Process for the preparation of gallolylated procyanidine dimers

Abstract

The present application relates to the technical field of plant functional component degradation and synthesis, and particularly relates to a preparation method of grape seed galloylated procyanidin dimer (B2-3'-O-gallate). Grape seed procyanidin polymer (PPCs) is used as raw material, and acid is used as catalyst to degrade grape seed procyanidin polymer (PPCs) into an extension unit containing carbon cation, and the extension unit is combined with nucleophilic reagent epicatechin gallate (ECG) to generate galloylated procyanidin dimer B2-3'-O-gallate. The preparation process is simple and feasible, and can quickly and massively synthesize and prepare the rare galloylated procyanidin dimer B2-3'-O-gallate, and provides a new way for preparation of rare procyanidins.

Description

Technical Field

[0001] This invention relates to the field of plant functional component degradation and synthesis technology, and in particular to a method for preparing galloylated proanthocyanidin dimer (B2-3'-O-gallate) from grape seeds. Background Technology

[0002] Proanthocyanidins are polyphenols polymerized from different flavan-3-ol structural units, possessing a variety of biological activities. Oligomeric proanthocyanidins, in particular, have received widespread attention from the international academic community in recent years due to their potential effects on human health, and have remained a research hotspot in the fields of medicine, health products, food, and cosmetics. Galloylated proanthocyanidin oligomers are proanthocyanidins with galloyl groups introduced into their structure. Compared to proanthocyanidins without galloyl groups, these oligomers exhibit more significant biological activities, demonstrating outstanding performance in antioxidation, anticancer, and anti-inflammation. However, the content of galloylated proanthocyanidin oligomers in nature is low. For example, the most abundant galloylated proanthocyanidin dimer in grape seed, B2-3'-O-gallate (B2-3'-G), accounts for less than 0.01% of the total oligomers. This is a major reason why, despite their high activity, galloylated proanthocyanidins are difficult to study in depth.

[0003] Oligomeric proanthocyanidins constitute only a small portion of the natural population; the majority exist as proanthocyanidin polymers, accounting for up to 65%. Due to their large molecular weight and complex molecular structure, proanthocyanidin polymers are difficult for the human body to absorb and utilize through cell membranes, resulting in extremely low bioavailability and often being treated as waste. This leads to a severe waste of proanthocyanidin resources. Therefore, how to efficiently utilize these wastes and transform them into bioactive small molecules has become a key issue in solving the rational utilization of proanthocyanidin resources.

[0004] Acid-catalyzed degradation of proanthocyanidins is a method used to identify the molecular structure of proanthocyanidins. Under acidic conditions, nucleophiles are used to degrade proanthocyanidins into a large number of haploids and a small number of oligomers. In recent years, our research group has innovatively introduced catalytic degradation methods into the preparation of proanthocyanidin oligomers, obtaining nearly 20 kinds of oligomeric proanthocyanidin monomers. However, the yield is limited, only a few milligrams, or even less than 1 mg. Although galloacylated proanthocyanidins can be prepared, the yield is extremely low. This is mainly because the initial degradation is non-targeted and aimless, and the products are mixtures of dozens or even more oligomers, making it difficult to increase the yield. Moreover, there are currently very few degradation and preparation methods specifically for galloacylated proanthocyanidins, leaving the field of large-scale preparation almost unexplored. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing galloylated proanthocyanidin dimers (B2-3'-O-gallate) from grape seeds to address the shortcomings of existing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing galloacylated proanthocyanidin dimers involves using grape seed proanthocyanidin polymers (PPCs) as raw materials, using acid as a catalyst to degrade the grape seed proanthocyanidin polymers (PPCs) into extended units containing carbocations, and then combining them with the nucleophile ECG to generate galloacylated proanthocyanidin dimers B2-3'-O-gallate.

[0008] The epicatechin gallate (ECG) and grape seed proanthocyanidin polymers (PPCs) are in a mass ratio of 0.5:1 to 4:1, preferably 3:1.

[0009] To elaborate

[0010] (1) Grape seed proanthocyanidin polymer and nucleophilic reagent epicatechin gallate were dissolved in acidified methanol to prepare an initial solution;

[0011] (2) The initial solution is degraded under closed conditions at 30-70℃ (preferably 55℃) with shaking for 0.5-2.5h (preferably 1.5h), and then the reaction is terminated by ice bath (0℃);

[0012] (3) The reaction solution produced by (2) degradation was repeatedly extracted with ethyl acetate. The resulting extract was then purified by passing it through a C18 solid-phase extraction column and a preparative reversed-phase high-performance liquid chromatography column to obtain the monomer compound B2-3'-O-gallate with a high extraction rate.

[0013] The methanol acidification step (1) is a methanol solution that has been treated with hydrochloric acid.

[0014] In step (3), the reaction solution obtained in (2) is repeatedly extracted with ethyl acetate, the extraction layers are combined, dried with anhydrous sodium sulfate, the ethyl acetate layer is concentrated under reduced pressure, and freeze-dried into powder, which is the ethyl acetate layer powder containing the degradation product oligomeric proanthocyanidins B2-3'-O-gallate; wherein, the volume ratio of the reaction solution to the ethyl acetate in (2) is 1:1, and the extraction is performed 2-3 times.

[0015] The ethyl acetate layer powder containing the degradation product oligomeric proanthocyanidins B2-3'-O-gallate obtained after repeated extraction is dissolved in water, and then eluted with ethyl acetate using a C18 solid-phase extraction column. The eluent is collected for later use. The volume ratio of ethyl acetate to the solution of the ethyl acetate layer powder containing the degradation product oligomeric proanthocyanidins B2-3'-O-gallate (2 mg / ml) is 3-5:1.

[0016] The collected fraction obtained after purification by a C18 solid-phase extraction column was dissolved in water and then separated by a preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain the monomer compound B2-3'-O-gallate. The semi-preparative HPLC was used with a reversed-phase C18 column (250×10mm, 5μm); the mobile phase was 25% methanol:water (v / v) with isocratic elution at a flow rate of 3.5mL / min; the column temperature was 30℃; the detection wavelength was 280nm; and the injection volume was 2mL.

[0017] During the above degradation reaction, the temperature should not be too high and the reaction time should not be too long. The structure of the degradation product B2-3'-O-gallate is unstable at high temperatures and is easily destroyed. If the reaction temperature is too high, the polymerization reaction of proanthocyanidins will also increase, resulting in a decrease in the amount of B2-3'-O-gallate generated. Therefore, it is necessary to carry out the reaction at a reasonable temperature. At the same time, prolonged heating under acidic conditions may destroy the structure of proanthocyanidin B2-3'-O-gallate, leading to a decrease in the amount of degradation product generated.

[0018] Advantages of this invention:

[0019] This invention innovatively uses epicatechin gallate (ECG) as a nucleophile to bind with a large number of carbocation-containing extended units generated from the degradation of proanthocyanidin polymers (PPCs) in grape seeds, synthesizing a highly active galloylated proanthocyanidin dimer B2-3'-O-gallate. The principle is described in [link to invention]. Figure 1 The B2-3'-O-gallate degradation method proposed in this invention can obtain high yields of B2-3'-O-gallate, enabling the development and utilization of PPC waste resources, and providing new ideas and methods for the research of highly active galloylated proanthocyanidins. Attached Figure Description

[0020] Figure 1 The principle of epicatechin gallate degradation and conversion of grape seed polymeric proanthocyanidins is provided in the embodiments of the present invention.

[0021] Figure 2 The image provided in this embodiment of the invention shows the spectrum of grape seed polymeric proanthocyanidins.

[0022] Figure 3The effect of degradation time on the amount of B2-3'-O-gallate(Y) generated, as provided in the embodiments of the present invention.

[0023] Figure 4 The effect of degradation temperature on the amount of B2-3'-O-gallate(Y) generated, as provided in the embodiments of the present invention.

[0024] Figure 5 The effect of acidity on the amount of B2-3'-O-gallate(Y) produced in the embodiments of the present invention

[0025] Figure 6 The effect of material ratio on the amount of B2-3'-O-gallate(Y) generated in the embodiments of the present invention

[0026] Figure 7A Three-dimensional response surface plot of the effects of reaction time, temperature, and material ratio on the amount of B2-3'-O-gallate(Y) produced.

[0027] Figure 7B Three-dimensional response surface plot of the effects of reaction time, temperature, and material ratio on the amount of B2-3'-O-gallate(Y) produced.

[0028] Figure 7C The three-dimensional response surface plot shows the effects of reaction time, temperature, and material ratio on the amount of B2-3'-O-gallate(Y) produced.

[0029] Figure 8 The spectrum of the obtained B2-3'-O-gallate is provided for an embodiment of the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments:

[0031] The present invention specifically targets galloylated proanthocyanidin dimers (B2-3'-O-gallate), preferably using epicatechin gallate (ECG) as a novel nucleophile, and discloses a method for large-scale preparation of B2-3'-O-gallate using waste polymerized proanthocyanidins as raw materials, and the obtained galloylated proanthocyanidin dimers (B2-3'-O-gallate) have higher activity.

[0032] Optimization of reaction conditions for degradation process:

[0033] Example 1

[0034] (1) Accurately transfer 16.5 mL of 37% hydrochloric acid solution into a 1 L volumetric flask, add methanol to the mark, i.e., 0.01 M hydrochloric acid methanol solution.

[0035] (2) Column chromatography separation of grape seed proanthocyanidins: Weigh an appropriate amount of grape seed extract, dissolve it in distilled water, and prepare a 30 mg / mL grape seed proanthocyanidin loading solution. Measure 100 mL of the loading solution and pass it through a YMC ODS-A-HG (200 x 30 mm id, 25-40 μm) chromatography column pretreated with deionized water. First, elute with 300 mL of pH 7.0 deionized water to remove excess phenolic acids and other interfering components. Then, elute with 200 mL of ethyl acetate to remove haploid proanthocyanidins (CATs) and oligomeric proanthocyanidins (OPCs). Finally, elute with 300 mL of methanol to remove polymeric proanthocyanidins (PPCs). Remove the organic solvent from PPCs by rotary evaporation at 25 °C, freeze-dry, and store the powder at -20 °C for later use (see [link to relevant documentation]). Figure 2 ).

[0036] (3) Weigh grape seed polymeric proanthocyanidins (PPCs) and epicatechin gallate (ECG) powders, dissolve them in 0.01M hydrochloric acid methanol solution, with a PPCs to ECG mass concentration ratio of 1:1, and a final PPCs concentration of 12 g / L. The solution was reacted in a sealed container at 30℃ for different reaction times (0.5, 1, 1.5, 2, 2.5 h), and then the reaction was terminated in an ice bath (0℃) to obtain the degradation reaction solution.

[0037] (4) The above reaction solutions were rotary evaporated at 30°C to obtain degradation reaction product powder (see Figure 3 ).

[0038] Depend on Figure 3 The results of the single-factor experiment showed that the amount of B2-3'-G generated gradually increased with the increase of degradation time, but the amount of B2-3'-G generated gradually decreased after the time exceeded 1 hour. This may be because the structure of proanthocyanidin B2-3'-G was destroyed by prolonged heating under acidic conditions, resulting in a decrease in the amount of degradation products generated. Therefore, a reaction time of 1 hour was selected as the optimal degradation time.

[0039] Example 2

[0040] The B2-3'-O-gallate was extracted according to the method in Example 1, with the following differences:

[0041] Step (3): Grape seed proanthocyanidins and epicatechin gallate were mixed at a mass ratio of 1:1 and dissolved in acidified methanol to prepare an initial solution. The solution was reacted at different temperatures (30, 40, 50, 60, 70°C) for 0.5 h for degradation treatment, and then the reaction was terminated in an ice bath (0°C) to obtain the degradation reaction solution. The rest is the same as in Example 1, and the results are shown in […]. Figure 4 .

[0042] Depend on Figure 4The results of the single-factor experiment show that the amount of B2-3'-G generated increases with increasing temperature. The maximum amount of B2-3'-G is generated at 50℃. This may be because the structure of the degradation product B2-3'-G is unstable at high temperatures and is easily destroyed. Therefore, 50℃ was selected as the optimal degradation temperature for this reaction.

[0043] Example 3

[0044] The B2-3'-O-gallate was extracted according to the method in Example 1, with the following differences:

[0045] Step (1) prepare hydrochloric acid methanol solutions with different hydrochloric acid concentrations (0.005, 0.01, 0.02, 0.05, 0.1M) and dissolve them; then, according to step (3), weigh the above grape seed proanthocyanidin polymer and epicatechin gallate powder, and dissolve them with different hydrochloric acid methanol solutions to obtain the initial solution.

[0046] The above solution was subjected to degradation treatment by reacting in a sealed environment at 30°C for 0.5 h, and then the reaction was terminated by an ice bath (0°C) to obtain the degradation reaction solution. The rest was the same as in Example 1, and the results are shown below. Figure 5 .

[0047] Depend on Figure 5 The results of the single-factor experiment show that the amount of B2-3'-G generated is not significantly affected by the concentration of hydrochloric acid. The optimal amount of B2-3'-G is generated when the concentration of hydrochloric acid is 0.01N. Therefore, 0.01N hydrochloric acid is selected as the optimal acid concentration for this reaction.

[0048] Example 4

[0049] The B2-3'-O-gallate was extracted according to the method in Example 1, with the following differences:

[0050] Step (3) Grape seed polymeric proanthocyanidins and epicatechin gallate were mixed in different mass ratios (1:2, 1:1, 2:1, 3:1, 4:1), and then reacted at 30°C for 0.5 h for degradation treatment. The reaction was then terminated by ice bath (0°C) to obtain the degradation reaction solution. The rest was the same as in Example 1, and the results are shown in [see Example 1]. Figure 6 .

[0051] Depend on Figure 6 The results of the single-factor experiment showed that as the mass concentration ratio of epicatechin gallate to polymeric proanthocyanidins increased, the amount of B2-3'-G generated gradually increased. When the mass concentration ratio increased to 3:1, the amount of B2-3'-G generated tended to level off. Therefore, in order to save resources, the optimal mass concentration ratio was selected as 3:1.

[0052] Example 5

[0053] Similar to Example 1, based on Examples 1-4 above, the content (Y) of proanthocyanidin B2-3'-O-gallate was used as the evaluation index, and the reaction time (X1), reaction temperature (X2), and the mass concentration ratio of the reactant epicatechin gallate to the proanthocyanidin polymer (X3) were used as the main factors to be investigated. A three-factor, three-level degradation process optimization experiment was conducted using Box-Behnken Design (BBD). The results are shown in Tables 1-2. Figure 7A -C.

[0054] Box-Benhnken Design Model Predictions and Statistical Analysis Results

[0055] Using Design Expert V 8.0.6.1 software, a quadratic multiple regression model was performed on the experimental results. The quadratic multiple regression model equation for the amount of proanthocyanidin B2-3'-O-gallate generated (Y) on degradation time (X1), degradation temperature (X2), and reactant ratio (X3) is as follows:

[0056] Y=-3.392+1.554X1+0.1651X2+0.4672X3-2.666×10 -3 X1X2+4.444×10 -3 X1X3+1.000×10 -3 X2X3-0.4702X1 2 -1.495×10 -3 X2 2 -0.09755X3 2

[0057] Analysis of variance and significance were performed on the experimental results, and the results are shown in Table 2. The model's p-value < 0.05 and the lack-of-fit p-value > 0.05 indicate that the model is significantly differentiable, with effective goodness of fit and reliability, and can accurately predict the optimal degradation process. The coefficient of determination R² and the correction coefficient R² are 0.9445 and 0.8731, respectively, indicating that the experimental and predicted results of the model are in good agreement. The coefficient of variation is less than 5% (CV%, 3.83), indicating the reliability of the experimental results and the repeatability of the model.

[0058] Table 1. Box-Behnken Design Experiment and Results

[0059]

[0060] Table 2. Factor analysis results of the binomial regression equation.

[0061]

[0062] The 3D diagram of the interactions of various factors is shown in Figure 7. The response surface graph is steep and opens downwards, indicating that the amount of degradation products generated decreases as the factor level increases. Based on single-factor and response surface optimization experiments, the optimal degradation process conditions were obtained as follows: degradation time 1.51 h, degradation temperature 54.77 °C, L-cysteine ​​to polymeric proanthocyanidins mass concentration ratio 2.71:1, and the highest generation amount of proanthocyanidin B2-3'-O-gallate was 2.936 mg / mL.

[0063] Example 6

[0064] Based on the optimal conditions obtained above, degradation was performed to obtain B2-3'-O-gallate:

[0065] (1) Accurately transfer 16.5 mL of 37% hydrochloric acid solution into a 1 L volumetric flask, add methanol to the mark, i.e., 0.01 M hydrochloric acid methanol solution.

[0066] (2) Column chromatography separation of grape seed proanthocyanidins: Weigh an appropriate amount of grape seed extract, dissolve it in distilled water, and prepare a 30 mg / mL grape seed proanthocyanidin loading solution. Measure 100 mL of the loading solution and pass it through a YMC ODS-A-HG (200 x 30 mm id, 25-40 μm) chromatography column pretreated with deionized water. First, elute with 300 mL of pH 7.0 deionized water to remove excess phenolic acids and other interfering components. Then, elute with 200 mL of ethyl acetate to remove haploid proanthocyanidins (CATs) and oligomeric proanthocyanidins (OPCs). Finally, elute with 300 mL of methanol to remove polymeric proanthocyanidins (PPCs). Remove the organic solvent from PPCs by rotary evaporation at 25 °C, freeze-dry, and store the powder at -20 °C for later use (see [link to relevant documentation]). Figure 1 ).

[0067] (3) Weigh grape seed polymeric proanthocyanidins (PPCs) and epicatechin gallate (ECG) powder, dissolve them in 0.01M hydrochloric acid methanol solution, with a mass concentration ratio of ECG to PPCs of 3:1. The final concentration of ECG in the system is 36 g / L, and the final concentration of PPCs in the system is 12 g / L. The solution is treated by reacting in a sealed environment at 55℃ for 1.5 h, and then the reaction is terminated by an ice bath (0℃) to obtain the degradation reaction solution. Three repeated tests were conducted.

[0068] (4) The above reaction solution was rotary evaporated at 30°C to obtain the degradation reaction product powder.

[0069] (5) The above solutions were extracted with ethyl acetate at a volume ratio of 1:3. The extraction was performed three times, and the extraction layers were combined. The layers were dried with anhydrous sodium sulfate, and the ethyl acetate layer was concentrated under reduced pressure and freeze-dried into powder for later use. The freeze-dried powder is the ethyl acetate layer powder containing galloylated proanthocyanidin dimer B2-3'-O-gallate.

[0070] (6) The above solution was extracted three times with ethyl acetate, the extract layers were combined, dried with anhydrous sodium sulfate, the ethyl acetate layer was concentrated under reduced pressure, and freeze-dried into powder for later use. The freeze-dried powder is the ethyl acetate layer powder containing galloylated proanthocyanidin dimer B2-3'-O-gallate.

[0071] (7) Dissolve the above ethyl acetate powders in water to prepare a 2 mg / ml solution, which is the sample loading solution.

[0072] (8) The sample solution of 2 mL was purified by elution through a C18 solid-phase extraction column (500 mg / 3 mL) pretreated with deionized water, and eluted sequentially with diethyl ether, ethyl acetate and methanol at a volume of at least 2 column volumes.

[0073] (9) Collect the ethyl acetate eluent, dry it with anhydrous sodium sulfate, concentrate it under reduced pressure to remove the ethyl acetate eluent, and freeze-dry the resulting precipitate (-80℃) to obtain a freeze-dried powder for later use; the obtained freeze-dried powder is the ethyl acetate layer powder containing galloylated proanthocyanidin dimer B2-3'-O-gallate.

[0074] (10) The powder obtained above was dissolved in water and then purified by a semi-preparative liquid chromatograph (reversed-phase C18 column). The column was a reversed-phase C18 (250×10mm, 5μm); the mobile phase was 25% methanol:water (v / v) with isocratic elution at a flow rate of 3.5mL / min; the column temperature was 30℃; the detection wavelength was 280nm; the injection volume was 2mL, and the obtained fraction was collected and rotary evaporated at 30℃ to obtain galloylated proanthocyanidin dimer B2-3'-O-gallate powder (see...). Figure 8 ).

[0075] Depend on Figure 8 It can be seen that the B2-3'-G chromatographic peak in the obtained substance is the galloacylated proanthocyanidin dimer B2-3'-O-gallate.

[0076] Based on practical considerations, three repeated experiments were conducted under the conditions of a degradation time of 1.5 h, a degradation temperature of 55 °C, and a mass concentration ratio of epicatechin gallate to PPCs of 3:1. The average maximum degradation yield was found to be 2.783 mg / mL (RSD < 5%). There was no significant difference between the experimental value and the theoretical prediction, indicating that the process of degrading grape seed proanthocyanidins with epicatechin gallate obtained using the response surface methodology is feasible. The yield of B2-3'-O-gallate obtained from the degradation and conversion of each gram of PPCs can reach as high as 231.9 mg, or 231.9 mg / g.

[0077] In summary, the degradation and transformation method of this invention, using the proanthocyanidin structural unit ECG as a nucleophile, can achieve a yield of up to 231.9 mg of B2-3'-O-gallate per gram of PPCs. The established degradation process is simple, feasible, and can rapidly and massively synthesize and prepare the scarce galloacylated proanthocyanidin dimer B2-3'-O-gallate.

Claims

1. A method for preparing galloacylated proanthocyanidin dimers, characterized in that, Using grape seed proanthocyanidin polymers (PPCs) as raw materials, the PPCs are degraded into extended units containing carbocations using acid as a catalyst. These extended units then combine with epicatechin gallate (ECG) to generate galloylated proanthocyanidin dimers B2-3'-. O -gallate; Specifically: (1) Grape seed proanthocyanidin polymer and nucleophilic reagent epicatechin gallate were dissolved in acidified methanol to obtain an initial solution; (2) The initial solution was degraded under closed conditions at 30-70 °C with shaking for 0.5-2.5 h, and then the reaction was terminated by ice bath; (3) The reaction solution obtained in step (2) was repeatedly extracted with ethyl acetate, the extract layers were combined, dried with anhydrous sodium sulfate, the ethyl acetate layer was concentrated under reduced pressure, and lyophilized into powder, which is the oligomeric proanthocyanidin B2-3'- containing the degradation product. O - Ethyl acetate layer powder of gallate; wherein, in step (2), the volume ratio of the reaction solution to the ethyl acetate extract is 1:1; containing the degradation product oligomeric proanthocyanidins B2-3'- O The ethyl acetate layer powder of the gallate was dissolved in water, and then eluted with ethyl acetate using a C18 solid-phase extraction column. The eluent was collected for later use. Among these eluents were ethyl acetate and oligomeric proanthocyanidins B2-3'- containing degradation products. O The volume ratio of the ethyl acetate layer powder of gallate to its solution is 3-5:1; The galloylated proanthocyanidin dimer B2-3'- O The structure of -gallate is 。 2. The method for preparing galloylated proanthocyanidin dimers according to claim 1, characterized in that, The epicatechin gallate (ECG) and grape seed proanthocyanidin polymers (PPCs) are present in a mass ratio of 0.5:1 to 4:

1.

3. The method for preparing galloylated proanthocyanidin dimers according to claim 1, characterized in that, The collected fraction, purified by C18 solid-phase extraction column, was dissolved in water and then separated by semi-preparative reversed-phase high-performance liquid chromatography to obtain the monomer compound B2-3'- O -gallate; wherein, a semi-preparative liquid chromatograph is used, the chromatographic column is a reversed-phase C18, the size is 250 × 10 mm, 5 μm; the mobile phase is 25% v / v methanol: water isocratic elution, the flow rate is 3.5 mL / min; the column temperature is 30 ºC; the detection wavelength is 280 nm; the injection volume is 2 mL.

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