Novel ester alkaloid compound in purslane as well as extraction and separation method and application of novel ester alkaloid compound

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and particularly relates to a novel ester alkaloid compound in purslane as well as an extraction and separation method and application thereof. The molecular formula of the new compound is C8H15NO7, and the new compound is named as 5-(1, 2-dihydroxyethylethyl)-3, 4-dihydroxyethylethyran-2-ylglycinate, and the structural formula of the new compound is shown in the description. The invention also provides an extraction and separation method of the novel compound, which sequentially adopts water extraction, macroporous resin adsorption and elution, ODS medium-pressure column preparation, Sephadex LH-20 and high performance liquid chromatograph preparation, and successfully extracts and separates the novel ester alkaloid compound. The structure is determined by mass spectrum, carbon spectrum, hydrogen spectrum and two-dimensional nuclear magnetic spectrum analysis methods. The novel compound has anti-inflammatory and anti-cholinesterase effects, and the novel compound and salts or derivatives thereof can be used as synthetic primers of other compounds and raw materials for new drug development and pharmacological activity research, and can be used for preparing anti-inflammatory and anti-cholinesterase drugs or health care products.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and in particular relates to a new ester alkaloid compound in purslane and an extraction and separation method and application thereof. Background Art

[0002] Portulaca oleracea L., also known as amaranth, melon seed vegetable, and grasshopper vegetable, is an annual herbaceous plant of the Portulacaceae family. Portulaca oleracea is widely distributed and rich in resources. It is one of the 78 wild plants with medicinal and edible properties specified by the former Ministry of Health of my country. Portulaca oleracea is included in the 2020 edition of the Pharmacopoeia of the People's Republic of China. It has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is used for heat-toxic bloody dysentery, carbuncle, furuncle, eczema, erysipelas, snake and insect bites, blood in stool, hemorrhoids, metrorrhagia, etc.

[0003] Modern pharmacological studies on purslane have shown that it has anti-inflammatory, antiviral, antihypertensive, antilipidemic, anti-glycemic, antioxidant, anticancer, skeletal and smooth muscle relaxation, immune function regulation and other effects. The main chemical components of purslane include flavonoids, coumarins, terpenes, steroids, alkaloids, amino acids, lignans, volatile oils, polysaccharides, various pigments and organic acid esters, which provide the material basis for its diverse pharmacological effects. Among them, alkaloid compounds are a major class of chemical components in purslane. The alkaloid components reported so far include norepinephrine, dopamine, uracil, adenine, adenosine, betaine, cyclodipeptide, N, N dicyclohexylurea, allantoin, oleraciamide F, oleracone L, oleracone A, oleracone M.

[0004] Currently, most of the chemical components isolated from Purslane are known and have low structural novelty. Therefore, the development and separation of new compounds in Purslane is urgently needed. Summary of the invention

[0005] In view of the above problems, the present invention provides a new compound extracted and separated from Portulaca oleracea. Studies have shown 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-mentioned object of the present invention, the present invention provides a new compound having the molecular formula C8H 15 NO7, named 5-(1,2-di hydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate, has the chemical structure:

[0007]

[0008] To achieve the above-mentioned object of the present invention, the present invention also provides a method for extracting and separating new ester alkaloid compounds in Portulaca oleracea, which specifically comprises the following steps:

[0009] Step 1, take dried purslane medicinal material, extract it with water, filter the water extract, combine the filtrate, concentrate it under reduced pressure, cool it to room temperature, and obtain the medicinal solution for use;

[0010] Step 2, evaporating the Chinese medicine solution in step 1 to dryness, adsorbing it with a macroporous resin, eluting it with a water-ethanol gradient, and concentrating the hot water portion under reduced pressure to obtain an extract, thereby obtaining a hot water extract;

[0011] Step 3, separating the hot water extract in step 2 through an ODS (octadecylsilane bonded silica gel filler) column, using a methanol-water gradient elution to obtain several elution fractions, detecting them through thin layer chromatography, developing colors, combining the elution fractions with developed colors, and concentrating the combined elution fractions to dryness under reduced pressure for later use;

[0012] Step 4, the obtained product in step 3 is further separated by chromatography on a pretreated Sephadex LH-20 column, and isocratically eluted with 20% methanol to obtain several elution fractions, which are detected by thin layer chromatography, developed, and the developed elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use;

[0013] Step 5, the obtained product in step 4 is further separated by chromatography on a pretreated Sephadex LH-20 column, isocratically eluted with 20% methanol to obtain several elution fractions, which are detected by thin layer chromatography, developed, and the developed elution fractions are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for later use;

[0014] Step 6: The concentrate obtained in step 5 is separated and prepared by HPLC (high performance liquid chromatography), and methanol: 0.1% formic acid is used as the mobile phase for isocratic elution to prepare the new ester alkaloid compound of the present invention.

[0015] Furthermore, in step 1, water reflux extraction is performed twice, each time for 2 hours, and the amount of water used is 10 times that of the medicinal material.

[0016] Furthermore, the mobile phase elution procedure used in step 2 is a gradient elution of cold water, hot water, 30% ethanol, 50% ethanol, and 70% ethanol.

[0017] Furthermore, in step 3, the volume ratio of water to methanol is 100:0, 95:5, 90:10, 80:20 and 100:0 for gradient elution.

[0018] Furthermore, in step 4, the volume ratio of methanol to water is 100:0, 95:5, 90:10, 80:20 and 100:0 for gradient elution.

[0019] Furthermore, in step 5, the methanol elution procedure is 20% methanol isocratic elution.

[0020] Furthermore, the pretreatment process of the ODS and dextran gel is: soaking in methanol for 24 hours, loading on the column, washing with methanol until there is no turbidity when dripped into water, and then balancing with the initial mobile phase.

[0021] Furthermore, in step 6, the volume ratio of methanol to water in the isocratic elution of methanol:0.1% formic acid is 45:55.

[0022] The novel ester alkaloid compound of the invention can be used for preparing anti-inflammatory and anti-cholinesterase drugs or health products.

[0023] Compared with the prior art, the invention has the beneficial effects.

[0024] The separation and pharmacological activity research of the new ester alkaloid compound of purslane described in the present invention has not been reported in existing paper journals; the present invention provides a new ester alkaloid compound derived from purslane and an extraction and separation method for the new compound of the present invention, which sequentially adopts water extraction, macroporous resin adsorption elution, ODS medium-pressure column, Sephadex LH-20 and high-performance liquid chromatography for separation, purification and preparation, and successfully extracts and separates the new compound. The method has only five operating steps, and the operation method is simple and fast. The extraction and separation process mainly adopts water extraction and ethanol elution, the process method is environmentally friendly, and the purity of the compound separated by the method is greater than 90%. In addition, studies have shown that the compound has anti-inflammatory and anti-cholinesterase effects. Therefore, the new compound of the present invention and its derivatives can be used as synthetic leads of other compounds, as well as raw materials for new drug development and pharmacological activity research, and can also be used to prepare anti-inflammatory and anti-cholinesterase drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a high-resolution mass spectrum of the new compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention.

[0026] Figure 2 This is the H-NMR spectrum of the new compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention.

[0027] Figure 3The novel compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention 13 C-NMR spectrum.

[0028] Figure 4 This is the DEPT spectrum of the new compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention.

[0029] Figure 5 This is the H MBC spectrum of the new compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention.

[0030] Figure 6 The novel compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention 1 H- 1 H COSY spectrum.

[0031] Figure 7 This is the H SQC spectrum of the new compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention.

[0032] Figure 8 The figure is the R OESY spectrum of the novel compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention. DETAILED DESCRIPTION

[0033] The present invention provides a new compound, the molecular formula of which is C8H 15 NO7, named 5-(1,2-dihydroxyethyl)-3,4-dihydroxy tetrahydrofuran-2-ylglycinate, has the chemical formula:

[0034]

[0035] Table 1 shows the NMR data of the new compound: 1 H-NMR and 13C-NMR in DMSO.

[0036] Table 1 NMR data of the novel compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate of the present invention.

[0037] The structural identification of the compounds of the present invention is shown in Figure 1-Figure 8 .

[0038] 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate: Pale yellow powder, easily soluble in water, slightly soluble in methanol. UHPLC-ESI-Q-TOF-MS gives m / z: 179.0561 [M-C2H4NO] - The fragment ion peak of , the molecular weight is 179.0560. 1 H-NMR, 13 C-NMR and DEPT data suggest that the possible molecular formula of the compound is C8H 15 NO7, unsaturation is 2. 13 The C-NMR spectrum and DEPT spectrum showed eight carbon signals, namely two methylene carbons (δ C :68.80;61.16)、5 methyl carbons (δ C :70.10; 73.66; 75.55; 76.74; 102.89), 1 carbonyl carbon (δ C :173.25).

[0039] 1 The H-NMR spectrum shows two methylene signals as δ H 3.05 (2H, m), δ H 3.63 (2H, d, J = 11.46 Hz); 5 methine signals are δ H 2.88 (1H, m), δ H 3.10 (1H, m), δ H 3.40 (1H, d, J=5.76Hz), δ H 3.60 (1H, m), δ H 4.12 (1H, dd, J = 3.48, 7.80 Hz). HSQC spectrum showed that H-2′ (δ H 4.12) and C-2′(δ C 102.89); and because the C-2′ chemical shift value is larger, it indicates that it is connected to two oxygen atoms. 1 H- 1The H COSY spectrum shows that H-2′(δ H 4.12) and H-3′(δ H 2.88) related, H-3′(δ H 2.88) and H-4′(δ H 3.10) related, H-4′(δ H 3.10) and H-5′(δ H 3.60) related, H-5′(δ H 3.60) and H-1″(δ H 3.40) related, H-1″(δ H 3.40) and H-2″(δ H 3.05), indicating that C-2′(δ C 102.89), C-3′(δ C 73.66), C-4′(δ C 75.55), C-5′(δ C 76.74), C-1″(δ C 70.10) and C-2″(δ C 68.80) are connected in sequence; combined with the HMBC spectrum showing that H-2′ and C-5′ are related, and H-5′ and C-2′ are related, it indicates the existence of a five-membered oxygen-containing heterocycle containing a substituent. The HMBC spectrum shows that H-2′ and C-1 (δ C 173.25), combined with the ROESY spectrum showing H-2′ and H-2(δ H 3.63) are correlated with each other, indicating that C-1 and C-2 (δ C 61.16) are connected. 13 The C-NMR spectrum shows that C-3′, C-4′, C-1″, and C-2″ are in the low field, and their chemical shift values ​​are slightly greater than C-2, indicating that C-3′, C-4′, C-1″, and C-2″ are all connected to hydroxyl groups, while C-2 is connected to amino groups. Therefore, based on the above information, it can be determined that this new compound has the above structure.

[0040] The present invention also provides a method for extracting and separating the above-mentioned compound, the specific steps of which are:

[0041] Step 1: Weigh 250 kg of dried purslane medicinal material, extract it with water, the amount of water is 10 times the amount of the medicinal material, extract it twice, each time for 2 hours, filter the water extract, combine the filtrate, concentrate it under reduced pressure to 250 L, cool it to room temperature, and obtain the medicinal solution for use.

[0042] Step 2: evaporate the drug solution obtained in step 1 to dryness, adsorb and elute with AB-8 resin, and isocratically elute with hot water. Recover the drug solution to an extract under reduced pressure at below 80°C to obtain a hot water extract.

[0043] Step 3: The hot water extract in step 2 is separated by medium- and low-pressure ODS columns, wherein the filler particle size is 40-70 μm, and gradient elution is performed using methanol-water (0 / 100, 5 / 95, 10 / 90, 20 / 80, 50 / 50, 100 / 0, v / v) to obtain 35 fractions in total (i.e., 35 bottles in total, each with 100 mL), which are detected by thin layer chromatography and color developed. The colored 6-15 eluted fractions are combined, and the combined 6-15 fractions are concentrated to dryness under reduced pressure below 75° C. for later use.

[0044] Step 4: The product obtained in step 3 is further separated by pretreated ODS medium-pressure column chromatography, wherein the filler particle size is 20-40 μm, and gradient elution is performed with methanol-water (0 / 100, 5 / 95, 10 / 90, 20 / 80, 50 / 50, 100 / 0, v / v) (pressurized to a flow rate of 1 mL / min and room temperature) to obtain 14 fractions (i.e., 18 bottles are obtained by gradient elution, each with 100 mL), which are detected by thin layer chromatography and color developed. The colored 4-8 fractions are retained and concentrated to dryness under reduced pressure below 50° C. for later use.

[0045] Step 5: The obtained in step 4 is separated by pre-treated dextran gel column chromatography (Sephadex LH-20), eluted with methanol, and 30 elution fractions are obtained (i.e., 30 bottles are obtained, each bottle is 50 mL), detected by thin layer chromatography, color is developed, and the 16-22 colored fractions are retained, and concentrated to dryness under reduced pressure below 50°C for standby use. The pre-treatment process of the ODS and dextran gel is to soak in methanol for 24 hours, load the column, wash with methanol until there is no turbidity when dripped into water, and then balance with the initial mobile phase.

[0046] Step 6: The product obtained in step 5 was separated and prepared by HPLC, using methanol:0.1% formic acid (45:55, v / v) as the mobile phase and detection wavelengths of 210nm and 254nm to separate and prepare the new compound of the present invention. The purity determined by normalization method was 90-99%.

[0047] The anti-inflammatory effect of the novel ester alkaloid compound of the present invention.

[0048] 1Main materials.

[0049] 1.1 Drugs and reagents: The new ester alkaloid compounds used in the experiment were prepared by the above method with a purity of 90-99%. They were accurately weighed and diluted with DMSO to the required solution for each dose group as follows: DMEM high-glucose medium, fetal bovine serum (Hyclone, USA); penicillin and streptomycin (Hangzhou Sijiqing Company); LPS (Sigma, USA); ELISA kits for IL-1β and TNF-α (Cayman, USA); cell lysate, Griess reagent (Biyuntian Biotechnology Co., Ltd.).

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

[0051] 1.3 Grouping: Divided into control group, LPS group and experimental group, one group each.

[0052] 2 Experimental methods.

[0053] 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.

[0054] 2.2 CCK-8 method to determine cell viability: The three groups of RAW264.7 macrophages in logarithmic growth phase were inoculated in 96-well culture plates at a cell density of 1×10 4 / mL, 100μL per well, 37℃, 5% CO2, after overnight culture, different concentrations of the compound of the present invention 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate (5μM~50μM) were added to the experimental group, and after incubation for 1h, LPS with a concentration of 1μg / mL was added to the LPS group and the experimental group, and a zero adjustment group (culture solution containing DMSO solvent) was set up, and 3 duplicate wells were set up in each group to investigate the effect of the addition of drugs on the cells. After the cells in the above groups were cultured for 24h, 10μL of CCK-8 was added to the cells in each well, and after further incubation for 2h at 37℃, 5% CO2, the absorbance value of each well was measured at a wavelength of 450nm by an enzyme marker.

[0055] 2.3ELISA method for determination of inflammatory factors IL-1β and TNF-α: RAW264.7 macrophages in the logarithmic growth phase were inoculated in 24-well culture plates at a cell density of 1×10 5 / mL, 1mL per well, cultured overnight at 37°C and 5% CO2, the experimental group added the new amide alkaloid compound 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate (1-20μM) of the present invention, cultured for 1h, added LPS (final concentration of 1μg / mL) to each well, incubated for 24h, and each group was treated with 3 wells. The ELISA method was used to determine the content of IL-1β and TNF-α secreted by RAW264.7 macrophages after being treated with the new alkaloid compound from Purslane.

[0056] 3 Experimental results.

[0057] The experimental results show that the new amide alkaloid compound of the present invention has no effect on the proliferation of LPS-induced macrophages RAW264.7 within 25 μM, is safe and non-toxic; and can effectively inhibit the excessive inflammatory cytokines IL-1β and TNF-α produced by LPS-induced macrophages RAW264.7 in a concentration-dependent manner.

[0058] The experimental results of the relative cell survival rate of the new amide alkaloid compounds are shown in Table 2.

[0059] Table 2 Effects of the compounds of the present invention on the relative survival rate of RAW264.7 macrophages.

[0060]

[0061] Note: * P<0.05 compared with the control group. # P<0.05 compared with LPS group.

[0062] The results of ELISA determination of inflammatory factors IL-1β and TNF-α are shown in Table 3.

[0063] Table 3 Effects of the compounds of the present invention on the levels of IL-1β and TNF-α secreted by RAW264.7 cells induced by LPS (mean±standard deviation, n=3).

[0064]

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

[0066] The anticholinesterase effect of the novel ester alkaloid compounds of the present invention.

[0067] 1Main materials.

[0068] 1.1 Drugs and reagents: The new compounds used in the experiment were prepared by the above method with a purity of 96%. Physostigmine (Shanghai Hanxiang Biotechnology Co., Ltd.), acetylcholine iodide (ATCI) and acetylcholinesterase (AChE) (Dalian Meilun Biotechnology Co., Ltd.), dithiodinitroformic acid (DTNB) (Shanghai Jinshui Biotechnology Co., Ltd.), disodium hydrogen phosphate and sodium dihydrogen phosphate (Shanghai Sinopharm Reagent Co., Ltd.).

[0069] 1.2 Experimental instruments and equipment: HBS-1096A96-well microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.), 1 / 100,000 balance (METTLER, Switzerland), HH-4 digital display constant temperature water bath (Jiangsu Jintan Ronghua Instrument Manufacturing Co., Ltd.).

[0070] 2 Experimental methods.

[0071] This experiment was conducted to determine the anticholinesterase activity of new amine alkaloid compounds according to the modified Ellman method. 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-ylglycinate and physostigmine were accurately weighed and prepared into five series of sample solutions with concentrations of 3.125μM, 6.25μM, 12.5μM, 25.0μM and 50.0μM using methanol. The specific operation is as follows: add 140 μL of phosphate buffered saline (0.1M, pH=8.0, containing 0.1 mol / L disodium hydrogen phosphate and sodium dihydrogen phosphate), 20 μL of sample solution, 15 μL of AChE (0.2U / mL) to a 96-well ELISA plate, incubate at 37°C for 10 minutes, add 10 μL of ATCI (4mmol / L) and 10 μL of DTNB (15mmol / L), incubate at 37°C for 20 minutes, place the 96-well plate in an ELISA instrument, and measure the absorbance of each group of samples at a wavelength of 405nm. Among them, methanol was used instead of the sample solution as the blank group, and physostigmine was used instead of the sample solution as the positive control group. The cholinesterase inhibition rate of each compound was calculated according to the following formula (A represents absorbance):

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

[0073] 3 Experimental results.

[0074] The experimental results show that the new compound of the present invention exhibits certain anticholinesterase activity, and the inhibitory effect on cholinesterase increases with the increase of the compound concentration, showing a dose-dependent trend.

[0075] The anticholinesterase activities of the novel amine alkaloid compounds of the present invention are shown in Table 4.

[0076] Table 4 Anticholinesterase activities of the compounds of the present invention.

[0077]

[0078]

[0079] In summary, the present invention provides a new ester alkaloid compound in purslane and an extraction and separation method thereof, wherein water extraction, macroporous resin adsorption elution, ODS medium pressure column, Sephadex LH-20 and high performance liquid chromatography are sequentially used for separation, purification and preparation, and the new compound is successfully separated and obtained. The method is simple, rapid and environmentally friendly, and the compound separated by the method has a high purity. Since the obtained compound has a unique chemical structure and is extracted from the commonly used traditional Chinese medicine purslane, it has anti-inflammatory and anticholinesterase effects; therefore, the new ester alkaloid compound and its derivatives of the present invention can be used as natural products to develop new traditional Chinese medicines, and have broad prospects.

Claims

1. A new ester alkaloid compound isolated from Portulaca oleracea. It is characterized in that The molecular formula is: C 8 H 15 NO 7 , and named according to the structure 5-(1,2-dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-yl glycinate, its chemical structure is as follows: 。 2. A method for extracting and separating a new ester alkaloid compound from Portulaca oleracea. It is characterized in that The specific steps include: Step 1, take dried purslane medicinal material, extract it with water, filter the water extract, combine the filtrate, concentrate it under reduced pressure, cool it to room temperature, and obtain the medicinal solution for use; Step 2, evaporating the Chinese medicine solution in step 1 to dryness, adsorbing it with a macroporous resin, eluting it with a water-ethanol gradient, and concentrating the hot water portion under reduced pressure to obtain an extract, thereby obtaining a hot water extract; Step 3, separating the hot water extract in step 2 through an ODS column, using a methanol-water gradient elution, evaporating the 20% methanol portion and applying it to a silica gel column, sequentially eluting with a methanol-water gradient elution to obtain several elution fractions, detecting by thin layer chromatography, developing color, combining the elution fractions that have developed color, and concentrating the combined elution fractions to dryness under reduced pressure for later use; Step 4, the obtained product in step 3 is further separated by pre-treated dextran gel column chromatography, isocratically eluted with 20% methanol to obtain several elution fractions, detected by thin layer chromatography, color developed, and the elution fractions with color developed are combined, and the combined elution fractions are concentrated to dryness under reduced pressure for standby use; Step 5: The concentrate obtained in step 4 is subjected to HPLC separation and preparation, with methanol:0.1% formic acid (v / v) as the mobile phase to prepare the new ester alkaloid compound of the present invention.

3. The extraction and separation method according to claim 2, It is characterized in that In step 1, water reflux extraction is performed twice, each time for 2 hours, and the amount of water used is 10 times that of the medicinal material.

4. The extraction and separation method according to claim 2, It is characterized in that The mobile phase elution program used in step 2 is cold water, hot water, 30% ethanol, 50% ethanol, 70% ethanol gradient elution.

5. The extraction and separation method according to claim 2, It is characterized in that In step 3, the volume ratio of water to methanol is 100:0, 95:5, 90:10, 80:20 and 100:0 for gradient elution.

6. The extraction and separation method according to claim 2, It is characterized in that In step 4, the volume ratio of methanol to water is 100:0, 95:5, 90:10, 80:20 and 100:0 for gradient elution.

7. The extraction and separation method according to claim 2, It is characterized in that The methanol elution procedure in step 5 is 20% methanol isocratic elution.

8. The extraction and separation method according to claim 2, It is characterized in that The pretreatment process of the ODS and dextran gel is as follows: soaking in methanol for 24 hours, loading onto the column, washing with methanol until there is no turbidity when dripped into water, and then balancing with the initial mobile phase.

9. The extraction and separation method according to claim 2, It is characterized in that The volume ratio of methanol to water in the methanol:0.1% formic acid isocratic elution used in step 6 is 45:

55.

10. Use of the new ester alkaloid compound as claimed in claim 1, It is characterized in that The compound can be used to prepare anti-inflammatory and anti-cholinesterase drugs or health products.

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