A method for separating and preparing steroidal ketones and saponins in radix chondroli ti chii

Abstract

The application discloses a separation and preparation method of steroidal ketones and saponins in Radix Achyranthis Bidentatae, and belongs to the technical field of extraction and preparation of effective components of traditional Chinese medicines. The separation and preparation method comprises the following steps: crushing Radix Achyranthis Bidentatae, adding a solvent to perform reflux extraction, concentrating, and preparing a crude extract of Radix Achyranthis Bidentatae; removing high-polarity components from the crude extract of Radix Achyranthis Bidentatae through a macroporous resin column chromatography to obtain an enriched crude product of Radix Achyranthis Bidentatae; separating the enriched crude product of Radix Achyranthis Bidentatae through high-speed counter-current chromatography to obtain three single components and a fourth component mixture; and preparing 25R-achyranthasterone and 25S-achyranthasterone from the fourth component sample through silver ion coordination high-speed counter-current chromatography. The separation and preparation method can quickly purify and prepare five steroidal ketones and saponins with high purity, is simple and convenient to operate, and is good in stability, and the purity of the five steroidal ketones and saponins reaches more than 90%.

Description

Technical Field

[0001] This invention relates to the field of extraction and preparation technology of effective components of traditional Chinese medicine, and in particular to a method for separating and preparing sterone and saponin components from Achyranthes bidentata. 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] Achyranthes bidentata Bl., a plant in the Amaranthaceae family, is the dried root of the plant. Also known as Huai Achyranthes, it is one of the "Four Great Huai Medicines" and is mainly cultivated in Henan Province, China. The 2020 edition of the *Pharmacopoeia of the People's Republic of China* records that Achyranthes bidentata has the effects of promoting blood circulation, tonifying the liver and kidneys, strengthening tendons and bones, promoting urination, and guiding blood downwards. Chemical studies have shown that Achyranthes bidentata contains polysaccharides, triterpenoid saponins, sterones, flavonoids, and alkaloids, among which polysaccharides, triterpenoid saponins, and sterones are the main active ingredients. It has been reported that the sterones have pharmacological activities including antitumor effects, analgesic and anti-inflammatory effects, anti-osteoporosis effects, hypoglycemic effects, and effects on the nervous system. Therefore, sterone compounds in Achyranthes bidentata play a very important role, and sterones are also characteristic components of the Achyranthes genus. Among them, 25R-Achyranthes bidentata and 25S-Achyranthes bidentata belong to the Achyranthes bidentata class and are a pair of diastereomers. The separation of this type of component often employs conventional silica gel column chromatography or high-pressure preparative column chromatography. However, column chromatography has the disadvantages of a long separation cycle, high solvent consumption, dead adsorption, and poor separation effect.

[0004] High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography technique that establishes a unique unidirectional hydrodynamic equilibrium between two solvent phases within a high-speed rotating spiral tube. One solvent phase serves as the stationary phase, while the other acts as the mobile phase. It is a continuous and highly efficient chromatographic separation technique. HSCCC is characterized by the absence of a solid support as the stationary phase, avoiding sample dead adsorption and structural changes caused by irreversible adsorption. It also boasts high sample recovery and allows for solvent system selection based on the properties of the target compound. HSCCC has become an effective method for separating and purifying various natural products, offering advantages such as high specificity, large-scale preparation, and high efficiency and speed. It has been widely applied in biochemistry, bioengineering, pharmaceuticals, natural product chemistry, environmental analysis, food, and materials science. However, traditional counter-current chromatography is limited to isomers with similar KD values. To separate isomers with similar KD values, the common practice is to extend the separation period, which often leads to peak broadening and excessive use of organic solvents. The inventors have discovered that there are currently no reports on the HSCCC isolation and preparation of 25R-auscinosterone, 25S-auscinosterone, and saponin components from Achyranthes bidentata. Summary of the Invention

[0005] In view of this, the present invention provides a method for separating and preparing sterone and saponin components from Achyranthes bidentata. The Achyranthes bidentata extract of the present invention is pretreated by macroporous resin column chromatography and then separated by high-speed countercurrent chromatography. The purity of each monomer component is detected by HPLC and can be above 90%. It can rapidly purify and prepare five kinds of sterone and saponin compounds with high purity. The operation is simple and has good stability.

[0006] This invention provides a method for separating and preparing sterone and saponin components from Achyranthes bidentata, comprising the following steps:

[0007] (1) Take Achyranthes bidentata powder, add solvent and reflux to extract, concentrate and obtain crude extract of Achyranthes bidentata;

[0008] (2) The crude extract of Achyranthes bidentata was subjected to macroporous resin column chromatography to remove highly polar components, thereby obtaining a crude enriched product of Achyranthes bidentata.

[0009] (3) The crude Achyranthes bidentata product was separated by high-speed countercurrent chromatography to obtain four components. The first component was ginsenoside IVa, as shown in formula (I); the second component was 2'-desugar-11-one-ginsenoside V, as shown in formula (II); the third component was β-ecdysterone, as shown in formula (III); and the fourth component was a mixture of 25S-Achyranthes bidentata and 25R-Achyranthes bidentata. The solvent system of the high-speed countercurrent chromatography was methyl tert-butyl ether / n-butanol / acetonitrile / water with a volume ratio of 4:2:3:8. The upper phase was the stationary phase and the lower phase was the mobile phase.

[0010]

[0011] (4) The fourth component was separated by high-speed countercurrent chromatography to obtain 25S-aushinone as shown in formula (IV) and 25R-aushinone as shown in formula (V); the solvent system of the high-speed countercurrent chromatography was containing Ag. + The volume ratio of methyl tert-butyl ether / n-butanol / acetonitrile / water is 4:2:3:8, with the upper phase being the stationary phase and the lower phase being the mobile phase;

[0012]

[0013] Preferably, in step (1), the solvent is an aqueous ethanol solution with an ethanol content of 60-80%, and the mass ratio of the Achyranthes bidentata to the aqueous ethanol solution is 1:3-5.

[0014] Preferably, in step (1), the extraction is performed 2 to 4 times, and the extraction time is 1 to 2 hours each time; after combining the filtrates, the crude extract of Achyranthes bidentata is obtained by rotary evaporation under reduced pressure.

[0015] Preferably, in step (2), the resin used in the macroporous resin column chromatography is selected from HP-10 or D-101.

[0016] Further, in step (2), water, 20%, 40%, 60%, 80% and 95% ethanol-water are used for elution in sequence, the 40% ethanol-water elution fraction is collected, the solvent is recovered, and crude product enriched in Achyranthes bidentata is obtained.

[0017] Preferably, in steps (3) and (4), the injection rate is 5 to 30 mL / min, the flow rate of the mobile phase is 0.5 to 5 mL / min, and the separation speed of the high-speed countercurrent chromatography is 600 to 900 r / min.

[0018] Preferably, in steps (3) and (4), the stationary phase retention rate is 60-80%, the detector is a UV detector and an evaporation photodetector connected in series, the UV detection wavelength is 250-260 nm, and the fraction is collected every 3-10 min.

[0019] Preferably, in step (4), the concentration of silver ions is 0.05 to 0.3 mol / L.

[0020] Preferably, in step (3), the running time of high-speed countercurrent chromatography is 4.5 to 6 hours; in step (4), the running time of high-speed countercurrent chromatography is 4 to 6 hours.

[0021] Preferably, the separation and preparation method further includes analyzing and detecting different components using high-performance liquid chromatography (HPLC). The specific HPLC conditions are as follows: acetonitrile is used as mobile phase A, and 0.1% formic acid aqueous solution is used as mobile phase B, with gradient elution performed as follows: 0-10 min, 5%-20% A; 10-20 min, 20% A; 20-30 min, 20%-40% A; 30-60 min, 40%-60% A; 60-65 min, 60%-100% A; 65-66 min, 100%-95% A; 66-73 min, 95% A.

[0022] Furthermore, the high-performance liquid chromatography uses a Waters-C18 column (250×4.6mm, 5μm), an evaporative light detector, a column temperature of 20–30℃, a flow rate of 0.5–2mL / min, and an injection volume of 5–20μL.

[0023] The present invention has achieved the following beneficial effects:

[0024] The *Achyranthes bidentata* extract of this invention, after pretreatment by macroporous resin column chromatography, was separated by high-speed countercurrent chromatography to obtain individual components. Purity analysis by HPLC yielded over 90% pure β-ecdysterone, *Sophora japonica* saponin IVa, and 2'-desugar-11-one-*Sophora japonica* saponin V. Furthermore, the addition of silver ions to the solvent system utilized the intermolecular forces between silver ions and the compounds to achieve the separation of 25R-*Achyranthes bidentata* and 25S-*Achyranthes bidentata*. This separation and preparation method is suitable for the rapid purification of five high-purity compounds, including sterones and saponins, from *Achyranthes bidentata*, and is simple to operate with good stability. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0026] Figure 1 This is a chromatogram of the fraction of Achyranthes bidentata eluted with 40% ethanol-water separated and purified by high-speed countercurrent chromatography in Example 1 of the present invention. In this chromatogram, 4 is ginsenoside IVa, 5 is 2'-desugar-11-keto-ginsenoside V, 1 is β-ecdysterone, and 2+3 is a mixture of 25S-Achyranthes bidentata and 25R-Achyranthes bidentata.

[0027] Figure 2This is the chromatogram of 25R-Achyranthesone and 25S-Achyranthesone separated and purified by high-speed countercurrent chromatography in Example 1 of the present invention; wherein, 2 is 25S-Achyranthesone and 3 is 25R-Achyranthesone.

[0028] Figure 3 This is an HPLC chromatogram of five monomeric components obtained by high-speed countercurrent chromatography separation and purification in Example 1 of the present invention; wherein, compound 1 is ginsenoside IVa, compound 2 is 25S-auscinone, compound 3 is 25R-auscinone, compound 4 is 2'-desugar-11-one-ginsenoside V, and compound 5 is β-ecdysterone. Detailed Implementation

[0029] 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.

[0030] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0031] Example 1

[0032] 1. Preparation of Achyranthes bidentata extract: Take 5 kg of dried Achyranthes bidentata root, crush it, and extract it by heating and reflux with 70% ethanol at a solid-liquid ratio of 1:4. Extract it three times for 2 h, 1 h and 1 h respectively. Combine the filtrates and evaporate under reduced pressure to obtain crude ethanol extract of Achyranthes bidentata (800 g).

[0033] 2. The crude ethanol extract of Achyranthes bidentata was pretreated by macroporous resin column chromatography to remove highly polar components such as polysaccharides:

[0034] A 70% ethanol extract of Achyranthes bidentata was dissolved in water, and sample pretreatment was performed using HP-10 macroporous resin column chromatography. Elution was performed sequentially with water, 20%, 40%, 60%, 80%, and 95% ethanol-water to remove highly polar components such as polysaccharides. HPLC analysis showed that sterones and saponins in Achyranthes bidentata were mainly found in the 40% ethanol-water elution fraction. Figure 3 The 40% ethanol eluent was collected, and the solvent was recovered to obtain a crude product (150g) enriched with sterones and saponins from Achyranthes bidentata.

[0035] 3. High-speed countercurrent chromatography separation and purification:

[0036] To enrich the crude product from Achyranthes bidentata eluted in 40% ethanol-water fraction, a solvent system was prepared according to the solvent ratio of methyl tert-butyl ether-n-butanol-acetonitrile-water (4:2:3:8, v / v). The system was placed in a separatory funnel, shaken well, and allowed to stand for separation. After equilibration, a two-phase solvent system was obtained, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. 5 mL of the upper and 5 mL of the lower phase were used to dissolve 150 mg of the 40% ethanol-water eluted Achyranthes bidentata fraction in these solutions to prepare the sample solution. A semi-preparative high-speed countercurrent chromatograph developed by Shanghai Tongtian Company was used. First, the stationary phase was pumped into the column at a flow rate of 20 mL / min. After the upper phase eluted, the column was rotated forward at 800 rpm. Once the rotation speed reaches 800 rpm, pump the mobile phase at a flow rate of 2.0 mL / min until the lower phase flows out. Set the UV detector wavelength to 254 nm and connect it in series with the evaporative light detector. Inject the prepared sample solution into the injection valve, changing the load to inject function, allowing the sample to enter the high-speed countercurrent chromatograph. Then, based on the evaporative light spectrum (e.g., ... Figure 1 (As shown) The target component was received, yielding four components. The high-speed countercurrent chromatograph ran for 6 hours.

[0037] Nuclear magnetic resonance (NMR) confirmed that the first component is ginsenoside IVa, as shown in formula (I); the second component is 2'-desugar-11-keto-ginsenoside V, as shown in formula (II); the third component is β-ecdysterone, as shown in formula (III); and the fourth component is a mixture of 25S-auscinone (formula (IV)) and 25R-auscinone (formula (V)).

[0038]

[0039] The separated components (component 1, component 2, and component 3) were analyzed using high-performance liquid chromatography (HPLC). HPLC conditions: Waters-C18 (250×4.6 mm, 5 μm), evaporative light detector, column temperature: 25℃, flow rate: 1.0 mL / min, injection volume: 10 μL. The mobile phase consisted of a gradient elution of acetonitrile (A) and 0.1% formic acid aqueous solution (B), with the following gradient conditions: 0–10 min, 5%–20% A; 10–20 min, 20% A; 20–30 min, 20%–40% A; 30–60 min, 40%–60% A; 60–65 min, 60%–100% A; 65–66 min, 100%–95% A; 66–73 min, 95% A. The purity of ginsenoside IVa was 93%; the purity of 2'-desugar-11-one-ginsenoside V was 91%; and the purity of β-ecdysterone was 95%. Figure 3 ).

[0040] The isolated first, second, and third fractions were analyzed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectra using a Burker 400MHz nuclear magnetic resonance spectrometer. The data obtained are as follows:

[0041] Bamboo rhizome saponin IVa methyl ester (component 1, formula (I)): (1) Electrospray ionization mass spectrometry (ESI-MS), m / z [M+H] + 808.4522,C 43 H 67 O 14 (2) 1 H NMR (600MHz, Pyridine-d5) δ (ppm): 6.26 (dd, J=8.2, 3.5Hz, H-1′), 5.36 (d, J=3.9Hz, H-12), 4.93 (s, H-1″), 1.23 (d, J=3.6H z,Me-27),1.20(s,Me-23),1.03(s,Me-25),0.91(s,Me-30),0.85(d,J=3.5Hz,Me-29),0.82(s,Me-24),0.76(s,Me-26); (3) 13 C NMR(151MHz,Pyridine-d5)δ176.21(C-28),170.58(C-6″),143.87(C-13),122. 58(C-12),107.01(C-1″),95.49(C-1′),88.88(C-3),79.06(C-5′),78.62(C-3′ ),77.62(C-3″),76.93(C-5″),75.11(C-2″),73.85(C-2′),72.92(C-4″),70.83 (C-4′),61.92(C-6′),55.47(C-5),51.77(OCH3),47.73(C-9),46.73(C-17),45 .91(C-19),41.84(C-14),41.46(C-18),39.62(C-8),39.24(C-4),38.36(C-1), 36.66(C-10),33.71(C-21),32.86(C-7),32.82(C-29),30.50(C-22),29.61(C- 20),27.97(C-15),27.88(C-23),26.31(C-2),25.83(C-27),23.48(C-16),23.3 7(C-11),23.11(C-30),18.19(C-6),17.19(C-26),16.64(C-24),15.25(C-25).

[0042] 2'-Desaccharide-11-keto-senna saponin V (second component, formula (II)): (1) ESI-MS, m / z [M+H] + 809.4323,C 36 H 54 O9; (2) 1 H NMR (400MHz, DMSO-d6) δ (ppm): 5.45 (s, H-1′), 3.55 (d, J = 9.7Hz, H-3), 2.85 (dd, J = 13.8, 4.5Hz, H-18), 2.29 (s), 1.3 1(s,Me-27),1.00(s,Me-23),0.97(s,Me-26),0.91(s,Me-25),0.88(s,Me-24),0.83(s,Me-29),0.76(s,Me-30); (3) 13 C NMR(101MHz,DMSO)δ199.76(C-11),175.51(C-28),171.03(C-6″),169.35(C -13),127.32(C-12),105.98(C-1″),94.82(C-1′),88.22(C-3),78.33(C-3′) ,76.99(C-5′),76.65(C-3″),75.96(C-5″),74.15(C-2″),72.88(C-2′),72.0 0(C-4″),70.03(C-4′),61.55(C-6′),61.14(C-5),54.74(C-9),46.03(C-17) ,45.18(C-19),44.10(C-14),43.70(C-18),41.62(C-8),40.93(C-4),38.90 (C-1),36.93(C-10),33.48(C-21),32.87(C-7),32.75(C-22),31.25(C-20), 30.77(C-23),27.93(C-15),27.63(C-2),26.08(C-27),23.63(C-29),23.51( C-30),22.82(C-16),19.08(C-6),17.27(C-26),16.86(C-24),16.58(C-25).

[0043] β-ecdysterone (third component, formula (III)): (1) ESI-MS, m / z [M+H] + 465.6634,C 27 H 44 O7; (2)1 HNMR (400MHz, Methanol-d4) δ (ppm): 5.80 (d, J = 2.5Hz, H-7), 3.94 (q, J = 3.0Hz, H-3), 3.83 (dt, J = 11.9, 3.8Hz, H-2), 3.33 (m, J = 1.6Hz, H-22), 3.15 (m, J = 1 0.4,7.2,2.6Hz,H-9),2.39(d,J=4.4Hz,H-5),2.36(d,J=4.5Hz,H-17),1.20 (s,H-21),1.19(s,H-27),1.18(s,H-26),0.96(s,H-19),0.89(s,H-18);(3) 13 C NMR(101MHz,Methanol-d4)δ35.98(C-1),67.13(C-2),67.31(C-3),31.45(C-4),50.40(C-5),205.05 (C-6),120.74(C-7),166.58(C-8),33.71(C-9),37.88(C-10),20.14(C-11),31.12(C-12),46.97(C-1 3),83.83(C-14),30.39(C-15),19.66(C-16),49.14(C-17),16.66(C-18),23.01(C-19),76.52(C-20 ),20.11(C-21),77.03(C-22),25.95(C-23),41.00(C-24),69.90(C-25),27.58(C-26),28.31(C-27).

[0044] 4. Preparation of 25R- and 25S-achysteine ​​from the fourth component sample using silver ion coordination and high-speed countercurrent chromatography:

[0045] Collect 20 mg of the fourth component and prepare a solvent system according to the solvent ratio of methyl tert-butyl ether-n-butanol-acetonitrile-water (4:2:3:8, v / v). Place the solvent system in a separatory funnel and add silver nitrate to the separatory funnel to make the concentration of silver nitrate 0.1 mol / L. Shake well and allow it to stand for separation. After equilibrium, a two-phase solvent system is obtained. The upper phase is used as the stationary phase and the lower phase is used as the mobile phase. Take 5 mL of the upper phase and 5 mL of the lower phase and dissolve 20 mg of the fourth component of Achyranthes bidentata in them to prepare the sample solution. Use a semi-preparative high-speed countercurrent chromatograph developed by Shanghai Tongtian Company. First, pump the stationary phase into the column at a flow rate of 20 mL / min. After the upper phase flows out, rotate the column forward at a speed of 800 rpm. Once the rotation speed reaches 800 rpm, pump the mobile phase at a flow rate of 2.0 mL / min until the lower phase flows out. Set the UV detector wavelength to 254 nm and connect it in series with the evaporative light detector. Inject the prepared sample solution into the injection valve, changing the load to inject function, allowing the sample to enter the high-speed countercurrent chromatograph. Then, based on the evaporative light spectrum (e.g., ... Figure 2 The target component was received (as shown), yielding 25S-auscinone (formula (IV)) and 25R-auscinone (formula (V)) sequentially. The high-speed countercurrent chromatography ran for 6 hours.

[0046] The separated precipitates were analyzed using high-performance liquid chromatography (HPLC). HPLC conditions: Waters-C18 (250×4.6 mm, 5 μm), evaporative light detector (ELSD), column temperature: 25℃, flow rate: 1.0 mL / min, injection volume: 10 μL. The mobile phase consisted of a gradient elution of acetonitrile (A) and 0.1% formic acid aqueous solution (B), with the following gradient conditions: 0-10 min, 5%-20% A; 10-20 min, 20% A; 20-30 min, 20%-40% A; 30-60 min, 40%-60% A; 60-65 min, 60%-100% A; 65-66 min, 100%-95% A; 66-73 min, 95% A. The purity of 25S-achyranthesone was 96%; the purity of 25R-achyranthesone was 95%. Figure 3 ).

[0047] Structural identification: The isolated monomeric components were analyzed using mass spectrometry and Burker 400MHz nuclear magnetic resonance spectrometry, respectively. The data obtained are as follows:

[0048] 25S-Achyranthesone (Formula (IV)): (1) ESI-MS, m / z [M+H] + 481.3453,C 27 H 44 O7; (2) 11H NMR (400 MHz, Methanol-d4) δ (ppm): 5.81 (d, J = 2.5 Hz, H-7), 3.95 (t, J = 3.1 Hz, H-2), 3.83 (dt, J = 12.0, 3.9 Hz, H-22), 3.46 (m, J = 10.7 Hz, H-26a), 3.3 (d, J = 3.4 Hz, H-26b), 3.16 (d, J = 8.5 Hz, H-9), 1.59 (dd, J = 14.0, 6.8 Hz, H-21), 1.17 (s, H-18), 0.96 (s, H-19), 0.94 (d, J = 6.6 Hz, H-27); (3) 13 13C NMR (101 MHz, Methanol-d4) δ 205.04 (C-6), 166.52 (C-8), 120.75 (C-7), 83.86 (C-14), 76.83 (C-22), 76.43 (C-20), 67.31 (C-2), 67.12 (C-3), 66.75 (C-26), 50.39 (C-5), 49.09 (C-17), 37.86 (C-10), 35.97 (C-1), 35.65 (C-25), 33.71 (C-9), 31.46 (C-4), 31.12 (C-15), 30.70 (C-12), 30.37 (C-23), 28.77 (C-24), 23.00 (C-19), 20.11 (C-11), 19.61 (C-16), 16.63 (C-18), 16.10 (C-27).

[0049] 25R-Inokosterone (Formula (V)): (1) ESI-MS, m / z [M + H] + 481.3453, C 27 H 44 O7; (2) 1 1H NMR (400 MHz, Methanol-d4) δ (ppm): 5.80 (d, J = 2.5 Hz, H-7), 3.94 (q, J = 2.9 Hz, H-2), 3.83 (dt, J = 12.0, 3.9 Hz, H-22), 3.42 (dd, J = 10.6, 6.0 Hz, H-26a), 3.39 (d, J = 3.4 Hz, H-26b), 3.20 (d, J = 8.5 Hz, H-9), 1.59 (dd, J = 14.0, 6.8 Hz, H-21), 1.18 (s, H-18), 0.96 (s, H-19), 0.92 (d, J = 6.7 Hz, H-27); (3) 13C NMR(101MHz,Methanol-d4)δ35.97(C-1),67.31(C-2),67.24(C-3),31.46(C-4),50.39(C-5),205.04 (C-6),120.74(C-7),166.53(C-8),33.7(C-9),37.86(C-10),20.09(C-11),30.42(C-12),47.93(C-1 3),83.85(C-14),31.12(C-15),19.60(C-16),49.07(C-17),16.62(C-18),23.00(C-19),76.38(C-20 ),20.14(C-21),76.41(C-22),30.37(C-23),28.58(C-24),35.42(C-25),67.12(C-26),15.50(C-27).

[0050] Example 2: Optimization of Macroporous Resin Columns

[0051] This example investigated the effects of four different resins (XDA-6, HP-10, D-101, and AB-8) on the separation and purification of Achyranthes bidentata extract.

[0052] A 70% ethanol extract of Achyranthes bidentata was added to the four resins mentioned above, and then eluted with water and different concentrations of ethanol-water (including 20%, 40%, 60%, 80%, and 95% ethanol-water, v / v). The results showed that HP-10 and D-101 could selectively separate triterpenoid saponins and sterones, mainly eluted with 40% ethanol. HPLC-MS / MS analysis revealed that the 40% ethanol eluent contained sterones and triterpenoid saponins. For HP-10, the 40% ethanol eluent was 4.5 g, while for D-101 it was 3.1 g, indicating that HP-10 was enriched with more triterpenoid saponins and sterones. Therefore, HP-10 was optimized as the best macroporous resin, with water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and 95% ethanol as separation solvents for further HSCCC testing.

[0053] Example 3: Screening for uric acid-lowering activity

[0054] 1. The survival rate of HK-2 cells by compounds of formulas (I) to (V) was detected by the MTT assay.

[0055] Logarithmically growing HK-2 cells were adjusted to a density of 10,000 / ml, with 100 μL seeded per well in 96-well plates. Cells adhered for 24 hours (density exceeding 80%), and were then starved with serum for 12 hours. A blank control group, a model group, and an experimental group were established. In the experimental groups, the original culture medium was replaced with different concentrations (10 μM) of culture medium containing compounds of formulas (I) to (V), with 6 replicates per group. After 24 hours of culture, 10 μL of 5 mg / ml MTT solution was added. Four hours later, the original culture medium was aspirated, and 100 μL of LDMSO was added to dissolve the purple crystals. The OD value was measured at 490 nm using a microplate reader, and cell viability was calculated.

[0056] 2. The uric acid-lowering activity of compounds of formulas (I) to (V) was detected by HPLC.

[0057] Logarithmically growing HK-2 cells were adjusted to a density of 10,000 / ml, with 1 ml per well, and seeded in 24-well plates. A blank control group, a model group, groups treated with compounds (I) to (V), and a positive control group were established. After 48 hours of culture, the blank control group and model group were replaced with fresh culture medium. The groups treated with compounds (I) to (V) were treated with 10 μM of compounds (I) to (V), and the positive control group was treated with 100 μM allopurinol and febuxostat solution. After 24 hours of culture, the original culture medium was aspirated, and each well was washed three times with PBS. The blank control group was treated with serum-free and adenosine-free medium, while the model group, treatment groups, and positive control group were treated with 1 mL of medium containing 1.25 mM adenosine. After 30 hours of incubation, 1 μL of 0.005 U / mg XOD solution was added to each well. After 8 hours of culture, the supernatant was collected, and the uric acid content was determined by HPLC. The results are shown in Table 1.

[0058] Table 1. Screening results of uric acid-lowering activity of compounds from formulas (I) to (V) of Achyranthes bidentata.

[0059]

[0060]

[0061] The survival rate of HK-2 cells by compounds of formulas (I) to (V) was determined using the MTT assay. Table 1 shows that all compounds had no significant inhibitory effect on HK-2 cell growth, with cell survival rates exceeding 90%. The uric acid-lowering activity of compounds of formulas (I) to (V) was measured using a hyperuricemic cell model. Table 1 shows that compounds of formula (I) (methyl baicalein IVa) and formula (II) (2'-desugar-11-one-baicalein V) exhibited better uric acid-lowering activity, with uric acid levels of 15.7 mg / dL and 21.4 mg / dL, respectively, both significantly lower than the uric acid levels in the model group.

[0062] 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 sterone and saponin components from Achyranthes bidentata, characterized in that, Includes the following steps: (1) Take Achyranthes bidentata powder, add solvent and reflux to extract, concentrate to obtain crude extract of Achyranthes bidentata; the solvent is an aqueous ethanol solution with an ethanol content of 60-80%, and the mass ratio of Achyranthes bidentata to aqueous ethanol solution is 1:3-5; the number of extractions is 2-4 times, and the extraction time for each extraction is 1-2 hours; after combining the filtrates, the crude extract of Achyranthes bidentata is obtained by rotary evaporation under reduced pressure. (2) The crude extract of Achyranthes bidentata was subjected to macroporous resin column chromatography to remove highly polar components. It was eluted sequentially with water, 20%, 40%, 60%, 80% and 95% ethanol-water, and the 40% ethanol-water elution fraction was collected. The solvent was recovered to obtain the enriched crude Achyranthes bidentata. The resin used in the macroporous resin column chromatography was selected from HP-10 or D-101. (3) The crude Achyranthes bidentata extract was separated by high-speed countercurrent chromatography to obtain four components: the first component was ginsenoside IVa, as shown in formula (I); the second component was 2'-desugar-11-keto-ginsenoside V, as shown in formula (II); the third component was β-ecdysterone, as shown in formula (III); and the fourth component was 25 S - Achyranthesinone and 25 R - A mixture of oxostosterone; the solvent system of the high-speed countercurrent chromatography is methyl tert-butyl ether / n-butanol / acetonitrile / water, with a volume ratio of 4:2:3:8, the upper phase is the stationary phase and the lower phase is the mobile phase; Formula (I); Equation (II); Equation (III); (4) The fourth component was separated by high-speed countercurrent chromatography to obtain 25 as shown in formula (IV). S - Achyranthesinone and 25 as shown in formula (V) R - Achyranthesone; the solvent system of the high-speed countercurrent chromatography is containing Ag. + The mixture of methyl tert-butyl ether / n-butanol / acetonitrile / water has a volume ratio of 4:2:3:8, with the upper phase being the stationary phase and the lower phase being the mobile phase; Ag + The concentration is 0.05~0.3mol / L; Formula (IV); Formula (V); In steps (3) and (4), the injection rate is 5-30 mL / min, the flow rate of the mobile phase is 0.5-5 mL / min, and the separation speed of the high-speed countercurrent chromatography is 600-900 r / min; the stationary phase retention rate is 60-80%, the detector is a combination of an ultraviolet detector and an evaporative light detector, and the ultraviolet detection wavelength is 250-260 nm; the fraction is collected every 3-10 min.

2. The separation and preparation method according to claim 1, characterized in that, In step (3), the running time of high-speed countercurrent chromatography is 4.5~6 h; in step (4), the running time of high-speed countercurrent chromatography is 4~6 h.

3. The separation and preparation method according to claim 1, characterized in that, The separation and preparation method further includes analyzing and detecting different components using high-performance liquid chromatography (HPLC). The specific HPLC conditions are as follows: acetonitrile is used as mobile phase A, and 0.1% formic acid aqueous solution is used as mobile phase B, with gradient elution performed as follows: 0-10 min, 5%-20% A; 10-20 min, 20% A; 20-30 min, 20%-40% A; 30-60 min, 40%-60% A; 60-65 min, 60%-100% A; 65-66 min, 100%-95% A; 66-73 min, 95% A.

4. The separation and preparation method according to claim 3, characterized in that, The high-performance liquid chromatography (HPLC) uses a Waters-C18 column (250 × 4.6 mm, 5 μm), an evaporative light detector, a column temperature of 20–30 °C, a flow rate of 0.5–2 mL / min, and an injection volume of 5–20 μL.

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