Triterpenoid saponin compounds as well as preparation method and application thereof

Abstract

The invention relates to a triterpenoid saponin compound, a preparation method thereof and application of the triterpenoid saponin compound in preparation of anti-neuroinflammation drugs. The triterpenoid saponin compound has a structure as shown in a formula I. An in-vitro anti-neuroinflammation activity experiment proves that the triterpenoid saponin compound has an obvious inhibition effect on lipopolysaccharide (LPS)-induced BV2 cell inflammation, and can be used as a novel anti-neuroinflammation drug and a neurodegenerative disease treatment drug. Formula I

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine manufacturing, specifically to a triterpenoid saponin compound, and also to its uses and preparation methods. Background Technology

[0002] Neuroinflammation, as a core pathological response of the central nervous system in states of injury or disease (such as Alzheimer's disease, Parkinson's disease, and stroke), is essentially a chronic inflammatory cascade triggered by the overactivation of microglia and astrocytes. These activated glial cells release large amounts of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6) and reactive oxygen species (ROS), leading to blood-brain barrier damage, neuronal death, and impaired synaptic plasticity. It is important to note that this process exhibits significant spatiotemporal differences in different diseases: multiple sclerosis and early stroke are mainly characterized by the infiltration of peripheral immune cells (such as T cells and neutrophils) after blood-brain barrier disruption; while Alzheimer's disease and Parkinson's disease are more prominently characterized by a pathological mechanism driven by the continuous activation of microglia in the central nervous system. Currently, there is still a significant gap in specific therapeutic drugs targeting this complex pathological process, and traditional anti-inflammatory drugs are insufficient to meet clinical needs due to insufficient central penetration or excessive suppression of immune function.

[0003] Astragalus is a legume plant, specifically Astragalus mongholicus (Astragalus membranaceus). Astragalus membranaceus var. mongholicus ) or Astragalus membranaceus ( Astragalus membranaceus The dried root of Astragalus membranaceus has the following main effects: tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing edema, generating fluids and nourishing blood, promoting blood circulation and relieving pain, promoting pus drainage and detoxification, and promoting tissue regeneration and wound healing. Given its application in the treatment of ischemic stroke, exploring highly effective and low-toxicity neuroinflammatory inhibitors derived from Astragalus membranaceus is of significant value and importance. Summary of the Invention

[0004] This invention isolates a class of triterpenoid saponins from the root of Astragalus membranaceus, which have a novel structure and exhibit activity in inhibiting neuroinflammation.

[0005] Therefore, in a first aspect, the present invention provides a triterpenoid saponin compound having the structure shown in Formula I: Formula I R1 is selected from hydrogen or... α -L-Ara, R2 is selected from one of the following structures: Preferably, the triterpenoid saponin compounds of the present invention are selected from the following compounds 1 and 2: A second aspect of the invention relates to a method for preparing the aforementioned triterpenoid saponins, particularly compounds 1 and 2, comprising: a. Extraction steps: Extract Mongolian Astragalus ( Astragalus membranaceus var. mongholicus The roots of the plant were extracted successively with anhydrous ethanol and ethanol-water by heating and reflux. The extracts were combined and concentrated under reduced pressure to obtain an extract. b. Separation steps: The suspension was added to water and then extracted with petroleum ether and ethyl acetate to obtain petroleum ether extract, ethyl acetate extract and water fraction. These were then separated by column chromatography to obtain compounds 1 and 2, respectively.

[0006] In one embodiment of the present invention, the extraction step includes: extracting the root of Astragalus membranaceus by heating and refluxing with anhydrous ethanol 2-3 times, extracting the residue by heating and refluxing with ethanol-water at a volume concentration of 30-70% 2-3 times, combining the extracts, and concentrating under reduced pressure to obtain an extract.

[0007] Preferably, the residue is extracted 2-3 times by heating and reflux with ethanol-water at a volume concentration of 50-60%.

[0008] In one embodiment of the present invention, the column chromatography includes one or more combinations of macroporous resin column chromatography, silica gel column chromatography, medium-low pressure ODS column chromatography, reversed-phase semi-preparative HPLC, and preparative HPLC.

[0009] Preferably, the separation step includes: Water samples were separated by macroporous resin column chromatography with a gradient elution of ethanol-water (0:10, 1:9, 3:7, 1:1, 19:1, v / v) to obtain five fractions C1–C5. Fraction C5 was separated by silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate (1:0, 100:1, 5:1, 1:1, 0:1, v / v) to obtain five fractions C5A–C5E. Fraction C5E was separated by silica gel column chromatography with a gradient elution of dichloromethane-methanol (100:1, 50:1, 30:1, 10:1, 5:1, 3:1, 1:1, 1:5, 1:10, v / v) to obtain 16 fractions C5E1–C5E16. Fraction C5E13 was further separated by medium... Compound 1 was obtained by separation by ODS column chromatography with a gradient elution of methanol-water (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0, v / v). Compound 1 was obtained by separation of C5E13A–C5E13R by reversed-phase semi-preparative HPLC with isocratic elution of acetonitrile-water (23:77, v / v) as the mobile phase. Compound 2 was obtained by separation of C5E13L by reversed-phase semi-preparative HPLC with isocratic elution of acetonitrile-water (25:75, v / v) as the mobile phase.

[0010] This invention has discovered that the triterpenoid saponins have a good effect on anti-neuroinflammatory effects, and can inhibit LPS-induced NO release from BV2 cells.

[0011] Therefore, a third aspect of the present invention is to provide the use of the triterpenoid saponin compound in the preparation of a medicament for the treatment of neuroinflammation.

[0012] Preferably, the drug is used to treat neurodegenerative diseases. Preferably, the neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, or Parkinson's disease.

[0013] A fourth aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned triterpenoid saponin compound.

[0014] The beneficial effects of this invention are: This invention isolates a novel class of triterpenoid saponins from the root of *Astragalus membranaceus*. These triterpenoid saponins possess a novel structure. The anti-inflammatory activity of these compounds was evaluated using an LPS-induced BV2 cell model. The results showed that the triterpenoid saponins of this invention exhibit significant anti-neuroinflammatory activity. Therefore, the triterpenoid saponins of this invention can serve as novel anti-neuroinflammatory drugs and therapeutic agents for neurodegenerative diseases. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Show the separation flow chart of compounds 1 and 2; Figure 2 For compound 1 1 H- 1 H COSY spectrum; Figure 3 The HSQC spectrum of compound 1; Figure 4 The HMBC spectrum of compound 1; Figure 5 For compound 2 1 H- 1 H COSY spectrum; Figure 6 The HSQC spectrum of compound 2; Figure 7 The HMBC spectrum of compound 2; Figure 8 The anti-neuroinflammatory activities of compounds 1 and 2 are evaluated. Results are expressed as mean ± SD, where This indicates that p < 0.05 compared to the model group; This indicates that p < 0.01 compared to the model group; This indicates that p < 0.001 compared to the model group. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. The embodiments are provided to better illustrate the content of the present invention and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0017] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0018] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0019] Example 1: Preparation of compounds 1 and 2 50 kg of Astragalus membranaceus root was mechanically pulverized and extracted twice with 500 L of anhydrous ethanol under reflux for 2 hours each time. The extracts were filtered, and the residue was further extracted twice with 400 L of ethanol-water (1:1) under reflux for 2 hours each time. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was suspended in water and extracted sequentially with petroleum ether and ethyl acetate to obtain 565 g of petroleum ether extract, 213 g of ethyl acetate extract, and 10847 g of aqueous fraction.

[0020] The water sample (10069 g) was separated by macroporous resin column chromatography with a gradient elution of ethanol-water (0:10, 1:9, 3:7, 1:1, 19:1, v / v) to obtain 5 fractions C1–C5; the C5 fraction (142 g) was separated by silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate (1:0, 100:1, 5:1, 1:1, 0:1, v / v) to obtain 5 fractions C5A–C5E; the C5E fraction (97 g) was separated by silica gel column chromatography with a gradient elution of dichloromethane-methanol (100:1, 50:1, 30:1, 10:1, 5:1, 3:1, 1:1, 1:5, 1:10, v / v) to obtain 16 fractions C5E1–C5E16; C5E13 (7.0 ... The fraction C5E13 (g) was separated by medium-pressure ODS column chromatography with a methanol-water gradient elution (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0, v / v) to obtain 18 fractions C5E13A–C5E13R; the fraction C5E13J (0.795 g) was separated by reversed-phase semi-preparative HPLC with isocratic elution using acetonitrile-water (23:77, v / v) to obtain compound 1 (t). R =25.8 min, 24.35 mg). The C5E13L (0.507 g) fraction was separated by reversed-phase semi-preparative HPLC, with isocratic elution using acetonitrile-water (25:75, v / v) as the mobile phase, to give compound 2 (t R =50.0 min, 34.25 mg).

[0021] Compound 1 Colorless powder -10 ( c 0.1, CH3OH); UV (CH3OH) λ max (log ε 201 (3.86)nm; ECD (MeOH) λ max (Δ ε ) 202 (+1.70), 215 (-5.14) nm; IR (KBr) ν max 3376, 2936 and 1046 cm -1 HRESIMS m / z 1152.6191 ([M - H] - The calculated value is C. 55 H 94 O 24N, 1152.6171); 1 H NMR(500 MHz, CD3OD), δ H : 1.44 (1H, m, H-1a), 1.07 (1H, m, H-1b), 1.92 (1H, m, H-2a), 1.71 (1H, m, H-2a), 3.16 (1H, m, H-3), 0.83 (1H, m, H-5), 1.58 (2H, m,H-6), 2.12 (1H, m, H-7a), 1.20 (1H, m, H-7b), 1.20 (1H, m, H-9), 2.09 (1H, m,H-11a), 1.47 (1H, m, H-11b), 4.61 (1H, m, H-12), 1.19 (1H, m, H-13), 1.73(1H, m, H-15a), 1.67 (1H, m, H-15b), 2.08 (2H, m, H-16), 5.21 (1H, m, H-17),1.25 (3H, s, H-18), 0.94 (3H, s, H-19), 1.64 (3H, s, H-21), 2.00 (2H, m, H-22), 1.67 (2H, m, H-23), 3.46 (1H, m, H-24), 3.51 (1H, m, H-26a), 3.45 (1H,m, H-26b), 1.11 (3H, s, H-27), 1.02 (3H, s, H-28), 0.87 (3H, s, H-29), 1.25(3H, s, H-30), 4.39 (1H, d, J =6.9 Hz, H-1′), 3.38 (1H, m, H-2′), 3.43 (1H, m,H-3′), 3.76 (1H, m, H-4′), 3.41 (1H, m, H-5′), 4.04 (1H, d, J =12.0 Hz, H-6′a),3.73 (1H, dd, J=12.0, 5.8 Hz, H-6′b), 5.57 (1H, brs, H-1′′), 4.02 (1H, brs, H-2′′), 3.31 (1H, m, H-3′′), 3.30 (1H, m, H-4′′), 3.90 (1H, m, H-5′′), 1.20(3H, s, H-6′′), 4.33 (1H, d, J =7.4 Hz, H-1′′′), 3.20 (1H, m, H-2′′′), 3.33(1H, m, H-3′′′), 3.49 (1H, m, H-4′′′), 3.87 (1H, m, H-5′′′a), 3.18 (1H, m, H-5′′′b), 4.59 (1H, d, J =8.4 Hz, H-1′′′′), 3.63 (1H, m, H-2′′′′), 3.46 (1H, m,H-3′′′′), 3.30 (1H, m, H-4′′′′), 3.31 (1H, m, H-5′′′), 3.89 (1H, m, H-6′′′′a), 3.66 (1H, m, H-6′′′′b), 2.00 (3H, s, H-1′′′′-NHCOC H 3); 13 C NMR (125 MHz,CD3OD), δ C: 40.1 (C-1), 27.2 (C-2), 90.3 (C-3), 40.3 (C-4), 57.8 (C-5), 19.9(C-6), 44.0 (C-7), 45.2 (C-8), 62.2 (C-9), 37.4 (C-10), 33.9 (C-11), 74.4 (C-12), 61.2 (C-13), 77.6 (C-14), 44.0 (C-15), 24.2 (C-16), 125.9 (C-17), 26.0(C-18), 16.5 (C-19), 135.9 (C-20), 16.2 (C-21), 37.4 (C-22), 30.7 (C-23),76.0 (C-24), 74.2 (C-25), 68.3 (C-26), 20.6 (C-27), 28.3 (C-28), 16.8 (C-29),17.6 (C-30), 105.5 (C-1′), 80.2 (C-2′), 79.5 (C-3′), 71.8 (C-4′), 76.8 (C-5′), 69.7 (C-6′), 101.3 (C-1′′), 81.6 (C-2′′), 71.8 (C-3′′), 74.2 (C-4′′),70.1 (C-5′′), 18.0 (C-6′′), 105.5 (C-1′′′), 74.9 (C-2′′′), 77.6 (C-3′′′), 71.2 (C-4′′′), 66.9 (C-5′′′), 104.6 (C-1′′′′), 58.1 (C-2′′′′), 76.3 (C-3′′′′), 72.1 (C-4′′′′), 78.0 (C-5′′′′), 62.9 (C-6′′′′), 174.6 (1′′′′-NH C OCH3), 23.1 (1′′′′-NHCO C H3).

[0022] By HRESIMS m / z 1152.6191 ([M - H] - The calculated value is C. 55 H 94 O 24 N, 1152.6171), determined that the molecular formula of compound 1 is C 55 H 95 O 24 N has 9 degrees of unsaturation. 11H NMR showed one olefinic hydrogen signal and nine methyl hydrogen signals. 13 C10 NMR and HSQC spectra revealed one carbonyl carbon signal, two double bond carbon signals, nine methyl carbon signals, thirteen methylene carbon signals, twenty-five methine carbon signals, and five quaternary carbon signals. These characteristic signals suggest that compound 1 is a tricyclic triterpenoid saponin. Further analysis of key signals from the COSY, HSQC, and HMBC spectra of compound 1 confirmed its chemical structure. Comparison of the specific rotation and ECD data of compound 1 with data from similar tricyclic triterpenoid saponins confirmed that the absolute configuration of compound 1 is 3... β , 12 β , 14 R , 17 E , twenty four R , 25 S .

[0023] Compound 2 Colorless powder -12 ( c 0.1, CH3OH); UV (CH3OH) λ max (log ε 201 (3.82)nm; ECD (MeOH) λ max (Δ ε ) 200 (+3.64), 217 (-2.35) nm; IR (KBr) ν max 3405 and 2937cm -1 HRESIMS m / z 1310.6378 ([M - H] - The calculated value is C. 61 H 100 O 29 N, 1310.6386); 1 1H NMR (500MHz, pyridine- d 5) δ H: 1.37 (1H, m, H-1a), 1.15 (1H, m, H-1b), 2.38 (1H, m, H-2a), 1.95 (1H, m, H-2a), 3.42 (1H, m, H-3), 0.76 (1H, m, H-5), 1.51 (2H, m,H-6), 2.19 (1H, m, H-7a), 1.13 (1H, m, H-7b), 0.88 (1H, m, H-9), 2.15 (1H, m,H-11a), 1.73 (1H, m, H-11b), 4.85 (1H, m, H-12), 1.21 (1H, m, H-13), 2.07(1H, m, H-15a), 1.95 (1H, m, H-15b), 2.43 (1H, m, H-16a), 2.29 (1H, m, H-16b), 5.50 (1H, m, H-17), 1.48 (3H, s, H-18), 0.90 (3H, s, H-19), 1.73 (3H,s, H-21), 2.57 (1H, dd, J =14.2, 7.6 Hz, H-22a), 2.45 (1H, dd, J =14.2, 5.2 Hz,H-22b), 5.18 (1H, m, H-23), 4.35 (1H, m, H-24), 1.31 (3H, s, H-26), 1.46 (3H,s, H-27), 1.24 (3H, s, H-28), 1.19 (3H, s, H-29), 1.57 (3H, s, H-30), 4.87(1H, d, J =6.7 Hz, H-1′), 4.14 (1H, m, H-2′), 4.15 (1H, m, H-3′), 4.04 (1H, m,H-4′), 4.00 (1H, m, H-5′), 4.77 (1H, m, H-6′a), 4.28 (1H, m, H-6′b), 6.64(1H, brs, H-1′′), 4.82 (1H, brs, H-2′′), 4.69 (1H, m, H-3′′), 4.21 (1H, m, H-4′′), 4.67 (1H, m, H-5′′), 1.65 (3H, s, H-6′′), 4.97 (1H, d, J=7.4 Hz, H-1′′′), 4.02 (1H, m, H-2′′′), 4.15 (1H, m, H-3′′′), 4.18 (1H, m, H-4′′′), 4.33(1H, m, H-5′′′a), 3.68 (1H, m, H-5′′′b), 5.58 (1H, d, J =8.2 Hz, H-1′′′′), 4.36(1H, m, H-2′′′′), 4.34 (1H, m, H-3′′′′), 3.88 (1H, m, H-4′′′′), 3.84 (1H, m,H-5′′′), 4.37 (1H, m, H-6′′′′a), 4.05 (1H, m, H-6′′′′b), 4.85 (1H, d, J =7.2Hz, H-1′′′′′), 4.40 (1H, m, H-2′′′′′), 4.10 (1H, m, H-3′′′′′), 4.23 (1H, m,H-4′′′′′), 4.23 (1H, m, H-5′′′′a), 3.72 (1H, m, H-5′′′′′b), 2.19 (3H, s, H-1′′′′-NHCOC H 3); 13 C NMR (125 MHz, pyridine- d 5), δ C: 39.6 (C-1), 27.3 (C-2), 89.6(C-3), 40.0 (C-4), 57.1 (C-5), 19.7 (C-6), 43.6 (C-7), 45.0 (C-8), 61.6 (C-9), 36.9 (C-10), 34.2 (C-11), 73.6 (C-12), 61.6 (C-13), 76.6 (C-14), 44.6 (C-15), 24.0 (C-16), 131.1 (C-17), 26.7 (C-18), 16.6 (C-19), 129.6 (C-20), 16.9(C-21), 45.8(C-22), 77.3 (C-23), 87.4 (C-24), 70.1 (C-25), 26.0 (C-26), 26.0(C-27), 28.4 (C-28), 17.2 (C-29), 18.0 (C-30), 156.0 (C-31), 105.7 (C-1′),78.9 (C-2′), 80.4 (C-3′), 72.2 (C-4′), 77.3 (C-5′), 70.3 (C-6′), 101.1 (C-1′′), 82.4 (C-2′′), 72.4 (C-3′′), 74.8 (C-4′′), 70.1 (C-5′′), 19.0 (C-6′′),106.4 (C-1′′′), 75.4 (C-2′′′), 78.7 (C-3′′′), 71.6 (C-4′′′), 67.6 (C-5′′′), 104.8 (C-1′′′′), 57.4 (C-2′′′′), 85.7 (C-3′′′′), 70.7 (C-4′′′′), 78.6 (C-5′′′′), 63.1 (C-6′′′′), 106.2 (C-1′′′′′), 72.6 (C-2′′′′′), 74.7 (C-3′′′′′), 69.8 (C-4′′′′′), 67.9 (C-5′′′′′), 172.6 (1′′′′-NHCOCH3), 24.1 (1′′′′-NHCOCH3).

[0024] By HRESIMS m / z 1310.6378 ([M - H] - The calculated value is C. 61 H 100 O 29N, 1310.6386), determined that the molecular formula of compound 2 is C 61 H 101 O 29 N has 12 degrees of unsaturation. 1 1H NMR showed one olefinic hydrogen signal and ten methyl hydrogen signals. 13 C10 NMR and HSQC spectra revealed two carbonyl carbon signals, two double bond carbon signals, ten methyl carbon signals, twelve methylene carbon signals, thirty methine carbon signals, and five quaternary carbon signals. These characteristic signals suggest that compound 2 is a tricyclic triterpenoid saponin. Further analysis of key signals from the COSY, HSQC, and HMBC spectra of compound 2 confirmed its chemical structure. Comparison of the specific rotation and ECD data of compound 2 with data from similar tricyclic triterpenoid saponins confirmed that the absolute configuration of compound 2 is 3. β , 12 β , 14 R , 17 E , twenty three S , twenty four R .

[0025] Example 2: Anti-neuroinflammatory activity test of compounds 1 and 2 (1) Cell culture BV2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 50 mg / mL streptomycin. All cells were cultured in an incubator at 37°C and 5% CO2.

[0026] (2) Determination of NO concentration released by LPS-induced BV2 cells BV2 cells in the logarithmic growth phase were seeded in 96-well cell culture plates at a cell density of 2 × 10⁶ cells / well. 4 Cells / well. After 12 hours of culture, the culture medium in the 96-well plate was discarded, and control, model, and drug-treated groups were set up. The positive control drug was dexamethasone (DX), and each sample was tested in triplicate. Complete DMEM medium was added to the control group, complete DMEM medium containing 1 μg / mL LPS was added to the model group, and complete DMEM medium containing 1 μg / mL LPS and 20 μM drug was added to the drug-treated group. Cultures were continued for 24 hours. Subsequently, 50 μL of cell supernatant was taken from each well, and the NO content in the cell supernatant was measured according to the instructions of the nitric oxide assay kit.

[0027] For details, please see [link / details]. Figure 8 .

[0028] Compounds 1-2 significantly reduced LPS-induced NO release from BV2 cells and inhibited neuroinflammatory responses.

[0029] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A triterpenoid saponin compound having the structure shown in Formula I: Formula I R1 is selected from hydrogen or... α -L-Ara, R2 is selected from one of the following structures: 。 2. The triterpenoid saponin compound according to claim 1, characterized in that, The compound is selected from the following compounds 1 and 2: 。 3. Use of the triterpenoid saponin compound according to any one of claims 1-2 in the preparation of a medicament for use in the treatment of neuroinflammation.

4. The use according to claim 3, characterized in that, The drug is used to treat neurodegenerative diseases.

5. The use according to claim 4, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, or Parkinson's disease.

6. A pharmaceutical composition comprising the triterpenoid saponin compound according to any one of claims 1-2.

7. The method for preparing the triterpenoid saponin compound according to claim 2, comprising: a. Extraction steps: Extract Mongolian Astragalus ( Astragalus membranaceus var. mongholicus The roots of the plant were extracted successively with anhydrous ethanol and ethanol-water by heating and reflux. The extracts were combined and concentrated under reduced pressure to obtain an extract. b. Separation steps: The extract was suspended in water and extracted with petroleum ether and ethyl acetate to obtain petroleum ether extract, ethyl acetate extract and water fraction, which were then separated by column chromatography to obtain compounds 1 and 2, respectively.

8. The method for preparing triterpenoid saponins according to claim 7, characterized in that, The extraction steps include: extracting the roots of Astragalus membranaceus by heating and refluxing with anhydrous ethanol 2-3 times, extracting the residue by heating and refluxing with ethanol-water at a volume concentration of 30-70% 2-3 times, combining the extracts, and concentrating under reduced pressure to obtain an extract. The column chromatography includes one or more combinations of macroporous resin column chromatography, silica gel column chromatography, medium-low pressure ODS column chromatography, reversed-phase semi-preparative HPLC, and preparative HPLC.

Images