A piperidine alkaloid compound in portulaca oleracea and extraction and separation method and use thereof

Abstract

This invention belongs to the field of traditional Chinese medicine extraction and separation technology, and particularly relates to a piperidine alkaloid compound extracted, separated, and identified from purslane, and its extraction and separation method. The novel alkaloid compound has the molecular formula C0. 10 H 14 N6O3, chemical name 2-(6-amino-9- H (-purin-9-yl)piperidine-3,4,5-triol. A method for extracting and separating the above-mentioned novel alkaloid compound is also provided, comprising sequential water decoction extraction, macroporous resin column chromatography, ODS column chromatography, dextran gel column chromatography (Sephadex LH-20), and high-performance liquid chromatography for separation, purification, and preparation. Its structure was determined to be a novel alkaloid compound by mass spectrometry, proton NMR, carbon NMR, and two-dimensional nuclear magnetic resonance spectroscopy. The novel alkaloid compound possesses anti-inflammatory, anticholinesterase, and antioxidant activities. The novel alkaloid compound and its salts or derivatives of this invention can be used as raw materials for drug development and pharmacological activity studies.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction and separation technology, and in particular relates to a piperidine alkaloid compound in purslane and its extraction and separation method and uses. Background Technology

[0002] Purslane, belonging to the Portulacaceae family and Portulacales order in phytochemical taxonomy, is an annual herb widely distributed in tropical and subtropical regions worldwide. It is named "purslane" because its leaves resemble horse teeth; and it is also known as "longevity vegetable" due to its cold resistance, strong vitality, and long lifespan.

[0003] Purslane contains a variety of chemical components, mainly including alkaloids, flavonoids, organic acids, terpenes, coumarins, polysaccharides, amino acids, and minerals. Among these, purslane contains a relatively large number of alkaloid compounds, such as uracil, adenine, norepinephrine, dopamine, adenosine, N,N-dicyclohexylurea, purslane amide, allantoin, N-trans-feruloyltyramine, Iseluxine, Oleracrylimide A, Oleracrylimide B, Oleracrylimide E, and Oleraisoquinoline A. The chemical components in purslane are closely related to its diverse pharmacological effects. Modern pharmacological studies have shown that purslane possesses antioxidant, neuroprotective, anti-inflammatory, antibacterial, antitumor, hypoglycemic, hepatoprotective, and immune-enhancing effects. Purslane contains a wide variety of chemical components with diverse pharmacological activities. The development and isolation of compounds in purslane can provide a foundation for in-depth research on purslane. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a piperidine alkaloid compound extracted from purslane. Studies have shown that the piperidine alkaloid compound of this invention possesses anti-inflammatory, anticholinesterase, and antioxidant activities. Furthermore, this invention provides a simple, rapid, environmentally friendly, and highly pure extraction and separation method for the compound.

[0005] To achieve the above-mentioned objectives of this invention, this invention provides a piperidine alkaloid compound with the molecular formula C0. 10 H 14 N6O3, chemically named 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol, has the following chemical structural formula:

[0006]

[0007] To achieve the above-mentioned objectives of this invention, this invention also provides a method for extracting and separating a piperidine alkaloid compound from purslane, specifically comprising the following steps:

[0008] Step 1: Take dried purslane, decoct it in water to extract the extract, concentrate the extract, and let it cool to room temperature to obtain the medicinal liquid for later use.

[0009] Step 2: Load the concentrate from Step 1 onto macroporous resin, elute with water and ethanol of different concentrations, and recover 30% of the ethanol fraction under reduced pressure to obtain a concentrate for later use.

[0010] Step 3: Separate the concentrate from Step 2 using an ODS column (Octadecylsilyl, octadecylsilane bonded silica gel packing material), using a methanol-water gradient elution, and perform thin-layer chromatography detection and color development. Combine the 30% methanol elution fraction and evaporate to dryness to obtain the concentrate for later use.

[0011] Step 4: The concentrate obtained in Step 3 is further separated by ODS column chromatography using methanol-water gradient elution. The concentrate is then detected by thin-layer chromatography and colorimetric analysis. The 30% methanol eluent is combined and evaporated to dryness to obtain the concentrate for later use.

[0012] Step 5: The product obtained in step 4 is further separated by pretreated dextran gel column (Sephadex LH-20) chromatography, eluted isocratically with 10% methanol to obtain several elution fractions, detected by thin-layer chromatography, colorimetrically developed, and the colored elution fractions are combined. The combined elution fractions are concentrated to dryness under reduced pressure for later use.

[0013] Step 6: The concentrate obtained in Step 5 is separated by HPLC (high performance liquid chromatography) and isocratic elution is performed using methanol-0.1% formic acid (volume percentage) as the mobile phase to finally obtain the piperidine alkaloid compound of the present invention.

[0014] Furthermore, in step 1, the water decoction is performed twice, each time for 2 hours, with the amount of water being 8 to 16 times that of the medicinal material.

[0015] Furthermore, in step 2, the macroporous resin is AB-8 macroporous resin, and the volume ratio of ethanol to water is 30:70, 50:50, 70:30, and 100:0 gradient elution.

[0016] Furthermore, in step 3, the volume ratio of methanol to water is eluted in gradients of 10:90, 30:70, 50:50, 70:30, and 100:0; the ODS particle size is 40–70 μm.

[0017] Furthermore, in step 4, the volume ratio of methanol to water is eluted in gradients of 10:90, 30:70, 50:50, 70:30, and 100:0; the ODS particle size is 40–70 μm.

[0018] Furthermore, in step 5, the methanol elution procedure is isocratic elution with 10% methanol.

[0019] Furthermore, the pretreatment process of the ODS and dextran gel is as follows: soaking in methanol for 24 hours, loading onto a column, washing with methanol until no turbidity is observed when dropped into water, and then equilibrating with the initial mobile phase.

[0020] Furthermore, in step 6, the volume ratio of methanol to 0.1% formic acid is 10:90, and the retention time of the compounds is 3.506 min.

[0021] The present invention also provides the application of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol isolated from purslane as described above in the preparation of anti-inflammatory drugs, anticholinesterase drugs and antioxidant drugs.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0023] The isolation and pharmacological activity studies of the compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol from purslane described in this invention have not been reported in other plants. This invention provides a novel alkaloid compound derived from purslane and a method for its extraction and separation. The method involves sequential extraction by water decoction, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20 dextran gel column chromatography, and high-performance liquid chromatography for separation, purification, and preparation. A novel alkaloid compound was successfully extracted and separated. This method involves only six steps, is simple and rapid, uses water for extraction, and is environmentally friendly. The compound obtained by this method has a high purity, greater than 90%. Furthermore, studies have shown that the above compound possesses anti-inflammatory, anticholinesterase, and antioxidant activities. Therefore, the novel compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol, its salts, and derivatives can be used as raw materials for drug development and pharmacological activity studies. Attached Figure Description

[0024] Figure 1 The novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol of this invention 1 H-NMR spectrum.

[0025] Figure 2 The novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol of this invention 13 C-NMR spectrum.

[0026] Figure 3 The DEPT spectrum of the novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol is shown.

[0027] Figure 4 The HSQC spectrum of the novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol is shown.

[0028] Figure 5 The HMBC spectrum of the novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol is shown.

[0029] Figure 6 The COESY spectrum of the novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol is shown.

[0030] Figure 7 The ROESY spectrum of the novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol is shown.

[0031] Figure 8 The high-resolution mass spectrum of the novel alkaloid compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol is shown. Detailed Implementation

[0032] The following embodiments will help to understand the present invention, but these embodiments are only for illustrative purposes and the present invention is not limited thereto. The operating methods in the embodiments are all conventional operating methods in this technical field.

[0033] Example 1: 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol.

[0034] This invention provides a piperidine alkaloid compound with the molecular formula C 10 H 14 N6O3, chemically named 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol, has the following chemical structural formula:

[0035]

[0036] Table 1 shows the NMR data of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol. The solvent used for NMR was DMSO-d6.

[0037] Table 1. NMR data of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol of this invention.

[0038]

[0039] 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol: A yellowish-brown oily substance, readily soluble in water. When spotted onto a silica gel thin-layer plate and sprayed with dilute potassium bismuth iodide solution, the spots turn orange-yellow, indicating that this compound is an alkaloid. 1 H-NMR, 13 Based on C10NMR and HR-ESI-TOF-MS signals, the possible molecular formula of this compound is C10. 10 H 14 N6O3, with an unsaturation degree of 7. HR-ESI-TOF-MS provides the m / z [MH]. - The quasi-molecular ion peak is at 265.1050, calculated value (C). 10 H 13 N6O3 - (265.1054). According to 13 The C10 NMR and DEPT spectra show that the compound has 10 carbon resonance peaks, including one methylene (δ) peak. C 61.26), 6 methines (δ C 88.30, 73.56, 70.54, 85.71, 151.97, 140.49), three quaternary carbon resonance peaks (δ C 118.90, 148.32, 155.14). The NMR data of the compounds are listed in Table 1. 1 The 1H NMR spectrum shows resonances of two separate heteroaryl methines (δ¹⁸ methyl groups). H According to 8.11, 8.24) 1 In H-NMR and HMBC spectra, H-2'(δ) H 8.11) respectively with C-4'(δ C 148.32) and C-5'(δ C 118.90) shows a strong correlation; H-8'(δ) H 8.24) shows strong correlations with C-4' and C-5' respectively, and according to 13The low-field chemical shifts of C-2', C-4', and C-8' in the C-NMR spectrum indicate their connection to N atoms; the even lower field of C-6' suggests its connection to -NH2; and the higher field of C-5' relative to the three C atoms indicates its connection to a C atom at the other end, thus confirming the presence of a purine structure. 1 H-NMR spectrum and HMBC spectrum, H-4(δ) H 4.35) and C-2(δ C 88.30), C-6 (δ) C 61.36) related, H-3(δ) H 4.69) and C-5(δ) C 85.71) related, H-6(δ) C 3.76, 3.83) and C-2(δ C The correlation with 88.30 indicates the existence of a six-membered ring. According to... 1 H- 1 The correlations between H-2 and H-3, H-3 and H-4, H-4 and H-5, and H-5 and H-6 in the HCOSY spectrum corroborate the above-mentioned six-membered ring. 13 The low-field chemical shifts of C-3, C-4, and C-5 in the C-NMR spectrum indicate their connection to hydroxyl groups. The low-field chemical shift of C-6 suggests a possible connection to a nitrogen atom, implying the presence of a piperidine structure. Furthermore, the strong correlation between H-2 and C-4' and C-8' in the HMBC spectrum indicates that the piperidine ring and purine ring are connected via a C-2-N-9' link. Based on this information, combined with the molecular weight from the HR-ESI-TOF-MS spectrum, the structure of this new alkaloid can be confirmed.

[0040] This invention also provides a method for the extraction and separation of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol, the specific steps of which are as follows:

[0041] Step 1: Weigh 250 kg of dried purslane and extract it by decoction with water. The amount of water used is 10 times that of the raw material. Extract twice, 2 hours each time. Combine the extracts, heat and concentrate, and cool to room temperature to obtain the medicinal liquid for later use.

[0042] Step 2: After evaporating the drug solution obtained in Step 1 to dryness, separate it by AB-8 macroporous resin column chromatography. Use ethanol-water gradient elution (30:70, 50:50, 70:30 and 100:0, v:v) to collect the 30% ethanol fraction, and recover it to the extract under reduced pressure to obtain the concentrate for later use.

[0043] Step 3: Separate the concentrate from Step 2 using a pretreated ODS column with a packing particle size of 40–70 μm. Elute with a methanol-water gradient (10:0, 30:70, 50:50, 70:30, and 100:0, v:v). Detect the elution by thin-layer chromatography and perform color development. Combine the elution fractions with 30% methanol, concentrate under reduced pressure to dryness, and set aside for later use.

[0044] Step 4: The product obtained in Step 3 is further separated by pretreated ODS column chromatography. The packing material has a particle size of 40–70 μm. Elution is performed using a methanol-water gradient (10:0, 30:70, 50:50, 70:30, and 100:0, v / v). Thin-layer chromatography is used for detection and color development. The 30% methanol elution fraction is combined and concentrated to dryness under reduced pressure for later use. The ODS pretreatment process involves soaking in methanol for 24 hours, loading onto the column, washing with methanol until no turbidity is observed when added to water, and then equilibrating with the initial mobile phase.

[0045] Step 5: The product obtained in Step 4 is further separated by pretreated dextran gel column chromatography (Sephadex LH-20). Elution is performed isocratically with 10% methanol to obtain several eluting fractions. These fractions are then detected by thin-layer chromatography, and the resulting eluting fractions are combined. The combined eluting fractions are then concentrated to dryness under reduced pressure for later use. The pretreatment process for the dextran gel involves soaking it in methanol for 24 hours, loading it onto the column, washing with methanol until no turbidity is observed when added to water, and then equilibrating with the initial mobile phase.

[0046] Step 6: The fraction obtained in Step 5 was separated and prepared by HPLC. Methanol and 0.1% formic acid in a volume ratio of 10:90 were used as the mobile phase, and the detection wavelengths were 210 and 254 nm. The new alkaloid compound of the present invention was obtained by separation and preparation, and the purity was determined to be 96% by normalization method.

[0047] Example 2: Anti-inflammatory effect of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol of the present invention.

[0048] 1. Main materials.

[0049] 1.1 Drugs and reagents: The novel alkaloids used in the experiment were prepared by the above method with a purity of 96%. DMEM high-glucose culture medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Sijiqing Pharmaceutical Co., Ltd.); LPS (Sigma, USA); ELISA kits for IL-1β, TNF-α and the inflammatory mediator PGE2 (Cayman, USA); cell lysis buffer.

[0050] 1.2 Cell line: RAW264.7 macrophages (ATCC Cell Bank, USA).

[0051] 1.3 Grouping: The group was divided into a control group, an LPS group, and an experimental group.

[0052] 2. Experimental methods.

[0053] 2.1 Cell culture: Add 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) to DMEM high glucose medium and store at 4°C.

[0054] 2.2 CCK-8 assay for cell viability: RAW264.7 macrophages in the logarithmic growth phase from the three groups described above were seeded in 96-well culture plates at a cell density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured overnight at 37°C and 5% CO2 at 100 μL / mL. Different concentrations of the compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol (5 μM–50 μM) were added to the experimental group. After incubation for 1 h, 1 μg / mL LPS was added to both the LPS group and the experimental group. A zeroing group (culture medium containing DMSO) was also set up, with three replicates per group to investigate the effect of drug addition on cells. After 24 h of cell culture, 10 μL of CCK-8 was added to each well, and the cells were incubated for another 4 h at 37°C and 5% CO2. The absorbance of each well was measured at 450 nm using a microplate reader.

[0055] 2.3 ELISA assay for inflammatory factors IL-1β, TNF-α, and inflammatory mediator PGE2: Log-growing RAW264.7 macrophages were seeded in 24-well culture plates at a cell density of 1×10⁻⁶ cells / well. 5 The concentration of LPS was 1 mL / mL, and the mixture was incubated overnight at 37°C with 5% CO2. For the experimental group, 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol (1 μM–20 μM) of this invention was added and incubated for 1 h. Then, LPS (final concentration 1 μg / mL) was added to each well, and the mixture was incubated for a total of 24 h. Each treatment was repeated in triplicate. The levels of IL-1β, TNF-α, and the inflammatory mediator PGE2 were determined by ELISA.

[0056] 3. Experimental results.

[0057] Experimental results show that the compound of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7; it can effectively inhibit the secretion of excessive inflammatory cytokines IL-1β, TNF-α and inflammatory mediator PGE2 produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0058] The results of the relative cell viability experiments of the invented compounds are shown in Table 2.

[0059] Table 2 shows the effect of the compounds of this invention on the relative survival rate of RAW264.7 macrophages.

[0060]

[0061] The results of ELISA measurements of inflammatory factors IL-1β, TNF-α, and inflammatory mediator PGE2 are shown in Table 3.

[0062] Table 3. Effects of the compounds of the present invention on the levels of IL-1β, TNF-α and the inflammatory mediator PGE2 secreted by LPS-induced RAW264.7 cells (mean ± standard deviation, n = 3).

[0063]

[0064]

[0065] Note: * P<0.05 compared with the control group, # P<0.05 compared with the LPS group.

[0066] Example 3: Anticholinesterase activity of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol of the present invention.

[0067] 1. Main materials.

[0068] 1.1 Drugs and Reagents: The novel alkaloids used in the experiment were prepared by the above method with a purity of 96%. Physostigmine (Shanghai Hanxiang Biotechnology Co., Ltd.), acetylcholine iodiosulfate (ATCI) and acetylcholinesterase (AChE) (Dalian Meilun Biotechnology Co., Ltd.), dithiodinitrobanoic acid (DTNB) (Shanghai Jinshui Biotechnology Co., Ltd.), disodium hydrogen phosphate and sodium dihydrogen phosphate (Shanghai Guoyao Holding Reagent Co., Ltd.).

[0069] 1.2 Experimental instruments and equipment: HBS-1096A96-well microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.), 0.001 g balance (METTLER, Switzerland), HH-4 digital display constant temperature water bath (Jiangsu Jintan Ronghua Instrument Manufacturing Co., Ltd.).

[0070] 2. Experimental methods.

[0071] In this experiment, the anticholinesterase activity of each compound was determined using a modified Ellman method. 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol and physostigmine were accurately weighed and prepared into five series of sample solutions with methanol concentrations of 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM. The specific procedure is as follows: Add 140 μL of phosphate buffer solution (0.1 M, pH = 8.0, containing 0.1 mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate), 20 μL of sample solution, and 15 μL of AChE (0.2 U / mL) to a 96-well microplate. Incubate at 37°C for 10 minutes, then add 10 μL of ATCI (4 mmol / L) and 10 μL of DTNB (15 mmol / L). Incubate at 37°C for 20 minutes. Place the 96-well plate in a microplate reader and measure the absorbance of each sample group at a wavelength of 405 nm. Methanol was used as the blank group instead of the sample solution, and physostigmine was used as the positive control group. The cholinesterase inhibition rate of each compound was calculated using the following formula (A represents absorbance):

[0072] Inhibition rate (%) = (A 空白 -A 样品 ) / A 空白 ×100%

[0073] 3. Experimental results.

[0074] Experimental results show that the novel alkaloid compound of this invention exhibits certain anticholinesterase activity, and the inhibitory effect on cholinesterase increases with increasing compound concentration, showing a dose-dependent trend.

[0075] The anticholinesterase activity of the novel alkaloids of this invention is shown in Table 4.

[0076] Table 4 shows the anticholinesterase activity of the compounds of this invention.

[0077]

[0078] Example 4: Antioxidant activity of 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol of the present invention.

[0079] 1. Main materials.

[0080] 1.1 Reagents and Chemicals: The novel alkaloids used in the experiment were prepared by the above method with a purity of 96%. 1,1-Diphenyl-2-picrylhydrazine radical (DPPH) (Sigma-Aldrich, USA), butylated hydroxyanisole (BHA) (Shanghai Xiangrui Biotechnology Co., Ltd.), and methanol (chromatographic grade, Tianjin Kaixin Chemical Industry Co., Ltd.).

[0081] 1.2 Experimental instruments and equipment: Hitachi UV-3010 UV-Vis spectrophotometer (Hitachi, Japan), and a 0.0001 g balance (METTLER, Switzerland).

[0082] 2. Experimental methods.

[0083] This experiment used the DPPH free radical scavenging method to determine the antioxidant activity of various compounds. Accurately weighed 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol and BHA were prepared into five series of sample solutions with concentrations of 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM using methanol. Furthermore, the DPPH solution was prepared fresh each time; an appropriate amount of DPPH was accurately weighed and prepared into an 80 μM solution using methanol. All procedures were performed in the dark. The specific procedures were as follows: 1 mL of sample solution was thoroughly mixed with 1 mL of DPPH solution, and the mixture was placed at room temperature in the dark for 10 min. The absorbance of the sample was measured after setting the UV spectrophotometer to 517 nm. Methanol was used as a blank group, BHA as a positive control group, and a mixture of 1 mL of methanol and 1 mL of sample solution was used as a control group. The DPPH scavenging rate of each compound was calculated using the following formula (A represents absorbance):

[0084] DPPH clearance rate (%) = (1-(A) 样品 -A 对照 ) / A 空白 )×100%

[0085] 3. Experimental results.

[0086] Experimental results show that the novel alkaloid compound of this invention exhibits certain antioxidant activity, and the antioxidant effect increases with increasing compound concentration, showing a dose-dependent trend.

[0087] The antioxidant activity of the novel alkaloid compounds of this invention is shown in Table 5.

[0088] Table 5 shows the antioxidant activity of the compounds of this invention.

[0089]

[0090] In summary, this invention provides compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol and its extraction and separation method. The method involves sequential extraction with water decoction, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20 chromatography, and high-performance liquid chromatography (HPLC) for separation, purification, and preparation. A novel alkaloid compound was successfully extracted and separated. This method is simple, rapid, and environmentally friendly, and the compound obtained by this method has high purity. Since the obtained compound is extracted from the commonly used traditional Chinese medicine purslane, it possesses anti-inflammatory, anticholinesterase, and antioxidant activities. Therefore, the novel compound 2-(6-amino-9H-purin-9-yl)piperidine-3,4,5-triol, its salts, and derivatives of this invention can provide insights for drug development and have broad development prospects.

Claims

1. A piperidine alkaloid compound isolated from purslane, characterized in that, The molecular formula is: C 10 H 14 N6O3, chemical structural formula is: 。 2. A method for extracting and separating piperidine alkaloids from purslane medicinal material as described in claim 1, characterized in that, Specifically, the steps include the following: Step 1: Take dried purslane, decoct it in water to extract the extract, concentrate the extract, and let it cool to room temperature to obtain the medicinal liquid for later use; Step 2: Load the concentrate from Step 1 onto a macroporous resin, elute with water and ethanol of different concentrations, and recover 30% of the ethanol fraction under reduced pressure to obtain a concentrate for later use; the macroporous resin is AB-8 macroporous resin, and the volume ratio of ethanol to water is 30:70, 50:50, 70:30 and 100:0 gradient elution. Step 3: Separate the concentrate from Step 2 using an ODS column with methanol-water gradient elution. Detect the concentrate using thin-layer chromatography and perform color development. Combine the 30% methanol eluent and evaporate to dryness to obtain the concentrate for later use. The methanol-water volume ratios were 10:90, 30:70, 50:50, 70:30, and 100:0 gradient elution. The ODS particle size was 40–70 μm. Step 4: The concentrate obtained in Step 3 is further separated by ODS column chromatography using a methanol-water gradient elution. Thin-layer chromatography is then performed for detection and color development. The 30% methanol eluent fraction is combined and evaporated to dryness to obtain the concentrate for later use. The methanol-water volume ratios are 10:90, 30:70, 50:50, 70:30, and 100:0 gradient elution. The ODS particle size is 40–70 μm. Step 5: The product obtained in step 4 is further separated by pretreated dextran gel column chromatography, eluted isocratically with 10% methanol to obtain several elution fractions, detected by thin-layer chromatography, colorimetrically developed, and the colorimetric elution fractions are combined. The combined elution fractions are concentrated to dryness under reduced pressure for later use. Step 6: The concentrate obtained in Step 5 was separated by HPLC and isocratic elution was performed using methanol-0.1% formic acid as the mobile phase to finally obtain the alkaloid compound.

3. The extraction and separation method according to claim 2, characterized in that, In step 1, the herbs are decocted and extracted twice, each time for 2 hours, with the amount of water being 8 to 16 times the amount of the herbs.

4. The extraction and separation method according to claim 2, characterized in that, The pretreatment process of the ODS and dextran gel is as follows: soak in methanol for 24 hours, load onto a column, wash with methanol until there is no turbidity when dropped into water, and then equilibrate with the initial mobile phase.

5. The extraction and separation method according to claim 2, characterized in that, In step 6, the volume ratio of methanol to 0.1% formic acid is 10:90, and the retention time of the compound is 3.506 min.

6. The use of the piperidine alkaloid compound as described in claim 1, characterized in that, The compound is used to prepare anti-inflammatory drugs, anticholinesterase drugs, and antioxidant drugs.

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