A method for separating components of peony pollen based on macroporous resin enrichment

Abstract

This invention relates to the field of natural product separation technology, and more particularly to a method for separating components of peony pollen based on macroporous resin enrichment. This invention uses macroporous resin combined with high-speed countercurrent chromatography to systematically separate peony pollen extract, obtaining paeoniflorin oxide, methyl gallate, paeoniflorin, tartrazine-3-O-sophoroside, 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside, and N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine, N 1 -(E)-N 5 -(E)-N 10 The separation method for seven compounds, namely (E)-tristigmoyl spermidine, is simple and yields high product purity.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, and in particular to a method for separating peony pollen components based on macroporous resin enrichment. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the 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] Pollen is the essence of life, possessing high nutritional and medicinal value. Peony pollen is rich in active substances, including flavonoids, polyphenols, plant proteins, and organic acids. Peony pollen has significant effects on blood sugar metabolism, improving blood circulation, and regulating blood lipid levels. As a natural health food, peony pollen has attracted much attention in the health supplement field due to its various physiological regulatory functions. Peony pollen has antioxidant, anti-aging, metabolism-promoting, cholesterol-degrading, and benign prostatic hyperplasia-regulating effects. However, peony pollen still suffers from low research levels and insufficient utilization. It has not received sufficient attention and resources, resulting in the waste of large quantities of pollen. Most existing research on the extraction and separation of peony pollen only focuses on the separation and purification of some compounds. Therefore, systematic extraction, separation, and structural identification of the active components of peony pollen are necessary. Summary of the Invention

[0004] In view of this, the present invention provides a method for separating peony pollen components based on macroporous resin enrichment.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a method for separating peony pollen components based on macroporous resin enrichment, comprising the following steps: (1) Peony pollen was extracted by shaking in a shaker to obtain peony pollen extract; (2) Peony pollen extract was adsorbed onto a macroporous resin column and eluted by a water-ethanol gradient to obtain components A, B and C; (3) Component A was separated by high-speed countercurrent chromatography using solvent system 1 to obtain compounds 1 and 2; (4) Component B was separated by high-speed countercurrent chromatography using solvent system 2 to obtain compounds 3, 4 and 5; (5) Component C was separated by high-speed countercurrent chromatography using solvent system 3 to obtain compounds 6 and 7; The structural formulas of each compound are as follows: .

[0006] Furthermore, in step (1), the extraction solvent is an alcohol, preferably methanol; the ratio of peony pollen to extraction solvent is 1:8-12, preferably 1:10.

[0007] Furthermore, in step (1), the extraction time is 1-3 hours and the number of extractions is 1-3 times.

[0008] Furthermore, in step (1), the concentration temperature is 30-50 ℃, preferably 40 ℃.

[0009] Furthermore, in step (2), the macroporous resin is selected from D101, AB-8, MQ-10 or HPD100, preferably MQ-10.

[0010] Furthermore, in step (2), a gradient elution of 10%, 20-30%, and 50-70% ethanol aqueous solution is used.

[0011] Furthermore, in step (2), the pH value is 3-5 during macroporous resin column adsorption.

[0012] Further, in step (3), solvent system 1 is a mixed solvent of n-butanol and water; the volume ratio of n-butanol to water is 1:1-1.5, preferably 1:1.

[0013] Furthermore, in step (3), a single injection of 50-60 mg is performed at a flow rate of 4-6 mL / min, and separation is carried out using the continuous injection EECCC mode.

[0014] Further, in step (4), solvent system 2 is a mixed solvent of ethyl acetate, n-butanol and water; the volume ratio of ethyl acetate, n-butanol and water is 1:1:1.5-2.5, preferably 1:1:2.

[0015] Furthermore, in step (4), a single injection of 50-60 mg is performed at a flow rate of 4-6 mL / min, and separation is carried out using the continuous injection EECCC mode.

[0016] Further, in step (5), the solvent system 3 is a mixed solvent of n-hexane, ethyl acetate, methanol and water; the volume ratio of n-hexane, ethyl acetate, methanol and water is 1:3-5:1-1.5:3-5, preferably 1:4:1:4.

[0017] Furthermore, in step (5), a single injection of 30-40 mg is performed at a flow rate of 1-3 mL / min, and separation is carried out using IRCCC mode.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention utilizes macroporous resin combined with high-speed countercurrent chromatography to systematically separate components of peony pollen, obtaining paeoniflorin oxide, methyl gallate, paeoniflorin, tartrazine-3-O-sophoroside, 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside, and N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine, N 1 -(E)-N 5 -(E)-N 10 The separation of seven compounds, including (E)-tristigmoyl spermidine, was achieved using a simple method. A specific solvent system and macroporous resin were employed, resulting in high purity (over 98%). This method enables the systematic separation of compounds from peony pollen. Attached Figure Description

[0019] 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.

[0020] Figure 1 This is a graph showing the adsorption and desorption performance of different types of macroporous adsorption resins for various compounds in peony pollen; where (A) is paeoniflorin oxide; (B) is methyl gallate; (C) is paeoniflorin; (D) is tartrazine-3-O-sophoroside; (E) is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; and (F) is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine; (G) for N 1 -(E)-N 5 -(E)-N 10 -(E)-Tricarboxyloylsemine; Figure 2 This is a graph showing the effect of different pH values ​​on the adsorption and desorption properties of various compounds in peony pollen by macroporous resins; where (A) is paeoniflorin oxide; (B) is methyl gallate; (C) is paeoniflorin; (D) is tartrazine-3-O-sophoroside; (E) is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; and (F) is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine; (G) for N 1 -(E)-N 5-(E)-N 10 -(E)-Tricarboxyloylsemine; Figure 3 These are graphs showing the effect of time on the adsorption and desorption properties of macroporous resins; where (A) is the effect of time on adsorption performance; and (B) is the effect of time on desorption performance. Figure 4 This is a graph showing the effect of different ethanol concentrations on the desorption of various compounds in peony pollen; where (A) is paeoniflorin oxide; (B) is methyl gallate; (C) is paeoniflorin; (D) is tartrazine-3-O-sophoroside; (E) is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; and (F) is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine; (G) for N 1 -(E)-N 5 -(E)-N 10 -(E)-Tricarboxyloylsemine; Figure 5 This is an HPLC chromatogram of the components of peony pollen extract after enrichment with macroporous resin. Figure 6 This is the IRCCC separation diagram of component A; Figure 7 This is the IRCCC separation diagram of component B; Figure 8 This is the IRCCC separation diagram of component C; Figure 9 This is an HPLC chromatogram of peony pollen extract and its separated compounds. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0023] Example 1 1. Extraction of peony pollen samples Weigh 500 g of peony pollen and extract it in a water bath shaker at a ratio of 1:10 (material to liquid) using methanol as the extraction solvent. The extraction time is 2 h, and the extraction is repeated twice. After filtration, the extracts are combined and concentrated at 40 °C. The extract is then freeze-dried by rotary evaporation to obtain 113 g of peony pollen alcohol extract for later use.

[0024] 2. HSCCC separation of peony pollen extract HSCCC Solvent System Selection: A two-phase solvent system was used for sample separation. 20 mL of solvent was prepared according to different solvent ratios, thoroughly shaken, and allowed to separate. 2 mL of each phase was transferred to centrifuge tubes, and an appropriate amount of sample powder to be separated was added. The mixture was thoroughly shaken or sonicated to dissolve the powder, and then centrifuged until the solvent completely evaporated. Subsequently, a certain amount of methanol solution was used for dissolution. After pretreatment with a 0.22 μm organic nylon filter membrane, the sample was transferred to a sample vial and analyzed using high-performance liquid chromatography (HPLC). The integrated peak areas of each target compound were recorded in the chromatogram, with the upper phase peak area designated as A1 and the lower phase peak area as A2. The partition coefficients of the target compounds were calculated. K D ),in K D = A1 / A2.

[0025] Preparation of solvent system and sample solution: Prepare a two-phase solvent system in a 2 L separatory funnel according to the specified ratio. Shake thoroughly to mix completely, then allow to stand for separation. Pour out the upper and lower layers separately; the upper phase is the stationary phase, and the lower phase is the mobile phase. Sonicate the solutions for 20 min to remove air bubbles. Dissolve the sample to be separated in equal volumes of the upper and lower phases of the selected solvent system for subsequent separation.

[0026] 3. High-speed countercurrent chromatography separation: According to different operating methods, it can be divided into the following 3 separation modes.

[0027] (1) Normal HSCCC separation mode Turn on the cold trap 15 minutes in advance to maintain the temperature at 25 °C and preheat the UV detector. First, pump the stationary phase of the system into the countercurrent chromatograph at a flow rate of 30 mL / min. When liquid streams out of the tube, stop the pump and place the pump head in the mobile phase bottle. Set the rotation speed to 800 rpm and the flow mode to "FWD-IN". Pump the system into the countercurrent chromatograph at a suitable flow rate. When the lower limit of the pump's set flow rate is stably flowing out of the end of the tubing, the internal system has reached equilibrium. Separate the pre-dissolved sample by injecting it into the countercurrent chromatograph. After separation, turn on the oil pump to purge the liquid from the tubing. The volume percentage of stationary phase is the retention rate. After each separation, clean the entire tubing with anhydrous ethanol.

[0028] (2) Continuous injection EECCC mode After the sample is separated according to the standard HSCCC countercurrent procedure, the same sample is injected again through the six-way valve. After three consecutive injections, the pump is stopped and the countercurrent mobile phase is replaced with the stationary phase. The other conditions are the same as the standard countercurrent procedure. After all samples have been eluted, the elution is stopped and the same components are combined.

[0029] (3) Cyclic IRCCC separation mode The injection procedure was performed as described in (1). Based on the real-time spectrum of the UV detector, before the required cyclic components were eluted, the eluent was pumped back into the countercurrent chromatograph tubing via an external six-way valve using a constant flow pump for the next separation. After observing successful separation of each component through the UV detection spectrum, the six-way valve mode was switched to end the cyclic separation mode, allowing the eluent to flow out. The eluent was collected and subsequent operations were performed according to the conventional HSCCC method.

[0030] The eluent collected in the centrifuge tubes of the fraction collector was filtered through a 0.22 µm nylon filter membrane and analyzed by HPLC. Identical components were combined and then evaporated to dryness by rotary evaporation or lyophilization.

[0031] 4. High-performance liquid chromatography and nuclear magnetic resonance mass spectrometry analysis: The detection conditions for liquid chromatography are as follows: the chromatographic column is an Agilent Extend-C10 ... 18 (250 mm × 4.6 mm, 5.0 µm); mobile phase was methanol (A)-water (B): 0 min, 5% A; 25 min, 95% A; 26 min, 5% A; 30 min, 5% A. Flow rate was 1.0 mL / min, injection volume was 10 µL, column temperature was 25 ℃, and detector wavelength was 254 nm. Mass spectrometry analysis was performed using an LC-MS-9030 Q-TOF quadrupole-time-of-flight mass spectrometer (Shimadzu).

[0032] 5. Screening and enrichment of macroporous resins Macroporous resin pretreatment: The macroporous resin was soaked in 95% ethanol solution for 24 h, then rinsed with ethanol of the same concentration until the eluent was clear, and then rinsed with ultrapure water until there was no alcohol odor. Subsequently, it was subjected to alkali treatment (5% NaOH, 2-4 h) and acid treatment (5% HCl, 2-4 h) in sequence, and then repeatedly washed with ultrapure water until neutral, and soaked and stored for later use.

[0033] Static adsorption and desorption performance of macroporous resins: Three portions of each pretreated resin were weighed and placed in conical flasks. 10 mL of a 20 mg / mL aqueous solution of peony pollen extract was added. The mixture was shaken for 24 h at a temperature of 30 ℃ and a flow rate of 150 r / min. After adsorption equilibrium, the resin was removed by filtration. The integral area S1 of each monomer compound in the supernatant was calculated by liquid chromatography, and the integral area S0 of each monomer compound before adsorption and desorption was also measured. After filtration and drying, the resin was added back to the conical flask, along with an equal volume of 95% ethanol solution. The mixture was shaken for 24 h at a temperature of 30 ℃ and a flow rate of 150 r / min. The resin was removed by filtration again, and the integral area S2 of each monomer compound in the supernatant was measured. The adsorption and desorption capacities of different macroporous resins for peony pollen extract were evaluated by adsorption and desorption rates. The formulas for calculating adsorption and desorption rates are as follows: ; .

[0034] Static adsorption-desorption kinetics experiment: Weigh 6 g of each of MQ-10 resin into a conical flask, and add 30 mL of peony pollen ethanol extract aqueous solution. Adsorption was carried out at 30 ℃ with constant shaking at 150 r / min for 6 h. Every 30 min, 0.5 mL of supernatant from one conical flask was taken for measurement, and a static adsorption curve was plotted to calculate the adsorption rate. After filtration and drying, the resin was added back into the conical flask, along with an equal volume of 95% ethanol solution. The temperature was controlled at 30 ℃, and the desorption rate at 150 r / min was maintained for 6 h with shaking. Every 30 min, 0.5 mL of supernatant was taken for measurement until the integral area of ​​each compound in the liquid chromatography spectrum showed no significant change, and a static desorption curve was plotted.

[0035] Effect of solution pH on the adsorption and desorption rate of macroporous resin: 10 mL of 20 mg / mL peony pollen extract aqueous solution was added to a 250 mL volumetric flask. The pH of the peony pollen alcohol extract aqueous solution was adjusted to 3.0, 5.0, 7.0, 9.0, and 11.0 with 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solutions, respectively. 2.0 g of MQ-10 macroporous resin was accurately weighed and placed in an Erlenmeyer flask. The solution was incubated at 30℃ with constant temperature shaking at 150 r / min for 3 h. 0.5 mL of the supernatant was taken and the integral area of ​​each monomer compound in the supernatant was calculated by liquid chromatography. The adsorption rate was calculated according to the formula.

[0036] Dynamic adsorption curves of macroporous resin: Dynamic adsorption experiments were conducted using a glass column with an inner diameter of 1.0 cm and a length of 10 cm. 4 g of macroporous resin was accurately weighed and loaded onto the column using a wet method. An aqueous solution of crude peony pollen extract was pumped in at a rate of 2 BV / h. Different ethanol concentration solutions (10%, 30%, 50%, 70%, and 90%) were prepared and eluted at a rate of 2 BV / h, with an elution volume of 5 BV. The elution rate of each compound was detected by liquid chromatography.

[0037] Preparation and pretreatment of the loading solution: Weigh 15 g of peony pollen extract, dissolve it in an appropriate amount of anhydrous ethanol, weigh 30 g of macroporous resin into the solution, let it stand for 3 h, then concentrate it by rotary evaporation to dry granules, and load it onto the column using the dry loading method. After the column is packed, let it stand for 2 h.

[0038] Macroporous resin column elution: Elute with pure water, 10%, 20%, 30%, 40%, 50%, 60%, and 70% ethanol-water sequentially at a flow rate of 10 mL / min for 5 BV. Collect the eluent, perform liquid phase analysis, and lyophilize by rotary evaporation.

[0039] 6. Results and Discussion 6.1 Screening and Enrichment of Macroporous Resins Figure 1 This is a graph showing the adsorption and desorption performance of different types of macroporous adsorption resins for various compounds in peony pollen; where (A) is paeoniflorin oxide; (B) is methyl gallate; (C) is paeoniflorin; (D) is tartrazine-3-O-sophoroside; (E) is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; and (F) is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine; (G) for N 1 -(E)-N 5 -(E)-N 10 -(E)-Tristigroylspermine. (From...) Figure 1 It can be seen that the adsorption capacities of the various macroporous resins for the seven compounds are not significantly different, but their desorption capacities vary considerably. Therefore, resins D101, AB-8, MQ-10, and HPD100 can be selected for subsequent experiments. Considering the adsorption and desorption capacities of each resin for the compounds, MQ-10 macroporous resin was ultimately chosen for further experiments.

[0040] Figure 2This is a graph showing the effect of different pH values ​​on the adsorption and desorption properties of various compounds in peony pollen by macroporous resins; where (A) is paeoniflorin oxide; (B) is methyl gallate; (C) is paeoniflorin; (D) is tartrazine-3-O-sophoroside; (E) is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; and (F) is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine; (G) for N 1 -(E)-N 5 -(E)-N 10 -(E)-Tristigroylspermine. (From...) Figure 2 As can be seen from (B), (F), and (G) in the data, the adsorption and desorption of these three compounds by MQ-10 decreased to varying degrees as the alkalinity of the solution increased, with the effect on desorption being more pronounced. Figure 2 As can be seen from (A), (C), (D), and (E), the other compounds all showed varying degrees of decrease. Therefore, considering all factors, a pH value of 5 was chosen for subsequent experiments.

[0041] Figure 3 These are graphs showing the effect of time on the adsorption and desorption properties of macroporous resins; where (A) shows the effect of time on adsorption performance; and (B) shows the effect of time on desorption performance. Figure 3 It can be seen that the adsorption capacity of the seven compounds reached saturation at 180 min and desorption was completed at 120 min. Therefore, the optimal adsorption and desorption times were determined to be 180 min and 120 min, respectively.

[0042] Figure 4 This is a graph showing the effect of different ethanol concentrations on the desorption of various compounds in peony pollen; where (A) is paeoniflorin oxide; (B) is methyl gallate; (C) is paeoniflorin; (D) is tartrazine-3-O-sophoroside; (E) is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; and (F) is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine; (G) for N 1 -(E)-N 5 -(E)-N 10 -(E)-Tricarboxyloylsemine. From Figure 4It can be seen that when the ethanol concentration is 50%, all components except compound 7 have reached the maximum desorption. When the ethanol concentration is 70%, all compounds have reached the maximum desorption. Therefore, the optimal ethanol concentration for desorption is determined to be 70%.

[0043] Through a series of adsorption and desorption experiments, the optimal macroporous resin was determined before subsequent enrichment.

[0044] Weigh 15 g of peony pollen ethanol extract, dissolve it in an appropriate amount of anhydrous ethanol, weigh 30 g of macroporous resin into the solution, let it stand for 3 h, then concentrate it by rotary evaporation to a dry granular state, and load it onto a column using a dry method. After column packing, let it stand for 2 h. Set the flow rate to 10 mL / min and equilibrate with 10% ethanol solution. Elute sequentially with 5 L of 10%, 20%, 30%, 40%, 50%, 60%, and 70% ethanol solutions, collecting the eluents in 500 mL vials. Perform high-performance liquid chromatography (HPLC) analysis on each collected vial, combine the eluents containing similar compounds, concentrate them by rotary evaporation, and then freeze-dry them to obtain multiple fractions containing trace amounts of the same compounds.

[0045] Figure 5 This is an HPLC chromatogram of the components of peony pollen extract after enrichment with macroporous resin. (Example:) Figure 5 As shown, using MQ-10 macroporous resin, the peony pollen extract was separated into three components, A, B, and C, by water-ethanol gradient elution (10%, 20-30%, 50-70% ethanol). Each component was collected, concentrated, and freeze-dried for subsequent separation.

[0046] 6.2 HSCCC Separation Process for Macroporous Resin Components Table 1. Partition coefficients of peony pollen samples in different solvent systems

[0047] Table 1 shows the partition coefficients of different compounds in peony pollen in a countercurrent chromatographic two-phase solvent system. K value), K The value represents the solubility and partitioning of the compound between different phases in a two-phase solvent system. K The value >2 indicates that the compound elutes more slowly, retains longer, and has lower separation efficiency in countercurrent chromatography with the above phase as the stationary phase. K <0.5 indicates that the compound has a short retention time and may be difficult to separate effectively; K When the value is between 0.5 and 2, separation is relatively reasonable. To determine whether two compounds can be separated, the separation coefficient α needs to be used. 1,2 = K 1 / K 2 (K 1> K 2) When α>1.5, it indicates that the compounds can be separated well. When α<1.5, countercurrent chromatography with a circulating method is required for separation in this study.

[0048] Solvent system selection: A series of solvent systems were selected as shown in Table 1. Among the selected solvent systems, compound 2 in component A... K The values ​​were consistently high, indicating that it could not be prepared using the normal HSCCC reverse elution mode. Therefore, a 1:1 (v / v) ratio of n-butanol:water was ultimately chosen for subsequent IRCCC separation of component A. Compounds 4 and 5 in component B... K The values ​​are quite similar in multiple systems, indicating that they are difficult to separate by cycling. However, in the system of ethyl acetate:n-butanol:water = 5:5:10 (v / v), compounds 4 and 5 show different values. K The values ​​were 0.95 and 1.42, respectively, and the separation factor α was 1.49, indicating that it could be separated by circulation in this system. Therefore, ethyl acetate: n-butanol: water = 5:5:10 (v / v) was finally selected for the subsequent separation of component B. The solvent system n-hexane: ethyl acetate: methanol: water = 2:8:2:8 (v / v) was selected for the IRCCC separation of compounds 6 and 7 in component C.

[0049] Component A separation: The solvent system was n-butanol:water at a ratio of 1:1 (v / v), the flow rate was set at 5 mL / min, and the retention rate was 57.6%. Compounds 1 and 2 were separated using continuous injection EECCC mode. Figure 6 This is the IRCCC separation diagram of component A, as shown below. Figure 6 As shown, a single injection of 50 mg yielded compound 1 (59.4 mg, purity > 98%) and compound 2 (35.2 mg, purity > 98%).

[0050] Component B separation: Using a solvent system of ethyl acetate: n-butanol: water 5:5:10 (v / v), with a flow rate of 5 mL / min, compounds 3, 4 and 5 were separated by continuous injection. Figure 7 This is the IRCCC separation diagram of component B, as shown below. Figure 7 As shown, a single injection of 50 mg yielded 15.7 mg of compound 3 monomer with a purity >98%, 56.4 mg of compound 4 with a purity >98%, and 42.9 mg of compound 5 with a purity >98%.

[0051] Component C separation: Using a solvent system of n-hexane:ethyl acetate:methanol:water = 2:8:2:8 (v / v), with a flow rate of 2 mL / min, compounds 6 and 7 were separated by circulation. Figure 8This is the IRCCC separation diagram of component C, as shown below. Figure 8 As shown, after injecting 30 mg and cyclic separation, the retention rate was 52.8%, yielding compound 6 (6.4 mg, purity > 98%) and compound 7 (17.2 mg, purity > 98%).

[0052] Figure 9 This is an HPLC chromatogram of peony pollen extract and its separated compounds. Seven compounds (HPLC purity >98%) were successfully isolated from the ethanol extract of peony pollen using HSCCC. The detection results are as follows. Figure 9 As shown.

[0053] By comparing the compounds with literature, the seven compounds isolated from peony pollen were identified as follows: compound 1 is paeoniflorin oxidase, compound 2 is methyl gallate, compound 3 is paeoniflorin, compound 4 is tartrazine-3-O-sophoroside, compound 5 is 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside, and compound 6 is N... 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine, compound 7 is N 1 -(E)-N 5 -(E)-N 10 -(E)-Tricarboxyloylspermine. Characterization data are as follows: Oxidized paeoniflorin (1): ESI-MS m / z 495.1507 [MH] - The molecular formula is C 23 H 28 O 12 , 1H NMR (400MHz, DMSO-d6) δ: 7.87-7.79 (2H, m, H-2'', 6''), 6.90-6.82 (2H, m, H-3'',5''), 5.29 (1H d, J = 17.4 Hz, H-9), 4.63-4.52 (2H, m, H-8), 4.38 (1H, dd, J= 21.0, 7.7 Hz, H-1'), 3.66 (1H, dd, J = 11.8, 2.1 Hz, H-6'a), 3.15-2.94 (4H,m, H-2'-5'b), 2.45-2.32 (2H, m, H-6'b ,H-5), 2.09-1.98 (1H, m, H-3a), 1.81 (1H, d, J = 10.6 Hz, H-7b), 1.68-1.60 (1H, m, H-3b), 1.28-1.24 (3H, d, J =19.1 Hz, H-10). 13 C NMR (101 MHz, DMSO-d6) δ: 166.05 (C-7''), 162.65 (C-4''), 132.02 (C-2'', 6''), 120.65 (C-1''), 115.84 (C-3'', C-5''), 105.19 (C-4), 100.56 (C-9), 99.08 (C-1'), 87.92 (C-1), 85.42 (C-2), 77.38 (C-3', 5'), 73.91(C-2'), 70.71 (C-6), 70.54 (C-4'), 61.69 (C-6'), 60.25 (C-8), 44.07 (C-3), 42.78 (C-5), 22.52 (C-7), 19.56 (C-10).

[0054] Methyl gallate (2): ESI-MS m / z 183.0294 [MH] - The molecular formula is C8H8O5. 1 H NMR (400MHz, DMSO-d6) δ: 6.94 (2H, s, H-2, 6), 3.74 (3H, s, 7-OCH3). 13C NMR (101 MHz, DMSO-d6) δ: 166.81 (7-COO-), 146.11 (C-3), 139.05 (C-4), 119.66 (C-1), 108.98 (C-2, 6), 52.02 (8-OCH3).

[0055] Paeoniflorin (3): ESI-MS m / z 479.4138 [MH] - The molecular formula is C 23 H 28 O 11 , 1 H NMR (600 MHz, DMSO-d6) δ: 7.99 (2H, d, J = 8.3 Hz, H-2'', 6''), 7.68 (1H, t, J = 7.4 Hz, H-4''), 7.55 (2H, t, J = 7.8 Hz, H-3'', 5''), 5.32 (1H, s, H-9), 4.68-4.59 (2H,m, H-8), 4.39 (1H, d, J = 7.7 Hz, H-1'), 3.64 (1H, m, H-6'a), 3.17-2.95 (5H,m, H-2'-6'b), 2.44 (1H, m, H-5), 2.37 (1H, m, H-7a), 2.05 (1H, d, J = 12.2Hz, H-3a), 1.82 (1H, d, J = 10.4 Hz, H-7b), 1.65 (1H, d, J = 12.0 Hz, H-3b), 1.24 (3H, s, H-10). 13C NMR (151 MHz, DMSO-d6) δ: 166.23 (C-7''), 133.92 (C-4''), 130.13 (C-1''), 130.10 (C-2'', 6''), 129.25 (C-3'', 5''), 105.20 (C-4), 100.50 (C-9), 99.09 (C-1'), 87.94 (C-1), 85.42 (C-2), 77.39 (C-3', 5'), 77.36 (C-3', 5'), 73.90 (C-2'), 70.70 (C-6), 70.42 (C-4'), 61.66 (C-6'), 60.91 (C-8), 44.05 (C-3), 42.75 (C-5), 22.45 (C-7), 19.57 (C-10).

[0056] Tartrazine-3-O-sophoroside (4): ESI-MS m / z 669.1663 [MH] - The molecular formula is C 29 H 34 O 18 , 1 HNMR (400 MHz, DMSO-d6) δ: 12.34 (1H, s, 5-OH), 8.10-8.03 (2H, m, H-2', H-6'), 6.98-6.91 (2H, m, H-3',H-5'), 6.26 (1H, s, H-8), 5.76-5.69 (1H, m, H-2''),5.48 (2H, s, H-1'', H-1'''), 5.19-4.96 (1H, m, H-1'''), 4.63 (3H, t, J = 8.4Hz, H-3'',H-4'',H-5''), 3.81 (3H, s, -OCH3), 3.56-3.44 (3H, m, -OCH3'). 13C NMR(101 MHz, DMSO-d6) δ: 178.00 (C-4), 156.4 (C-2), 155.74 (C-7), 148.96 (C-4'), 133.29 (C-3), 131.23 (C-2'), 128.00 (C-6'), 121.53 (C-1'), 115.86 (C-3', C-5'), 104.54 (C-1''), 103.97 (C-1'''), 99.52 (C-6), 98.45 (C-8), 82.90 (C-2''), 77.97 (C-5''), 77.46 (C-5'''), 77.02 (C-3'''), 74.84 (C-2'''), 70.15(C- 3''), 70.03 (C-4'''), 61.38 (C-6''), 61.28 (-OCH3), 61.00 (C-6''').

[0057] 5,7,4'-Trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside (5): ESI-MS m / z 639.1547 [MH] - The molecular formula is C 28 H 32 O 17 , 1 H NMR (400 MHz, DMSO-d6) δ: 7.83 (1H, d, J = 2.1 Hz,H-2'), 7.67 (1H, dd, J = 8.4, 2.1 Hz, H-6'), 6.97 (1H, d, J = 8.5 Hz, H-5'), 6.28 (1H, s, H-6), 5.82-5.75 (1H, m, H-1'''), 4.63 (1H, d, J = 7.7 Hz, H-1''), 3.83 (3H, s, 8-OCH3), 3.55-3.51 (3H, m, H-3'', H-4'', H-5''), 3.43 (1H,dd, J = 11.5, 4.7 Hz, H-6''), 3.33 (1H, dd, J = 12.7, 3.5 Hz, H-6'''), 3.14(2H, s, H-5'', H-5'''). 1313C NMR (101 MHz, DMSO-d6) δ: 177.94 (C-4), 158.07 (C-7), 156.39 (C-5), 155.90 (C-2), 150.01 (C-4'), 148.97 (C-9), 147.51 (C-8), 133.32 (C-3), 103.98 (C-1'''), 99.56 (C-1''), 82.53 (C-2''), 77.89 (C-3'',C-5''), 77.29 (C-3''',C-5'''), 76.98 (C-4''), 74.76 (C-4'''), 70.25 (C-6''), 70.00 (C-6'''), 56.12 (8-OCH3).

[0058] N 1 ,N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine (6): ESI-MS m / z 598.26 [M-H] - , molecular formula C 34 H 37 N3O7, 1 1H NMR (400 MHz, DMSO-d6) δ: 9.47 (br, s, OH), 8.15-7.91 (br, m, NH-1, NH-10), 7.43-7.27 (7H, m, H-7'', 7''', 7', 2''', 6''', 2', 6'), 7.09 (1H, d, J = 5.6 Hz, H-6''), 6.98 (m, H-2''), 6.86-6.654 (6H, m, 8'', 3', 5', 3''', 5''', 3''), 6.46-6.33 (2H, m, H-8''', 8'), 3.48 (2H, m, H-4, 6), 3.19 (2H, m, H-2, 9), 1.73 (1H, m, H-3), 1.48 (2H, m, H-7, 8). 13C NMR(101 MHz, DMSO-d6) δ: 166.05 / 166.01 / 165.91 / 165.88 / 165.84 / 165.79 (C-9', 9'',9'''), 159.26 / 159.23 (C-4', 4'''), 147.83 / 147.77 (C-4''), 145.88 (C-5''), 142.39 (C-7''), 139.29 / 139.13 / 139.09 / 139.00 (C-7', 7'''), 129.67 / 129.62 (C-2', 6', 2''', 6'''), 127.17 (C-1''), 126.39 (C-1', 1'''), 121.10 (C-3''), 120.76 (C-2''), 119.27 / 119.22 / 119.13 / 118.95 (C-8', 8'''), 116.19 / 116.13 (C-3', 3''', 5', 5'''), 115.61 / 115.27 (C-8''), 115.06 (C-6''), 47.38 / 45.98 (C-6), 45.42 / 44.28 (C-4), 38.93 / 38.72 (C-9), 37.08 / 36.76 (C-2), 30.23 / 28.44 (C-3), 27.42 / 27.26 (C-8), 27.09 / 25.67 (C-7).

[0059] N 1 -(E)-N 5 -(E)-N 10 -(E)-Tricarboxyloylspermine (7): ESI-MS m / z 582.26 [MH] - The molecular formula is C 34 H 37 N3O6, 1H NMR (400 MHz, DMSO-d6) δ: 9.88(br, s, OH), 8.16-7.98 (br, m,NH-1, NH-10), 7.58-7.27 (9H, m, H-7’’, 7’’’, 7’, 2’’, 6’’, 2’’’, 6’’’, 2’ ,6’), 6.90 (1H, d, J = 15.5 Hz, H-8’’) / 6.84 (1H, d, J = 15.5 Hz, H-8’’), 6.81-6.75 (4H, m, H-3’, 5’, 3’’’, 5’’’), 6.73 (2H, d, J = 8.2 Hz, H-3’’, 5’’),6.48-6.36 (2H, m, H-8’’’, 8’), 3.48 (2H, m, H-4, 6), 3.20 (2H, m, H-2, 9),1.78 (1H, q, J = 7.1 Hz, H-3) / 1.69 (1H, q, J = 7.1 Hz, H-3), 1.56 (1H, m, H-7), 1.50 / 1.42 (1H, m, H-8). 13C NMR (101 MHz, DMSO-d6) δ: 166.08 / 166.02 / 165.91 / 165.86 / 165.80 (C-9', 9'', 9'''), 159.45 / 159.36 / 159.29 (C-4', 4'', 4'''), 141.99 (C-7''), 139.28 / 139.11 / 139.02 (C-7', 7'''), 130.25 / 130.18 (C-2'',6''), 129.66 / 129.61 (C-2', 6', 2''', 6'''), 126.65 / 126.59 (C-1''), 126.39 / 126.36 / 126.31(C-1',1'''), 119.25 / 119.19 (C-8'''), 119.14 / 118.93 (C-8'),116.25 / 16.21 / 116.08 (C-3', 3'', 3''', 5', 5'', 5'''), 115.18 / 115.07 (C-8''), 47.37 / 45.94 (C-6), 45.40 / 44.26 (C-4), 38.94 / 38.65 (C-9), 37.09 / 36.69 (C-2), 30.20 / 28.44 (C-3), 27.36 / 27.27 (C-8), 27.07 / 25.68 (C-7).

[0060] 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 separating peony pollen components based on macroporous resin enrichment, characterized in that, Includes the following steps: (1) Peony pollen was extracted by shaking in a shaker to obtain peony pollen extract; (2) Peony pollen extract was adsorbed onto a macroporous resin column and eluted by a water-ethanol gradient to obtain components A, B and C; (3) Component A was separated by high-speed countercurrent chromatography using solvent system 1 to obtain paeoniflorin oxide and methyl gallate; (4) Component B was separated by high-speed countercurrent chromatography using solvent system 2 to obtain paeoniflorin, tartrazine-3-O-sophoroside and 5,7,4'-trihydroxy-8-methoxyflavonol-3-O-β-D-sophoroside; (5) Component C was separated by high-speed countercurrent chromatography using solvent system 3 to obtain N. 1 N 10 -di-p-coumaroyl-N 5 -caffeoylspermidine and N 1 -(E)-N 5 -(E)-N 10 -(E)-Tricarboxyloylsemine; Solvent system 1 is a mixed solvent of n-butanol and water, with a volume ratio of n-butanol to water of 1:1-1.5; solvent system 2 is a mixed solvent of ethyl acetate, n-butanol, and water, with a volume ratio of ethyl acetate, n-butanol, and water of 1:1:1.5-2.5; solvent system 3 is a mixed solvent of n-hexane, ethyl acetate, methanol, and water, with a volume ratio of n-hexane, ethyl acetate, methanol, and water of 1:3-5:1-1.5:3-5. In step (2), the macroporous resin is selected from MQ-10; In step (2), a gradient elution of 10%, 20-30%, and 50-70% ethanol aqueous solution is used; when the macroporous resin column is used for adsorption, the pH value is 3-5.

2. The separation method as described in claim 1, characterized in that, In step (1), the extraction solvent is methanol; the ratio of peony pollen to extraction solvent is 1:8-12.

3. The separation method as described in claim 1, characterized in that, In step (1), the extraction time is 1-3 hours and the number of extractions is 1-3 times.

4. The separation method as described in claim 1, characterized in that, The volume ratio of n-butanol to water is 1:

1.

5. The separation method as described in claim 1, characterized in that, The volume ratio of n-hexane, ethyl acetate, methanol, and water is 1:4:1:

4.

6. The separation method as described in claim 1, characterized in that, The volume ratio of ethyl acetate, n-butanol, and water is 1:1:

2.

7. The separation method as described in claim 1, characterized in that, In steps (3) and (4), a single injection of 50-60 mg is performed at a flow rate of 4-6 mL / min, and separation is carried out using the continuous injection EECCC mode.

8. The separation method as described in claim 1, characterized in that, In step (5), a single injection of 30-40 mg is performed at a flow rate of 1-3 mL / min, and separation is carried out using IRCCC mode.

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