Separation of alkaloids from pH zoned Astragalus brachytagus by countercurrent chromatography and its application

Abstract

The application discloses a method for separating alkaloid compounds in pH zone banding horsebean by counter-current chromatography and application. Horsebean seeds are crushed to 40-60 meshes, 95% ethanol is used for reflux extraction for three times, each time for 2 hours, the extract is combined, filtered, concentrated under reduced pressure, evaporated to be alcohol-free, water is added, 1% hydrochloric acid aqueous solution is added to adjust pH to 2.0, petroleum ether is added, ammonia solution is added to adjust pH to 9.5, chloroform is added for extraction for 5 times, chloroform extract is obtained, and the chloroform extract is evaporated under reduced pressure to obtain crude alkaloids. The crude alkaloids are separated by using a first-end elution mode of PZRCCC, concentrated under reduced pressure to obtain alkaloid compounds. The alkaloid compounds in horsebean can be effectively separated.

Description

Technical Field

[0001] This invention relates to a countercurrent chromatography method and its application for the separation of alkaloid compounds in the pH zone of *Phyllostachys edulis*. Background Technology

[0002] *Vigna edulis* is a valuable medicinal plant that grows in valleys and grasslands of Central Asia, Europe, Siberia, Russia, and China. The entire plant is rich in quinolinidine alkaloids, accounting for approximately 3% of the seeds. Pharmacological studies have shown that the alkaloids in *Vigna edulis* possess antibacterial and anti-inflammatory activities. Recently, tobacco use has become a global public health concern. As a typical quinolinidine alkaloid in tobacco, nicotine dependence is a major cause of smoking behavior, and smoking has a significant impact on public health diseases, including cardiovascular disease, lung cancer, and chronic obstructive pulmonary disease. Therefore, there is great interest in developing drug therapies for smoking cessation and stimulating nicotinic receptor neurons. Varenicline tartrate is a well-known partial agonist of the α4β2 nicotinic receptor and was approved by the US FDA as a smoking cessation treatment on May 39, 2006. Similar to varenicline, cytosine-rich seeds, accounting for more than 50% of the total alkaloids, are a potential α4β2 partial nicotinic receptor with the structure of quinolinidine alkaloids. It is considered safe and inexpensive by medical experts and consumers. In addition, cytisine has nootropic and antidepressant effects and can be used to treat hepatitis and liver cancer. Besides cytisine, the seeds of *T. muscatus* are also rich in quinolinic acid alkaloids and many similar alkaloid components. Therefore, an effective method for isolating quinolinic acid alkaloids from *T. muscatus* seeds is urgently needed. Summary of the Invention

[0003] This invention provides a countercurrent chromatography method and application for the separation of alkaloids in the pH zone of *Phyllostachys edulis*, and solves the technical problem of effectively separating alkaloids from *Phyllostachys edulis*.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The countercurrent chromatography method for separating alkaloids from *Phyllostachys edulis* in the pH zone is performed according to the following steps: The seeds of the pea plant were crushed to 40-60 mesh and extracted three times by reflux with ethanol for 2 hours each time. The extracts were combined, filtered, concentrated under reduced pressure, and evaporated until no alcohol odor was detected. Water was added, and the pH was adjusted to 2.0 with HCl aqueous solution. Petroleum ether was added, and the pH was adjusted to 9.5 with ammonia solution. Chloroform was added and extracted five times to obtain a chloroform extract. The chloroform extract was evaporated to dryness under reduced pressure to obtain crude alkaloids. The crude alkaloids were separated using the first-to-last elution mode of PZRCCC, and then concentrated under reduced pressure to obtain alkaloid compounds. The mass ratio of the pea seeds, ethanol, water, petroleum ether, and chloroform is 1:10-15:0.25-1:10-15:0.75-1.5:1-3. The molecular structure of the alkaloid compound is as follows: The ethanol is a 95% aqueous solution; the HCl aqueous solution contains 1% HCl by mass; and the ammonia solution is a 1:1 aqueous solution by volume.

[0005] The first and last elution mode of PZRCCC uses solvent to separate the crude alkaloids. The steps are as follows: The upper and lower phases are added to the crude alkaloid to prepare a sample solution; Using the upper phase as the stationary phase and the lower phase as the mobile phase, the column was degassed by sonication. The cold hydrazine was turned on, and the stationary phase was pumped into the column at a temperature of 25℃ and a flow rate of 30 mL / min. After the column was completely filled, the rotation speed was adjusted to 800 rpm to achieve hydrodynamic equilibrium. The sample solution was injected into the chromatographic column through the injection valve, and the absorbance of the UV detector was continuously monitored at 280 nm. The chromatographic data was recorded by turning on the recorder. The eluent is continuously collected using an automatic collector, and each peak fraction is collected into a reagent bottle according to the chromatogram. After the desired peak is eluted, stop the rotation speed and elution. Use a vacuum pump to collect the components in the column in a graduated cylinder. Concentrate the collected fraction under reduced pressure to obtain alkaloid compounds. The mass-to-volume ratio of crude alkaloids, upper phase, and lower phase is 1:10:10 (g / mL / mL). The solvent is a composition of chloroform, methanol, and water in a volume ratio of 4:3:3, and its preparation method is as follows: Place chloroform, methanol and water in a separatory funnel, shake thoroughly, and let stand for 30 minutes to obtain the solvent; The upper layer of the solvent is the upper phase, and the lower layer of the solvent is the lower phase.

[0006] The method also includes high-performance liquid chromatography (HPLC) analysis of the alkaloid compounds, with the following analytical conditions: Chromatographic column: Waters Symmetry (250 × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) and 0.05% TEA aqueous solution (B); gradient elution conditions: 0–7 min, 90–70% A, 10–30% B; 7–12 min, 70–65% A, 30–35% B; 12–13 min, 65–90% A, 35–10% B; and 13–20 min, 90% A, 10% B; flow rate: 1 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 μL.

[0007] It also includes the structural identification of terpenoid compounds, the method of which is as follows: Through ESI-MS, 1 H and 13 The separated compounds were identified by C10 NMR spectroscopy. ESI-MS (positive and negative ion modes) mass spectrometry analysis was performed on a Xevo TQ-XS spectrometer, and NMR spectra were recorded on a Bruker AV-400 spectrometer. DMSO- d 6 was used as a solvent, and tetramethylsilane (TMS) was used as an internal standard. Chemical shift ( δ The value is expressed in ppm, and the coupling constant ( J (in Hz)

[0008] The application of pH-zone countercurrent chromatography to separate alkaloids from *Phragmites australis* for the treatment of inflammation and reduction of central nervous system damage.

[0009] Compound structure identification and analysis: Compound 1: N-formylcytisine: ESI-MS m / z 219.2619 [M+H] + The molecular formula is C 12 H 14 N2O2. 1 H-NMR (400 MHz, DMSO-d6): δ H 7.88 / 7.59 (1H, s, CHO), 7.37 / 7.33 (1H,dd, J = 7.2, 8.8 Hz, H-4), 6.22 (2H, dd, J = 1.2, 8.8 Hz, H-3), 6.15 (2H, dd,J = 1.2, 7.2 Hz, H-5), 4.31 / 4.11 (1H, d, J = 13.6 Hz, H-10β), 3.93 / 3.81 (1H, d, J = 13.6 Hz, H-10α), 3.13 (2H, d, J = 1.6 Hz, H-7), 2.51 (2H, br.s, H-9). 13 C-NMR (100 MHz, DMSO-d6): δ C162.6 / 162.5 (C-14), 161.6 / 161.5 (C-2), 150.2 / 149.9 (C-6), 139.4 (C-4), 116.5 / 116.3 (C-3), 105.2 / 105.1 (C-5), 53.2 / 51.6 (C-13),49.2 / 48.7 (C-10), 46.9 / 45.9 (C-11), 34.4 / 33.7 (C-7), 27.0 / 26.8 (C-9),25.9 / 25.8 (C-8). Compound 2: N-acetycytisine: ESI-MS m / z 233.2814 [M+H] + The molecular formula is C 13 H 16 N2O2. 1 H-NMR (400 MHz, DMSO-d6): δ H 7.38 / 7.33 (1H, dd, J = 7.2, 8.4 Hz, H-4), 6.24 (2H, dd, J = 1.2, 8.4 Hz, H-3), 6.14 (2H, dd, J = 1.2, 7.2 Hz, H-5), 3.97 / 3.79 (1H, d, J = 14.2 Hz, H-10β), 3.66 (2H, m, H-10α), 3.15 (1H, br.s,H-7), 2.42 (1H, br.s, H-9),1.88 / 1.56 (3H, s, H-15); 13 C-NMR (100 MHz, DMSO-d6): δ C 169.1 / 168.9 (C-14), 162.6 / 162.5 (C-2), 150.4 / 150.1 (C-6), 139.4 / 139.2(C-4), 116.5 / 116.2 (C-3), 105.2 / 105.1 (C-5), 53.7 / 52.1 (C-13), 49.1 / 48.9 (C-10), 47.3 (C-11), 34.8 / 34.2 (C-7), 27.5 / 27.4 (C-9), 25.7 / 25.5 (C-8), 21.6 / 20.9 (C-15). Compound 3: (-)-Cytisine: ESI-MS m / z 191.2414 [M+H] + The molecular formula is C 11 H14 N2O. 1 H-NMR (400 MHz, DMSO-d6): δ H 7.32 (1H, dd, J = 6.8, 8.8 Hz, H-4), 6.20 (1H, dd, J = 1.2, 8.8 Hz, H-3), 6.04 (1H, dd, J = 1.2, 6.8 Hz, H-5), 3.79 (1H, d, J = 15.6 Hz, H-10β), 3.70 (1H, d, J = 6.8 Hz, H-10α), 2.93~2.76 (5H, m, J = 6.8 Hz, H-11α, 11β, H-13α, 13β, 7), 2.23 (1H, s, NH), 1.86 (1H, m, H-9),1.80 (2H, m, H-8α, 8β). 13 C-NMR (100 MHz, DMSO-d6): δ C 162.7(C-2), 152.8(C-6), 139.1(C-4), 115.5(C-3), 104.3(C-5), 53.9(C-13), 53.1(C-11), 49.9(C-10), 35.2(C-7), 27.7(C-9), 26.3 (C-8). Compound 4:13-β-hydroxylthermopsin: ESI-MS m / z 261.3314 [M+H] + , scientistsC 15 H 20 N2O2. 1 H-NMR (400 MHz, CDCl3): δ H7.30 (1H, d, J = 8.0 Hz, H-4), 6.46(1H, dd, J = 4.0, 8.0 Hz, H-3), 6.01 (1H, d, J = 8.0 Hz, H-5), 4.24 (1H, d, J= 16.0 Hz, H-10β), 3.63 (1H, m, H-10α), 3.35 (1H, dd, J = 4.0, 8.0 Hz, H-17α), 2.97 (1H, s, H-7), 2.60 (1H, m, H-15α), 2.45 (1H, m, H-17β), 2.12 (1H, m,H-9), 2.08 (1H, m, H-11), 1.96 (1H, m, H-8α), 1.95 (1H, m, H-15β), 1.82 (1H,m, H-8β). 13 C-NMR (100 MHz, CDCl3): δ C 163.7 (C-2), 151.9 (C-6), 138.8 (C-4),116.6 (C-3), 105.0 (C-5), 68.8 (C-13), 63.7 (C-11), 52.1 (C-17), 54.1 (C-15),44.9 (C-10), 39.2 (C-12), 35.3 (C-7), 34.5 (C-14), 32.6 (C-9), 27.2 (C-8). Compound 5: N-methylcytisine: ESI-MS m / z 205.2714 [M+H] + , with the molecular formula C 12 H 16 N2O . 1 H-NMR (400 MHz, DMSO-d6): δ H7.31 (1H, dd, J = 6.8, 8.8 Hz, H-4), 6.20 (1H, dd, J = 1.2, 8.8 Hz, H-3), 6.06 (1H, dd, J = 1.2, 6.8 Hz, H-5), 3.79 (1H, d, J = 15.6 Hz, H-10β), 3.67 (1H, d, J = 6.8 Hz, H-10α), 2.99 (1H, d, J = 1.6 Hz, H-7), 2.82 (1H, d, J = 10.8 Hz, H-11α), 2.72 (1H, d, J = 10.8 Hz, H-13α), 2.36 (1H, d, J = 2.4 Hz, H-9), 2.17 (1H, d, J = 10.8 Hz, H-11β), 2.15 (1H, d, J = 10.8 Hz, H-13β), 2.04 (3H, s, N-CH3), 1.76 (1H, d, J = 12.4 Hz, H-8α), 1.63 (1H, d, J = 12.4 Hz, H-8β). 13 C-NMR (100 MHz, DMSO-d6): δ C 162.7 (C-2), 152.6 (C-6), 139.2 (C-4), 115.8 (C-3), 104.2 (C-5), 62.7 (C-11), 62.2 (C-13), 50.1 (C-10), 46.3 (C-14), 34.9 (C-7), 27.7 (C-9), 25.1 (C-8). Compound 6: Thermopsine: ESI-MS m / z 244.3314 [M+H] + , with the molecular formula C 15 H 20 N2O. 1 H-NMR (400 MHz, DMSO-d6): δ H7.31 (1H, dd, J = 6.8, 8.8 Hz, H-4), 6.20 (1H, dd,J = 1.2, 8.8 Hz, H-3), 6.06 (1H, dd, J = 1.2, 6.8 Hz, H-5), 3.97 (1H, d, J =16.0 Hz, H-10β), 3.48 (1H, dd, J = 6.4, 16.0 Hz, H-10α), 2.99 (1H, d, J = 2.4Hz, H-7), 2.57 (1H, d, J = 8.8 Hz, H-17α), 2.28 (1H, d, J = 8.8 Hz, H-17β),1.95 (1H, m, H-15α), 1.88 (1H, m, H-11), 1.82 (1H, m, H-12α), 1.78 (1H, m, H-15β), 1.78 (1H, m, H-8α), 1.74 (1H, m, H-13α), 1.68 (1H, m, H-12β), 1.45 (1H,m, H-14α), 1.41 (1H, m, H-14β), 1.29 (1H, m, H-8β), 1.23 (1H, m, H-13β). 13 C-NMR (100 MHz, DMSO-d6): δ C 162.6 (C-2), 152.7 (C-6), 139.3 (C-4), 115.8 (C-3), 104.2 (C-5), 65.8 (C-11), 63.5 (C-17), 55.9 (C-15), 44.8 (C-10), 34.8 (C-7), 32.6 (C-9), 29.8 (C-12), 27.4 (C-8), 25.6 (C-14), 24.5 (C-13).

[0010] The invention has the following beneficial technical effects: High-speed countercurrent chromatography (HSCCC) is a type of liquid-liquid partition chromatography that avoids irreversible adsorption of samples on a solid support, offering high recovery, easy scale-up, good separation capability, rapid separation, and low solvent consumption. Therefore, this method has become an effective approach for the preparation, separation, and purification of various natural products. pH zone countercurrent chromatography (PZRCCC), developed based on HSCCC, can be used to separate ionic compounds, including organic acids and alkaloids. Based on pKa values ​​and hydrophobicity, the chromatographic peaks are rectangular, indicating high sample concentrations. Compared to traditional CCC, PZRCCC increases sample loading by approximately 10 times, resulting in higher peak concentrations and fewer impurities. In this study, PZRCCC was used to separate alkaloids from *Pterocarya stenoptera* seeds. Six quinocyridine alkaloids were successfully separated by nuclear magnetic resonance and electrospray ionization mass spectrometry (ESI-MS), and their structures were characterized. Attached Figure Description

[0011] Figure 1 This is a high-performance liquid chromatogram of alkaloid compounds in *Phyllostachys edulis*. Figure 2 Six compounds were separated by countercurrent chromatography in the pH zone; Figure 3 It is a two-dimensional (2D) diagram of the docking between the compound and the receptor protein molecule. Detailed Implementation

[0012] The invention will be further illustrated below with specific examples.

[0013] Example 1 The countercurrent chromatographic separation method for alkaloids in the pH zone of *Phyllostachys edulis* was performed according to the following steps: The seeds of the pea plant were crushed to 50 mesh and extracted three times by reflux with ethanol for 2 hours each time. The extracts were combined, filtered, concentrated under reduced pressure, and evaporated until no alcohol odor was detected. Water was added, and the pH was adjusted to 2.0 with HCl aqueous solution. Petroleum ether was added, and the pH was adjusted to 9.5 with ammonia solution. Chloroform was added and extracted five times to obtain chloroform extract. The chloroform extract was evaporated to dryness under reduced pressure to obtain crude alkaloids. The crude alkaloids were separated using the first-to-last elution mode of PZRCCC, and then concentrated under reduced pressure to obtain alkaloid compounds. The mass ratio of the pea seeds, ethanol, water, petroleum ether, and chloroform is 1:12:0.5:12:0.8:2. The molecular structure of the alkaloid compound is as follows:

[0014] The ethanol is an aqueous solution with a mass fraction of 95%; the HCl aqueous solution has a mass fraction of 1% HCl; and the ammonia solution is an aqueous solution with a volume ratio of 1:1.

[0015] The first and last elution mode of PZRCCC uses solvent to separate the crude alkaloids. The steps are as follows: The upper and lower phases are added to the crude alkaloid to prepare a sample solution; Using the upper phase as the stationary phase and the lower phase as the mobile phase, the column was degassed by sonication. The cold hydrazine was turned on, and the stationary phase was pumped into the column at a temperature of 25℃ and a flow rate of 30 mL / min. After the column was completely filled, the rotation speed was adjusted to 800 rpm to achieve hydrodynamic equilibrium. The sample solution was injected into the chromatographic column through the injection valve, and the absorbance of the UV detector was continuously monitored at 280 nm. The chromatographic data was recorded by turning on the recorder. The eluent is continuously collected using an automatic collector, and each peak fraction is collected into a reagent bottle according to the chromatogram. After the desired peak is eluted, stop the rotation speed and elution. Use a vacuum pump to collect the components in the column in a graduated cylinder. Concentrate the collected fraction under reduced pressure to obtain alkaloid compounds. The mass-to-volume ratio of crude alkaloids, upper phase, and lower phase is 1:10:10 (g / mL / mL). The solvent is a composition of chloroform, methanol, and water in a volume ratio of 4:3:3, and its preparation method is as follows: Place chloroform, methanol and water in a separatory funnel, shake thoroughly, and let stand for 30 minutes to obtain the solvent; The upper layer of the solvent is the upper phase, and the lower layer of the solvent is the lower phase.

[0016] The method also includes high-performance liquid chromatography (HPLC) analysis of the alkaloid compounds, with the following analytical conditions: Chromatographic column: Waters Symmetry (250 × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) and 0.05% TEA aqueous solution (B); gradient elution conditions: 0–7 min, 90–70% A, 10–30% B; 7–12 min, 70–65% A, 30–35% B; 12–13 min, 65–90% A, 35–10% B; and 13–20 min, 90% A, 10% B; flow rate: 1 mL / min; column temperature: 25 °C; detection wavelength: 280 nm; injection volume: 10 μL.

[0017] It also includes the structural identification of terpenoid compounds, the method of which is as follows: Through ESI-MS, 1 H and 13The separated compounds were identified by C10 NMR spectroscopy. ESI-MS (positive and negative ion modes) mass spectrometry analysis was performed on a Xevo TQ-XS spectrometer, and NMR spectra were recorded on a Bruker AV-400 spectrometer. DMSO- d 6 was used as a solvent, and tetramethylsilane (TMS) was used as an internal standard. Chemical shift ( δ The value is expressed in ppm, and the coupling constant ( J (in Hz)

[0018] The application of pH-zone countercurrent chromatography to separate alkaloids from *Phragmites australis* for the treatment of inflammation and reduction of central nervous system damage.

[0019] Molecular docking of compounds Based on literature reports, the 3D structures of the compounds were downloaded from the PubChem database. Two proteins were selected: COX-2 (PDB ID: 5F1A), an inflammation-related protein, and MMP-2 (PDB ID: 1QIB), a neuroprotective protein. The 3D structures of the target proteins were downloaded from the PDB (http: / / www.rcsb.org) database for docking. Affinity was used as the evaluation index; the higher the absolute value of the affinity, the more stable the binding between the ligand and the receptor, and the higher the probability of interaction. Generally, a binding energy less than 0 indicates that spontaneous binding is possible, with a minimum binding energy ≤ -5.0 kJ·mol⁻¹. -1 This indicates that the molecules docked well with the proteins, and the minimum binding energies of the six compounds to the two receptor proteins were all less than -5.0 kJ·mol⁻¹. -1 Both have strong binding ability and may exert corresponding effects through these two targets, making them potential active compounds.

[0020] Table 1. Docking results of compounds with receptor proteins Table 1

[0021] From a two-dimensional planar diagram of compound-protein interactions ( Figure 3 It can be seen that the interaction force is mainly hydrogen bonding, suggesting that hydrogen bonding may enhance the binding ability of the compound to the receptor, thereby increasing the activity of the compound. All compounds showed good affinity for the target protein. In binding to COX-2, hydrogen bonding was the main interaction, primarily forming hydrogen bonds with GlnA461 and CysA47. Compound 6 showed the strongest binding affinity to the anti-inflammatory protein COX-2, with a minimum binding energy of -8.97 kcal / mol. In binding to MMP-2, hydrogen bonding was the main interaction, primarily forming hydrogen bonds with Thr229. Compound 3 showed the strongest binding affinity to MMP-2, with a minimum binding energy of -11.17 kcal / mol.

[0022] Inhibiting COX-2 synthesis is an effective method for treating inflammation. MMP-2 is considered an important indicator of central nervous system diseases, and docking results suggest that the compounds may serve as potential drugs for treating inflammation and reducing central nervous system damage. Molecular docking, as a computer-aided screening technique, provides a rapid and efficient method for screening target compounds with specific structures and activities.

[0023] in conclusion Alkaloids from *Pterocarya stenoptera* seeds were separated using pH-zone countercurrent chromatography. The optimal solvent system was chloroform-methanol-water (4:3:3, v / v). 40 mM HCl was added to the stationary phase, and 10 mM TEA was added to the mobile phase, yielding six compounds. Molecular docking analysis showed that all six compounds exhibited good affinity for two protein receptors related to anti-inflammation and neuroprotection, primarily through hydrogen bonding interactions, indicating potential activity. This study provides a reference for the isolation of chemical components from *Pterocarya stenoptera* seeds and the further discovery of active compounds.

Claims

1. A countercurrent chromatographic separation method for alkaloid compounds in *Phaseolus vulgaris* in the pH zone, characterized in that, Follow these steps: The seeds of the pea plant were crushed to 40-60 mesh and extracted three times by reflux with ethanol for 2 hours each time. The extracts were combined, filtered, concentrated under reduced pressure, and evaporated until no alcohol odor was detected. Water was added, and the pH was adjusted to 2.0 with HCl aqueous solution. Petroleum ether was added, and the pH was adjusted to 9.5 with ammonia solution. Chloroform was added and extracted five times to obtain a chloroform extract. The chloroform extract was evaporated to dryness under reduced pressure to obtain crude alkaloids. The crude alkaloids were separated using the first-to-last elution mode of PZRCCC, and then concentrated under reduced pressure to obtain alkaloid compounds. The mass ratio of the pea seeds, ethanol, water, petroleum ether, and chloroform is 1:10-15:0.25-1:10-15:0.75-1.5:1-3. The molecular structure of the alkaloid compound is as follows: ； The first and last elution mode of PZRCCC uses solvent to separate the crude alkaloids. The steps are as follows: The upper and lower phases are added to the crude alkaloid to prepare a sample solution; Using the upper phase as the stationary phase and the lower phase as the mobile phase, the column was degassed by sonication. The cold hydrazine was turned on, and the stationary phase was pumped into the column at a temperature of 25°C and a flow rate of 30 mL / min. After the column was completely filled, the rotation speed was adjusted to 800 rpm to achieve hydrodynamic equilibrium. The sample solution was injected into the chromatographic column through the injection valve, and the absorbance of the UV detector was continuously monitored at 280 nm. The chromatographic data was recorded by turning on the recorder. The eluent is continuously collected using an automatic collector, and each peak fraction is collected into a reagent bottle according to the chromatogram. After the desired peak is eluted, stop the rotation speed and elution. Use a vacuum pump to collect the components in the column in a graduated cylinder. Concentrate the collected fraction under reduced pressure to obtain alkaloid compounds. The mass-to-volume ratio of the crude alkaloid, the upper phase, and the lower phase was 1 g: 10 mL: 10 mL. The solvent is a composition of chloroform, methanol, and water in a volume ratio of 4:3:3, and its preparation method is as follows: Place chloroform, methanol and water in a separatory funnel, shake thoroughly, and let stand for 30 minutes to obtain the solvent; The upper layer of the solvent is the upper phase, and the lower layer of the solvent is the lower phase.

2. The countercurrent chromatographic separation method for alkaloids in *Phaseolus vulgaris* in the pH zone as described in claim 1, characterized in that, The ethanol is an aqueous solution with a mass fraction of 95%; the HCl aqueous solution has a mass fraction of 1% HCl; and the ammonia solution is an aqueous solution with a volume ratio of 1:

1.

3. The countercurrent chromatographic separation method for alkaloids in *Phragmites australis* in the pH zone as described in claim 1, characterized in that, The method also includes high-performance liquid chromatography (HPLC) analysis of the alkaloid compounds, with the following analytical conditions: Chromatographic column: Waters Symmetry 250×4.6 mm, 5 μm; mobile phase: acetonitrile A and 0.05% TEA aqueous solution B; gradient elution conditions: 0–7 min, 90–70% A, 10–30% B; 7–12 min, 70–65% A, 30–35% B; 12–13 min, 65–90% A, 35–10% B; and 13–20 min, 90% A, 10% B; flow rate: 1 mL / min; column temperature: 25℃; detection wavelength: 280 nm; injection volume: 10 μL.

Images