Preparation method and application of polyphenol compounds in peony pods

Abstract

The invention discloses a preparation method and application of polyphenol compounds in peony pods, and belongs to the technical field of effective extraction of polyphenol compounds. The preparation method comprises the following steps: extracting polyphenol compounds in peony fruit pods by adopting ethanol to obtain a crude fruit pod extract; dissolving the capsule crude extract by adopting a high-speed counter-current chromatography solvent system to obtain a sample solution; separating the sample solution in an elution-sweeping-ejection mode of high-speed counter-current chromatography to obtain a polyphenol monomer and a mixture; and separating the mixture by adopting internal circulation counter-current chromatography. The polyphenol compounds in the peony pods are separated and purified by adopting an elution-sweeping-ejection combined internal circulation counter-current chromatography mode, and a plurality of polyphenol compounds are obtained through nuclear magnetic resonance spectrum and mass spectrum structure characterization. The preparation method established by the invention can be used for effectively separating polyphenol compounds with wide distribution coefficients in peony pods, and can also be used for separating phenolic components in other complex plants.

Description

Technical Field

[0001] This invention belongs to the field of effective extraction technology of polyphenolic compounds, specifically relating to a method for preparing and applying polyphenolic compounds from peony pods. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Polyphenolic compounds have broad application prospects in the food, health product, cosmetic, and pharmaceutical fields due to their significant antibacterial, antioxidant, and anti-inflammatory biological activities. Currently, polyphenols are mainly found in tea leaves, grape seeds, fruit and vegetable peels and pomace, and the roots, stems, and leaves of certain specific plants. However, the development and utilization of plant resources still faces limitations, and finding new, readily available plant raw materials rich in polyphenols has significant economic and scientific research value.

[0004] Peony pods, which enclose peony seeds, are typically used as firewood after the seeds are removed, and their full potential is not realized. Peony pods contain various polyphenols, but there are no reports in existing technologies regarding the systematic extraction of their chemical components, especially polyphenols, from peony pods. This means that peony pods, as a potential new resource of polyphenols, have not yet been effectively identified and utilized, and their potential biological activity and economic value remain untapped. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing and applying polyphenolic compounds from peony pods. The preparation method provided by this invention can effectively separate polyphenolic compounds with a wide partition coefficient from peony pods, and can also be used for the separation of phenolic components from other complex plants.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of this invention provides a method for preparing polyphenolic compounds from peony pods, comprising: Ethanol was used to extract polyphenolic compounds from peony pods to obtain crude extract of the pods; The crude extract of the fruit pods was dissolved in a high-speed countercurrent chromatography solvent system to obtain a sample solution; The sample solution was separated in the elution-sweep-top mode of high-speed countercurrent chromatography to obtain polyphenol monomers and mixtures; the mixture was then separated using internal circulation countercurrent chromatography.

[0007] In some embodiments of the present invention, the extraction of polyphenolic compounds from peony pods using ethanol includes: Peony pods were extracted with anhydrous ethanol at a temperature of 50-60℃ and a material-to-liquid ratio of 1:9-11 to obtain crude extract of the pods.

[0008] In some embodiments of the present invention, the polyphenolic compounds include compounds 1-8, whose structural formulas are as follows: .

[0009] In some embodiments of the present invention, the sample solution is separated in the elution-sweep-top mode of high-speed countercurrent chromatography to obtain a mixture of compounds 1, 2, 3, 4, and 5, and a mixture of compounds 6, 7, and 8. The mixture of compounds 4 and 5 was separated by internal circulation countercurrent chromatography to obtain compounds 4 and 5. The mixture of compounds 6, 7 and 8 was separated by countercurrent chromatography to obtain a mixture of compounds 6, 7 and 8. The mixture of compounds 7 and 8 was separated by internal circulation countercurrent chromatography to obtain compounds 7 and 8.

[0010] In some embodiments of the present invention, the high-speed countercurrent chromatography solvent system of the elution-sweep-top mode is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of (1-2):(8-9):(1-2):(8-9).

[0011] Preferably, the volume ratio is 1:9:1:9.

[0012] In some embodiments of the present invention, when separating the mixture of compounds 4 and 5, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of (1-2):(8-9):(1-2):(8-9).

[0013] Preferably, the volume ratio is 1:9:1:9.

[0014] In some embodiments of the present invention, when separating a mixture of compounds 6, 7 and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of 5:5:(2-3):(7-8).

[0015] Preferably, the volume ratio is 5:5:2.5:7.5.

[0016] In some embodiments of the present invention, when separating the mixture of compounds 7 and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of 5:5:(2-3):(7-8).

[0017] Preferably, the volume ratio is 5:5:2.5:7.5.

[0018] In a second aspect, the present invention provides an application of the above-mentioned method for preparing polyphenolic compounds from peony pods in the extraction of active ingredients from peony pods.

[0019] The beneficial effects of this invention are as follows: This invention provides a method for preparing polyphenolic compounds from peony pods. The method employs an elution-sweep-top-ejection combined with internal circulation countercurrent chromatography to separate and purify the polyphenolic compounds from peony pods. Structural characterization by nuclear magnetic resonance spectroscopy and mass spectrometry yielded multiple polyphenolic compounds. The preparation method established in this invention can effectively separate polyphenolic compounds with a wide partition coefficient from peony pods and can also be used for the separation of phenolic components from other complex plants. Furthermore, this invention uses high-speed countercurrent chromatography to separate and extract various polyphenolic compounds from peony pods, transforming agricultural waste (peony pods) into high-value-added products, which aligns with the concepts of circular economy and green chemistry.

[0020] Based on nuclear magnetic resonance (NMR) and mass spectrometry structural characterization, this invention isolated eight polyphenolic compounds from peony pods, with the following structures: methyl gallate (1), 1,2,3,4,6-pentagalloglucopyranose (2), methyl gallate isomers (me- or para-) (3), 1,2,3,6-tetra-O-galloyl-β-D-glucopyranose (4), 3-O-dicarboxylo-1,2,4,6-tetra-O-galloyl-β-D-pyranoseranose (5), (+)-ε-viniferin (6), amurensin B (7), and gnetin H (8). The purity of all eight extracted polyphenolic compounds was higher than 98%. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a flowchart illustrating the separation of polyphenolic compounds from peony pods in Example 1 of the present invention. Figure 2 This is a total sample chromatogram of countercurrent chromatography separation in Example 1 of the present invention, wherein A is a total sample chromatogram of separation using conventional countercurrent chromatography, and B is a total sample chromatogram of separation using elution-sweep-ejection mode; Figure 3The diagrams show the effects of secondary separation of the mixture in Example 1 of the present invention. In the diagrams, A represents the effect of separating compounds 6+7+8 using internal circulation countercurrent chromatography, B represents the effect of separating compounds 7+8 using internal circulation countercurrent chromatography, and A represents the effect of separating compounds 4+5 using internal circulation countercurrent chromatography. Figure 4 This is a high-performance liquid chromatography (HPLC) analysis of eight polyphenolic compounds isolated from peony pods in Example 1 of the present invention. Detailed Implementation

[0023] Given the lack of reports in the existing technology on the systematic extraction of chemical components, especially polyphenolic compounds, from peony pods, this invention proposes a method for preparing and applying polyphenolic compounds from peony pods.

[0024] A first typical embodiment of the present invention provides a method for preparing polyphenolic compounds from peony pods, comprising: Ethanol was used to extract polyphenolic compounds from peony pods to obtain crude extract of the pods; The crude extract of the fruit pods was dissolved in a high-speed countercurrent chromatography (HSCCC) solvent system to obtain a sample solution; The sample solution was separated in the elution-sweep-top mode of high-speed countercurrent chromatography to obtain polyphenol monomers and mixtures; the mixture was then separated using internal circulation countercurrent chromatography.

[0025] HSCCC is a highly efficient liquid-liquid partition chromatography method with advantages such as high capacity, high recovery, no irreversible adsorption, and low solvent consumption. Various elution-sweep-top elution, linear elution, and internal circulation methods have been established. This invention utilizes HSCCC to separate polyphenolic compounds from peony pods. A combination of elution-sweep-top elution and internal circulation countercurrent chromatography was employed to separate and purify the polyphenolic components from the peony pods. Multiple polyphenolic compounds were successfully obtained through NMR and mass spectrometry structural characterization.

[0026] The elution-sweep-elution mode fully utilizes the stationary phase of countercurrent chromatography, enabling the separation of polyphenolic compounds with large partition coefficients. Internal circulation countercurrent chromatography separates polyphenolic compounds with similar partition coefficients. The combination of these two methods effectively separates complex natural products. The preparation method provided by this invention exhibits good separation performance for polyphenolic compounds and can also be applied to the separation of polyphenolic compounds from other complex plants.

[0027] In some embodiments of this implementation, the extraction of polyphenolic compounds from peony pods using ethanol includes: Peony pods were extracted with anhydrous ethanol at a temperature of 50-60℃ and a material-to-liquid ratio of 1:9-11 to obtain crude extract of the pods.

[0028] This invention utilizes anhydrous ethanol to extract polyphenolic compounds from peony pods, achieving effective utilization of this waste resource and successfully obtaining a crude extract rich in polyphenols. Based on the moderate polarity, good safety, low cost, and ease of operation of anhydrous ethanol, this invention can efficiently and gently extract target polyphenols, reducing excessive dissolution of strongly polar and non-polar impurities. This is not only groundbreaking in raw material utilization but also provides a foundation for subsequent high-purity separation and purification of complex polyphenols using HPCCC, offering a relatively controllable impurity composition, good solvent compatibility, and a high content of target active ingredients.

[0029] In some embodiments of this implementation, the extraction of polyphenolic compounds from peony pods using ethanol includes: Peony pods were extracted with anhydrous ethanol at 55℃ and a solid-liquid ratio of 1:10. w / v ), filter, concentrate under reduced pressure, and obtain crude extract of the pods.

[0030] As is understandable, the term "material-to-liquid ratio" refers to the ratio of the mass (w) of the solid "material" to the volume (v) of the "liquid" used as the extractant. For example, a material-to-liquid ratio of 1:10 means that the ratio of the mass of peony pod material added per unit time to the volume of anhydrous ethanol is 1:10, that is, the volume of anhydrous ethanol is 10 times the mass of the added peony pods.

[0031] In some embodiments of this implementation, the polyphenolic compounds include compounds 1-8, whose structural formulas are as follows: .

[0032] By characterizing the structure using nuclear magnetic resonance spectroscopy and mass spectrometry, this invention isolated eight polyphenolic compounds from peony pods, with the following structures: methyl gallate (1), 1,2,3,4,6-pentagalloglucopyranose (2), methyl gallate isomers (me or para) (3), 1,2,3,6-tetra-O-galloyl-β-D-glucopyranose (4), 3-O-dicarboxylo-1,2,4,6-tetra-O-galloyl-β-D-pyranoseranose (5), (+)-ε-viniferin (6), amurensin B (7), and gnetin H (8).

[0033] It should be noted that compound 3 is an isomer of methyl gallate, and it was determined through testing to be either a meta or para isomer.

[0034] In some embodiments of this implementation, the sample solution is separated in the elution-sweep-top mode of high-speed countercurrent chromatography to obtain compounds 1, 2, 3, a mixture of compounds 4 and 5, and a mixture of compounds 6, 7 and 8. The mixture of compounds 4 and 5 was separated by internal circulation countercurrent chromatography to obtain compounds 4 and 5. The mixture of compounds 6, 7, and 8 was separated by internal circulation countercurrent chromatography to obtain a mixture of compounds 6, 7, and 8. The mixture of compounds 7 and 8 was separated by internal circulation countercurrent chromatography to obtain compounds 7 and 8.

[0035] This invention separates sample solutions using the elution-sweep-top mode of high-speed countercurrent chromatography, saving time and solvent. While the elution-sweep-top mode ensures the elution of all compounds, it cannot achieve complete separation of all compounds, and the polarities of the compounds in the mixture are relatively similar. Therefore, internal circulation countercurrent chromatography is used to further separate mixtures with similar partition coefficients, achieving separation through multiple cycles.

[0036] For HSCCC separation, selecting a suitable two-phase solvent system is crucial. A smaller partition coefficient ( K D A value <0.5 may lead to incomplete separation of compounds, while a larger value may result in incomplete separation of compounds. K D Values ​​(>2.0) may increase elution duration and solvent consumption, and also result in a wider peak. K D A value between 0.5 and 2.0 is more suitable. Meanwhile, the separation factor coefficient (α value) (α= K D1 / K D2 , K D1 > K D2 A value ≥1.5 is also beneficial for the separation effect of countercurrent chromatography.

[0037] In some embodiments of this implementation, the high-speed countercurrent chromatography solvent system for the elution-sweep-top mode is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of (1-2):(8-9):(1-2):(8-9). Studies have shown that the elution-sweep-top mode in this solvent system can elute all compounds.

[0038] In some embodiments of this implementation, the high-speed countercurrent chromatography solvent system for the elution-sweep-top mode is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 1:9:1:9. Studies have shown that this solvent system provides better separation of all compounds.

[0039] In the elution-sweep-top countercurrent chromatography elution mode, ethyl acetate forms a two-phase system with the aqueous phase (methanol-water), where ethyl acetate and methanol act as "bridging solvents" that significantly influence the partitioning behavior of polyphenols. At this ratio, a high proportion of ethyl acetate enhances the solubility of polyphenols in the organic phase, while the combination of methanol and water provides a sufficiently polar environment for the polyphenols to achieve suitable partitioning between the two phases. K D This ratio ensures adequate interfacial tension and polar gradient between the two phases, promoting the dynamic distribution of polyphenols and improving separation efficiency.

[0040] In some embodiments of this implementation, when separating the mixture of compounds 4 and 5, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of (1-2):(8-9):(1-2):(8-9). Studies have shown that compounds 4 and 5 can be separated by internal circulation countercurrent chromatography using this solvent system. In some embodiments of this implementation, when separating the mixture of compounds 4 and 5, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 1:9:1:9. Studies have shown that this solvent system provides better separation of compounds 4 and 5.

[0041] In some embodiments of this implementation, when separating the mixture of compounds 6, 7, and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:(2-3):(7-8). Studies have shown that compound 6 can be separated from the mixture using internal circulation countercurrent chromatography in this solvent system.

[0042] In some embodiments of this implementation, when separating the mixture of compounds 6, 7, and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:2.5:7.5. Studies have shown that compound 6 is separated better under this solvent system.

[0043] In some embodiments of this implementation, when separating the mixture of compounds 7 and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:(2-3):(7-8). Studies have shown that compounds 7 and 8 can be separated using internal circulation countercurrent chromatography in this solvent system.

[0044] In some embodiments of this implementation, when separating the mixture of compounds 7 and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 5:5:2.5:7.5. Studies have shown that this solvent system provides better separation of compounds 7 and 8.

[0045] A second typical embodiment of the present invention provides an application of the above-mentioned method for preparing polyphenolic compounds from peony pods in the extraction of active ingredients from peony pods.

[0046] A third typical embodiment of the present invention provides a method for preparing polyphenolic compounds from peony pods, wherein a crude extract of the pods is obtained by extracting from the peony pods, and the crude extract of the pods is processed using the above-described preparation method.

[0047] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0048] Example 1: A method for preparing polyphenolic compounds from peony pods I. Materials and Methods 1. Reagents and materials The organic solvents used in HSCCC include anhydrous ethanol, n-butanol, n-hexane, ethyl acetate, and methanol, all of which are of analytical grade, and the water is ultrapure water.

[0049] The peony pods were harvested from the Peony Garden in Heze City, Shandong Province.

[0050] 2. Preparation of crude extract from fruit pods Take 100 g of peony pods and mix them with ethanol at 55℃ at a ratio of 1:10 ( w / v Extraction was performed using a material-liquid ratio, followed by filtration and vacuum concentration to obtain 32 g of crude extract from the fruit pods, with a yield of 32%. The sample was stored in a 4°C refrigerator for further separation.

[0051] 3. Determination of distribution coefficient Prepare 5 mL solutions of the pre-defined solvents (n-hexane, ethyl acetate, methanol, and water) according to the different volume ratios shown in Table 1. Shake thoroughly to separate the layers. Take 2 mL of each phase into centrifuge tubes, add 3 mg of crude fruit extract, and shake thoroughly to dissolve. Take 1 mL of each phase into 1.5 mL centrifuge tubes and centrifuge to concentrate the solution until the solvent has completely evaporated. Add 1 mL of methanol to each of the two centrifuge tubes to fully dissolve the sample. Filter the solution through a 0.22 μm organic nylon filter membrane and transfer it to a liquid chromatography vial. Analyze the solution using HPLC and record the peak areas of each target compound. The partition coefficients of each peak (…) are also recorded. K D The value is calculated using the following formula: K D= A U / A L ,in A U (Photographed) and A L (Lower phase) indicates the peak area of ​​the target compound.

[0052] 4. Two-phase solvent system and sample solution preparation Conventional HSCCC separation uses n-hexane / ethyl acetate / methanol / water (HEMWat) (1:9:1:9). v / v Two-phase solvent system, in the above ratio (1:9:1:9, v / v Prepare 2 L of solvent in a separatory funnel, shake thoroughly to mix, let stand to separate the layers, take out the upper and lower phases separately, and place the upper and lower phases in an ultrasonic instrument for 20 min to remove air bubbles. The upper phase is the stationary phase and the lower phase is the mobile phase.

[0053] Weigh 200 mg of crude extract from the pods, add 5 mL of stationary phase and 5 mL of mobile phase, and sonicate for 5 min to fully dissolve the sample solution for countercurrent chromatography separation.

[0054] 5. High-speed countercurrent chromatography separation Figure 1 A separation flowchart for the preparation method provided by the present invention.

[0055] In the conventional high-speed countercurrent chromatography (HSCCC) separation process, hexane / ethyl acetate / methanol / water (HEMWat) (1:9:1:9, v / v The upper phase (stationary phase) of the two-phase solvent system was pumped into the countercurrent chromatography column at a rate of 30 mL / min. After the column was full, the rotation speed was set to 800 r / min, the separation temperature was 25℃, and the mobile phase was pumped at a flow rate of 2 mL / min in a "head-to-tail" mode. After hydrodynamic equilibrium was reached, the peony pod sample solution was injected into the countercurrent chromatography injection valve, and the detection wavelength was set to 313 nm. The sample solutions were collected in separate tubes according to the chromatographic elution results. After separation, the solvent was purged from the column using nitrogen. The stationary phase retention rate was defined as the ratio of the stationary phase volume after purging to the total volume of the separation column.

[0056] The elution-sweep-top-ejection separation process is the same as that of conventional countercurrent chromatography in the initial stage. During the "sweep" stage, the stationary phase is pumped in, while other parameters remain unchanged. In this stage, an "incorrect" mobile phase is generated, resulting in the elution of a mixture of upper and lower phases. When only the stationary phase elutes, this is recorded as the start of the "top-ejection" mode, and collection continues until all components have been successfully eluted.

[0057] The elution method in internal circulation countercurrent chromatography mode is as follows: The upper phase (stationary phase) of the two-phase solvent system is pumped into the separation column. Once the column is full, the rotation speed is set to 800 r / min, and the separation temperature is 25℃. The lower phase (mobile phase) is then pumped into the column in a "head-to-tail" mode (5 mL / min). After hydrodynamic equilibrium is reached, the sample solution is injected into the countercurrent chromatography injection valve, and the detection wavelength is set to 313 nm. After the sample has completely entered the separation column, the sample injection valve is switched to circulation mode. When the internal circulation separation achieves the desired effect, the sample injection valve is switched to collection mode, and the sample solution is collected in separate tubes. After separation, nitrogen gas is used to purge the solvent from the column.

[0058] 6. HPLC analysis and structural determination HPLC conditions are as follows: column C 18 (250 mm × 4.6 mm, 5.0 µm); mobile phase was acetonitrile (A)-water (B): 0 min, 15% A; 17 min, 25% A; 22 min, 50% A; 27 min, 15% A; 30 min, 15% A; flow rate was 1.0 mL / min; detector wavelength was 313 nm.

[0059] The conditions for nuclear magnetic resonance spectroscopy are: DMSO- d 6 is a deuterated solvent, chemical shift ( δ ) and coupling constant ( J ) respectively ppm and Hz express.

[0060] II. Results and Discussion 1. Selection of two-phase solvent system As shown in Table 1, a series of HEMWat solvent systems were tested for the separation of polyphenolic components from peony pods. K D value.

[0061] Table 1. Partition coefficients of compounds in different solvent systems

[0062] As shown in Table 1, when using HEMWat(1:1:1:1, v / v ) and (5:5:3:7, v / v In solvent systems, the target compound's K D A low value (<0.5) indicates that the target compound elutes too quickly and cannot be completely separated.

[0063] Using HEMWat (3:7:3:7, v / v When using a solvent system, K D The values ​​increase for compounds 1, 3, and 6. K D The values ​​were 0.62, 0.93, and 1.86, respectively, for compounds 2, 4, and 5. K D The value is <0.5, while the mixture of compounds 7+8... K D The value is 3.23, which is not suitable for sample separation.

[0064] Using HEMWat (2:8:2:8, v / v ) and (1:9:1:9, v / v When using a solvent system, K D The value increases as the elution intensity decreases, HEMWat (1:9:1:9, v / v Solvent systems for compounds 1, 3, 6, and 7+8 K D When the value is greater than 2, the separation time is longer. Therefore, the "elution-cleaning-ejection" mode is adopted to save time and solvent.

[0065] Furthermore, an internal loop mode is adopted, with HEMWat (5:5:2.5:7.5, v / v Solvent system for separating compounds 6 and 7+8, HEMWat (1:9:1:9, v / v Compounds 4 and 5 were separated using a solvent system.

[0066] 2. Separation steps like Figure 1 As shown, the elution-sweep-ejection mode of this invention separates compounds 1, 2, 3, a mixture of compounds 4 and 5, and a mixture of compounds 6, 7, and 8 from the crude extract of the fruit pods. Compound 4 and compound 5 are separated from the mixture of compounds 4 and 5 using an internal circulation mode. Compound 6 is separated from the mixture of compounds 6, 7, and 8 using a high-speed countercurrent chromatography mode, yielding a mixture of compounds 7 and 8. Compound 7 and compound 8 are then separated from the mixture of compounds 7 and 8 using an internal circulation mode.

[0067] like Figure 2 As shown, using HEMWat(1:9:1:9, v / v The solvent system was used to separate 200 mg of crude extract from the fruit pods using elution-sweep-top mode and conventional countercurrent chromatography.

[0068] Figure 2 Figure A shows the conventional countercurrent chromatography (CCC) mode, in which the major compounds 2, 4 and 5 were eluted within 7 hours.

[0069] When using the elution-purge-ejection separation mode ( Figure 2 (Figure B) Without changing the separation temperature, flow rate, or rotation speed, a sweep elution and extrusion stage was added, and all compounds were successfully eluted. According to HPLC results, five main peaks were effectively generated, including three monomeric compounds 1, 2, and 3, with yields of 6.3 ± 0.2, 10.5 ± 0.4, and 7.1 ± 0.2 mg, respectively, and purities exceeding 98%. Figure 4 Curves D, E, and F were used to obtain two mixtures of compounds 4+5 and 6+7+8, with yields of 21.3 ± 0.2 and 34.8 ± 0.4 mg, respectively, and purities exceeding 98%. Figure 4 (Curves B and C). The recoveries of compounds 1, 2, 3, 4+5 and 6+7+8 were 3.2%, 5.3%, 3.6%, 10.7% and 17.4%, respectively.

[0070] While the elution-purge-ejection mode ensures the elution of all compounds, it cannot achieve complete separation of all compounds, and the compounds in the fraction are relatively similar in polarity. Therefore, an internal circulation separation mode is used to continue the separation.

[0071] Figure 3 Figure A shows Figure 2 Compounds 6, 7, and 8 in Figure B are in HEMWat (5:5:2.5:7.5). v / v The conventional CCC mode separation effect in the solvent system, with an injection volume of 60 mg, resulted in the successful separation of compound 6 after approximately 1.2 hours, while compounds 7 and 8 were simultaneously eluted as a mixture. Figure 3 The results in Figure A show that, after one separation, 21.6 ± 0.3 mg of compound 6 was successfully separated with a purity > 98%. Figure 4 The recoveries of compounds 6 and 7+8 were 36.0% and 49.8%, respectively, according to curve I.

[0072] Figure 3 Figure B shows Figure 3 The internal circulation separation effect of compounds 7 and 8 in Figure A is shown, with an injection volume of 50 mg. After approximately 1.5 hours, the six-way valve was adjusted to allow internal circulation separation of the sample solution. After 5 internal circulations, compound 7 was separated. Figure 4 (middle curve J) and compound 8 ( Figure 4The concentrations obtained from the curve (K) were 17.6 ± 0.4 and 22.0 ± 0.3 mg (purity > 98%), respectively, and the recoveries of compounds 7 and 8 were 35.2% and 44.0%, respectively.

[0073] Figure 3 Figure C shows Figure 2 Figure B shows the internal circulation separation effect of compounds 4 and 5. The injection volume was 50 mg, and after 7 internal circulation separations, compound 4 ( Figure 4 (Curve G) and compound 5 ( Figure 4 The compounds were completely separated in curve H), yielding 15.3 ± 0.3 and 20.6 ± 0.4 mg, respectively, with a purity >98%. The recoveries of compounds 4 and 5 were 30.6% and 41.3%, respectively.

[0074] The separation results of internal circulation mode and conventional countercurrent chromatography showed that the peak capacities of compounds 4 and 5 increased from 13 (compounds 4+5), respectively. Figure 2 The number of compounds (in Figure B) increased to 15 (compound 4, Figure 3 (Figure C) and 23 (compound 5, Figure 3 (Figure C). The theoretical plate numbers range from 1929 (compounds 4 and 5). Figure 2 The number of compounds (in Figure B) increased to 3466 (compound 4). Figure 3 (Figure C) and 3588 (compound 5, Figure 3 (See Figure C). Similarly, the peak capacities of compounds 7 and 8 increased from 11 (compounds 7+8, ...). Figure 3 The number of compounds in Figure A has increased to 19 (compound 7). Figure 3 Figure B) and 22 (compound 8, Figure 3 Figure B in the middle. The theoretical plate number is 3770 (compounds 7+8). Figure 3 A) Increased to 6852 (compound 7, respectively) Figure 3 (Figure B) and 7427 (compound 8, Figure 3 (See Figure B). The results show that the preparation method established in this invention can achieve good separation results.

[0075] 3. Structural identification Compound 1 (methyl gallate, Figure 4 Mid-peak 1): C8H8O5, ESI-MS m / z 183.03 [MH] - . 13 C NMR (101 MHz, DMSO) δ:166.79 (C-7), 146.05 (C-3, 5), 138.90 (C-4), 119.75 (C-1), 108.97 (C-2, 6), 52.04 (C-Me). 1 H NMR (400 MHz, DMSO- d 6) δ : 9.17 (3H, s, OH), 6.94 (2H, s, H-2, 6), 3.75 (3H, s, Me). Compound 2 (1,2,3,4,6-pentagalloglucose, Figure 4 Zhongfeng 2): C 41 H 32 O 26 ESI-MS m / z 939.12 [MH] - . 13 C NMR (101 MHz, DMSO) δ : 165.88 (C-G6-7), 165.27 (C-G3-7), 165.05 (C-G2-7), 164.92 (C-G4-7), 164.39 (C-G1-7), 146.14 (C-G1-3,5), 146.02 (C-G6-3,5), 145.96 (C-G4-3,5), 145.92 (C-G2-3,5), 145.84 (C-G3-3,5), 140.13 (C-G1-4), 139.61 (C-G2-4), 139.59 (C-G4-4), 139.38 (C-G3-4), 139.23 (C-G6-4), 119.34(C-G6-1), 118.55 (C-G3-1), 118.51 (C-G4-1), 118.38 (C-G2-1), 117.79 (C-G1-1), 109.48 (C-G1-2,6), 109.35 (C-G4-2,6), 109.25 (C-G6-2,6), 109.21 (C-G2-2,6), 109.18 (C-G3-2,6), 92.15 (C-Glc-1), 72.58 (C-Glc-5), 72.38 (C-Glc-3), 71.02(C-Glc-2), 68.21 (C-Glc-4), 61.87 (C-Glc-6). 1 H NMR (400 MHz, DMSO) δ: 6.97 (2H, s, G-6), 6.91 (2 H, s, G-1), 6.85 (2 H, s, G-4), 6.82 (1 H, s, G-2), 6.77 (2 H, s, G-3), 6.38 (1 H, d, J = 8.3, Glc-1), 5.96 (1 H, t, J = 9.7, Glc-3), 5.43(2 H, dd, J = 19.0, 9.6Glc-2,4), 4.59 (1 H, d, J 9.7, Glc-5), 4.30 (2 H, brs,Glc-6). Compound 3 (methyl gallate isomer (meta or para)). Figure 4 Zhongfeng 3): C 15 H 12 O9, ESI-MS m / z 335.0436 [MH] - . 13 C NMR (101 MHz, DMSO- d 6) δ : 166.37, 166.15, 164.54, 163.91, 151.01, 146.84, 146.13, 146.09, 143.35, 139.60, 139.43, 139.17, 131.58, 127.30, 120.30, 118.84, 118.58, 115.61, 113.90, 109.72, 109.68, 108.50, 52.59, 52.32. 1 H NMR (400 MHz, DMSO- d 6) δ : 7.33 (s, 1H), 7.16 (s, 1H), 7.09 (s,2H), 7.08 (s, 2H), 7.03 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H). Compound 4 (1,2,3,6-tetra-O-galloyl-β-D-glucose, Figure 4 Zhongfeng 4): C 48 H 36 O 30 ESI-MS m / z 1091.1 [MH] - .13 C NMR (101 MHz, DMSO- d 6) δ : 165.89 (C-G1-7), 165.85 (C-G2-7),165.07 (C-G3-7), 164.99 (C-G4-7), 164.96 (C-G5-7), 163.89 (C-G2-7’), 150.87(C-G2-4), 146.73 (C-G2-3’,5’), 146.14 (C-G3-3,5), 146.07 (C-G4-3,5), 146.00(C-G5-3,5), 145.96 (C-G1-3,5), 143.86 (C-G2-5), 140.11 (C-G2-3), 140.09 (C-G2-4’), 139.65 (C-G1-4), 139.43(C-G3-4), 139.27 (C-G4-4), 139.22 (C-G5-4),126.24 (C-G2-1’), 118.82 (C-G1-1), 118.67 (C-G3-1), 118.56 (C-G4-1), 118.51(C-G5-1), 117.81 (C-G2-1), 116.22 (C-G2-2), 114.05 (C-G2-6), 109.75 (C-G1-2,6), 109.49 (C-G5-2,6), 109.40 (C-G4-2,6), 109.30 (C-G3-2,6), 109.22 (C-G1-2’,6’), 92.09 (C-Glc-5), 72.96 (C-Glc-4), 72.53 (C-Glc-3), 71.03 (C-Glc-1),68.29 (C-Glc-2), 63.57 (C-Glc-6). 1 H NMR (400 MHz, DMSO- d 6) δ : 7.14-6.68 (m,12H, galloyl group), 6.41 (dd, J = 12.0, 8.2 Hz, 1H, Glc-5), 6.00 (q, J = 10.3Hz, 1H, Glc-2), 5.45 (m, 2H, Glc-1,3), 4.62 (s, 1H, Glc-4), 4.31 (d, J= 12.8Hz, 2H, Glc-6). Compound 5 (3-O-dicarboxylo-1,2,4,6-tetra-O-galloyl-β-D-pyranose, Figure 4 Zhongfeng 5): C 48 H 36 O 30 ESI-MS m / z 1091.1 [MH] - . 13 C NMR (101 MHz, DMSO) δ : 165.49 (C-G1-7), 165.29 (C-G2-7), 165.06 (C-G3-7), 165.02 (C-G4-7), 164.95 (C-G5-7), 164.55(C-G3-7'), 151.01 (C-G3-4), 146.91 (C-G3-5), 146.16 (C-G1-3,5), 145.98 (C-G2-3,5), 145.94 (C-G4-3,5), 145.85 (C-G5-3,5), 145.72 (C-G3-3',5'), 140.23 (C-G3-4'), 139.65 (C-G1-4), 139.63 (C-G2-4) 139.49 (C-G4-4), 139.41 (C-G5-4), 126.99 (C-G3-1'), 118.87 (C-G1-1), 118.74 (C-G2-1), 118.53 (C-G4-1), 118.47(C-G5-1), 118.37 (C-G3-1), 116.30 (C-G3-2), 113.97 (C-G3-6), 109.78 (C-G3-2',6'), 109.49 (C-G1-2,6), 109.38 (C-G2-2,6), 109.27(C-G4-2,6), 109.18 (C-G5-2,6), 92.19 (C-Glc-5), 72.96 (C-Glc-3,4), 71.04 (C-Glc-1), 68.06 (C-Glc-2), 63.57 (C-Glc-6). 1 H NMR (400 MHz, DMSO- d 6) δ : 7.24-6.67 (m, 12H, galloyl group),6.38 (t, J= 7.8 Hz, 1H, Glc-1), 5.96 (q, J = 10.2 Hz, 1H, Glc-3), 5.52 - 5.36 (m, 2H, Glc-2, Glc-4), 4.61 (s, 1H, Glc-5), 4.43 - 4.23 (m, 2H, Glc-6). Compound 6 ((+)-ε-viniferin, Figure 4 peak 6): C 28 H 22 O6, ESI-MS m / z 453.1384 [M-H] - . 13 13C NMR (101 MHz, DMSO- d 6) δ : 161.19 (C-13’), 159.14 (C-11,13), 158.98 (C-4’), 157.86 (C-4), 157.77 (C-11’), 146.48 (C-9), 135.28 (C-9’), 132.19 (C-1), 129.39 (C-3’,6’), 128.41 (C-2,5), 128.22 (C-11’), 127.55 (C-7’), 122.52 (C-8’), 118.92 (C-14’), 115.96 (C-3,5), 115.72 (C-3’,5’), 105.97 (C-10,14), 103.60 (C-10’), 101.51 (C-12), 96.41 (C-7), 92.85 (C-12’), 55.56 (C-8). 1 1H NMR (400 MHz, DMSO- d 6) δ : 7.13 (4H, d, J = 8.4 Hz, H-2,6,2’,6’), 6.84 (1H, d, J = 16.4 Hz, H-7’), 6.78 - 6.72 (2H, m, H-3,5), 6.68 (2H, d, J = 8.5 Hz, H-3’,5’), 6.63 - 6.55 (2H, m, H-10,14), 6.24 (1H d, J = 2.0 Hz, H-12’), 6.06 - 5.95 (3H q,J = 2.0Hz, H-12, 8',10'), 5.34 (1H, d, J = 5.2 Hz, H-7), 4.42 (1H, d, J = 5.2 Hz, H-8). Compound 7 (amurensin B, Figure 4 Zhongfeng 7): C 42 H 32 O9, ESI-MS m / z 679.2039 [MH] - . 13 C NMR (101 MHz, DMSO-) d 6) δ : 161.18 (C-11',13'), 159.25 (C-11,13,11'',13''), 157.84(C-4,4',4''), 146.23 (C-9,9''), 133.04 (C-9'), 132.04(C-1,1''), 131.66(C-1'),128.61 (C-7'), 127.88 (C-2',6'), 127.62 (C-2,6,2'',6''), 121.35(C-8'), 120.25(C-10',14'), 115.78 (C-3,5,3',5',3'',5''), 106.05 (C-10,14,10'',14''), 101.74(C-12,12''), 93.09 (C-7,7''), 91.45 (C-12'), 56.30 (C-8,8''). 1 H NMR (400 MHz, DMSO- d 6) δ 7.21 (4H, d, J = 8.5, H-2,6,2'',6''), 6.80(2H, d, J = 8.6, H-2',6'), 6.79 (4H, d, J = 8.5, H-3,5,3'',5''), 6.56 (2H, d, J = 8.6, H-3',5'), 6.51 (1H,s, H-12'), 6.48 (1H, s, H-7'), 6.43 (1H, s, H-8'), 6.05 (4H, d, J= 1.6, H-10,14,10'',14''), 6.01 (2H, s, H-12, 12''), 5.37 (2H, d, J = 5.0, H-7,7''), 4.50(2H, d, J = 5.0, H-8,8''). Compound 8 (gnetin H, Figure 4 Zhongfeng 8): C 42 H 32 O9, ESI-MS m / z 679.2045 [MH] - . 13 C NMR (101 MHz, DMSO-) d 6) δ 161.24 (C-11',13'), 159.17 (C-11, 13,11'',13''), 157.78(C-4,4',4''), 146.15(C-9,9''), 133.19 (C-9'), 132.15 (C-1,1''), 131.95 (C-1'), 128.60 (C-7'), 127.96 (C-2',6'), 127.53 (C-2,6,2'',6''), 121.29 (C-8'),119.42 (C-10',14'), 115.77 (C-3,5,3',5',3'',5''), 105.98 (C-10, 14, 10'',14''), 101.53 (C-12,12''), 93.10 (C-7, 7''), 91.02 (C-12'), 56.48 (C-8,8''). 1 H NMR (400 MHz, DMSO- d 6) δ 7.19 (4H, d, J = 8.5, H-2, 6,2'',6''), 6.81 (2H, d, J = 8.6, H-2',6'), 6.78 (4H, d, J = 8.5, H-3,5,3'',5''), 6.56 (2H, d, J= 8.6, H-3',5'), 6.50 (1H, s, H-12'), 6.46 (1H, s, H-7'), 6.45 (1H, s, H-8'), 6.05(4H, d, J = 1.6, H-10, 14,10'',14''), 6.01 (2H, s, H-12,12''), 5.38 (2H, d, J =5.0, H-7,7''), 4.49 (2H, d, J = 5.0, H-8,8''). The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing polyphenolic compounds from peony pods, characterized in that, include: Ethanol was used to extract polyphenolic compounds from peony pods to obtain crude extract of the pods; The crude extract of the fruit pods was dissolved in a high-speed countercurrent chromatography solvent system to obtain a sample solution; The sample solution was separated in the elution-sweep-top mode of high-speed countercurrent chromatography to obtain polyphenol monomers and mixtures; the mixture was then separated using internal circulation countercurrent chromatography.

2. The preparation method according to claim 1, characterized in that, The polyphenolic compounds extracted from peony pods using ethanol include: Peony pods were extracted with anhydrous ethanol at a temperature of 50-60℃ and a material-to-liquid ratio of 1:9-11 to obtain crude extract of the pods.

3. The preparation method according to claim 1, characterized in that, The polyphenolic compounds include compounds 1-8, whose structural formulas are as follows: 。 4. The preparation method according to claim 3, characterized in that, The sample solution was separated in the elution-sweep-top mode of high-speed countercurrent chromatography to obtain compounds 1, 2, 3, a mixture of compounds 4 and 5, and a mixture of compounds 6, 7 and 8. The mixture of compounds 4 and 5 was separated by internal circulation countercurrent chromatography to obtain compounds 4 and 5. The mixture of compounds 6, 7 and 8 was separated by countercurrent chromatography to obtain a mixture of compounds 6, 7 and 8. The mixture of compounds 7 and 8 was separated by internal circulation countercurrent chromatography to obtain compounds 7 and 8.

5. The preparation method according to claim 4, characterized in that, The high-speed countercurrent chromatography solvent system in the elution-sweep-ejection mode is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of (1-2):(8-9):(1-2):(8-9); Preferably, the volume ratio is 1:9:1:

9.

6. The preparation method according to claim 4, characterized in that, When separating the mixture of compounds 4 and 5, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of (1-2):(8-9):(1-2):(8-9); Preferably, the volume ratio is 1:9:1:

9.

7. The preparation method according to claim 4, characterized in that, When separating the mixture of compounds 6, 7 and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of 5:5:(2-3):(7-8); Preferably, the volume ratio is 5:5:2.5:7.

5.

8. The preparation method according to claim 4, characterized in that, When separating the mixture of compounds 7 and 8, the high-speed countercurrent chromatography solvent system is a mixed solution of n-hexane, ethyl acetate, methanol and water in a volume ratio of 5:5:(2-3):(7-8).

9. The preparation method according to claim 8, characterized in that, The volume ratio is 5:5:2.5:7.

5.

10. The application of the method for preparing polyphenolic compounds from peony pods according to any one of claims 1-9 in the extraction of active ingredients from peony pods.

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