage is 250～500mL, preferably 350mL; the eluent used in elution gradient 4 is a formic acid water mixture with a volume concentration of 0.2% and methanol, the volume ratio of the formic acid water mixture to methanol is 0:100, and the dosage is 250～500mL, preferably 350mL.

After obtaining the four elution components, the four elution components are detected by high performance liquid chromatography to determine the component where the dimerized catechin pigment is located. In the present invention, the conditions of the high performance liquid chromatography detection method are the same as the above conditions, which will not be repeated here.

The present invention separates the components containing dimerized catechin pigment by medium-low pressure preparation column to obtain dimerized catechin pigment with structures shown in formulas I to VI.

In the present invention, the chromatographic column used for the medium- and low-pressure preparative column separation is an ODS-C ₁₈ reverse chromatographic column, the mobile phase includes phase A and phase B, phase A is a formic acid aqueous solution with a volume concentration of 0.2% (v/v, water containing 0.2% formic acid), and phase B is methanol; the elution conditions for the medium- and low-pressure preparative column separation are: 0-5min, 25%-42% methanol aqueous solution; 5-10min, 42%-80% methanol aqueous solution; 10-18min, 80%-25% methanol aqueous solution; 18-23min, 25% methanol aqueous solution.

In the present invention, after obtaining the dimeric catechin pigment having the structure shown in formula I to VI, the present invention performs purity and mass spectrometry identification on six monomer compounds; the detection method for the purity identification of the dimeric catechin pigment is high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The HPLC detection method is the same as the conditions for determining the presence of the dimeric catechin pigment by high performance liquid chromatography detection of the ODS-C ₁₈ reversed chromatography column separation fraction, which will not be repeated here. In the present invention, the purity identification of the dimeric catechin pigment having the structure shown in formula I to VI is preferably performed by UPLC-MS. The detection flow rate is preferably 0.25 mL/min, and the mobile phase includes phase A and phase B: phase A is a formic acid aqueous solution with a volume concentration of 0.1% (v/v, water containing 0.1% formic acid), phase B is acetonitrile, and the elution conditions are: 0-2.5 min, 2% methanol aqueous solution; 2.5-3 min, 2% methanol aqueous solution to 5% methanol aqueous solution; 3-8 min, 5% methanol aqueous solution to 15% methanol aqueous solution; 8-15 min, 15% methanol aqueous solution to 18% methanol aqueous solution; 15-19 min, 18% methanol aqueous solution to 25% methanol water; 19-21 min, 25% methanol water to 2% methanol aqueous solution; 21-25 min, 2% methanol aqueous solution.

In the present invention, the structural formula of the dimerized catechin pigment having the structure shown in Formulas I to VI is:

The present invention also provides the use of the dimerized catechin natural pigment described in the above technical solution or the dimerized catechin natural pigment prepared by the preparation method described in the above technical solution in the field of coloring of tea beverages, new tea beverages or food. The present invention preferably uses the dimerized catechin natural pigment as a colorant. In the present invention, the dimerized catechin natural pigment is an orange-yellow or orange-red amorphous powder. The present invention preferably uses the dimerized catechin natural pigment as a tea beverage colorant and adds it to the tea beverage, so that the yellowness or redness of the product increases, significantly changing the color of the product.

In the following examples, the purity of the phenotypic catechins was analytically pure, with a purity of ≥95% respectively;

The following Examples 1 to 7 are examples of synthesis, separation and purification, identification and color characteristics of dimerized catechin pigments. The prepared polymer product is directly dissolved in methanol and then detected by high performance liquid chromatography or UPLC-MS.

Example 1

Isolation, purification and identification of dimerized catechin pigment (Formula I)

(1) Weigh 45.8 mg EGCG (analytical standard, purity 95%) in a test tube, add 1 mL of pure water, vortex for 3 minutes, ultrasonic for 3 minutes, and after fully dissolving, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL of pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amount of EGCG is 4.58 g, and combine the crude reaction solutions.

(2) Add an equal volume of ethyl acetate to the crude reaction solution, shake well, let stand for 5 minutes, and extract to obtain an aqueous phase and an ethyl acetate phase, respectively. Repeat the above steps to extract the aqueous phase three times, combine the ethyl acetate phases obtained by the three extractions, and evaporate and concentrate to obtain a crude product rich in dimerized catechin pigment.

(3) The crude product rich in dimerized catechin pigment was redissolved with 5 mL of 10% methanol water (v/v, water containing 0.2% formic acid), and the mixed solution after redissolution was mixed evenly and separated by ODS- _C18 reversed phase chromatography column, and eluted with 0.2% formic acid water mixed solution (v/v, water containing 0.2% formic acid) and methanol in volume ratios of 90:10, 75:25, 50:50 and 0:100 as eluents, each elution gradient was 350 mL, and the eluents were collected respectively to obtain 4 components.

(4) Component 3 was separated by a medium-low pressure preparative column, and gradient eluted with a 5% to 80% methanol-water (water containing 0.2% formic acid) solution. The eluate was collected according to the peak position of the ultraviolet absorption signal, and the components obtained by gradient elution were detected and analyzed by LC-DAD-MS to obtain the component containing the dimerized catechin pigment (Formula I). The separation steps are shown in Figure 1, and its pure mass spectrum is shown in Figure 2. Finally, the component was concentrated and dried to obtain an orange-red amorphous powder with a purity greater than 80%.

Example 2

Isolation, Purification and Identification of Dimeric Catechin Pigment (Formula Ⅱ)

(1) Weigh 30.6 mg of EGC (analytical standard, purity 95%) in a test tube, add 1 mL of pure water, vortex for 3 minutes, and sonicate for 3 minutes. After fully dissolved, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL of pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amount of EGC is 3.06 g, and combine the crude reaction solutions.

(2) The crude reaction solution was extracted, separated by ODS-C ₁₈ reversed phase chromatography column, separated by medium and low pressure preparative columns, and detected by LC-DAD-MS, and the methods were the same as (2), (3), and (4) in Example 1. Finally, the component containing the dimerized catechin pigment (Formula II) had a mass spectrum as shown in FIG3. Finally, the component was concentrated and dried to obtain an orange-yellow amorphous powder with a purity greater than 80%.

Example 3

Isolation, Purification and Identification of Dimeric Catechin Pigment (Formula III)

(1) Weigh 29.0 mg EC (analytical standard, purity 95%) into a test tube, add 1 mL pure water, vortex for 3 minutes, sonicate for 3 minutes, and after fully dissolving, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amount of EC is 2.90 g, and combine the crude reaction solutions.

(2) The crude reaction solution was extracted, separated by ODS-C ₁₈ reversed phase chromatography column, separated by medium and low pressure preparative columns, and detected by LC-DAD-MS, and the methods were the same as those in (2), (3), and (4) in Example 1. Finally, the component containing the dimerized catechin pigment (Formula III) had a mass spectrum as shown in FIG4. Finally, the component was concentrated and dried to obtain an orange-yellow amorphous powder with a purity greater than 80%.

Example 4

Isolation, Purification and Identification of Dimeric Catechin Pigment (Formula Ⅳ)

(1) Weigh 44.2 mg ECG (analytical standard, purity 95%) into a test tube, add 1 mL pure water, vortex for 3 minutes, ultrasonic for 3 minutes, and after fully dissolving, transfer the test tube to a 150°C oil bath and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amount of ECG is 4.42 g, and combine the crude reaction solutions.

(2) The crude reaction solution was extracted, separated by ODS-C ₁₈ reversed phase chromatography column, separated by medium and low pressure preparative columns, and detected by LC-DAD-MS, and the methods were the same as those in (2), (3), and (4) in Example 1. Finally, the component containing the dimerized catechin pigment (Formula IV) had a mass spectrum as shown in FIG5. Finally, the component was concentrated and dried to obtain an orange-red amorphous powder with a purity greater than 80%.

Example 5

Isolation, Purification and Identification of Dimeric Catechin Pigment (Formula V)

(1) Weigh 44.2 mg ECG and 30.6 mg EGC (analytical standard, purity 95%) in a test tube, add 1 mL pure water, vortex for 3 minutes, ultrasonic for 3 minutes, and after fully dissolving, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amounts of ECG and EGC are 4.42 g and 3.06 g, respectively, and combine the crude reaction solutions.

(2) The crude reaction solution was extracted, separated by ODS-C ₁₈ reversed phase chromatography column, separated by medium and low pressure preparative columns, and detected by LC-DAD-MS, and the methods were the same as those in (2), (3), and (4) in Example 1. The mass spectrum of the component containing the dimerized catechin pigment (Formula V) is shown in Figure 6. Finally, the component was concentrated and dried to obtain an orange-red amorphous powder with a purity greater than 80%.

Example 6

Isolation, Purification and Identification of Dimeric Catechin Pigment (Formula VI)

(1) Weigh 29.0 mg EC and 45.8 mg EGCG (analytical standard, purity 95%) in a test tube, add 1 mL pure water, vortex for 3 minutes, ultrasonic for 3 minutes, and after fully dissolving, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amounts of EC and EGCG are 2.90 g and 4.58 g, respectively, and combine the crude reaction solutions.

(2) The crude reaction solution was extracted, separated by ODS-C ₁₈ reversed phase chromatography column, separated by medium and low pressure preparative columns, and detected by LC-DAD-MS, and the methods were the same as those in (2), (3), and (4) in Example 1. The mass spectrum of the component containing the dimerized catechin pigment (Formula VI) is shown in Figure 7. Finally, the component was concentrated and dried to obtain an orange-red amorphous powder with a purity greater than 80%.

Test Example 1

Isolation, purification and identification of dimeric catechin pigments (Formula I to Formula VI)

(1) Weigh 29.0 mg EC, 30.6 mg EGC, 44.2 mg ECG and 45.8 mg EGCG (analytical standard, purity 95%) in a test tube, add 3 mL pure water, vortex for 3 minutes, ultrasonic for 3 minutes, and after fully dissolving, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is completed, re-dissolve with 3 mL pure water to obtain a crude reaction solution containing dimerized catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amounts of EC, EGC, ECG and EGCG are 2.90 g, 3.06 g, 4.42 g and 4.58 g, respectively, and combine the crude reaction solutions.

(2) The crude reaction solution is extracted, separated by ODS-C ₁₈ reversed phase chromatography column, separated by medium and low pressure preparative columns, and detected by LC-DAD-MS, and the methods are the same as those in (2), (3), and (4) in Example 1. The mass spectra of the component containing the dimerized catechin pigment (Formula I to Formula VI) are shown in Figures 2 to 7. Finally, the component is concentrated and dried to obtain an orange-yellow or orange-red amorphous powder with a purity greater than 80%.

Test Example 2

Verification of the properties of the six dimerized catechin pigments prepared in Examples 1 to 6

Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) techniques were used to perform mass spectrometry identification and nuclear magnetic resonance analysis on the six monomer compounds separated; the mass spectrometry detection used a _C18 reverse chromatographic column, the mobile phase A used 0.1% formic acid water (v/v, water containing 0.2% formic acid), the mobile phase B used acetonitrile, the flow rate was 0.25 mL/min, the column temperature was 40°C, the elution gradient was 0-25 min, 2-25% B, the six dimerized catechin pigment structures and their C positions are shown in FIG8 :

The properties of the six dimerized catechin pigments detected are as follows:

(1) The six dimerized catechin pigments are orange-yellow or orange-red amorphous powders;

(2) UV detection wavelengths of six dimerized catechin pigments: UV _λmax (nm): 278 and 490;

(3) LC-MS (negative ion mode):

EGCG dimer catechin pigment (Formula I, m/z=745.1433), EGC dimer catechin pigment (Formula II, m/z=593.1335), EC dimer catechin pigment (Formula III, m/z=561.1472), ECG dimer catechin pigment (Formula IV, m/z=713.1556), EGC-ECG dimer catechin pigment (Formula V, m/z=729.1557) and EGCG-EC dimer catechin pigment (Formula VI, m/z=729.1644).

(4) The NMR spectrum data are as follows:

Table 1 H-NMR and C-NMR data of six dimerized catechin pigments

Continued

Note: ¹ H NMR and ¹³ C NMR were tested at 600 MHz, δ unit is ppm, solvent is deuterated methanol, and the “-” in the table indicates that there is no data here. s: singlet; m: multiplet; d: doublet; dd: quartet.

As shown in Table 1, the present invention successfully synthesized, separated, purified and identified six dimeric catechin pigments having structures shown in Formula I to Formula VI.

Application Examples

Application of six dimerized catechin pigments

(1) Weigh 1 g of dry tea and brew it with 50 mL of boiling water for 5 min. Pour the tea into an evaluation bowl and cool it to room temperature. Repeat this process to prepare 18 servings of tea.

(2) Addition group 1: Weigh 4 mg of the six kinds of dimerized catechin pigments obtained by separation and preparation, add them to three portions of tea soup respectively, and stir evenly. Addition group 2: Weigh 10 mg of the six kinds of dimerized catechin pigments obtained by separation and preparation, add them to three portions of tea soup respectively, and stir evenly. Addition group 3: Weigh 50 mg of the six kinds of dimerized catechin pigments obtained by separation and preparation, add them to three portions of tea soup respectively, and stir evenly. The color comparison of the tea soup before and after addition is shown in Figure 9. The color difference analysis of the three groups of dimerized catechin pigments with different addition amounts is shown in Figure 10. After adding the six kinds of dimerized catechin pigments, the a value (redness) and b value (yellowness) of the tea soup increased significantly.

According to the comparison results of Figures 9 and 10, adding six new natural pigments (dimeric catechin pigments) into tea soup can significantly change the color of the tea soup, increasing the redness and yellowness of the tea soup. The added amount is 20 mg/L to 1 g/L.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. A preparation method of natural dimeric catechin pigment comprises the following steps:

(1) Crude reaction product preparation: mixing phenotype catechin, a pH regulator and water for non-enzymatic oxidation reaction to obtain a crude reaction product containing dimeric catechin pigment;

(2) Extraction, concentration and crude separation: dissolving the crude reaction product in Shui Fu, extracting with ethyl acetate, removing the water phase to obtain an ethyl acetate extract phase, and evaporating and concentrating to obtain a concentrated and dried ethyl acetate extract phase;

(3)ODS-C ₁₈ reverse chromatographic column separation: the ethyl acetate extract phase after concentration and drying is redissolved with 10% methanol water (water contains 0.2% formic acid), and then the aqueous solution is passed through ODS-C ₁₈ Separating by reverse chromatographic column, gradient eluting with methanol water solutions of different concentrations, and collecting eluate to obtain crude product rich in six dimeric catechin pigments or monomeric dimeric catechin pigments;

(4) Separating a medium-low pressure preparation column: separating the crude product rich in six dimeric catechin pigments or monomeric dimeric catechin pigments by using a medium-low pressure preparation column, performing gradient elution by using a methanol aqueous solution containing 0.2% formic acid, collecting eluent containing dimeric catechin pigments according to the position of an ultraviolet absorption signal peak, concentrating and drying the collected eluent to obtain six compounds with purity of more than 80%, wherein the structures are respectively shown as a formula I (EGCG), a formula II (EGC), a formula III (EC), a formula IV (ECG), a formula V (EGC-ECG) and a formula VI (EGCG-EC):

2. the method of manufacturing according to claim 1, wherein:

the phenotype catechin in the step (1) is selected from any one or a mixture of four phenotype catechins in EGCG, ECG, EGC, EC.

3. The method of manufacturing according to claim 1, wherein:

the mass of the water in the step (1) is 50-1000000% of the mass of the phenotypic catechin; the pH regulator is acid or alkali.

4. The method of manufacturing according to claim 1, wherein:

the pH value of the non-enzymatic oxidation reaction in the step (1) is 3-12, the reaction temperature is 20-200 ℃, and the reaction time is 10-180 min.

5. The method of manufacturing according to claim 1, wherein:

the filler used for separation in the step (3) is ODS-C ₁₈ Particles, the ODS-C ₁₈ The particle size is 5-200 μm.

6. The method of manufacturing according to claim 1, wherein:

the gradient elution of step (3) comprises:

the eluent used in the elution gradient 1 is 0.2% concentration formic acid water (water contains 0.2% formic acid) and methanol, and the volume ratio of the 0.2% formic acid water mixed solution to the methanol is 90:10;

the eluent used in the elution gradient 2 is 0.2% concentration formic acid water (water contains 0.2% formic acid) and methanol, and the volume ratio of the 0.2% formic acid water mixed solution to the methanol is 75:25;

the eluent used in the elution gradient 3 is 0.2% concentration formic acid water (water contains 0.2% formic acid) and methanol, and the volume ratio of the 0.2% formic acid water mixed solution to the methanol is 50:50;

the eluent used in elution gradient 4 was pure methanol.

7. The method of manufacturing according to claim 1, wherein:

the stuffing used for the medium-low pressure preparation column in the step (4) is ODS-C ₁₈ Particles with the particle size range of 5-100 mu m;

the eluent is 5% -80% methanol water solution (v/v).

8. A natural dimeric catechin pigment produced according to the production process of any one of claims 1-7.

9. Use of a natural dimeric catechin as defined in claim 8 in the manufacture of a tea beverage.

10. The use according to claim 9, comprising:

the dimeric catechin pigment is added into the tea beverage with the addition amount of 20mg/L to 1g/L.