A New Diimine Alkaloid Compound in Portulaca oleracea L., and Its Extraction, Isolation Method and Application

Abstract

The present invention belongs to the technical field of traditional Chinese medicine extraction and separation, and particularly relates to a diimine alkaloid compound in purslane, and its extraction, separation method and application. The diimine alkaloid compound has a molecular formula of C 12 H 10 N2O6, and is named (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9 H -1,3,6-trioxonin-9-one. The present invention also provides a method for extracting and separating the above new compound. By successively using ethanol reflux extraction, ODS medium-pressure column, Sephadex LH-20 column chromatography and UPLC preparation, a diimine alkaloid compound is successfully extracted and separated. Its structure is determined by methods such as mass spectrometry, carbon spectrum, hydrogen spectrum and two-dimensional nuclear magnetic spectroscopy. This compound has anti-inflammatory and anti-cholinesterase effects. The compound of the present invention and its salts or derivatives can be used as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research, and are used for preparing anti-inflammatory and anti-cholinesterase drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine extraction and separation, and particularly relates to a new diimine alkaloid compound in Portulaca oleracea L., and its extraction, separation method and application. Background Art

[0002] Portulaca oleracea L., also known as Portulaca oleracea, Longevity Vegetable, and Ant Vegetable, is an annual herbaceous plant of the genus Portulaca in the Portulacaceae family. Portulaca oleracea prefers acidic soil, is drought and waterlogging tolerant, and is distributed in various regions of China. Portulaca oleracea is included in the Pharmacopoeia of the People's Republic of China (2020 Edition), and has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and relieving dysentery. It is mainly used for treating heat-toxic blood dysentery, carbuncles and sores, eczema, erysipelas, snake and insect bites, hematochezia, hemorrhoids, metrorrhagia and other symptoms.

[0003] Modern pharmacological studies have shown that Portulaca oleracea has anti-inflammatory, antibacterial, antiviral, hypoglycemic, hypotensive, hypolipidemic, antioxidant, anticancer, antitumor, relaxing skeletal and smooth muscles, and regulating immune function. The main chemical components of Portulaca oleracea include flavonoids, coumarins, terpenoids, steroids, alkaloids, amino acids, lignans, volatile oils, polysaccharides, various pigments and minerals, etc., which provide a material basis for its diverse pharmacological effects. Among them, imine alkaloids are a class of traditional Chinese medicine chemical components in Portulaca oleracea. In this paper, the isolation and identification of Portulaca oleracea are carried out, and the anti-inflammatory and anti-cholinesterase activities of its chemical components are evaluated, providing a technical route and theoretical basis for the study of the effective substances of Portulaca oleracea, clinical application and new drug research and development.

[0004] At present, most of the chemical components isolated from Portulaca oleracea are known, and the structural novelty is relatively low. Therefore, the development and separation of new compounds from Portulaca oleracea are urgently needed. Summary of the Invention

[0005] In view of the above problems, the present invention provides a diimine alkaloid compound extracted and separated from Portulaca oleracea. Through research, it is found that the new compound of the present invention has anti-inflammatory and anti-cholinesterase effects. At the same time, a simple, rapid, environmentally friendly and high-purity extraction and separation method for the new compound of the present invention is provided.

[0006] To achieve the above object of the present invention, the present invention provides a diimine alkaloid compound with the molecular formula C 12 H 10 N2O6, named as (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one according to the structural formula, and the chemical structural formula is:

[0007]

[0008] To achieve the above object of the present invention, the present invention also provides a method for extracting and separating a new diimine alkaloid compound from Portulaca oleracea L., which specifically comprises the following steps:

[0009] Step 1: Take the dried medicinal materials of Portulaca oleracea L., extract by water decoction, concentrate the extract, and cool it to room temperature to obtain a medicinal liquid for standby.

[0010] Step 2: Pour the water extract obtained in Step 1 into macroporous resin and make it adsorbed, and elute with a volume ratio of water to ethanol of 70:30 to obtain an ethanol extract.

[0011] Step 3: After evaporating the ethanol extract obtained in Step 2 to dryness, subject it to column chromatography separation on a pretreated ODS column (Octadecylsilyl, octadecylsilyl-bonded silica gel packing), elute successively with a methanol-water gradient to obtain several elution fractions, detect by thin-layer chromatography, develop color, combine the developed elution fractions, and concentrate the combined elution fractions to dryness under reduced pressure for standby.

[0012] Step 4: Subject the extract obtained in Step 3 to column chromatography separation on a pretreated ODS column again, elute successively with a methanol-water gradient to obtain several elution fractions, detect by thin-layer chromatography, develop color, combine the developed elution fractions, and concentrate the combined elution fractions to dryness under reduced pressure for standby.

[0013] Step 5: Subject the product obtained in Step 4 to column chromatography separation on a pretreated Sephadex LH-20 column, elute isocratically with methanol to obtain several elution fractions, detect by thin-layer chromatography, develop color, combine the developed elution fractions, and concentrate the combined elution fractions to dryness under reduced pressure for standby.

[0014] Step 6: Perform HPLC (high performance liquid chromatography) separation and preparation on the concentrate obtained in Step 5, use methanol: 0.1% formic acid as the mobile phase, and prepare the compound of the present invention.

[0015] Further, in Step 1, the water decoction extraction is carried out twice, each time for 2 hours, and the water dosage is 8 to 16 times that of the medicinal materials.

[0016] Further, in Step 3, the gradient elution is carried out with a volume ratio of methanol to water of 10:90, 30:70, 50:50 and 70:30, and the ODS particle size is 40 to 70 μm.

[0017] Further, in Step 4, the gradient elution is carried out with a volume ratio of methanol to water of 10:90, 30:70, 40:60 and 50:50.

[0018] Further, in Step 5, the methanol elution program is an isocratic elution.

[0019] Further, in the step 4, the pretreatment process of ODS and Sephadex is soaking in methanol for 24 hours, loading onto the column, washing with methanol until no turbidity appears when dropped into water, and then equilibrating with the initial mobile phase.

[0020] Further, in the step 6, the volume ratio of methanol to water in the isocratic elution with methanol∶0.1% formic acid is 25∶75.

[0021] The diimine alkaloid compound of the present invention can be used for preparing drugs with anti-inflammatory and anti-cholinesterase effects.

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

[0023] The separation and pharmacological activities of the new diimine compounds from Portulaca oleracea L. in the present invention have not been reported in existing papers and periodicals; the present invention provides new diimine compounds derived from Portulaca oleracea L. and an extraction and separation method for the new compounds of the present invention. The macroporous resin column, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatograph are used for separation, purification and preparation in sequence, and new compounds are successfully extracted and separated. The operation steps of this method are only six steps, the operation method is simple and fast, the extraction and separation process mainly uses ethanol extraction and methanol elution, the process method is environmentally friendly, and the purity of the compounds separated by this method is relatively high, greater than 90%. In addition, studies have shown that this compound has anti-inflammatory and antioxidant effects. Therefore, the new compounds of the present invention can be used as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research, and can also be used for preparing anti-inflammatory and antioxidant drugs. Description of the Drawings

[0024] Figure 1 It is the high-resolution mass spectrum of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention.

[0025] Figure 2 It is the 1 1H-NMR spectrum of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention.

[0026] Figure 3For the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention 13 C-NMR spectrogram.

[0027] Figure 4 For the DEPT135 spectrogram of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention.

[0028] Figure 5 For the HMBC spectrogram of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention.

[0029] Figure 6 For the HSQC spectrogram of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention.

[0030] Figure 7 For the 1 H- 1 H COSY spectrogram of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention.

[0031] Figure 8 For the ROESY spectrogram of the diimine compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention. Detailed implementation mode

[0032] The present invention provides a diimine compound with the molecular formula C 12 H 10N2O6, named as (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9 according to the structural formula

[0033] -one, and its chemical structural formula is:

[0034]

[0035] Table 1 shows the NMR data of this compound: 1 1H-NMR and 13 13C-NMR in dimethyl sulfoxide

[0036] Table 1 NMR data of the compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-on

[0037] e

[0038]

[0039]

[0040] The structural identification of the compound of the present invention refers to Figures 1-8 .

[0041] (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one: brown paste, soluble in methanol and water. After spotting on a silica gel thin layer plate and spraying with Dragendorff's reagent, it shows color, indicating that the compound contains alkaloids. After spraying with ferric trichloride test solution, the spot shows cyan, indicating that the compound contains phenolic hydroxyl groups. UV (methanol) λ max : 249, 302 nm, IR (potassium bromide): 1515, 1542, 1558, 1633, 3440 cm -1 . UHPLC-ESI-QTOF-MS gives m / z: 278.0541 [M-H] - of the quasi-molecular ion peak, and the molecular weight is 278.0539. Combining 1 1H-NMR, 13 13C-NMR and DEPT data, it is speculated that the molecular formula of this compound is C 12 H 10 N2O6, and the degree of unsaturation is 9

[0042] 13The \(^{13}\)C-NMR spectrum and DEPT spectrum show 10 carbon signals, including 1 vinylic carbon (\(\delta\) C : 152.89), 5 methine carbons (\(\delta\) C : 115.19, 130.24, 126.46, where 115.19 and 130.24 are overlapping peaks), and 4 quaternary carbons (1 vinylic carbon, \(\delta\) C : 157.51; 1 carbonyl carbon, \(\delta\) C : 175.18; 2 imine carbons, \(\delta\) C : 161.04, 171.51).

[0043] 1 The \(^{1}\)H-NMR spectrum shows 4 aromatic hydrocarbon signals \(\delta\) H 6.81 (2H, s), \(\delta\) H 7.39 (2H, s), where both sets of hydrogens are overlapping peaks; 1 vinylic signal \(\delta\) H 8.34 (1H, s), 1 methine signal \(\delta\) H 7.94 (1H, s, \(J = 8.76\)). From the 1 \(^{1}\)H-NMR spectrum, it can be seen that there is an AABB system of a benzene ring. The corresponding signals are H-3' / H-5' \(\delta\) H 6.81 (2H, s, \(J = 8.58\)), H-2' / H-6' \(\delta\) H 7.39 (2H, s, \(J = 8.52\)) and 13 the \(^{13}\)C-NMR spectrum signals \(\delta\) C 115.19 (C-3' and C-5' are overlapping), \(\delta\) C 130.24 (C-2' and C-6' are overlapping). The chemical shift of H-4' is at a low field, indicating that a hydroxyl group is attached to C-4'. According to the HMBC spectrum, H-7 (\(\delta\) H 8.34) is correlated with C-8 (\(\delta\) C 123.26), and H-7 is at a low field, indicating that H-7 is connected to an oxygen. At the same time, according to the HMBC spectrum, it is known that H-7 is correlated with C-9 (\(\delta\) C 175.18), H-2 (\(\delta\) H 7.94) is correlated with C-9, and C-9 is at an even lower field, indicating that H-2 and H-8 are connected to a carbonyl group. According to the 1 \(^{1}\)H-NMR spectrum, H-2 is a methine, and H-2 is at a low field, so it can be determined that H-2 is connected to a hydroxyl group and an oxygen. According to the HMBC spectrum, H-7 is correlated with C-5 (\(\delta\) C 171.51), H-2 is correlated with C-4 (\(\delta\) CIt is related to 161.04). The infrared spectrum shows two absorption peaks of imine bonds, indicating that there is one imino group on each of C-4 and C-5. To meet the requirements of the molecular formula, C-4 and C-5 are connected. Based on the above information, it can be determined that there is a nine-membered oxygen-containing heterocyclic structure containing an imine group and a carbonyl group. According to the correlation between H-2' and H-6' and C-8 in the HMBC spectrum, the benzene ring can be connected to this nine-membered oxygen-containing heterocyclic structure. Thus, based on the above information, this new compound can be determined to have the above structure.

[0044] The present invention also provides a method for extracting and separating a new diimine alkaloid compound from Portulaca oleracea L., which specifically includes the following steps:

[0045] Step 1: Weigh the dried medicinal materials of Portulaca oleracea L., extract by boiling with water, concentrate the extract, and cool it to room temperature to obtain a medicinal liquid for standby.

[0046] Step 2: Pour the water extract obtained in Step 1 into macroporous resin and continuously stir to adsorb it, and elute with ethanol and water in a volume ratio of 30 / 70. The macroporous resin column is 100-200 mesh to obtain an ethanol extract.

[0047] Step 3: After evaporating the ethanol extract with a volume ratio of 30 / 70 of ethanol and water in Step 2 to dryness, dissolve it with methanol and separate it by medium-pressure column chromatography on pretreated ODS. The filler particle size is 40-60 μm, and gradient elution is carried out with methanol-water (10 / 90, 30 / 70, 50 / 50, 70 / 30, v / v) (under pressure, with a flow rate of 1 mL / min and a temperature of room temperature) to obtain a methanol extract.

[0048] Step 4: Separate the product obtained in Step 3 by medium-pressure column chromatography on pretreated ODS again. The filler particle size is 40-60 μm, and gradient elution is carried out with methanol-water (10 / 90, 30 / 70, 40 / 60, 50 / 50, v / v) (under pressure, with a flow rate of 1 mL / min and a temperature of room temperature) to obtain 15 fractions (that is, 15 bottles are obtained by gradient elution, 240 mL per bottle). Detect by thin-layer chromatography, develop color, combine the colored parts, and concentrate to dryness under reduced pressure below 50 °C for standby.

[0049] Step 5: Separate the product obtained in Step 4 by gel permeation chromatography on pretreated Sephadex LH-20, and elute with methanol-water 30 / 70 to obtain 14 elution fractions (that is, a total of 14 bottles are obtained, 50 mL per bottle). Detect by thin-layer chromatography, develop color, and concentrate to dryness under reduced pressure below 50 °C for standby. The pretreatment process of the ODS and Sephadex is to soak in methanol for 24 h, load the column, wash with methanol until there is no turbidity when dripping into water, and then equilibrate with the initial mobile phase.

[0050] Step 6: The product obtained in Step 5 was separated and prepared by HPLC. Methanol: 0.1% formic acid (25:75, v / v) was used as the mobile phase, and the detection wavelengths were 210 nm and 254 nm. The new compound of the present invention was separated and prepared, and the purity was determined by the normalization method to be 85-95%.

[0051] Anti-inflammatory effect of the novel diimine alkaloid compound of the present invention.

[0052] 1 Main materials.

[0053] 1.1 Drugs and reagents: The novel diimine alkaloid compounds used in the experiment were prepared by the above method, with a purity of 85-95%. They were accurately weighed and diluted with DMSO to the solutions required for the following dose groups. DMEM high-glucose medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Siqing Company); LPS (Sigma, USA); ELISA kits for IL-1β and TNF-α (Cayman, USA); cell lysate, Griess reagent (Beyotime Biotechnology Co., Ltd.).

[0054] 1.2 Cell line: RAW264.7 macrophages (ATCC cell bank, USA).

[0055] 1.3 Grouping: Divided into a control group, an LPS group, and an experimental group, with one group each.

[0056] 2 Experimental methods.

[0057] 2.1 Cell culture: DMEM high-glucose medium was added with 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin), and cultured in a 37.5% CO2 incubator.

[0058] 2.2 MTT colorimetric method for determining cell viability: RAW264.7 macrophages in the logarithmic growth phase were inoculated into 96-well culture plates in the above three groups, and the cell density was 1×10 4At a density of 1×10 cells / mL, 100 μL per well, cultured overnight at 37°C under 5% CO2 conditions. After that, different concentrations of the novel diimine alkaloid compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention were added to the experimental group. After incubation for 1 h, LPS with a final concentration of 1 μg / mL was added to both the LPS group and the experimental group. Additionally, a blank control group (culture medium containing DMSO solvent) was set up, with 3 replicate wells in each group, to investigate the effect of the added drug on the cells. After culturing the cells in each group for 24 h, 20 μL of MTT at 5 mg / mL was added to the cells in each well, and incubation was continued for 4 h at 37°C under 5% CO2 conditions. Then, the culture was terminated, the liquid in the wells was aspirated, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was shaken for 10 min to fully dissolve the crystals inside the cells. The absorbance value of each well was measured at a wavelength of 570 nm using an ELISA reader.

[0059] 2.3 Determination of inflammatory factors IL-1β and TNF-α by ELISA: RAW264.7 macrophages in the logarithmic growth phase were seeded in a 24-well culture plate <9000043>at a density of 1×10 cells / mL, 1 mL per well, cultured overnight at 37°C under 5% CO2 conditions. The novel diimine alkaloid compound (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonin-9-one of the present invention was added to the experimental group. After incubation for 1 h, LPS (final concentration of 1 μg / mL) was added to each well, and the cells were co-incubated for 24 h. Each treatment was repeated in 3 wells. The contents of IL-1β and TNF-α secreted by RAW264.7 macrophages treated with the novel diimine alkaloid compound derived from Portulaca oleracea were determined by ELISA.

[0060] 3 Experimental results.

[0061] The experimental results showed that the novel diimine alkaloid compound of the present invention had no effect on the proliferation of LPS-induced macrophages RAW264.7, was safe and non-toxic, and could effectively inhibit the excessive inflammatory cytokines IL-1β and TNF-α produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0062] The experimental results of the relative cell viability are shown in Table 2.

[0063] Table 2 Effect of the compound of the present invention on the relative viability of RAW264.7 macrophages.

[0064]

[0065] Note: * P < 0.05 compared with the control group (significant difference in the high-concentration group).

[0066] The results of measuring inflammatory factors IL-1β and TNF-α by ELISA are shown in Table 3.

[0067] Table 3 Effects of the compounds of the present invention on the contents of IL-1β and TNF-α secreted by LPS-induced RAW264.7 cells (mean ± standard deviation, n = 3).

[0068]

[0069]

[0070] Note: *P < 0.05 compared with the control group, #P < 0.05 compared with the LPS group, mean ± SD, n = 3.

[0071] Anticholinesterase effect of the novel diimine alkaloid compounds of the present invention.

[0072] 1 Main materials.

[0073] 1.1 Drugs and reagents: The novel diimine alkaloid compounds used in the experiment were prepared by the above method, with a purity of 85-95%. Sodium dihydrogen phosphate, disodium hydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd.), physostigmine (HanXiang Biotechnology Co., Ltd.), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Shanghai Jinsui Biotechnology Co., Ltd.), acetylcholinesterase (AChE) and acetylthiocholine iodide (ATCI, Dalian Meilun Biotechnology Co., Ltd.).

[0074] 1.2 Grouping: Divided into a negative control group, a positive control group and an experimental group, one group each.

[0075] 2 Experimental methods.

[0076] 2.1 Sample preparation: Accurately weigh 1 mg of the sample and physostigmine separately. Using methanol as the solvent, prepare five gradient concentrations of 359.42, 179.71, 89.85, 44.92, and 22.46 μM respectively. Accurately weigh 7.039 g of sodium dihydrogen phosphate and 5.996 g of disodium hydrogen phosphate, make up to 50 mL with distilled water. Take 3.40 mL of sodium dihydrogen phosphate and 46.6 mL of disodium hydrogen phosphate to prepare 50 mL of PBS; accurately weigh 0.0588 g of DTNB, add 10 mL of PBS to prepare a DTNB solution (15 mmol / L); accurately weigh 0.01 g of AChE, add 10 mL of PBS to prepare an AChE solution (0.2 u / mL); accurately weigh 0.042 g of ATCI, make up to 10 mL with distilled water to prepare an ATCI solution (15 mmol / L).

[0077] 2.2 Determination of anticholinesterase activity by the improved Ellman method: In a 96-well microplate, sequentially add 140 μL of PBS (0.1 M, pH = 8.0), 10 μL of DTNB (15 mmol / L), 15 μL of AchE (0.2 u / mL), and 20 μL of the sample solution. In the negative control group experiment, methanol is used instead of the sample, and in the positive control group experiment, physostigmine is used instead of the sample. After incubation at 37 °C for 10 min, add 10 μL of ATCI (15 mmol / L). After incubation at 20 °C for 10 min, measure the absorbance value at 410 nm using a microplate reader. Calculate the inhibition rate according to the following formula: Inhibition rate (%) = (blank group - sample) / blank group × 100%.

[0078] 3 Experimental results.

[0079] The experimental results show that the novel diimine compounds of the present invention have anticholinesterase effects.

[0080] The experimental results are shown in Table 4.

[0081] Table 4 Anticholinesterase inhibitory activity of the compounds of the present invention.

[0082]

[0083] In summary, the present invention provides a novel diimine alkaloid compound and its extraction and separation method. By successively using macroporous resin column chromatography, ODS medium-pressure column chromatography, Sephadex LH-20 column chromatography and HPLC for separation and preparation, the novel diimine alkaloid compound is successfully separated. This method is simple, rapid, environmentally friendly, and the compound separated by this method has a high purity. Since the obtained compound has a unique chemical structure and is extracted from the common traditional Chinese medicine Portulaca oleracea L., it has anti-inflammatory and anti-cholinesterase effects. Therefore, the novel diimine alkaloid compound of the present invention can be developed as a new traditional Chinese medicine from natural products and has broad prospects.

Claims

1. A diimine alkaloid compound isolated from Portulaca oleracea L. medicinal materials, characterized in that, Molecular formula: C 12 H 10 N2O6, and named as (Z)-2-hydroxy-8-(4-hydroxyphenyl)-4,5-diimino-4,5-dihydro-9H-1,3,6-trioxonon-9-one according to the structure, and its chemical structural formula is as follows: 。 2. A method for extracting and separating a diimine alkaloid compound from Portulaca oleracea L., characterized in that, Specifically, it includes the following steps: Step 1: Take the dried Portulaca oleracea L. medicinal materials, extract them by water decoction, concentrate the extract, and cool it to room temperature to obtain a medicinal liquid for standby; Step 2: Pour the water extract obtained in Step 1 into macroporous resin and let it adsorb, elute with a 70:30 volume ratio of water and ethanol, and recover the extraction ethanol under reduced pressure to obtain an extract paste, thereby obtaining an ethanol extract; Step 3: After evaporating the ethanol extract obtained in Step 2 to dryness, subject it to column chromatography separation on a pretreated ODS column, elute successively with a methanol-water gradient to obtain several elution fractions, detect them by thin-layer chromatography, develop color, combine the elution fractions that develop color, and concentrate the combined elution fractions to dryness under reduced pressure for standby; Step 4: Subject the extract obtained in Step 3 to column chromatography separation on a pretreated ODS column again, elute successively with a methanol-water gradient to obtain several elution fractions, detect them by thin-layer chromatography, develop color, combine the elution fractions that develop color, and concentrate the combined elution fractions to dryness under reduced pressure for standby; Step 5: Subject the product obtained in Step 4 to column chromatography separation on a pretreated Sephadex gel column, elute with isocratic methanol, obtain several elution fractions, detect them by thin-layer chromatography, develop color, combine the elution fractions that develop color, and concentrate the combined elution fractions to dryness under reduced pressure for standby; Step 6: Perform HPLC separation and preparation on the concentrate obtained in Step 5, use methanol: 0.1% formic acid (v / v) as the mobile phase for isocratic elution, and prepare the said compound.

3. The extraction and separation method according to claim 2, characterized in that, In Step 1, the water decoction extraction is carried out twice, each time for 2 hours, and the water consumption is 8 - 16 times that of the medicinal materials.

4. The extraction and separation method according to claim 2, wherein In Step 3, the gradient elution is carried out with a volume ratio of methanol and water of 10:90, 30:70, 50:50, and 70:30, and the ODS particle size is 40 - 70 μm.

5. The extraction and separation method according to claim 2, wherein In Step 4, the gradient elution is carried out with a volume ratio of methanol and water of 10:90, 30:70, 40:60, and 50:

50.

6. The extraction and separation method according to claim 2, wherein In Step 4, the pretreatment process of ODS and Sephadex gel is to soak them in methanol for 24 hours, load the column, wash with methanol until no turbidity appears when dripping into water, and then equilibrate with the initial mobile phase.

7. The extraction and separation method according to claim 2, characterized in that, In Step 6, the volume ratio of methanol and water in the isocratic elution with methanol: 0.1% formic acid is 25:

75.

8. Use of a diimine alkaloid compound as described in claim 1, characterized in that, The said use is for preparing anti-inflammatory and anti-cholinesterase drugs.

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