A method for preparing procyanidin C1

Abstract

The present invention discloses a method for preparing procyanidin C1. In the present invention, reverse-phase column combined with preparative high performance liquid chromatography is used to separate and purify procyanidin C1. Through the optimization of the elution program and chromatographic conditions, the purity of the prepared procyanidin C1 can reach more than 98%. The product has high purity. The preparation method has a simple process. An aqueous solvent is used as the eluent throughout the process. The eluent is simple to obtain and can be recycled, which is green and environmentally friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and separation, and particularly relates to a preparation method of procyanidin C1. Background Art

[0002] Procyanidin is a very strong in-vivo active functional factor that has been continuously researched and developed in the medical and nutritional fields, and has the functions of antioxidant and free radical scavenging. In 2021, the Institute of Nutrition and Health of the Chinese Academy of Sciences released that procyanidin C1 in grape seed extract has the effect of specifically scavenging senescent cells.

[0003] At present, procyanidin C1 is often extracted and separated by extraction methods using organic solvents such as ether. However, such organic solvents are all volatile, flammable or explosive, and toxic. Whether in the extraction process or the extracted product, it is extremely easy to cause harm to the human body and environmental pollution. In addition, supercritical extraction method is also used, but its economic cost is relatively high; thin layer chromatography and column chromatography techniques are also common methods. Currently, the main separation methods include: silica gel thin layer chromatography, Sephadex G-25 column chromatography, BSA-Sepharose CL-4B, Sephadex LH-20 column chromatography, etc. These methods have the disadvantages of being too cumbersome, consuming a large amount of time, using a large amount of organic reagents, and the column chromatography packing being expensive.

[0004] Therefore, there is an urgent need to research and develop an extraction and separation method of procyanidin C1 with simple process, low cost and environmental friendliness. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a preparation method of procyanidin C1. The purity of procyanidin C1 prepared by this method can reach more than 98%. The product has high purity, simple process, uses an aqueous solvent as the eluent throughout the process, the eluent is simple to obtain and can be recycled, and it is environmentally friendly.

[0006] The present invention is achieved through the following technical solutions:

[0007] A preparation method of procyanidin C1 includes the following steps:

[0008] (1) Dissolve the grape seed extract and adsorb it on a reverse-phase column, and perform isocratic elution with methanol aqueous solutions with mass concentrations of 10%, 20%, 30%, and 40% in sequence. Collect the eluate with 30% methanol aqueous solution, and concentrate it under reduced pressure at low temperature to obtain solid powder A;

[0009] (2) Dissolve solid powder A and adsorb it on a reverse-phase column, and perform gradient elution with methanol aqueous solution. Collect the corresponding elution fractions, and concentrate them under reduced pressure at low temperature to obtain solid powder B;

[0010] (3) Dissolve solid powder B, then separate and purify it by preparative high performance liquid chromatography I. Collect the corresponding elution fractions according to the detection chromatogram, concentrate under reduced pressure at low temperature to obtain solid powder C, and dry it to obtain procyanidin C1.

[0011] In the method of the present invention, first, a reverse-phase chromatographic column is screened, and a reverse-phase ODS chromatographic column is selected. Using a methanol aqueous solution as the eluent, the grape seed extract is preliminarily enriched, and small polar impurities can be removed. The procyanidin fraction (the content of procyanidin C1 is about 10%) is enriched. Then, the procyanidin fraction is further separated and purified by a reverse-phase ODS column to obtain a total fraction mainly rich in procyanidin C1 (the content of procyanidin C1 is increased to about 50%). Then, by preparative high performance liquid chromatography, using an acetonitrile aqueous solution as the mobile phase, the total fraction rich in procyanidin C1 is separated and purified. Through the optimization of the chromatographic conditions in each step, the purity of the prepared procyanidin C1 can reach more than 98%, and the product purity is high.

[0012] Preferably, in step (1), the reverse-phase column is an ODS reverse chromatographic column, and the mass ratio of the sample amount to the mass of the reverse-phase column packing (loading amount) is 1:60 - 1:120, and the flow rate is 25 - 30 ml / min.

[0013] Preferably, in step (2), the reverse-phase column is an ODS reverse chromatographic column, the mass ratio of the sample amount to the mass of the reverse-phase column packing is 1:40 - 1:80, the flow rate is 25 - 30 ml / min, and the gradient elution program is:

[0014]

[0015] Further, in step (3), the chromatographic conditions of preparative high performance liquid chromatography I are: Thermo chromatographic column, 250

[0016] ×10 mm, 5 μm; using an acetonitrile - aqueous solution as the mobile phase, the elution program is:

[0017]

[0018] Further, the preparation method further includes step (4). Dissolve solid powder C, then separate and purify it by preparative high performance liquid chromatography II. Collect the corresponding elution fractions according to the detection chromatogram, concentrate under reduced pressure at low temperature to obtain solid powder D; the chromatographic conditions of preparative high performance liquid chromatography II are: Agilent chromatographic column, 250×9.4 mm, 5 μm; using an acetonitrile - aqueous solution as the mobile phase, the elution program is:

[0019]

[0020] Further, the preparation method further includes step (5) of dissolving solid powder D and separating and purifying it by preparative high performance liquid chromatography III, collecting corresponding elution fractions according to the detection chromatogram, and drying to obtain procyanidin C1; the chromatographic conditions of the preparative high performance liquid chromatography III are as follows: Agilent chromatographic column, 250×9.4 mm, 5 μm; using acetonitrile-aqueous solution as the mobile phase, the elution program is:

[0021]

[0022] Preferably, in steps (3) and (4), the injection volume is 0.4 - 1 ml, and the flow rate is 4 - 5 ml / min.

[0023] Preferably, in step (5), the injection volume is 0.1 - 0.2 ml, and the flow rate is 4 - 5 ml / min.

[0024] Procyanidin C1 has poor stability under high temperature conditions. To prevent the structure of procyanidin C1 from being damaged by high temperature, preferably, the temperature of the low-temperature vacuum concentration is not higher than 35°C.

[0025] Preferably, the drying is by freeze-drying method.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a preparation method of procyanidin C1. This method separates and purifies procyanidin C1 by using a reverse-phase column combined with preparative high performance liquid chromatography. Through the optimization of the elution program and chromatographic conditions, the purity of the prepared procyanidin C1 can reach more than 98%. The product has a high purity. The preparation method has a simple process, uses a water-containing solvent as the eluent throughout the process, the eluent is simple to obtain and can be recycled, which is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the 1H NMR spectrum of procyanidin C1;

[0029] Figure 2 is the 13C NMR spectrum of procyanidin C1. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further described below through specific embodiments. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the following embodiments.

[0031] Grape seed extract: Purchased commercially. After HPLC detection, the content of procyanidin C1 in the grape seed extract is 0.75%.

[0032] Main instruments: LC3000 preparative liquid phase, rapid preparative liquid chromatography (Changzhou Santai Preparation method of procyanidin C1

[0033] (1) Preliminary enrichment by reversed-phase ODS column

[0034] Take the grape seed extract sample powder and dissolve it in a methanol aqueous solution (10 times the amount of 30% methanol aqueous solution, ultrasonic for 5 minutes); pack the column by wet method and load the sample by wet method; sample loading ratio: 1 / 80 (g / g); flow rate: 30 mL / min; elution gradient: elute successively with 10%, 20%, 30%, and 40% methanol aqueous solution, elute for 5 column volumes (CV) at each ratio, collect the 30% methanol elution part, concentrate under reduced pressure at low temperature (not higher than 35 °C) to obtain a reddish-brown solid powder A (immediately store it in a -20 °C refrigerator) for use, and the content of procyanidin C1 detected by HPLC is about 10%;

[0035] (2) Reversed-phase gradient separation

[0036] Take the solid powder A and dissolve it in a methanol aqueous solution (10 times the amount of 10% methanol aqueous solution, ultrasonic for 5 minutes); pack the column by wet method and load the sample by wet method; sample loading ratio: 1 / 40 (g / g); flow rate: 30 mL / min; the elution solution is a methanol aqueous solution, and the gradient elution program is as follows:

[0037]

[0038] Collect the elution fractions of 8.5 - 13.5 CV (18% - 24.5% methanol), concentrate under reduced pressure at low temperature (not higher than 35 °C) to obtain a yellowish-brown solid powder B (immediately store it in a -20 °C refrigerator) for use, and the content of procyanidin C1 detected by HPLC is about 50%.

[0039] (3) Separation and purification by preparative high-performance liquid chromatography I

[0040] Take the solid powder B and dissolve it in a 10% acetonitrile aqueous solution, filter it through a 0.22 microporous filter membrane, and further purify the filtrate by an LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Thermo, 250*10, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; injection volume: 1 mL; mobile phase: acetonitrile - aqueous solution; elution program is as follows:

[0041]

[0042] Collect the elution fractions from 13 min to 25 min, concentrate under reduced pressure at low temperature (not higher than 35 °C) to obtain a yellow solid powder C (immediately store it in a -20 °C refrigerator) for use, and the content of procyanidin C1 detected by HPLC is about 70%;

[0043] (4) Separation and purification by preparative high-performance liquid chromatography II

[0044] Dissolve solid powder C in 10% acetonitrile aqueous solution, filter through a 0.22 μm microporous filter membrane, and further purify the filtrate with an LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Agilent, 250*9.4, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; injection volume: 1 mL; mobile phase: acetonitrile - aqueous solution; elution program:

[0045]

[0046] Collect the elution fraction of the 3rd large absorption peak, concentrate it under reduced pressure at low temperature (not higher than 35 °C) to obtain light yellow solid powder D (store it immediately in a -20 °C refrigerator) for use. The content of procyanidin C1 detected by HPLC is about 85%;

[0047] (5) Separation and purification by preparative high performance liquid chromatography III

[0048] Dissolve solid powder D in 10% acetonitrile aqueous solution, filter through a 0.22 μm microporous filter membrane, and detect the filtrate with an LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Agilent, 250*9.4, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; injection volume: 1 mL; mobile phase: acetonitrile - aqueous solution; elution program:

[0049]

[0050] Collect the elution fraction of the 2nd absorption peak, freeze-dry it, and identify it by nuclear magnetic resonance. The obtained product is procyanidin C1 (store it immediately in a -20 °C refrigerator), and the purity is about 98%.

[0051] The 1H NMR spectrum of procyanidin C1 is shown in Figure 1 , and the 13C NMR spectrum is shown in Figure 2 , and the data of 1H NMR and 13C NMR are as follows.

[0052] 1 H NMR(600MHz,CD3OD)δ7.13(s,1H),7.02(s,1H),6.91(d,J=6.7Hz,2H),6.84 - 6.54(m,6H),6.22 - 5.81(m,4H),5.22(s,1H),5.07(s,1H),4.99(s,1H),4.71(s,2H),4.32(s,1H),4.00(d,J=8.9Hz,2H),2.99 - 2.91(m,1H),2.81(d,J=16.5Hz,1H).

[0053] 1313C NMR (150 MHz, CD3OD) δ 158.4, 157.9, 157.3, 156.9, 156.6, 154.9, 154.6, 146.0, 145.8, 145.7, 145.6, 145.5, 132.7, 132.1, 119.3, 119.1, 118.9, 116.0, 115.3, 115.1, 107.7, 107.2, 107.1, 102.2, 101.5, 100.6, 97.6, 96.6, 96.3, 79.7, 77.1, 73.5, 73.0, 66.9, 37.4, 37.3, 29.9。

[0054] Example 2: Optimization of the Enrichment Conditions of the Reverse-Phase Chromatographic Column

[0055] Since the content of procyanidin C1 in grape seed extract is low, in order to isolate pure procyanidin C1, it is necessary to enrich the components. In this study, the single-factor variable method was used to investigate the effects of the chromatographic column, elution concentration, sample loading amount, and flow rate on the enrichment effect in turn.

[0056] 2.1 Investigation of the Enrichment Chromatographic Column Dissolve the grape seed extract sample powder in a methanol aqueous solution (10-fold amount of 30% methanol aqueous solution, sonicate for 5 minutes); pack the column by the wet method and load the sample by the wet method; Chromatographic columns: silica gel column, MCI resin column, Sephadex LH-20 column, reverse-phase ODS column; Sample loading ratio: 1 / 80 (g / g); Flow rate: 30 mL / min; Elute with 30% methanol aqueous solution for 5 column volumes (CV).

[0057] The effects of different chromatographic columns on the enrichment effect were investigated, and the results are shown in Table 1:

[0058] Table 1

[0059]

[0060]

[0061] The results showed that the reverse-phase ODS column could remove small-polarity impurities and enrich the procyanidin fraction, and the content of procyanidin C1 was about 10%. The enrichment effect was obvious. Therefore, the reverse-phase ODS column was preferably used in the present invention for the preliminary enrichment of procyanidin C1.

[0062] 2.2 Investigation of Different Elution Fractions

[0063] Dissolve the grape seed extract sample powder in an aqueous methanol solution (10 times the amount of 30% aqueous methanol solution, ultrasonic for 5 minutes); pack the column by the wet method and load the sample by the wet method; sample loading ratio: 1 / 80 (g / g); flow rate: 30 mL / min; elution gradient: elute successively with 10%, 20%, 30%, and 40% aqueous methanol solutions, and elute with each ratio for 5 column volumes (CV).

[0064] Examine the content of procyanidin C1 in different elution parts, and the results are shown in Table 2 below:

[0065] Table 2

[0066]

[0067] The results show that in the eluate of 30% aqueous methanol solution, the content of procyanidin C1 is relatively high and the impurities are less. Therefore, in the present invention, the eluate of 30% aqueous methanol solution is collected.

[0068] 2.3 Influence of sample loading amount on separation effect

[0069] Dissolve the grape seed extract sample powder in an aqueous methanol solution (10 times the amount of 30% aqueous methanol solution, ultrasonic for 5 minutes); pack the column by the wet method and load the sample by the wet method; sample loading ratio: 1 / 40, 1 / 80, 1 / 120 (g / g); flow rate: 30 mL / min; elute with 30% aqueous methanol solution for 5 column volumes (CV).

[0070] Examine the influence of sample loading amount on separation effect, and the results are shown in Table 3 below:

[0071] Table 3

[0072]

[0073] The results show that the separation effect of the sample loading ratio of 1 / 80 is better. With the increase of the amount of ODS, the separation effect has no obvious change. Therefore, the preferred sample loading ratio is 1 / 80.

[0074] 2.4 Influence of flow rate on separation effect

[0075] Dissolve the grape seed extract sample powder in an aqueous methanol solution (10 times the amount of 30% aqueous methanol solution, ultrasonic for 5 minutes); pack the column by the wet method and load the sample by the wet method; sample loading ratio: 1 / 80 (g / g); flow rates: 25 mL / min, 30 mL / min, 35 mL / min; elute with 30% aqueous methanol solution for 5 column volumes (CV).

[0076] Examine the influence of flow rate on separation effect, and the results are shown in Table 4 below:

[0077] Table 4

[0078]

[0079] The results showed that the separation effect was better at a flow rate of 30 mL / min. As the flow rate increased, the column pressure increased and overpressure occurred.

[0080] Example 3: Optimization of reverse-phase gradient separation conditions

[0081] 3.1 Optimization of the elution program

[0082] Dissolve solid powder A in a methanol aqueous solution (10 times the amount of 10% methanol aqueous solution, ultrasonic for 5 minutes); pack the column by the wet method and load the sample by the wet method; use the methanol aqueous solution as the eluent to investigate the influence of different gradient elution programs on the separation effect of procyanidin C1. The gradient elution programs are as follows:

[0083] Elution program 1:

[0084]

[0085] Elution program 2:

[0086]

[0087] Elution program 3:

[0088]

[0089] Elution program 4:

[0090]

[0091]

[0092] The separation results of each elution program are shown in Table 5 below:

[0093] Table 5

[0094]

[0095] The results showed that compared with elution programs 1 and 2, elution programs 3 and 4 had higher resolution and higher procyanidin C1 content. Elution program 4 significantly shortened the time compared with program 3, improved the separation efficiency, and saved solvents. Therefore, elution program 4 was preferred.

[0096] 3.2 Influence of sample loading amount on separation effect

[0097] Dissolve solid powder A in a methanol aqueous solution (10 times the amount of 10% methanol aqueous solution, ultrasonic for 5 minutes); pack the column by the wet method and load the sample by the wet method; use the methanol aqueous solution as the eluent and elute with elution program 4 in 3.1 to investigate the influence of the sample loading amount on the separation effect. The results are shown in Table 6:

[0098] Table 6

[0099]

[0100] The results showed that the sample loading ratios of 1 / 40 and 1 / 80 (g / g) had similar effects and good resolution.

[0101] 3.3 Influence of flow rate on separation effect

[0102] Dissolve solid powder A in methanol aqueous solution (10 times the amount of 10% methanol aqueous solution, ultrasonic for 5 minutes); pack the column by wet method and load the sample by wet method; use methanol aqueous solution as the eluent, select the elution program 4 in 3.1 for elution, and investigate the influence of flow rate on the separation effect. The results are shown in Table 7:

[0103] Table 7

[0104]

[0105] The results showed that with the increase of the flow rate, the resolution of procyanidin C1 increased, and the optimal flow rate was 30 mL / min.

[0106] Example 4: Optimization of chromatographic conditions for preparative high performance liquid chromatography I

[0107] Due to the inability to obtain high-purity procyanidin C1 by conventional reverse chromatographic column separation, we carried out preparative high performance liquid chromatography I separation and purification on the solid powder B obtained in the previous step.

[0108] 4.1 Optimization of elution program

[0109] Dissolve solid powder B in 10% acetonitrile aqueous solution, filter through a 0.22 microporous membrane, and detect the filtrate by LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Thermo, 250*10, 5μm; flow rate: 5 mL / min; column temperature: room temperature; injection volume: 1 mL; mobile phase: acetonitrile-aqueous solution; investigate the influence of different gradient elution programs on the separation effect of procyanidin C1. The gradient elution programs are as follows:

[0110] Elution program 1:

[0111]

[0112] Elution program 2:

[0113]

[0114] Elution program 3:

[0115]

[0116] The separation results of each elution program are shown in Table 8 below:

[0117] Table 8

[0118]

[0119] The results showed that for elution procedure 3, the content of procyanidin C1 was relatively high and the resolution was relatively high.

[0120] 4.2 Influence of injection volume on separation effect

[0121] Solid powder B was dissolved in 10% acetonitrile aqueous solution, filtered through a 0.22 μm microporous filter membrane, and the filtrate was detected by an LC3000 preparative liquid chromatograph. The chromatographic conditions were as follows: chromatographic column: Thermo, 250*10, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; mobile phase: acetonitrile - aqueous solution. The influence of different injection volumes on the separation effect of procyanidin C1 was investigated, and the results are shown in Table 9:

[0122] Table 9

[0123]

[0124] The results showed that when the injection volume was 0.4 - 1 mL, the resolution of procyanidin C1 was relatively high, and when the injection volume was 1.2 mL, the resolution decreased.

[0125] 4.3 Influence of flow rate on separation effect

[0126] Solid powder B was dissolved in 10% acetonitrile aqueous solution, filtered through a 0.22 μm microporous filter membrane, and the filtrate was detected by an LC3000 preparative liquid chromatograph. The chromatographic conditions were as follows: chromatographic column: Thermo, 250*10, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; mobile phase: acetonitrile - aqueous solution. The influence of different flow rates on the separation effect of procyanidin C1 was investigated, and the results are shown in Table 10:

[0127] Table 10

[0128]

[0129] The results showed that when the flow rate was 5 mL / min, the resolution of procyanidin C1 was relatively high, and when the flow rate was 6 mL / min, the column pressure increased and exceeded the pressure.

[0130] Example 5: Optimization of chromatographic conditions for preparative high performance liquid chromatography II

[0131] To further purify procyanidin C1, the chromatographic column was replaced, and solid powder C obtained in the previous step was separated and purified by preparative high performance liquid chromatography II.

[0132] 5.1 Optimization of elution procedure

[0133] Dissolve solid powder C in 10% acetonitrile aqueous solution, filter through a 0.22 μm microporous filter membrane, and detect the filtrate by an LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Agilent, 250*9.4, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; injection volume: 1 mL; mobile phase: acetonitrile - aqueous solution. Examine the effect of different gradient elution programs on the separation effect of procyanidin C1. The gradient elution programs are as follows:

[0134] Elution program 1:

[0135]

[0136] Elution program 2:

[0137]

[0138] The separation results of each elution program are shown in Table 11 below:

[0139] Table 11

[0140]

[0141] The results show that for elution program 2, the resolution of procyanidin C1 is better.

[0142] 5.2 Effect of injection volume on separation effect

[0143] Dissolve solid powder C in 10% acetonitrile aqueous solution, filter through a 0.22 μm microporous filter membrane, and detect the filtrate by an LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Agilent, 250*9.4, 5 μm; flow rate: 5 mL / min; column temperature: room temperature; mobile phase: acetonitrile - aqueous solution. Examine the effect of different injection volumes on the separation effect of procyanidin C1. The results are shown in Table 12 below:

[0144] Table 12

[0145]

[0146] The results show that the resolution is better for an injection volume of 1 mL, and the resolution decreases as the injection volume increases.

[0147] 5.3 Effect of flow rate on separation effect

[0148] Dissolve solid powder C in 10% acetonitrile aqueous solution, filter through a 0.22 μm microporous filter membrane, and detect the filtrate by an LC3000 preparative liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Agilent, 250*9.4, 5 μm; injection volume: 1 ml; column temperature: room temperature; mobile phase: acetonitrile - aqueous solution. Examine the effect of different flow rates on the separation effect of procyanidin C1. The results are shown in Table 13 below.

[0149] Table 13

[0150]

[0151] The results show that when the flow rate is 5 mL / min, the resolution of procyanidin C1 is relatively high. When the flow rate exceeds 5 mL / min, the column pressure increases and exceeds the pressure limit.

[0152] In summary, through the optimization of the chromatographic column and chromatographic conditions in each step, the purity of the prepared procyanidin C1 can reach over 98%. The product has a high purity. The entire process avoids the use of excessive organic solvents, only using methanol and acetonitrile aqueous solution as the eluent. The eluent is simple to obtain and can be recycled, which is green and environmentally friendly.

Claims

1. A method for preparing procyanidin C1, characterized in that, It includes the following steps: (1) Dissolve grape seed extract and adsorb it on a reversed-phase column, elute it successively with methanol aqueous solutions with mass concentrations of 10%, 20%, 30%, and 40%, collect the eluate of the 30% methanol aqueous solution, and concentrate it under reduced pressure at low temperature to obtain solid powder A; (2) Dissolve solid powder A and adsorb it on a reversed-phase column, elute it with a gradient of methanol aqueous solution, collect the corresponding elution fractions, and concentrate it under reduced pressure at low temperature to obtain solid powder B; In step (2), the gradient elution program is: ； (3) Dissolve solid powder B and separate and purify it by preparative high-performance liquid chromatography I. Collect the corresponding elution fractions according to the detection chromatogram, and concentrate it under reduced pressure at low temperature to obtain solid powder C; The chromatographic conditions of the preparative high-performance liquid chromatography I are: Thermo chromatographic column, 250×10mm, 5μm; Using acetonitrile-aqueous solution as the mobile phase, the elution program is: ； (4) Dissolve solid powder C and separate and purify it by preparative high-performance liquid chromatography II. Collect the corresponding elution fractions according to the detection chromatogram, and concentrate it under reduced pressure at low temperature to obtain solid powder D; The chromatographic conditions of the preparative high-performance liquid chromatography II are: Agilent chromatographic column, 250×9.4mm, 5μm; Using acetonitrile-aqueous solution as the mobile phase, the elution program is: ； (5) Dissolve solid powder D and separate and purify it by preparative high-performance liquid chromatography III. Collect the corresponding elution fractions according to the detection chromatogram, and dry it to obtain procyanidin C1; The chromatographic conditions of the preparative high-performance liquid chromatography III are: Agilent chromatographic column, 250×9.4mm, 5μm; Using acetonitrile-aqueous solution as the mobile phase, the elution program is: 。 2. The preparation method of procyanidin C1 according to claim 1, wherein: In step (1), the reversed-phase column is an ODS reversed-phase chromatographic column, the mass ratio of the sample amount to the mass of the reversed-phase column packing is 1:60 - 1:120, and the flow rate is 25 - 30 ml / min.

3. The preparation method of procyanidin C1 according to claim 1, characterized in that: In step (2), the reversed-phase column is an ODS reversed-phase chromatographic column, the mass ratio of the sample amount to the mass of the reversed-phase column packing is 1:40 - 1:80, and the flow rate is 25 - 30 ml / min.

4. The preparation method of procyanidin C1 according to claim 1, characterized in that: In steps (3) and (4), the injection volume is 0.4 - 1 ml, and the flow rate is 4 - 5 ml / min.

5. The preparation method of procyanidin C1 according to claim 1, characterized in that: In step (5), the injection volume is 0.1 - 0.2 ml, and the flow rate is 4 - 5 ml / min.

6. The preparation method of procyanidin C1 according to claim 1, characterized in that: The temperature of the concentration under reduced pressure at low temperature is not higher than 35°C, and the drying is freeze-drying.

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