A type of A ring hydroxyl fully substituted furostanol type steroidal saponin compound and its preparation method and use

Abstract

The present invention relates to the technical field of natural medicines, and specifically to a class of A-ring hydroxyl fully substituted furostanol-type steroidal saponin compounds in the rhizomes of the open-mouthed arrow, and separation and preparation methods and uses thereof. The present invention obtains three new furostanol-type steroidal saponin compounds by separating and purifying the crude methanol reflux extract of the medicinal plant open-mouthed arrow. Molecular docking experiments have shown that the aglycones of these compounds have good binding activity with iNOS and COX‑2, and may become potential inhibitors of iNOS and COX‑2. The method of the present invention is relatively simple and has good practical value. At the same time, it serves as a reference for continuing to discover such components from the open-mouthed arrow, and is of great significance to the comprehensive development and application of the open-mouthed arrow.

Description

Technical Field

[0001] The invention relates to the technical field of natural medicines, and in particular to a class of A-ring hydroxyl fully substituted furostanol-type steroidal saponin compounds in the rhizome of Tripterygium wilfordii and a separation and preparation method thereof and pharmaceutical use thereof. Background Art

[0002] Tupistra chinensis Baker is a plant of the genus Tupistra in the family Liliaceae. It is also known as Tupistra, Bamboo Root Seven, and Dieffenbachia. Tupistra chinensis Baker is a perennial herb. The roots and whiskers are removed and the dried rhizomes are used as medicine. It is cold in nature and tastes sweet and slightly bitter. Ancient medical books record that Tupistra chinensis Baker has the effects of clearing away heat and detoxifying, dispersing blood stasis and relieving pain. It is often used to treat sore throat, diphtheria, trauma, snake bites and rabies. Modern pharmacological studies have also shown that Tupistra chinensis Baker has good anti-tumor, anti-inflammatory, antibacterial, antioxidant, anti-cardiac hypertrophy and other pharmacological activities. Studies have found that the main chemical component of Tupistra chinensis Baker is steroidal saponins, which are also the main active ingredients in Tupistra chinensis. Steroidal saponin compounds are divided into spirostanol-type steroidal saponins, isostanol-type steroidal saponins, furostanol-type steroidal saponins and deformed spirostanol-type steroidal saponins according to the parent nucleus structure of the aglycone. Summary of the invention

[0003] The invention provides a furostanol-type steroidal saponin compound separated from open-mouthed arrowroot and a preparation method thereof.

[0004] The present invention provides a class of furostanol-type steroidal saponin compounds, which are all extracted and separated from the rhizomes of Eupatorium sphaerocephalum. The structural formula of the furostanol-type steroidal saponin compounds (1-3) is as follows:

[0005]

[0006] The preparation method of the above-mentioned steroidal saponin compound comprises the following steps:

[0007] S1. Weigh the dried rhizome of the open arrowroot, slice it and crush it, add methanol, reflux it at 65±3°C for 3 times, each time for 1-2 hours, combine the extracts after 3 refluxes, filter it and concentrate the filtrate under reduced pressure to obtain a crude methanol extract;

[0008] S2, the crude methanol extract is dissolved in water, extracted with petroleum ether, ethyl acetate and n-butanol in sequence, the petroleum ether and ethyl acetate extractions are discarded, and the remaining extract is concentrated under reduced pressure to obtain n-butanol extractions and aqueous extractions;

[0009] S3, the aqueous phase extraction part uses D101 macroporous adsorption resin column chromatography, ethanol-water is used as the mobile phase for gradient elution, and after thin layer G plate chromatography detection, similar components are combined to obtain 8 components Fr.A-Fr.H;

[0010] S4, the Fr.C component was subjected to reverse phase ODS column chromatography, with methanol-water as the mobile phase for gradient elution, and after TLC detection and merging of similar components, 5 components Fr.C1-Fr.C5 were obtained;

[0011] S5, separating the component Fr.C4 by high performance liquid chromatography and isocratic elution to obtain five components Fr.C4a-Fr.C4e;

[0012] S6. Purify the component Fr.C4d by high performance liquid chromatography and isocratic elution to obtain compound 1;

[0013] The component Fr.C4e was purified by high performance liquid chromatography and isocratic elution to obtain compound 2;

[0014] The component Fr.C5 was purified by high performance liquid chromatography and isocratic elution to obtain compound 3;

[0015] Further, in step S1, the liquid-to-solid ratio of methanol to the open arrow is 3:1-6:1 (mL:g);

[0016] Further, in step S2, the volumes of the extraction solvents petroleum ether, ethyl acetate, and n-butanol are 1-2 times the volume of the methanol extract aqueous solution;

[0017] Further, in step S3, the conditions for gradient elution of the aqueous phase extraction part are: gradient elution is performed at a volume ratio of ethanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1, and 1:0;

[0018] Further, in step S4, the conditions for gradient elution of the Fr.C component are: gradient elution is performed at a volume ratio of methanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 7:3, 9:1, and 1:0;

[0019] Further, in step S5, the conditions for isocratic elution of component Fr.C4 are as follows: isocratic elution is performed at a flow rate of 3 mL / min at a volume ratio of 14:86 of acetonitrile and formic acid aqueous solution (in the formic acid aqueous solution: the volume content of formic acid is 1‰, and the balance is water);

[0020] Further, in step S6, the conditions for isocratic elution of component Fr.C4d are as follows: isocratic elution is performed at a flow rate of 3 mL / min at a volume ratio of acetonitrile to formic acid aqueous solution (the volume content of formic acid is 1‰ and the balance is water) of 21:79;

[0021] Further, in step S6, the conditions for isocratic elution of component Fr.C4e are as follows: isocratic elution is performed at a flow rate of 3 mL / min at a volume ratio of acetonitrile to water of 23:77;

[0022] Furthermore, in step S6, the conditions for isocratic elution of component Fr.C5 are as follows: isocratic elution is performed at a flow rate of 3 mL / min in a volume ratio of acetonitrile to formic acid aqueous solution (the volume content of formic acid is 1‰ and the balance is water) of 26:74.

[0023] The technical solution provided by the present invention has the following beneficial effects:

[0024] The present invention separates and purifies the crude methanol reflux extract of the medicinal plant Rhizoma Cibotii, and obtains three new compounds. The compounds are determined to be furostanol-type steroidal saponin compounds by combining and applying a variety of modern spectral techniques. The method for extracting new furostanol-type steroidal saponin compounds from Rhizoma Cibotii is relatively simple and has good practical value, and at the same time serves as a reference for continuing to discover such components from Rhizoma Cibotii, and has important significance for the comprehensive development and application of Rhizoma Cibotii. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of extraction and separation of furostanol-type steroidal saponin compounds prepared in Example 1 of the present invention;

[0026] Figure 2 Compound 1 obtained in Example 1 of the present invention 1 H-NMR (600MHz, CD3OD) spectrum;

[0027] Figure 3 Compound 1 obtained in Example 1 of the present invention 13 C-NMR (150 MHz, CD3OD) spectrum;

[0028] Figure 4 DEPT (Distortionless Enhancement by Polarization Transfer) (θ=135°) spectrum of compound 1 prepared in Example 1 of the present invention;

[0029] Figure 5 HSQC (Heteronuclear Singular Qantum Correlation) spectrum of compound 1 prepared in Example 1 of the present invention;

[0030] Figure 6The HMBC (1H detected heteronuclear multiple bond correlation) spectrum of compound 1 prepared in Example 1 of the present invention;

[0031] Figure 7 Compound 1 obtained in Example 1 of the present invention 1 H- 1 H COSY (Correlation spectroscopy) spectrum;

[0032] Figure 8 ROESY (Rotating Frame Overhauser Effect Spectroscopy, rotating coordinate system NOE spectrum) spectrum of compound 1 prepared in Example 1 of the present invention;

[0033] Fig. 9 The UV spectrum of compound 1 prepared in Example 1 of the present invention;

[0034] Fig.10 HR-ESI-MS (High Resolution Electrospray Ionization Mass Spectroscopy) spectrum of compound 1 prepared in Example 1 of the present invention;

[0035] Fig.11 Compound 2 obtained in Example 1 of the present invention 1 H-NMR (600MHz, CD3OD) spectrum;

[0036] Fig.12 Compound 2 obtained in Example 1 of the present invention 13 C-NMR (150 MHz, CD3OD) spectrum;

[0037] Fig.13 DEPT (θ=135°) spectrum of compound 2 prepared in Example 1 of the present invention;

[0038] Fig.14 HSQC spectrum of compound 2 prepared in Example 1 of the present invention;

[0039] Fig.15 This is the HMBC spectrum of compound 2 prepared in Example 1 of the present invention;

[0040] Fig.16 Compound 2 obtained in Example 1 of the present invention 1 H- 1 H COSY spectrum;

[0041] Fig.17 ROESY spectrum of compound 2 prepared in Example 1 of the present invention;

[0042] Fig.18 The UV spectrum of compound 2 prepared in Example 1 of the present invention;

[0043] Fig.19 HR-ESI-MS spectrum of compound 2 prepared in Example 1 of the present invention;

[0044] Fig. 20 Compound 3 obtained in Example 1 of the present invention 1 H-NMR (600MHz, Pyridine-d5) spectrum;

[0045] Fig.21 Compound 3 obtained in Example 1 of the present invention 13 C-NMR (150 MHz, Pyridine-d5) spectrum;

[0046] Fig. 22 DEPT (θ=135°) spectrum of compound 3 prepared in Example 1 of the present invention;

[0047] Fig.23 HSQC spectrum of compound 3 prepared in Example 1 of the present invention;

[0048] Fig.24 is the HMBC spectrum of compound 3 prepared in Example 1 of the present invention;

[0049] Fig.25 Compound 3 obtained in Example 1 of the present invention 1 H- 1 H COSY spectrum;

[0050] Fig.26 ROESY spectrum of compound 3 prepared in Example 1 of the present invention;

[0051] Fig. 27 The UV spectrum of compound 3 prepared in Example 1 of the present invention;

[0052] Fig.28 HR-ESI-MS spectrum of compound 3 prepared in Example 1 of the present invention;

[0053] Fig.29 This is a three-dimensional effect diagram of the aglycone of compound 1 prepared in Example 1 of the present invention docking with iNOS;

[0054] Fig.30 This is a three-dimensional effect diagram of the aglycone of compound 2 prepared in Example 1 of the present invention docking with iNOS;

[0055] Fig.31 It is a three-dimensional effect diagram of the aglycone of compound 3 prepared in Example 1 of the present invention docking with iNOS;

[0056] Fig.32 This is a three-dimensional effect diagram of the aglycone of compound 1 prepared in Example 1 of the present invention docking with COX-2;

[0057] Fig.33 This is a three-dimensional effect diagram of the aglycone of compound 2 prepared in Example 1 of the present invention docking with COX-2;

[0058] Fig.34 This is a three-dimensional effect diagram of the docking of the aglycone of compound 3 prepared in Example 1 of the present invention with COX-2. DETAILED DESCRIPTION

[0059] In order to more clearly demonstrate the purpose, technical solutions and advantages of the present invention, the embodiments of the present invention will be further described below in conjunction with the accompanying drawings and examples.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] Embodiment 1:

[0063] This Example 1 provides a method for preparing a class of furostanol-type steroidal saponin compounds isolated from open arrowheads, and the specific steps are as follows:

[0064] Step S1, weigh 500g of dried rhizome of Tupistra chinensis, slice and crush into powder, place in a three-necked flask, add anhydrous methanol (Shanghai Titan Technology Co., Ltd.), place on an electric heating jacket for heating, reflux three times at 65°C, the volume of methanol added each time is 3L, 1.5L, 1.5L, and the reflux time is set to 2h, 1h, and 1h, respectively. After the reflux is completed, the crude extract after three refluxes is combined, and the filtrate is concentrated under reduced pressure after suction filtration to obtain a total of 140.4g of methanol crude extract; the rhizome of Tupistra chinensis Baker used in this Example 1 was collected from Yichang City, Hubei Province, and was identified by the School of Pharmacy of South-Central University for Nationalities as the rhizome of Tupistra chinensis Baker. of the genus Tupistra of the family Liliaceae.

[0065] Step S2, adding 2L of water to the crude methanol extract to dissolve, extracting with 2.3L of petroleum ether, 4.0L of ethyl acetate and 3.7L of n-butanol in sequence, the volume of the extraction solvent being 1-2 times of the aqueous solution of the extract, then discarding the petroleum ether and ethyl acetate extraction parts, and concentrating the remaining extract under reduced pressure to recover the solvent, to obtain 56.3g of n-butanol extraction part and 81.1g of aqueous phase extraction part;

[0066] Step S3, taking 30 g of the aqueous phase extraction part for D101 macroporous adsorption resin column chromatography, using ethanol and water as eluents, and performing gradient elution in a volume ratio of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1, and 1:0, and using TLC (thin-layer chromatography) to detect and merge similar components, wherein the developing solvent is chloroform: methanol: water = 7:3:1 (v / v / v, after mixing, standing and separating, and taking the lower layer as the developing solvent), and obtaining 8 components Fr.A-Fr.H in the order of elution;

[0067] Step S4, performing reverse phase ODS column chromatography on the Fr.C component (the component eluted when the volume ratio of ethanol to water is 3:7, 3.5 g), using methanol and water as eluents, and performing gradient elution in a ratio of 0:1, 1:9, 2:8, 3:7, 1:1, 7:3, 9:1, and 1:0, respectively, and combining similar components by TLC detection, wherein the developing solvent is chloroform: methanol: water = 7:3:1 (v / v / v, after mixing, standing to separate layers, and taking the lower layer as the developing solvent), and obtaining 5 components Fr.C1-Fr.C5 in order of elution, wherein the Fr.C4 component is the component eluted when the volume ratio of methanol to water is 1:1, and has a mass of 971.4 mg;

[0068] Step S5, performing high performance liquid chromatography separation on the Fr.C4 component, using a semi-preparative column YMC-Pack ODS-A (250×10 mm, 5 μm), using acetonitrile and formic acid aqueous solution (in the formic acid aqueous solution: the volume content of formic acid is 1‰, and the balance is water) as the mobile phase, performing isocratic elution at a volume ratio of 14:86 and a flow rate of 3 mL / min, and obtaining five components Fr.C4a-Fr.C4e in the order of elution;

[0069] Step S6: Component Fr.C4d(t R=32.8min, m=88.6mg) was purified by reverse phase HPLC using a semi-preparative column YMC-Pack ODS-A (250×10mm, 5μm), with acetonitrile and formic acid aqueous solution (the volume content of formic acid in the formic acid aqueous solution was 1‰, and the balance was water) as the mobile phase, and the volume ratio of the two was 21:79, and the flow rate was 3mL / min for isocratic elution. The retention time t R =25.6min component, that is, compound 1, with a mass of 8.8 mg;

[0070] For the component Fr.C4e(t R =36.5min, m=29.1mg) was purified by reverse phase HPLC using a semi-preparative column YMC-Pack ODS-A (250×10mm, 5μm) with acetonitrile and water as the mobile phases, at a volume ratio of 23:77 and a flow rate of 3mL / min. The retention time t R =23.3min component, that is, compound 2, with a mass of 3.0 mg;

[0071] The component Fr.C5 (the components eluted when the volume ratio of methanol to water was 7:3 and the volume ratio of methanol to water was 9:1, 2.1 g) was purified by reverse phase HPLC using a semi-preparative column YMC-Pack ODS-A (250×10 mm, 5 μm) with acetonitrile and formic acid aqueous solution (the volume content of formic acid in the formic acid aqueous solution was 1‰, and the balance was water) as the mobile phase. The volume ratio of the two was 26:74 and the flow rate was 3 mL / min. The retention time t R =30.0min component, that is, compound 3, with a mass of 2.7 mg;

[0072] The flowchart of the above steps is shown in Figure 1 .

[0073] Example 2: The structures of compounds 1-3 are identified as follows:

[0074] The compound 1-3 obtained in Example 1 was subjected to high-resolution mass spectrometry, ultraviolet spectrum, and nuclear magnetic resonance analysis to determine the structure of the compound 1-3.

[0075] The physicochemical data and spectral data of compounds 1-3 are as follows:

[0076] Compound 1: white amorphous powder; HR-ESI-MS m / z 671.32751[M+H] + (calcd for C 33 H 51 O 14 ,671.32733); UV(MeOH)λ max(logε):210(3.77); structural formula:

[0077]

[0078] The Chinese and English names of compound 1 are shown in Table 1, and the NMR data are shown in Table 2 and Table 3. 1 H-NMR (600MHz, CD3OD) spectrum see Figure 2 , 13 C-NMR (150MHz, CD3OD) spectrum Figure 3 , DEPT (θ=135°) spectrum see Figure 4 , HSQC spectrum see Figure 5 , HMBC spectrum see Figure 6 , 1 H- 1 H COSY spectrum see Figure 7 , ROESY spectrum see Figure 8 , UV spectrum see Fig. 9 , HR-ESI-MS spectrum see Fig.10 .

[0079] Compound 2: white amorphous powder; HR-ESI-MS m / z 695.32489[M+Na] + (calcd for C 33 H 52 O 14 Na, 695.32493); UV(MeOH)λ max (logε):210(3.76); structural formula:

[0080]

[0081] The Chinese and English names of compound 2 are shown in Table 1, and the NMR data are shown in Table 2 and Table 3. 1 H-NMR (600MHz, CD3OD) spectrum see Fig.11 , 13 C-NMR (150MHz, CD3OD) spectrum Fig.12 , DEPT (θ=135°) spectrum see Fig.13 , HSQC spectrum see Fig.14 , HMBC spectrum see Fig.15 , 1 H- 1 H COSY spectrum see Fig.16 , ROESY spectrum see Fig.17 , UV spectrum see Fig.18 , HR-ESI-MS spectrum see Fig.19 .

[0082] Compound 3: white amorphous powder; HR-ESI-MS m / z 713.33563[M+Na] + (calcd for C 33 H 54 O 15 Na,713.33549);UV(MeOH)λ max (logε):205(3.74),250(3.62); structural formula is

[0083]

[0084] The Chinese and English names of compound 3 are shown in Table 1, and the NMR data are shown in Table 2 and Table 3. 1 H-NMR (600MHz, Pyridine-d5) spectrum see Fig. 20 , 13 C-NMR (150MHz, Pyridine-d5) spectrum see Fig.21 , DEPT (θ=135°) spectrum see Fig. 22 , HSQC spectrum see Fig.23 , HMBC spectrum see Fig.24 , 1 H- 1 H COSY spectrum see Fig.25 , ROESY spectrum see Fig.26 , UV spectrum see Fig. 27 , HR-ESI-MS spectrum see Fig.28 .

[0085] Table 1: Chinese and English names of Compound 1, Compound 2, and Compound 3

[0086]

[0087]

[0088] Table 2: Compounds 1-3 13 C-NMR data (δ in ppm)

[0089]

[0090]

[0091] a:Measured in CD3OD,b:Measured in Pyridine-d5.

[0092] Table 3: Compounds 1-3 1 H-NMR data (δ in ppm, J in Hz)

[0093]

[0094]

[0095] a:Measured in CD3OD,b:Measured in Pyridine-d5.

[0096] Example 3

[0097] Since glycoside compounds are decomposed into sugars and aglycones in the body, the molecular docking method is used to verify and explain the binding modes of the aglycones of the three new compounds obtained in Example 1 with inducible nitric oxide synthase and cyclooxygenase 2:

[0098] Step 1. The database used in the experiment is PDB database (http: / / www.rcsb.org / ), and the software is SYBYL-X 1.10 (Tripos, USA), Discovery Studio 4.1 Client (DS, Accelrys, USA), and ChemOffice.

[0099] Step 2: Use ChemDraw software to draw the aglycone structural formula of the three compounds and save them in mol2 format. Import the mol2 format file into SYBYL-X 1.10 software, use the Minimize module for structural optimization, perform energy minimization calculations based on the Tripos force field on the compounds, load the Gasteiger-Hückel charge, save the optimized structure in mol2 format, and establish a ligand small molecule compound library to prepare for molecular docking.

[0100] Step 3. Download nitric oxide synthase (PDB ID: 3E6T) from the PDB database (http: / / www.rcsb.org) 1 、Cyclooxygenase 2 (PDB ID:4OTJ) 2 The crystal structure of the target protein was modified, hydrogenated, and loaded with AMBER FF99 charges in the SYBYL software using the Define function in the Docking module in Application, and its active site was located.

[0101] Step 4: After processing, dock the ligand small molecule compound library in step 2 with the target protein in the Docking module, merge the small molecules in the small molecule compound library with the target protein in turn, and save them as a PDF file.

[0102] Step 5: Use the Receptor-Ligand Interactions module in Discovery Studio software to analyze the molecular docking results and create a three-dimensional rendering.

[0103] iNOS is one of the three isoforms of nitric oxide synthase. iNOS exists in myocardial cells, vascular smooth muscle cells, fibroblasts, endothelial cells and inflammatory cells. It is a calcium-independent enzyme. Once iNOS is induced, it will induce the production of a large amount of NO, mediating the occurrence of various diseases. Therefore, it is of great significance to find potential inhibitors of iNOS. The iNOS molecular docking experiment refers to the following reference 1.

[0104] The aglycone of compound 1 is stabilized by hydrogen bonding interactions with Lys82, Tyr367, Glu371, and Asp376, and hydrophobic interactions with residues Ala276 and Trp340.

[0105] The aglycone of compound 2 is stabilized by forming hydrogen bond interactions with Lys82, Tyr367, Glu371, Asp376, and Gln381, and hydrophobic interactions with Trp84, Ala276, and Trp340.

[0106] The aglycone of compound 3 is stabilized by forming hydrogen bond interactions with Arg260 and Ala276 and hydrophobic interactions with residues Ala276, Pro344, and Arg375.

[0107] Cyclooxygenase 2 (COX-2) is a key enzyme in the biosynthesis of prostaglandins. It acts as a dioxygenase and peroxidase, mediating the formation of prostaglandins from arachidonic acid, and plays a special role in the inflammatory response. Targeted delivery of chemotherapeutic drugs to tumors has been explored as a means, and a related approach is to target intracellular proteins through ligands that bind to anticancer drugs. An attractive target for this approach is COX-2, which is highly expressed in a range of malignant tumors. The COX-2 molecular docking experiment refers to the following reference 2.

[0108] The aglycone of compound 1 is stabilized by forming hydrogen bond interactions with Leu82, Lys83, and Tyr115, and hydrophobic interactions with residues Lys83, Pro86, Val89, and Tyr115.

[0109] The aglycone of compound 2 is stabilized by COX-2 through the formation of hydrophobic interactions with residues Lys83, Pro84, Pro86, Val89, Tyr122, and Glu524.

[0110] The aglycone of compound 3 is stabilized by forming hydrogen bond interactions with Ser119 and hydrophobic interactions with residues Val89, Val349, Leu352, Tyr355, Leu359, Val523, and Ala527.

[0111] These hydrogen bonds and hydrophobic bonds play an important role in the binding of these three new compounds to iNOS and COX-2. Figures 29 to 34 The binding conformations in the samples and the docking scores indicate that these compounds have good binding activity with iNOS and COX-2 and can become potential inhibitors of iNOS and COX-2.

[0112] Table 4: Docking scores of compounds 1-3

[0113]

[0114] References:

[0115] 1.Garcin, ED; Arvai, AS; Rosenfeld, RJ; Kroeger, MD; Crane, BR; Andersson, G.; Andrews, G.; Hamley, PJ; Mallinder, PR; Nicholls, DJ; St-Gall ay,SA;Tinker,AC;Gensmantel,NP;Mete,A.;Cheshire,DR;Connolly,S.;Stuehr,DJ;Aberg,A.;Wallace,AV;Tainer,JA;Getzoff,ED,Anchored plasticity opensdoors for selective inhibitor design in nitric oxide synthase. Nat Chem Biol2008,4(11),700-7.

[0116] 2. Uddin, MJ; Crews, BC; Xu, S.; Ghebreselasie, K.; Daniel, CK; Kingsley, PJ; Banerjee, S.; Marnett, LJ, Antitumor Activity of Cytotoxic Cyclooxygenase-2Inhibitors. ACS Chem Biol 2016, 11(11), 3052-3060.

Claims

1. A method for preparing a furostanol-type steroidal saponin compound, wherein the furostanol-type steroidal saponin compound has the structure shown in compound 1, 2 or 3: The preparation method comprises the following steps: S1. Weigh the dried rhizome of the arrowroot, slice it, and then crush it, extract it with methanol, then filter the extract, and concentrate the filtrate under reduced pressure to obtain a crude methanol extract; S2, adding water to the crude methanol extract to obtain a methanol extract aqueous solution, extracting with petroleum ether, ethyl acetate and n-butanol in sequence, discarding the petroleum ether and ethyl acetate extraction parts, and concentrating the remaining extract under reduced pressure to obtain the n-butanol extraction part and the aqueous phase extraction part; S3, subjecting the aqueous phase extraction portion to D101 macroporous adsorption resin column chromatography and gradient elution to obtain 8 components Fr.A to Fr.H; S4, performing reverse phase ODS column chromatography on the Fr.C component and gradient elution to obtain five components Fr.C1 to Fr.C5; S5, separating the component Fr.C4 by high performance liquid chromatography and isocratic elution to obtain five components Fr.C4a to Fr.C4e; S6. Purify the component Fr.C4d by high performance liquid chromatography and isocratic elution to obtain compound 1; The component Fr.C4e was purified by high performance liquid chromatography and isocratic elution to obtain compound 2; The component Fr.C5 was purified by high performance liquid chromatography and isocratic elution to obtain compound 3; In step S3, the conditions for gradient elution of the aqueous phase extraction part are: using ethanol-water as the eluent, gradient elution is performed at a volume ratio of ethanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1, and 1:0, and similar components are combined by TLC detection, wherein a mixed solvent is prepared at a volume ratio of chloroform: methanol: water of 7:3:1, and after mixing, the mixture is allowed to stand for stratification, and the lower layer is taken as the developing solvent; In step S4, the conditions for gradient elution of the Fr.C component are as follows: using methanol-water as the eluent, gradient elution is performed at a volume ratio of methanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 7:3, 9:1, and 1:0, and similar components are combined by TLC detection, wherein a mixed solvent is prepared at a volume ratio of chloroform: methanol: water of 7:3:1, and after mixing, the mixture is allowed to stand for stratification, and the lower layer is taken as the developing solvent; In step S5, the conditions for isocratic elution of component Fr.C4 are as follows: ODS column, acetonitrile and formic acid aqueous solution are used in a volume ratio of 14:86, and isocratic elution is performed at a flow rate of 3 mL / min; wherein the volume content of formic acid in the formic acid aqueous solution is 1‰, and the remainder is water; In step S6, the conditions for isocratic elution of component Fr.C4d are as follows: ODS column, acetonitrile and formic acid aqueous solution in a volume ratio of 21:79, isocratic elution at a flow rate of 3 mL / min; wherein the volume content of formic acid in the formic acid aqueous solution is 1‰, and the remainder is water; The conditions for isocratic elution of component Fr.C4e were as follows: ODS column, isocratic elution at a flow rate of 3 mL / min in a volume ratio of acetonitrile to water of 23:77; The conditions for isocratic elution of component Fr.C5 are as follows: ODS column, isocratic elution at a flow rate of 3 mL / min with a volume ratio of acetonitrile to formic acid aqueous solution of 26:74; wherein the volume content of formic acid in the formic acid aqueous solution is 1‰, and the remainder is water.

2. The preparation method according to claim 1, characterized in that: In step S1, the liquid-to-solid ratio of methanol to open arrowroot is 3:1-6:1, mL:g; The extraction method is as follows: methanol is added to the crushed open arrowroot stems in three times, and then refluxed at 65±3°C for 1-2 hours each time, and the extracts are combined.

3. The preparation method according to claim 1, characterized in that: In step S2, the volumes of petroleum ether, ethyl acetate and n-butanol are 1-2 times the volume of the methanol extract aqueous solution.

Images