A method for separating and preparing chemical components from peony petals

Abstract

This invention relates to the field of natural product separation technology, and more particularly to a method for separating and preparing components from peony petals. The invention employs high-speed countercurrent chromatography combined with preparative liquid chromatography to separate compounds present in high concentrations in peony petals, yielding five compounds: paeoniflorin, kaempferol-3,7-O-β-D-diglucoside, robinin, kaempferol-7-O-β-D-glucoside, and apigenin-7-O-β-D-glucoside. The separation method is simple and produces products with high purity.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, and in particular to a method for separating and preparing chemical components from peony petals. Background Technology

[0002] Peony petals are a major byproduct of peony cultivation, rich in various functional and active ingredients such as monoterpenoid glycosides, flavonoids, phenolic acids, tannins, and polysaccharides. They possess multiple effects including antioxidant, antibacterial, anti-inflammatory, and deodorizing properties, and are produced in large quantities. The strong antioxidant activity of peony petals has led to their widespread application in the pharmaceutical industry. Numerous studies have found that flavonoids are abundant and important components of peony flowers, exhibiting a wide range of biological and pharmacological activities, including antioxidant, antibacterial, anti-inflammatory, and antitumor activities. Furthermore, volatile essential oils (EO) have been isolated and analyzed from peony flowers and have been used in the preparation of cosmetics, food, and pharmaceuticals.

[0003] Natural products refer to various substances derived from or produced within the bodies of animals, plants, and microorganisms or during their metabolism. Due to their wide range of sources and diverse structures, these substances hold irreplaceable value in the field of new drug development. The main extraction methods for natural products include: maceration extraction, percolation extraction, decoction extraction, reflux extraction, ultrasonic extraction, enzyme extraction, microwave-assisted extraction, and supercritical fluid extraction. After extraction, natural products yield a mixture of various components. To study the pharmacological effects of specific monomeric compounds, meticulous separation of the extract is necessary. Chromatographic separation is a commonly used method, achieving separation based on the principle that the substances to be separated interact differently between the mobile and stationary phases.

[0004] Literature review revealed that while there are currently many methods for separating peony petal extracts, the number of compounds obtained is relatively limited, and complete separation has not been achieved. Furthermore, peony petals contain various types of compounds, with significant differences in solubility between different types. Compounds of the same type may have similar solubility properties, making it difficult to separate monomers with high purity. Summary of the Invention

[0005] In view of this, the present invention provides a method for separating and preparing chemical components from peony petals. The present invention employs high-speed countercurrent chromatography (HSCCC) combined with preparative liquid chromatography, and by controlling the proportions of different solvents, achieves high-purity separation of compounds from peony petal extracts.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a method for separating and preparing chemical components from peony petals, comprising the following steps: (1) Peony petals were extracted to obtain peony petal extract; (2) Peony petal extract was separated by high-speed countercurrent chromatography using a solvent system to obtain fractions A, B and C; among them, fraction B is the compound rhubarb glycoside. (3) The fraction A was separated by cyclic countercurrent chromatography using a solvent system to obtain the compounds paeoniflorin oxidase and kaempferol-3,7-O-β-D-diglucoside. (4) Liquid chromatography was used to separate fraction C to obtain compounds kaempferol-7-O-β-D-glucoside and apigenin-7-O-β-D-glucoside.

[0007] Furthermore, in steps (2) and (3), the solvent system is a mixed solution of ethyl acetate, n-butanol, methanol, and water.

[0008] Further, the volume ratio of ethyl acetate, n-butanol, methanol, and water is 7-9:1-3:1:8-10; preferably, the volume ratio of ethyl acetate, n-butanol, methanol, and water is 8:2:1:9.

[0009] Furthermore, in step (1), ethanol is used for heating and reflux extraction; preferably, the number of extractions is 1-3 times, and the extraction time for each extraction is 1-3 hours.

[0010] Further, the specific operation of step (1) is as follows: add ethanol to the peony petals, heat to a slight boiling state and then reflux to extract 1-3 times, each time for 1-3 hours, filter and combine the extracts for concentration to obtain the final product.

[0011] Furthermore, the concentration temperature is 35-55 ℃.

[0012] Furthermore, in step (2), the flow rate of high-speed countercurrent chromatography is 4-8 mL / min, the single injection volume is 500-700 mg, and the retention rate is 50-60%.

[0013] Furthermore, in step (3), the flow rate of the cyclic countercurrent chromatography separation is 4-6 mL / min, the single injection volume is 50-70 mg, and the retention rate is 50-60%.

[0014] Further, in step (4), the mobile phase is a mixture of acetonitrile and formic acid aqueous solution; the formic acid aqueous solution has a formic acid mass concentration of 0.1-0.15%.

[0015] Further, the volume ratio of the acetonitrile and formic acid aqueous solution is 20-25:75-80; preferably, the volume ratio of the acetonitrile and formic acid aqueous solution is 21:79.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention uses an ethyl acetate / n-butanol / methanol / water solvent system in a single countercurrent separation to obtain one monomer and multiple combinations of compounds with similar polarity from the ethanol extract of peony petals in a single step. Then, each combination of compounds is subjected to a corresponding form of countercurrent separation mode. Combined with preparative liquid chromatography, five monomers are finally obtained. The separation method is simple and the separation purity is above 95%. Attached Figure Description

[0017] 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.

[0018] Figure 1 This is a chromatogram of the total peony petal extract sample separated by countercurrent chromatography in Example 1 of the present invention; Figure 2 This is a chromatogram of the separation of paeoniflorin oxidase and kaempferol-3,7-O-β-D-diglucoside by cyclic countercurrent chromatography in Example 1 of the present invention; Figure 3 This is a liquid chromatography chromatogram showing the separation of kaempferol-7-O-β-D-glucoside and the compound apigenin-7-O-β-D-glucoside in Example 1 of this invention; Figure 4 This is an HPLC chromatogram of the peony petal extract and various compounds in Example 1 of the present invention. Detailed Implementation

[0019] 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.

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0021] Example 1 1. Preparation of peony petal extract Weigh 0.5 kg of dried peony petals and place them in a 10 L three-necked flask. Add 5 L of 95% ethanol, heat to a gentle boil, and then reflux for extraction twice, 2 hours each time. Filter the peony petals with gauze and combine the extracts. Concentrate the extracts at 52 °C to obtain 126 g of crude product, which is then refrigerated for later use.

[0022] 2. Determination of distribution coefficient Take 2 mg of sample into a 10 mL centrifuge tube, add 4 mL each of the upper and lower phases of the test solvent system, shake thoroughly, and allow to separate into layers. Then, take 100 µL of the upper and lower phase solutions into separate 1.5 mL centrifuge tubes. After the upper phase has evaporated to dryness, add 1 mL of methanol to dissolve it. Add 900 µL of methanol to the lower phase to ensure complete dissolution. Detect using HPLC. The peak area occupied by the target peak in the upper phase is A. 上 The peak area occupied by the target peak in the lower phase is A. 下 Distribution coefficient K D =A 上 / A 下 .

[0023] 3. Solvent system and sample solution preparation According to the preset ratio, the selected solvent components were placed in a 2 L separatory funnel and thoroughly mixed. After vigorous shaking to ensure uniform mixing, the mixture was allowed to stand until clear two-phase separation was formed. After separation, the upper organic phase (stationary phase) and the lower aqueous phase (mobile phase) were collected separately and transferred to 1 L volumetric flasks for storage. To remove dissolved gases, the two phase solutions were subjected to ultrasonic degassing for 20 min each for later use. During sample preparation, the substances to be separated were dissolved in equal volumes of stationary and mobile phase solutions for subsequent separation experiments.

[0024] 4. Countercurrent Chromatography Separation Program HSCCC Separation Mode: Preheat the cold trap to 25 °C and turn on the UV detector for preheating. Select the upper phase of the corresponding solvent system as the stationary phase and pump it into the tubing at 30 mL / min. Once the corresponding flow rate of the upper phase exits the tubing, it indicates that the column is filled with stationary phase. Select the forward-to-forward (FWD-IN) mode and turn on the pump to inject the lower phase of the corresponding solvent system at the set flow rate for pre-injection equilibration. Once the set flow rate of the lower phase steadily exits the tubing, the separation system has reached equilibrium. When the solvent system reaches dynamic equilibrium within the HSCCC column, record the volume of stationary phase lost. Inject the prepared sample solution into the tubing through the six-way injection valve to complete the injection and start separation at the set flow rate. After injection, the sample solution sequentially passes through the internal tubing of the countercurrent chromatograph and the UV detector, and is collected in the centrifuge tube of the fraction collector. After each separation, blow out the liquid in the countercurrent chromatograph; the volume percentage of the stationary phase is the retention rate for that separation. After each separation, clean the tubing with ethanol.

[0025] Circulating countercurrent chromatography: In this mode, the equilibration and injection methods are the same as in conventional countercurrent chromatography. Based on the real-time image detected by the UV detector, just before the components requiring circulation are eluted, the eluent is pumped back into the countercurrent chromatograph tubing via an external six-way valve using a constant flow pump for the next separation. After successful separation of each component is observed through UV detection, the six-way valve mode is switched off, ending the circulating separation mode, allowing the eluent to flow out. The eluent is collected according to the HSCCC method for subsequent operations.

[0026] 5. HSCCC segmented collection and testing The optimal solvent system for HSCCC was selected, and its retention rate at different flow rates was calculated. The eluent (10 mL per tube) was collected at the maximum flow rate with a retention rate of 50%. The collected components were analyzed by high-performance liquid chromatography (HPLC). Eluents containing the same components were combined and concentrated by rotary evaporation. The resulting concentrate was freeze-dried to obtain a powdered product, and its purity was determined by HPLC.

[0027] 6. HPLC analysis of peony petals This experiment uses Agilent Extend-C 18 The analysis was performed using a chromatographic column (250 mm × 4.6 mm, 5.0 µm). The mobile phase consisted of acetonitrile and an aqueous solution containing 0.1% formic acid, with a flow rate set at 0.8 mL / min and an injection volume of 10 µL. The column temperature was maintained at 20 °C, and the detection wavelength was 254 nm. The specific gradient elution program is shown in Table 1.

[0028] Table 1. HPLC mobile phase detection conditions for peony petals

[0029] 7. Results and Discussion 7.1 Selection of Solvent System A key difference between high-speed countercurrent chromatography (HSCCC) and traditional chromatography lies in the use of a liquid as the stationary phase. Since different compounds exhibit specific partition patterns in the two solvent phases, the appropriate selection of the solvent system is a prerequisite for achieving efficient separation. In the HSCCC separation process, the partition coefficient (K) of the compounds... D K is a key parameter affecting the separation effect. Studies have shown that when K... D When the value is between 0.5 and 2.0, the target compound can be effectively separated within a suitable time. If K D If K < 0.5, the compound will elute rapidly due to its weak retention capacity, leading to separation failure; conversely, if K > 0.5, the compound will elute rapidly. DWhen α > 2, excessive retention significantly prolongs separation time and reduces separation efficiency. Furthermore, the separation coefficient (α = K1 / K2, where K1 > K2) is an important indicator for evaluating separation selectivity. When α < 1.5, target compounds with similar retention behaviors are prone to co-elution. To address this, K1 / K2 can be adjusted by optimizing the solvent system. D To improve separation efficiency, we used a high-speed countercurrent chromatography (HSCCC) system with an ethyl acetate / n-butanol / methanol / water solvent system, taking into account factors such as the polarity of the peony petal extract.

[0030] The K value represents the solubility and partitioning of a compound between different phases in a two-phase solvent system. A K value greater than 2 indicates slower elution of the compound in countercurrent chromatography using the stationary phase as the primary phase. A K value less than 0.5 indicates a short retention time, potentially making effective separation difficult. A K value between 0.5 and 2 indicates relatively reasonable separation. Determining whether two compounds can be separated requires using the separation coefficient α. 1,2 = K1 / K2 (K1>K2), when α>1.5, it indicates that the compounds can be separated well, and when α<1.5, the cyclic method of countercurrent chromatography is used for separation.

[0031] The high-speed countercurrent chromatography separation of compounds in peony petals requires the K value of each component in different solvent systems as a basis. The K value results of each compound detected by HPLC are shown in Table 2. The K values ​​show that the polarity of each compound in peony petals is quite different, making it difficult to separate all target components in one go. After classifying and collecting compounds with similar polarity in segments, the separation of single compounds is then carried out.

[0032] Table 2. Partition coefficients of peony petal samples in different solvent systems.

[0033] Table 2 shows that for compounds with high content in the ethanol extract of peony petals, the optimal solvent system is ethyl acetate / n-butanol / methanol / water (8:2:1:9, v / v). The compounds with high content in the ethanol extract of peony petals include compounds 1, 2, 3, 4, and 5. The K values ​​of these five compounds are... DThe values ​​are 0.62, 0.76, 1.67, 2.97, and 2.94, respectively. After one HSCCC separation, fraction A (mixture of compounds 1 and 2), fraction B (monomer of compound 3), and fraction C (mixture of compounds 4 and 5) are obtained. The α values ​​for compounds 1 and 2, and compounds 4 and 5 are < 1.5, indicating that a single HSCCC separation is insufficient. Based on the α value of 1.23 for compounds 1 and 2, compounds 1 and 2 can be separated using cyclic HSCCC to obtain monomers 1 and 2. A solvent system of ethyl acetate / n-butanol / methanol / water (8:2:1:9, v / v) is selected for cyclic HSCCC separation of mixtures 1 and 2 to obtain monomers 1 and 2.

[0034] 7.2 Countercurrent chromatography separation of ethanol extract from peony petals The ethanol extract of peony petals was separated using a solvent system of ethyl acetate / n-butanol / methanol / water (8:2:1:9, v / v) at a flow rate of 5 mL / min. The separation results are as follows: Figure 1 As shown, a single injection volume of 600.0 mg was used for separation, with a retention rate of 50%, yielding fraction A (a mixture of compounds 1 and 2) of 104.8 mg, fraction B (monomer of compound 3) of 110.6 mg with a purity greater than 95%, and fraction C (a mixture of compounds 4 and 5) of 181.7 mg.

[0035] 7.3 Separation of fraction A (mixture of compounds 1 and 2) Compounds 1 and 2 have similar polarities, making single-pass separation insufficient; therefore, a cyclic separation method can be used. A solvent system of ethyl acetate / n-butanol / methanol / water (8:2:1:9, v / v) was used for cyclic separation of compounds 1 and 2 at a flow rate of 5 mL / min. The separation results are as follows: Figure 2 As shown, after injecting 50.0 mg of the sample and cyclically separating it, the retention rate was 50%, yielding 5.9 mg of compound 1 monomer and 37.4 mg of compound 2 monomer, with a purity greater than 95%.

[0036] 7.4 Separation of Flow Segment C (a mixture of compounds 4 and 5) In preparative liquid chromatography, compounds 4 and 5 were separated using a mobile phase of (21% acetonitrile and 79% 0.1% formic acid aqueous solution). The separation results are as follows: Figure 3 As shown, after separation, 8.2 mg of compound 4 monomer and 21.0 mg of compound 5 monomer were obtained from 50.4 mg of sample, with a purity greater than 95%.

[0037] 7.5 HPLC Detection of Compounds in Peony Petals Peony petals were extracted and separated into five compounds using different modes of HSCCC preparative HPLC. The HPLC results of each compound are shown below. Figure 4 As shown.

[0038] By comparing each compound with relevant literature, the five compounds isolated from peony petals were identified as follows: Compound 1 is paeoniflorin oxidase, Compound 2 is kaempferol-3,7-O-β-D-dextrose glycoside, Compound 3 is kaempferol glycoside, Compound 4 is kaempferol-7-O-β-D-dextrose glycoside, and Compound 5 is apigenin-7-O-β-D-dextrose glycoside. Characterization data are as follows: Oxidized paeoniflorin (1): ESI-MS m / z: 497 [M + H] + The molecular formula is C 23 H 28 O 12 . 1 H NMR (400MHz, DMSO-d6) δ: 7.84 (2H, d, J = 8.7 Hz, H-2'', 6''), 6.87 (2H, d, J = 8.7Hz, HH-3'', 5''), 5.29 (1H, s, H-9), 4.57 (2H, d, J = 3.9 Hz, H-8), 4.38(1H, d, J = 7.5 Hz, H-1'), 3.65 (1H, dd, J = 10.2 Hz, 5.2, H-6'a), 3.18-2.93(5H, m, H-2'-6'b), 2.41 (1H, d, J = 6.0 Hz, H-5), 2.34 (1H, m, H-7a), 2.04(1H, d, J = 12.0 Hz, H-3a), 1.80 (1H, d, J = 10.5 Hz, H-7b), 1.64 (1H, d, J =12.0 Hz, H-3b), 1.23 (3H, s, H-10). 13C NMR (101 MHz, DMSO-d6) δ: 166.04 (C-7''), 162.57 (C-4''), 132.03 (C-2'', 6''), 120.71 (C-1''), 115.82 (C-3'', C-5''), 105.19 (C-4), 100.56 (C-9), 99.09 (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'), 61.68 (C-6'),60.26 (C-8), 44.07 (C-3), 42.78 (C-5), 22.52 (C-7), 19.57 (C-10).

[0039] Kaempferol-3,7-O-β-D-diglucoside (2): ESI-MS m / z: 633.14 [M + Na] + The molecular formula is C 27 H 30 O 16 . 1 H NMR (400 MHz, DMSO-d6) δ: 8.06 (2H, d, J = 8.8 Hz, H-2', 6'), 6.90 (2H, d, J = 8.8 Hz, H-3', 5'), 6.80 (1H, d, J = 1.0 Hz, H-8), 6.45 (1H, d, J= 1.0 Hz, H-6), 5.49 (1H, d, J = 7.2 Hz, Glc-1), 5.08 (1H, d, J = 7.2 Hz, Glc-1'), 3.03-3.75 (12H, m, Glc-2-6, Glc'-2-6). 13C NMR (101 MHz, DMSO-d6) δ:178.11 (C-4), 163.30 (C-7), 161.33 (C-5), 160.63 (C-9), 157.29 (C-2), 156.49(C-4'), 133.94 (C-3), 131.45 (C-2', 6'), 121.22 (C-1'), 115.63 (C-3', 5'),106.13 (C-10), 101.19 (Glc-1), 100.21 (C-6), 99.82 (C-1'''), 94.94 (C-8),78.02 (Glc-5), 77.64 (C-5'''), 76.91 (Glc-3), 76.91 (C-3'''), 74.69 (Glc-2), 73.56 (C-2'''), 70.39 (Glc-4), 70.06 (C-4'''), 61.31 (Glc-6), 61.10 (C-6''').

[0040] Rhus glycoside (3): ESI-MS m / z: 577.16 [M - H] - The molecular formula is C 27 H 30 O 14 . 1 HNMR (400MHz, DMSO-d6) δ: 7.94 (2H, d, J = 7.6 Hz, H-2', 6'), 6.95 (2H, d, J = 7.6 Hz, H-3', 5'), 6.88 (1H, d, J = 2.0 Hz, H-3), 6.80 (1H, d, J = 2.0 Hz, H-8), 6.38(1H, s, H-6), 5.24 (1H, d, J = 7.2 Hz, H-1''), 5.14 (1H, brs, H-1'''), 3.17-3.82 (9H, m, H-2''-6'', H-2'''-5'''), 1.21 (3H, d, J = 6.2 Hz, H-6'''). 13C NMR(101 MHz, DMSO-d6) δ: 182.45 (C-4), 164.76 (C-2), 163.01 (C-7), 161.90 (C-4'), 161.59 (C-5), 157.45 (C-9), 129.04 (C-2', 6'), 121.45 (C-1'), 116.51 (C-3', 5'), 105.90 (C-10), 103.65 (C-3), 100.93 (C-1''), 99.80 (C-6), 98.30 (C-1'''), 94.99 (C-8), 77.69 (C-2''), 77.50 (C-3''), 76.74 (C-5''), 72.35 (C-4'''), 70.96 (C-3'''), 70.87 (C-2'''), 70.13 (C-4''), 68.81 (C-5'''), 60.96(C-6''), 18.55 (C-6''').

[0041] Kaempferol-7-O-β-D-glucoside (4): ESI-MS m / z: 447 [MH] - The molecular formula is C 21 H 20 O 11 . 1 HNMR (400 MHz, DMSO-d6) δ: 12.62 (s,1H, 5-OH), 8.04 (d, J = 8.8 Hz, 2H, H-2',6'), 6.91 (2H, d, J = 8.9 Hz, H-3', 5'), 6.43 (1H, d, J = 2.1 Hz, H-6). 3.58-3.08(6H, m. Glc). 13 C NMR(101MHz, DMS0-d6) δ: 177.90(C-4), 164.82(C-5), 161.67(C-7), 160.43(C-4'), 156.86(C-9),156.67(C-2), 133.65(C-3), 131.33(C-2'6'), 121.36(C-1'), 104.40(C-10), 99.21(C-6), 94.15(C-8), 77.97(C-3'5').

[0042] Apigenin-7-O-β-D-glucoside (5): ESI-MS m / z: 431.13 [M - H]- , 433.11 [M +H] + The molecular formula is C 21 H 20 O 10 . 1 H NMR (400 MHz, DMSO-d6) δ: 7.96 (2H, d, J = 8.5 Hz, H-2', 6'), 6.95 (2H, d, J = 8.5 Hz, H-3', 5'), 6.87 (1H, s, H-3), 6.84 (1H, s,H-8), 6.45 (1H, s, H-6), 5.08 (1H, d, J = 7.0 Hz, H-1''), 3.11-3.78 (6H, m,H-2''-6''). 13 C NMR (101 MHz, DMSO-d6) δ: 182.46 (C-4) 164.74 (C-7) 163.43 (C-2) 161.92 (C-5) 161.60 (C-9) 157.42 (C-4') 129.08 (C-2', 6') 121.44 (C-1')116.49 (C-3', 5') 105.82 (C-3) 103.56 (C-10) 100.39 (C-1''), 100.00 (C-6)95.31 (C-8), 77.66 (C-5''), 76.92 (C-3''), 73.58 (C-2''), 70.03 (C-4''), 61.08 (C-6'').

[0043] .

[0044] 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 and preparing chemical components from peony petals, characterized in that, Includes the following steps: (1) Peony petals were extracted to obtain peony petal extract; (2) Peony petal extract was separated by high-speed countercurrent chromatography using a solvent system to obtain fractions A, B and C; among them, fraction B is the compound rhubarb glycoside. (3) The fraction A was separated by cyclic countercurrent chromatography using a solvent system to obtain the compounds paeoniflorin oxidase and kaempferol-3,7-O-β-D-diglucoside. (4) Liquid chromatography was used to separate fraction C to obtain compounds kaempferol-7-O-β-D-glucoside and apigenin-7-O-β-D-glucoside; In steps (2) and (3), the solvent system is a mixed solution of ethyl acetate, n-butanol, methanol, and water; The volume ratio of ethyl acetate, n-butanol, methanol, and water is 7-9:1-3:1:8-10; In step (1), ethanol is used for extraction by heating and reflux.

2. The separation and preparation method according to claim 1, characterized in that, The volume ratio of ethyl acetate, n-butanol, methanol, and water is 8:2:1:

9.

3. The separation and preparation method according to claim 1, characterized in that, In step (1), the number of extractions is 1-3 times, and the extraction time for each extraction is 1-3 hours.

4. The separation and preparation method according to claim 1, characterized in that, The specific operation of step (1) is as follows: add ethanol to the peony petals, heat to a slight boiling state and then reflux to extract 1-3 times, each time for 1-3 hours, filter and combine the extracts for concentration to obtain the final product.

5. The separation and preparation method according to claim 1, characterized in that, In step (2), the flow rate of high-speed countercurrent chromatography is 4-8 mL / min, the single injection volume is 500-700 mg, and the retention rate is 50-60%.

6. The separation and preparation method according to claim 1, characterized in that, In step (3), the flow rate of the cyclic countercurrent chromatography separation is 4-6 mL / min, the single injection volume is 50-70 mg, and the retention rate is 50-60%.

7. The separation and preparation method according to claim 1, characterized in that, In step (4), the mobile phase is a mixture of acetonitrile and formic acid aqueous solution.

8. The separation and preparation method according to claim 7, characterized in that, The formic acid aqueous solution has a formic acid mass concentration of 0.1-0.15%.

9. The separation and preparation method according to claim 7, characterized in that, The volume ratio of the acetonitrile and formic acid aqueous solution is 20-25:75-80.

10. The separation and preparation method according to claim 9, characterized in that, The volume ratio of the acetonitrile and formic acid aqueous solution is 21:79.

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