Natural dimeric catechin pigment, and preparation method and application thereof

Abstract

This invention provides a method for synthesizing novel natural pigments using phenotypic catechins. Various epicatechins, epigallocatechins, epicatechin gallate esters, and epigallocatechin gallate esters are used as substrates. These are mixed and subjected to a non-enzymatic oxidation reaction under humid and hot conditions to generate a new class of tea pigment compounds. The non-enzymatic oxidation products of the phenotypic catechins are then concentrated under reduced pressure, freeze-dried, extracted, and purified by column chromatography to obtain dimeric catechin pigments. The six novel dimeric catechin pigments obtained by this invention are synthesized using a simple method. The six novel dimeric catechin pigments obtained according to the method of this invention are orange-yellow or orange-red amorphous powders with good color-adjusting capabilities, and can be applied to coloring tea beverages and new-style tea drinks. The discovery of this type of natural pigment contributes to the research on coloring substances during the thermal processing of tea, the improvement of tea liquor color quality, and the development and application in the field of food coloring.

Description

Technical Field

[0001] This invention relates to the field of natural product chemical synthesis and separation and purification technology, specifically to a natural dimeric catechin pigment and its preparation method and application. Background Technology

[0002] Catechins are the main polyphenols in tea, possessing a distinct bitter and astringent taste, and are one of the main substances contributing to the overall flavor of tea. Furthermore, catechins exhibit strong biological activity, making them a key health-promoting active ingredient in tea. Heat processing (high-temperature drying or roasting) is a crucial step in tea production, not only reducing moisture content but also significantly improving flavor. High-temperature roasting deepens the color of the tea liquor, resulting in a brownish-yellow or even dark brown hue for the dried tea leaves. This is related to Maillard reactions and caramelization, but may also be due to non-enzymatic oxidation of polyphenols, forming more stable polymeric pigments, thus promoting the overall color of the tea liquor. These natural pigments are more suitable for development and application in food.

[0003] In recent years, studies on the transformation products of catechins using in vitro models or real tea matrices have shown that catechins mainly undergo isomerization, degradation, and polymerization reactions during thermal reactions. Current research primarily focuses on the transformation products of EC, EGC, EGCG, or other catechins in in vitro models using aqueous solutions or solid powders, but the temperature of these models is below 90℃. In 2003, Jack M. Miller's research group studied the transformation products of catechins using EC and C in aqueous solutions at room temperature (pH 6.5) 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 studied the transformation products of catechins by reacting EC and C in aqueous solution at room temperature (pH 6) for 24 hours, and identified some dehydrogenated dimers of catechins using LC-MS (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 studied the structural stability and color changes of EGCG using in vitro aqueous solution or solid model conditions at 85℃. They found that the color gradually deepened over time and identified two major EGCG dimers (polyester-type catechins 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 eight catechins (EC, C, EGC, GC, ECG, CG, EGCG, and GCG) in an in vitro aqueous solution model at 90℃ to study the transformation products of catechins. They also identified some catechin dimers, isomerization products, and degradation products 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.).

[0004] In 2019, Thomas Hofmann's research group studied catechin transformation products in vitro using catechin EC or C in alkaline solution and under 90°C heating conditions. They also identified some catechin dimer compounds through separation and purification (Daniel Germann, Timo D. Stark, and Thomas Hofmann. Formation and characterization of polyphenol-derived red chromophores: Enhancing the color of processed cocoapowders: Part 1 [J]. Journal of Agricultural and Food Chemistry, 2019). In 2020, Jean-Paul Vincken's research group used EC and EGC aqueous solution models to study the main products and solution color changes at different reaction times at 80℃, and identified some catechin dimers or trimers by LC-MS (Junfeng Tan., Wouter JCde Bruijn., Annemiek van Zadelhoff., et al. Browning of Epicatechin (EC) and Epigallocatechin (EGC) by Auto-Oxidation[J]. Journal of Agricultural and Food Chemistry, 2020.).

[0005] Researchers have successively studied the structural stability, transformation products, and color changes of catechins under in vitro heating conditions. However, the main polymerization products were identified primarily by LC-MS, but the reaction temperature was relatively low, the time for producing coloring substances was long, and the content was low. Furthermore, efficient synthesis, separation, and purification methods have not been established. Additionally, the structures of key coloring substances have not been isolated, purified, and identified, and their color characteristics have not been studied. This makes the separation of polymerized pigments from catechins after high-temperature heating extremely difficult, greatly limiting the development and application of this type of natural polymerized pigment in tea beverages, novel tea drinks, and food coloring. Summary of the Invention

[0006] In view of this, the present invention provides a natural dimer catechin pigment, its preparation method and application. The present invention uses in vitro synthesis, separation, purification and identification to obtain a natural dimer catechin pigment, which can promote the formation of red and yellow colors in tea soup, and promote the development and application of natural dimer catechin pigment in the fields of coloring tea beverages, new tea drinks and food coloring.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing natural dimer catechin pigment, comprising the following steps:

[0009] Step 1, Preparation of crude reaction product: Phenotype catechin, pH adjuster and water are mixed and subjected to non-enzymatic oxidation reaction under open and heated conditions to obtain crude reaction product containing dimer catechin pigment;

[0010] Step 2, extraction, concentration and crude separation: Water is added to the crude reaction product for redissolution, and then ethyl acetate is used for extraction to remove the aqueous phase, resulting in an ethyl acetate extract phase. This extract phase is then evaporated and concentrated to obtain a concentrated and dried ethyl acetate extract phase.

[0011] Step 3, ODS-C 18 Reverse chromatography column separation: The concentrated and dried ethyl acetate extract phase was reconstituted with 10% methanol-water (v / v, water containing 0.2% formic acid), and the reconstituted methanol-water solution was subjected to ODS-C chromatography. 18 Separation was performed using a reverse chromatography column, and different concentrations of methanol aqueous solution were sampled for gradient elution. The eluent was collected to obtain crude products rich in six dimeric catechin pigments or monomeric dimeric catechin pigments.

[0012] Step 4, medium-low pressure preparative column separation: The crude product rich in six dimer catechin pigments or monomeric dimer catechin pigments was separated using a medium-low pressure preparative column. Gradient elution was performed with a methanol-water solution containing 0.2% formic acid (v / v, water containing 0.2% formic acid). The eluent containing dimer catechin pigments was collected according to the position of the ultraviolet absorption signal peak. The collected eluent was dried under reduced pressure to obtain six compounds with a purity greater than 80%. Their structures are shown as Formula I (EGCG), Formula II (EGC), Formula III (EC), Formula IV (ECG), Formula V (EGC-ECG), and Formula VI (EGCG-EC), respectively. The compounds were then analyzed.

[0013]

[0014] Furthermore, the reaction substrate phenotypic catechin in step 1 is selected from any one or a mixture of four of EGCG, ECG, EGC, and EC.

[0015] Further, the mass of the water in step 1 is 50 to 1,000,000% of the phenotypic catechin mass.

[0016] Further, the pH adjuster in step 1 is an acid or a base, and the pH value of the non-enzymatic oxidation reaction is 3 to 12.

[0017] Furthermore, the heating temperature in step 1 is 20–200°C, and the non-enzymatic oxidation reaction time is 10–180 min.

[0018] Furthermore, the packing material used for separation in step 3 is ODS-C. 18 Particles, the ODS-C 18 The particles have a diameter range of 5–200 μm.

[0019] Further, step 3, the gradient elution, includes:

[0020] The eluent used in elution gradient 1 is 0.2% formic acid water (v / v, water contains 0.2% formic acid) and methanol, with a volume ratio of 0.2% formic acid water mixture to methanol of 90:10;

[0021] The eluent used in elution gradient 2 is 0.2% formic acid water (v / v, water containing 0.2% formic acid) and methanol, with a volume ratio of 75:25 between the 0.2% formic acid water mixture and methanol.

[0022] The eluent used in elution gradient 3 is 0.2% formic acid water (v / v, water containing 0.2% formic acid) and methanol, with a volume ratio of 50:50 between the 0.2% formic acid water mixture and methanol.

[0023] The eluent used in elution gradient 4 is pure methanol.

[0024] Furthermore, the packing material used in the medium-low pressure preparative column separation described in step 4 is ODS-C. 18 The particles, with a particle size range of 5–100 μm, were eluted using a gradient elution method with a 5%–80% methanol aqueous solution (v / v, water containing 0.2% formic acid).

[0025] Furthermore, the chromatographic column used in the detection method described in step 4 is C18. 18 The reverse-phase chromatographic column has two mobile phases: phase A and phase B. Phase A is a 0.1% (v / v) aqueous solution of formic acid (containing 0.2% formic acid in water), 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.

[0026] The present invention also discloses a natural dimeric catechin pigment prepared according to any of the above preparation methods.

[0027] The present invention also discloses the application of the above-mentioned natural dimer catechin pigment in the production of tea beverages.

[0028] Furthermore, the application includes:

[0029] Dimeric catechin pigment is added to tea beverages at a concentration of 20 mg / L to 1 g / L.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) This invention has low cost and high product purity.

[0032] The solvent used for extraction, ODS-C 18 The solvents used in reverse chromatography and reversed-phase chromatography can be recycled; the materials used in reversed-phase chromatography can be reused. The recycled solvents and reused separation materials ensure a low average separation cost.

[0033] (2) The preparation method of the present invention can realize the needs of large-scale production.

[0034] Raw material requirements are not high and costs are low; generally, commercially available analytical grade catechins are sufficient, facilitating large-scale preparation. Concentration and extraction operations are simple; separation is achieved using ODS-C. 18 Rough fractionation by reverse chromatography column, this ODS-C 18 Reverse phase chromatography column packing can be packed into medium-pressure column chromatography systems, can be reused, has a large separation capacity, and is easy to scale up for production; further separation using a reverse phase chromatography column after coarse separation is also very suitable for large-scale production. Attached Figure Description

[0035] Figure 1 This refers to the separation step of dimeric catechin pigment in Example 1;

[0036] Figure 2 The mass spectrum for the separation of EGCG dimer catechin pigment in Example 1;

[0037] Figure 3 The mass spectrum for the separation of dicatechin pigments from EGC in Example 2;

[0038] Figure 4 The mass spectrum for the preparation and separation of dicatechin pigment from EC in Example 3;

[0039] Figure 5 The mass spectrum for the separation of dicatechin pigments from ECG in Example 4;

[0040] Figure 6 This is the mass spectrum of the separation of dicatechin pigments from EGC-ECG in Example 5;

[0041] Figure 7 The mass spectrum for the preparation and separation of dicatechin pigments from EGCG-EC in Example 6;

[0042] Figure 8 Here are schematic diagrams of the structures of the six dimeric catechin pigments in Experimental Example 2;

[0043] Figure 9 This is a comparison chart of the tea liquor color before and after the addition of the six dimeric catechin pigments in Example 1;

[0044] Figure 10 The image shows the color difference of the tea liquor before and after the addition of the six dimeric catechin pigments in Example 1; the numbers a, b, c, and d in the image indicate significance (P < 0.05). Detailed Implementation

[0045] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0046] This invention involves mixing phenotypic catechins, a pH adjuster, and water, and then subjecting the mixture to a non-enzymatic oxidation reaction under open and heated conditions to obtain a polymer product.

[0047] In this invention, the purity of the phenotypic catechin is preferably ≥95% independently.

[0048] In this invention, during the non-enzymatic oxidation reaction, the mass of water is 50-1,000,000% of the phenotypic catechin mass, preferably 100-100,000%.

[0049] In this invention, the pH adjuster is an acid or a base. The acid is preferably a hydrochloric acid solution with a concentration of 0.02 mol / L, and the base is preferably a sodium hydroxide solution with a concentration of 0.02 mol / L. The pH value of the non-enzymatic oxidation reaction is 3 to 12.

[0050] In this invention, the method of mixing phenotypic catechin mixtures or monomeric catechins with water includes: sequential vortex mixing and ultrasonic mixing; the vortex mixing rate is 500 r / min, the time is 1 to 3 min, preferably 2.5 min; the ultrasonic mixing power is 200 W, the frequency is 40 kHz, the time is 1 to 3 min, preferably 2.5 min.

[0051] This invention does not specifically limit the method of opening; any method that achieves the opening effect is acceptable. In embodiments of this invention, the open reaction apparatus is a test tube, beaker, or glass bottle. In this invention, the temperature of the non-enzymatic oxidation reaction is 20–200°C, preferably 120–150°C; the reaction time is 10–180 min, preferably 30–120 min, and more preferably 80–100 min. After high-temperature heating, the phenotypic catechin yields a crude reaction product containing dimer catechin pigment. This product is redissolved in water, then extracted three times with an equal volume of ethyl acetate. The aqueous phase is removed to obtain the ethyl acetate extract phase. The dimer catechin pigment is detected by high-performance liquid chromatography to confirm its presence in the ethyl acetate extract phase, and then concentrated by rotary evaporation.

[0052] In this invention, the chromatographic column used for the high-performance liquid chromatography detection is preferably C18. 18 The reverse-phase chromatographic column used a mobile phase consisting of phase A and phase B. Phase A was a 0.2% formic acid aqueous solution (v / v, water containing 0.2% formic acid), and phase B was methanol. The flow rate was 1 mL / min, the detection wavelength was 278 nm, the detector was a DAD, the column temperature was 30 °C, and the elution conditions were as follows: 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.

[0053] After obtaining the ethyl acetate extract phase containing dimeric catechin pigment, further purification by column chromatography and rotary evaporation concentration were performed to obtain dimeric catechin pigment with the structures shown in formulas I to VI. The rotary evaporation concentration process is not specifically limited in this invention and can be carried out according to procedures well known in the art.

[0054] In this invention, the column chromatography purification process includes:

[0055] The concentrated and dried ethyl acetate extract phase was redissolved in 10% methanol-water (v / v, water containing 0.2% formic acid) to obtain the separated product, which was then subjected to ODS-C analysis. 18 The reverse column chromatography separation includes four elution gradients, yielding four elution fractions.

[0056] In this invention, the packing material used for column chromatography separation is preferably ODS-C. 18 The column is a reverse-phase glass column with dimensions of 8 × 40 cm. The chromatography separation preferably includes four elution gradients. Elution gradient 1 uses a 0.2% (v / v) formic acid-water mixture and methanol, with a volume ratio of 90:10, and a volume of 250–500 mL, preferably 350 mL. Elution gradient 2 uses a 0.2% (v / v) formic acid-water mixture and methanol, with a volume ratio of 75:25, and a volume of 25 mL. The eluent used in elution gradient 3 is a mixture of formic acid and water with a volume concentration of 0.2% and methanol, wherein the volume ratio of the formic acid and water mixture to methanol is 50:50, and the amount used is 250-500 mL, preferably 350 mL; the eluent used in elution gradient 4 is a mixture of formic acid and water with a volume concentration of 0.2% and methanol, wherein the volume ratio of the formic acid and water mixture to methanol is 0:100, and the amount used is 250-500 mL, preferably 350 mL.

[0057] After obtaining four elution fractions, high-performance liquid chromatography (HPLC) was used to detect them and determine the fraction containing the dimeric catechin pigment. In this invention, the HPLC detection conditions are the same as those described above and will not be repeated here.

[0058] This invention separates the components containing dimeric catechin pigments using a medium-low pressure preparative column to obtain dimeric catechin pigments with structures shown in formulas I to VI.

[0059] In this invention, the chromatographic column used for medium- and low-pressure preparative column separation is an ODS-C column. 18 The reverse-phase chromatographic column uses a mobile phase consisting of phase A and phase B. Phase A is a 0.2% (v / v) aqueous solution of formic acid, and phase B is methanol. The elution conditions for separation using a medium-low pressure preparative column are as follows: 0–5 min, 25%–42% aqueous methanol solution; 5–10 min, 42%–80% aqueous methanol solution; 10–18 min, 80%–25% aqueous methanol solution; 18–23 min, 25% aqueous methanol solution.

[0060] In this invention, after obtaining dimeric catechin pigments with structures shown in formulas I to VI, the purity of six monomeric compounds is determined by mass spectrometry. The detection method for determining the purity of the dimeric catechin pigments is high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The HPLC detection method is similar to the method described above for ODS-C... 18 The conditions for determining the presence of dimeric catechin pigments by high-performance liquid chromatography (HPLC) detection of fractions separated by reverse chromatography are the same and will not be repeated here. In this invention, the purity identification of dimeric catechin pigments having structures shown in formulas I to VI is preferably performed using UPLC-MS. The preferred detection flow rate is 0.25 mL / min, and the mobile phase includes phase A and phase B: phase A is a 0.1% (v / v) formic acid aqueous solution (water contains 0.1% formic acid), and phase B is acetonitrile. The elution conditions are as follows: 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 aqueous solution; 19–21 min, 25% methanol aqueous solution to 2% methanol aqueous solution; 21–25 min, 2% methanol aqueous solution.

[0061] In this invention, the structural formula of the dicatechin pigment having the structures shown in formulas I to VI is as follows:

[0062]

[0063]

[0064] This invention also provides the application of the dimeric catechin natural pigment described in the above-described technical solutions or the dimeric catechin natural pigment prepared by the above-described preparation methods in the field of coloring tea beverages, new-style tea drinks, or food coloring. Preferably, this invention uses the dimeric catechin natural pigment as a coloring agent. In this invention, the dimeric catechin natural pigment is an orange-yellow or orange-red amorphous powder. Preferably, this invention uses the dimeric catechin natural pigment as a tea beverage coloring agent, adding it to tea drinks to increase the yellowness or redness of the product, significantly changing the product's color.

[0065] In the following examples, the phenotypic catechins were all of analytical grade, with a purity of ≥95%.

[0066] Examples 1-7 below are examples of the synthesis, separation, purification, identification, and color characteristics of dimeric catechin pigments. The prepared polymer products were directly dissolved in methanol and detected by high performance liquid chromatography or UPLC-MS.

[0067] Example 1

[0068] Isolation, purification and identification of dicatechin pigment (Formula I)

[0069] (1) Weigh 45.8 mg of EGCG (analytical grade standard, purity 95%) into a test tube, add 1 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve the EGCG in 3 mL of pure water to obtain a crude reaction solution containing dimeric catechin pigment. Repeat the preparation of multiple reaction solutions to make the final amount of EGCG 4.58 g, and combine the crude reaction solutions.

[0070] (2) Add an equal volume of ethyl acetate to the crude reaction solution, shake well, let stand for 5 min, and extract to obtain an aqueous phase and an ethyl acetate phase. Repeat the above steps to extract the aqueous phase three times in total. Combine the ethyl acetate phases obtained from the three extractions and evaporate and concentrate to obtain a crude product rich in dimer catechin pigment.

[0071] (3) The crude product rich in dimeric catechin pigment was reconstituted with 5 mL of 10% methanol-water (v / v, water containing 0.2% formic acid). The reconstituted solution was mixed thoroughly and then passed through ODS-C. 18 Separation was performed using a reverse chromatography column, with elution sequentially using a mixture of 0.2% formic acid and water (v / v, water containing 0.2% formic acid) and methanol at volume ratios of 90:10, 75:25, 50:50, and 0:100. Each elution gradient was 350 mL, and the eluent was collected to obtain four fractions.

[0072] (4) Component 3 was separated by a medium-low pressure preparative column, using a gradient elution with 5%–80% methanol-water (water containing 0.2% formic acid) solution. The eluent was collected based on the position of the UV absorption signal peak. The components obtained by gradient elution were detected and analyzed by LC-DAD-MS to obtain the component containing dimeric catechin pigment (Formula I). ​​The separation steps are as follows: Figure 1 As shown, its purity test mass spectrum is as follows: Figure 2 As shown. Finally, after concentrating and drying the component, an orange-red amorphous powder with a purity greater than 80% was obtained.

[0073] Example 2

[0074] Isolation, purification and identification of dicatechin pigment (Formula II)

[0075] (1) Weigh 30.6 mg of EGC (analytical grade standard, purity 95%) into a test tube, add 1 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve with 3 mL of pure water to obtain a crude reaction solution containing dimeric 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.

[0076] (2) Extraction and ODS-C analysis of the crude reaction solution. 18 The steps of separation by reverse chromatography, separation by medium- and low-pressure preparative column, and detection by LC-DAD-MS were all the same as those in (2), (3), and (4) of Example 1. The mass spectrum of the component containing the dimeric catechin pigment (Formula II) is shown below. Figure 3 As shown. Finally, after concentrating and drying the component, an orange-yellow amorphous powder with a purity greater than 80% was obtained.

[0077] Example 3

[0078] Isolation, purification and identification of dicatechin pigment (Formula III)

[0079] (1) Weigh 29.0 mg EC (analytical grade standard, purity 95%) into a test tube, add 1 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve the EC in 3 mL of pure water to obtain a crude reaction solution containing dimeric catechin pigment. Repeat the preparation of multiple reaction solutions to make the final amount of EC 2.90 g, and combine the crude reaction solutions.

[0080] (2) Extraction and ODS-C analysis of the crude reaction solution. 18 The steps of separation by reverse chromatography, separation by medium- and low-pressure preparative column, and detection by LC-DAD-MS were all the same as those in (2), (3), and (4) of Example 1. The mass spectrum of the component containing the dimeric catechin pigment (Formula III) is shown below. Figure 4 As shown. Finally, after concentrating and drying the component, an orange-yellow amorphous powder with a purity greater than 80% was obtained.

[0081] Example 4

[0082] Isolation, purification and identification of dimeric catechin pigment (Formula IV)

[0083] (1) Weigh 44.2 mg of ECG (analytical grade standard, purity 95%) into a test tube, add 1 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tube to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve the ECG with 3 mL of pure water to obtain a crude reaction solution containing dimeric catechin pigment. Repeat the preparation of multiple reaction solutions so that the final amount of ECG used is 4.42 g, and combine the crude reaction solutions.

[0084] (2) Extraction and ODS-C analysis of the crude reaction solution. 18 The steps of separation by reverse chromatography, separation by medium- and low-pressure preparative column, and detection by LC-DAD-MS were all the same as those in (2), (3), and (4) of Example 1. The mass spectrum of the component containing the dimeric catechin pigment (Formula IV) is shown below. Figure 5 As shown. Finally, after concentrating and drying the component, an orange-red amorphous powder with a purity greater than 80% was obtained.

[0085] Example 5

[0086] Isolation, purification and identification of dicatechin pigment (Formula V)

[0087] (1) Weigh 44.2 mg ECG and 30.6 mg EGC (analytical grade standard, purity 95%) into test tubes, add 1 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tubes to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve with 3 mL of pure water to obtain a crude reaction solution containing dimeric 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.

[0088] (2) Extraction and ODS-C analysis of the crude reaction solution. 18 The steps of separation by reverse chromatography, separation by medium- and low-pressure preparative column, and detection by LC-DAD-MS were all the same as those in (2), (3), and (4) of Example 1. The mass spectrum of the component containing the dimeric catechin pigment (Formula V) is shown below. Figure 6 As shown. Finally, after concentrating and drying the component, an orange-red amorphous powder with a purity greater than 80% was obtained.

[0089] Example 6

[0090] Isolation, purification and identification of dicatechin pigment (Formula VI)

[0091] (1) Weigh 29.0 mg EC and 45.8 mg EGCG (analytical grade standard, purity 95%) into test tubes, add 1 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tubes to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve with 3 mL of pure water to obtain a crude reaction solution containing dimeric 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.

[0092] (2) Extraction and ODS-C analysis of the crude reaction solution. 18 The steps of separation by reverse chromatography, separation by medium- and low-pressure preparative column, and detection by LC-DAD-MS were all the same as those in (2), (3), and (4) of Example 1. The mass spectrum of the component containing the dimeric catechin pigment (Formula VI) is shown below. Figure 7 As shown. Finally, after concentrating and drying the component, an orange-red amorphous powder with a purity greater than 80% was obtained.

[0093] Experimental Example 1

[0094] Isolation, purification and identification of dimeric catechin pigments (Formulas I to VI)

[0095] (1) Weigh 29.0 mg EC, 30.6 mg EGC, 44.2 mg ECG, and 45.8 mg EGCG (analytical grade standard, purity 95%) into test tubes, add 3 mL of pure water, vortex for 3 minutes, sonicate for 3 minutes, and after complete dissolution, transfer the test tubes to an oil bath at 150°C and heat for 120 minutes. After the reaction is complete, redissolve with 3 mL of pure water to obtain a crude reaction solution containing dimer 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.

[0096] (2) Extraction and ODS-C analysis of the crude reaction solution. 18 The steps of separation by reverse chromatography, separation by medium- and low-pressure preparative column, and detection by LC-DAD-MS were all the same as those in (2), (3), and (4) of Example 1. The mass spectrum of the component containing the dimeric catechin pigments (Formulas I to VI) is shown below. Figures 2-7 As shown. Finally, after concentrating and drying the component, an orange-yellow or orange-red amorphous powder with a purity greater than 80% was obtained.

[0097] Experimental Example 2

[0098] Verification of the properties of the six dimeric catechin pigments prepared in Examples 1-6

[0099] Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) were used to identify the six isolated monomeric compounds by mass spectrometry and perform NMR analysis; mass spectrometry detection was performed using C1... 18 A reverse-phase chromatographic column was used. Mobile phase A consisted of 0.1% formic acid in water (v / v, water containing 0.2% formic acid), and mobile phase B consisted of acetonitrile. The flow rate was 0.25 mL / min, the column temperature was 40℃, and the elution gradient was 0–25 min, 2–25% B. The structures of the six dimeric catechin pigments and their C-positions are shown below. Figure 8 As shown:

[0100] The characteristics of the six dimeric catechin pigments obtained from the test are as follows:

[0101] (1) The six dimeric catechin pigments are orange-yellow or orange-red amorphous powders;

[0102] (2) UV detection wavelengths of six dimeric catechin pigments λmax (nm): 278 and 490;

[0103] (3) LC-MS (negative ion mode):

[0104] 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).

[0105] (4) The nuclear magnetic resonance spectroscopy data are as follows:

[0106] Table 1. 1H NMR and 1C NMR data of six dimeric catechin pigments

[0107]

[0108]

[0109] Continued table

[0110]

[0111]

[0112] Note: 1 H NMR and 13C NMR was performed at 600 MHz. δ values ​​are in ppm. The solvent was deuterated methanol. "-" in the table indicates no data is available for that location. s: singlet; m: multiplet; d: doublet; dd: quartet.

[0113] As shown in Table 1, the present invention successfully synthesized, isolated, purified and identified six dimeric catechin pigments with structures shown in Formulas I to VI.

[0114] Application examples

[0115] Application of six dimeric catechin pigments

[0116] (1) Weigh 1g of dry tea, steep it in 50mL of boiling water for 5 minutes, pour the tea into the tasting bowl, cool it to room temperature, and repeat the preparation of 18 tea infusions.

[0117] (2) Addition Group 1: Weigh 4 mg of each of the six dimeric catechin pigments prepared separately and add them to three portions of tea infusion, stirring well. Addition Group 2: Weigh 10 mg of each of the six dimeric catechin pigments prepared separately and add them to three portions of tea infusion, stirring well. Addition Group 3: Weigh 50 mg of each of the six dimeric catechin pigments prepared separately and add them to three portions of tea infusion, stirring well. Comparison of tea infusion color before and after addition: Figure 9 As shown in the figure. Color difference analysis was performed on three groups of dimeric catechin pigments with different addition amounts, as shown in the figure. Figure 10 As shown, the a value (redness) and b value (yellowness) of the tea soup increased significantly after adding six kinds of dimeric catechin pigments.

[0118] according to Figure 9 and Figure 10 The comparative results showed that the addition of six novel natural pigments (dimeric catechin pigments) to tea soup could significantly change the color of the tea soup, increasing the redness and yellowness of the tea soup, with an addition amount of 20 mg / L to 1 g / L.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a natural dimeric catechin pigment, comprising the following steps: (1) Preparation of crude reaction product: Phenotype catechin, pH adjuster and water are mixed and subjected to non-enzymatic oxidation reaction under open and heated conditions to obtain crude reaction product containing dimer catechin pigment. The phenotypic catechin is selected from any one or a mixture of four phenotypic catechins from EGCG, ECG, EGC and EC. The mass of water is 50-1,000,000% of the mass of phenotypic catechin. The pH adjuster is an acid or a base. The pH value of the non-enzymatic oxidation reaction is 3-12, the reaction temperature is 120-150℃, and the reaction time is 80-120 min. (2) Extraction, concentration and crude separation: The crude reaction product is redissolved in water, then extracted with ethyl acetate and the aqueous phase is removed to obtain the ethyl acetate extract phase. The extract phase is then evaporated and concentrated to obtain the concentrated and dried ethyl acetate extract phase. (3) ODS-C18 reverse chromatography column separation: The concentrated and dried ethyl acetate extract phase was reconstituted with methanol-water at a volume ratio of 10%, wherein the methanol-water contained 0.2% formic acid. The reconstituted methanol-water solution was then separated by an ODS-C18 reverse chromatography column. Methanol-water solutions of different concentrations were sampled for gradient elution, and the eluent was collected to obtain crude product rich in six dimer catechin pigments or monomeric dimer catechin pigments. (4) Separation by medium- and low-pressure preparative column: The crude products rich in six dimer catechin pigments or monomeric dimer catechin pigments were separated using a medium- and low-pressure preparative column. Gradient elution was performed using a methanol-water solution containing 0.2% formic acid. The eluent containing dimer catechin pigments was collected based on the position of the ultraviolet absorption signal peak. The collected eluent was concentrated and dried to obtain six compounds with a purity greater than 80%, and their structures are shown as Formula I (EGCG), Formula II (EGC), Formula III (EC), Formula IV (ECG), Formula V (EGC-ECG), and Formula VI (EGCG-EC), respectively. (Formula I); (Formula II); (Formula III); (Formula IV); (Form V); (Form VI).

2. The preparation method according to claim 1, wherein: The packing material used for separation in step (3) is ODS-C18 particles, and the ODS-C18 particles have a particle size range of 5~200μm.

3. The preparation method according to claim 1, wherein: Step (3) of gradient elution includes: The eluent used in elution gradient 1 is a mixture of 0.2% formic acid water and methanol, with a volume ratio of 90:10 between the 0.2% formic acid water mixture and methanol. The eluent used in elution gradient 2 is a mixture of 0.2% formic acid water and methanol, with a volume ratio of 75:25 between the 0.2% formic acid water mixture and methanol. The eluent used in elution gradient 3 is a mixture of 0.2% formic acid water and methanol, with a volume ratio of 50:50 between the 0.2% formic acid water mixture and methanol. The eluent used in elution gradient 4 is pure methanol.

4. The preparation method according to claim 1, wherein: The packing material used in the medium and low pressure preparation column in step (4) is ODS-C18 particles with a particle size range of 5~100μm; The eluent is a methanol aqueous solution with a volume ratio of 5% to 80%.

5. A natural dimeric catechin pigment prepared by any one of claims 1 to 4.

6. The application of the natural dimer catechin pigment according to claim 5 in the production of tea beverages.

7. The application according to claim 6, comprising: Dimeric catechin pigment is added to tea beverages at a concentration of 20 mg / L to 1 g / L.

Images