CN110256326B

CN110256326B - Indoline carboxylic acid alkaloid in purslane and extraction and separation method and application thereof

Info

Publication number: CN110256326B
Application number: CN201910640364.9A
Authority: CN
Inventors: 英锡相; 徐纹; 张文洁
Original assignee: Liaoning University of Traditional Chinese Medicine
Current assignee: Liaoning University of Traditional Chinese Medicine
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2022-05-13
Anticipated expiration: 2039-07-16
Also published as: CN110256326A

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a novel alkaloid compound extracted, separated and identified from purslane and an extraction and separation method thereof. The novel compound has a molecular formula of C₉H₉NO₄The chemical name is 4, 5-dihydroindoline-1-carboxylic acid. Also provides an extraction and separation method of the new compound, which adopts water decoction extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column and Sephadex LH-20 and high performance liquid chromatograph for separation and preparation in sequence, and successfully extracts and separates out a new alkaloid compound. The structure of the alkaloid compound is identified as a novel alkaloid compound by a method of mass spectrum, carbon spectrum, hydrogen spectrum and two-dimensional nuclear magnetic spectrum analysis. The new compound and the salt or the derivative thereof can be used as a lead for synthesizing other compounds, and raw materials for developing new medicines and researching pharmacological activity, and are used for preparing medicines for resisting tumors and protecting nerves.

Description

Indoline carboxylic acid alkaloid in purslane and extraction and separation method and application thereof

Technical Field

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to an alkaloid compound extracted, separated and identified from a purslane medicinal material and an extraction and separation method thereof, and particularly relates to an indoline carboxylic acid alkaloid in purslane and an extraction and separation method thereof.

Background

Herba Portulacae (Portulaca oleracea L.), also called herba Portulacae and herba Portulacae, is a plant of Portulacaceae. Purslane is favored to be fertile soil, has drought and waterlogging resistance, strong vitality, wide distribution and rich resources, and is more common in northeast of China. The purslane can be used as a medicine and can be eaten, and is one of wild plants which are determined by the Ministry of health and have homology of medicine and food. 2015, pharmacopoeia of the people's republic of China, which contains dry aerial parts of herba Portulacae, has effects of clearing away heat and toxic materials, cooling blood, stopping bleeding, and stopping dysentery, and can be used for treating toxic heat, bloody dysentery, carbuncle, furuncle, eczema, erysipelas, snake and insect bite, hematochezia, hemorrhoidal bleeding, metrorrhagia, metrostaxis, etc.

Modern pharmacological studies show that the purslane has the effects of reducing blood fat, reducing blood sugar, resisting inflammation, resisting oxidation, resisting tumors, resisting atherosclerosis, relaxing or exciting smooth muscles, enhancing immunity and the like. Research shows that various chemical components contained in purslane are closely related to various pharmacological effects of purslane, and the main chemical components of the purslane comprise: flavones, alkaloids, terpenoids, coumarins, organic acids, volatile oil, polysaccharides, amino acids, various pigments and minerals, etc. Wherein, alkaloid is a large active ingredient in purslane, while amide alkaloid accounts for the vast majority. The alkaloid components reported at present comprise norepinephrine, dopamine, a small amount of dopa, adenosine, uracil, adenine, N-dicyclohexylurea, allantoin and N-trans-feruloyl tyramine; cyclic dipeptide alkaloids and amide alkaloids are also present: oleracein A-I, K, L, N-S.

Most of the chemical components separated from purslane are known and have low structural one property, so the development and separation of new compounds in purslane are urgently needed.

Disclosure of Invention

In order to solve the problems, the invention provides an alkaloid compound extracted from purslane, and researches show that the alkaloid compound has the effects of resisting tumors and protecting nerves, and simultaneously provides a simple, convenient, rapid, environment-friendly and high-purity extraction and separation method for the alkaloid compound.

In order to achieve the aim, the invention provides indoline carboxylic acid alkaloid in the purslane, wherein the molecular formula is C₉H₉NO₄The chemical name is 4, 5-dihydroindoline-1-carboxylic acid, and the chemical structural formula is shown in the specification.

In order to realize the purpose, the invention also provides a method for extracting and separating indoline carboxylic acid alkaloid from purslane, which comprises the following specific steps.

Step 1, taking a purslane dried medicinal material, extracting twice with water (the amount of water is 8-16 times of the medicinal material), filtering the water extract, combining the filtrates, directly heating and concentrating, and cooling to room temperature to obtain a liquid medicine for later use.

Step 2, evaporating the liquid medicine obtained in the step 1 to dryness, putting the liquid medicine on a silica gel column, eluting the liquid medicine by using ethyl acetate, and recovering the ethyl acetate under reduced pressure to obtain an extract so as to obtain an ethyl acetate extract;

step 3, separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, performing gradient elution by using ethanol-water, evaporating water and 30% alcohol, putting the water and the alcohol on a silica gel column, performing gradient elution by using ethyl acetate and ethyl acetate-methanol in sequence to obtain a plurality of elution parts, detecting by using a thin-layer chromatography, developing, combining the developed elution parts, and concentrating the combined elution parts under reduced pressure until the combined elution parts are dry for later use;

step 4, separating the product obtained in the step 3 by a polyamide column, performing gradient elution by adopting ethanol-water, and evaporating 30% ethanol part to dryness for later use;

step 5, subjecting the product obtained in the step 4 to chromatographic separation by a pretreated ODS (Octadecylsilyl silica gel filler), performing gradient elution by methanol-water to obtain a plurality of elution parts, detecting by thin-layer chromatography, developing, and concentrating the developed elution parts under reduced pressure to dryness to obtain a concentrate for later use;

step 6, subjecting the product obtained in the step 5 to chromatographic separation by a pretreated Sephadex LH-20 column, eluting by methanol to obtain a plurality of elution parts, detecting by thin-layer chromatography, developing, combining the developed elution parts, and concentrating the combined elution parts under reduced pressure to dryness for later use;

and 7, performing HPLC (high performance liquid chromatography) separation preparation on the concentrate obtained in the step 6, and taking acetonitrile and 0.1% formic acid as mobile phases to prepare the indoline carboxylic acid alkaloid compound.

The pretreatment process of the ODS and Sephadex LH-20 gel comprises the steps of soaking in methanol for 24 hours, loading on a column, washing with methanol until no turbidity exists in dripping water, and balancing with an initial mobile phase.

Compared with the prior art, the invention has the beneficial effects.

The separation and pharmacological activity research of indoline carboxylic acid alkaloid in the purslane is not reported in the journal of the prior paper; the invention provides a new alkaloid compound from purslane and an extraction and separation method aiming at the new compound, which sequentially adopts water decoction extraction, silica gel column chromatography, polyamide column, ODS medium-pressure column, Sephadex LH-20 and high performance liquid chromatograph for separation, purification and preparation, so as to successfully extract and separate out the new compound, the method has seven operation steps, the operation method is simple and rapid, the extraction and separation process mainly adopts water extraction and ethyl acetate elution, the process method is environment-friendly, and the purity of the compound separated by the method is higher than 90 percent, in addition, the research shows that the compound has the effects of anti-tumor and neuroprotection, so the new compound and the salt and the derivative thereof can be used as the raw materials for synthesizing a guide substance from other compounds, developing new drugs and researching pharmacological activity, and can also be used for preparing anti-tumor drugs, Neuroprotective agents.

Drawings

FIG. 1 is a drawing showing the preparation of the novel alkaloid compounds of the present invention¹H-NMR spectrum chart.

FIG. 2 is a schematic representation of the novel alkaloid compounds of the present invention¹³C-NMR spectrum chart.

FIG. 3 is a nuclear magnetic resonance carbon spectrum (DEPT) spectrum of the novel alkaloid compound.

FIG. 4 shows NMR of the novel alkaloid compounds of the present invention¹H-¹HCOSY spectrum.

FIG. 5 is a nuclear magnetic resonance HMBC spectrum of the novel alkaloid compound of the present invention.

FIG. 6 is a diagram of the NMR HSQC spectrum of the novel alkaloid compounds of the present invention.

FIG. 7 is a ROESY spectrum of the novel alkaloid compounds of the present invention.

FIG. 8 is a high resolution mass spectrum of the novel alkaloid compounds of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1.

The invention provides indoline carboxylic acid alkaloid in purslane, the molecular formula is C₉H₉NO₄The chemical name is 4, 5-dihydroindoline-1-carboxylic acid, and the chemical structural formula is shown in the specification.

Table 1 shows the nuclear magnetic data for one alkaloid compound:¹H-NMR of¹³C-NMR in deuterated DMSO.

TABLE 1 Nuclear magnetic data for novel alkaloid compounds of the invention

The invention relates to the structure identification and derivation of indoline carboxylic acid alkaloid in purslane.

4, 5-dihydroindoline-1-carboxylic acid: brown powder, easily soluble in methanol, insoluble, slightly soluble in water. After the sample is applied to a silica gel thin layer plate, a spot sprayed with diluted bismuth potassium iodide solution shows orange color, which indicates that the compound is an alkaloid component. HR-ESI (-) -TOF-MS gives [ M-H ] M/z 194.0465]^-Has an excimer ion peak of 195.0532 molecular weight, binds¹H-NMR，¹³C-NMR and DEPT data, presuming that the possible molecular formula of the compound is C₉H₉NO₄The unsaturation degree was 6.¹³The C-NMR spectrum and the DEPT spectrum showed 9 carbon signals, respectively 2 CH₂(delta: 26.48, 39.60), 2 CH (delta: 116.63, 119.35), 5 quaternary carbons (1 carbonyl carbon, delta: 170.00; 4 olefinic carbons, delta: 111.18, 128.96, 144.03, 149.78)

¹H-NMR spectrum showed that the compound had 2 CH₂Signals are δ 2.76(2H, t, J ═ 6.72Hz), δ 3.35(2H, td, J ═ 6.82, 2.34Hz), respectively; 2 CH signals δ 6.52(1H, d, J ═ 7.92Hz), δ 6.84(1H, d, J ═ 7.86 Hz). According to¹H-¹The H COSY spectrum shows 2 CH₂δ 2.76 and δ 3.35 in 2 CH, δ6.52 and delta 6.84; HMBC spectra showing aromatic proton H-6 coupled to olefinic quaternary carbons C-3a, C-4, C-5, C-7a, aromatic proton H-7 coupled to olefinic quaternary carbons C-3a, C-4, C-5, C-6, C-7a, confirms the presence of a tetra-substituted benzene ring, and the presence of C4 (delta 149.78), C5 (delta 144.03) in the low field, and active hydrogens delta 8.36, delta 8.98, demonstrate that C-4, C-5 are attached to hydroxyl groups, respectively. In addition, the HMBC spectrum shows that H-2 is coupled with C-2, C-3a, C-7a, H-3 is coupled with C-2, C-3, C-3a, C-4, C-7a, H-7 is coupled with C-3, C-3a, C-7a, and the DEPT spectrum shows that C-2(39.60) is a carbon typically connected with N, which shows that the tetra-substituted benzene ring is fused with a five-membered ring containing N through C-3a, C-7a to form an indoline ring structure. C-1' (delta 170.00) is located at low field, and active hydrogen delta 12.77, demonstrating that the compounds contain a carboxyl group in common. H-2, H-7 are coupled to C-1 ', presumably N-1 is attached to C-1'. Finally, according to the information, the structure of one indoline carboxylic acid alkaloid in the purslane can be determined.

The invention also provides an extraction and separation method of the compound, which comprises the following specific steps.

Step 1: weighing 150kg of dry herba Portulacae, extracting with water under reflux with water amount (v/v) 10 times of the medicinal materials, extracting under reflux twice, each for 2 hr, heating at 90-100 deg.C for concentrating, and cooling to room temperature to obtain medicinal liquid.

Step 2: evaporating the liquid medicine obtained in the step 1 to dryness, performing chromatographic separation by using a silica gel column, isocratically eluting by using ethyl acetate (115L), wherein the silica gel is 100-200 meshes, and recovering the ethyl acetate to obtain an extract under reduced pressure below 40 ℃ to obtain an ethyl acetate extract.

And step 3: separating the ethyl acetate extract obtained in the step 2 by using a polyamide column, performing gradient elution by using ethanol-water (0/100, 30/70, 50/50, 70/30, 100/0, v/v), evaporating a water part and a 30% ethanol part at 90-100 ℃, performing chromatography separation by using a silica gel column, wherein the silica gel is 200-300 meshes, performing gradient elution by using ethyl acetate and ethyl acetate-methanol (5:1, 2:1, 1:2, v: v) sequentially to obtain 30 parts (namely obtaining 30 bottles in total, wherein each bottle is 300mL), detecting by using a thin layer chromatography, performing color development, combining the developed 1-15 elution parts, and performing reduced pressure concentration on the combined 1-15 parts at the temperature of below 40 ℃ until the parts are dried for later use.

And 4, step 4: separating the product obtained in step 3 with polyamide column, gradient eluting with ethanol-water (0/100, 30/70, 50/50, 70/30, 100/0, v/v), and evaporating 30% ethanol at 90-100 deg.C.

And 5: and (3) performing pretreated ODS (octadecylsilane chemically bonded silica) medium-pressure column chromatography separation on the product obtained in the step (4), wherein the granularity of the filler is 20-40 mu m, performing gradient elution (pressurizing to enable the flow rate to be 1mL/min and the temperature to be room temperature) by using methanol-water (30/70, 40/60, 50/50, 60/40, 70/30, 100/0, v/v) to obtain 12 parts (namely performing gradient elution to obtain 12 bottles, wherein each bottle is 100mL), detecting by using thin-layer chromatography, developing, reserving 3-4 parts for developing, and concentrating under reduced pressure below 50 ℃ until the parts are dry for later use.

Step 6: and (3) subjecting the product obtained in the step (5) to chromatographic separation by a pretreated Sephadex LH-20 column, eluting by methanol to obtain 17 elution parts (namely, 17 bottles are obtained in total and each bottle is 50mL), detecting by thin-layer chromatography, developing, reserving the developed 10-17 parts, and concentrating under reduced pressure below 50 ℃ until the parts are dry for later use to obtain a new compound. The pretreatment process of the ODS and the sephadex comprises the steps of soaking in methanol for 24 hours, loading on a column, washing with methanol until no turbidity exists in dropwise added water, and balancing with an initial mobile phase.

And 7: the new compound obtained in step 6 was prepared by HPLC separation with acetonitrile: 0.1% formic acid (13: 87, v/v) is used as a mobile phase, the detection wavelength is 210nm and 280nm, the new compound is obtained by separation and preparation, and the purity measured by a normalization method is 90-99%.

The invention relates to an antitumor effect of indoline carboxylic acid alkaloid in purslane.

1 main material.

1.1 drugs and reagents: the new alkaloid compound used in the experiment is prepared by the method, the purity of the compound is 90-99%, the compound is precisely weighed and diluted by DMSO to be a solution required by each dosage group. DMEM high-glucose medium, fetal bovine serum (Hyclone, usa); penicillin and streptomycin (Hangzhou Sijiqing Co., Ltd.).

1.2 cell lines: human colon cancer cell Caco-2, human breast cancer cell MCF-7, human gastric cancer cell BGC-823, human lung adenocarcinoma cell SPC-A1, human liver cancer cell BEL-7402, human cervical cancer cell Hela-229, ovarian cancer cell Ho-8910, and human oral epidermoid carcinoma cell KB (Shanghai cell Bank of China academy of sciences).

1.3 grouping: divided into a control group, an experimental group and a zero-adjustment group (culture solution containing DMSO solvent).

2 experimental methods.

2.1 cell culture, DMEM high sugar medium, added with l 0% fetal bovine serum, l% antibiotics (100U/mL penicillin and 100. mu.g/mL streptomycin), placed at 37 ℃ with 5% CO₂Culturing in an incubator.

2.2MTT method for detecting cell proliferation, inoculating cells in logarithmic growth phase into 96-well culture plate with cell density of 1 × 104/mL, 100 μ L per well, temperature of 37 deg.C, and 5% CO₂After overnight culture under the conditions, the experimental groups were added with the new alkaloid compounds of the present invention at different concentrations, each group was provided with 3 multiple wells, and after adding the drug, the mixture was placed at 37 ℃ with 5% CO₂Culturing in an incubator for 48 h. Absorbing the culture solution containing the medicine, and adding the mixture into the culture solution in a volume ratio of 4: 1 and MTT (5 mg/mL) for 4 hours, carefully absorbing the supernatant, adding 150 mu L of DMSO into each hole, placing the hole on a shaker to shake so as to completely dissolve crystals (5min), and detecting the absorbance (A) value of each hole by a microplate reader at the wavelength of 570 nm. Then, the inhibition rate of each concentration of compound on cell growth is calculated, and the inhibition rate formula is as follows: inhibition of cell growth rate ═ 1-A_{Medicine feeding hole}/A_{Control well}) X 100%, processing data with SPSS software, plotting inhibition rate against drug concentration, and calculating IC₅₀The value is obtained.

3, experimental results.

Experimental results show that the novel alkaloid compound has an inhibitory effect on proliferation of human colon cancer cells Caco-2, human breast cancer cells MCF-7, human gastric cancer cells BGC-823, human lung adenocarcinoma cells SPC-A1, human liver cancer cells BEL-7402, human cervical cancer cells Hela-229, ovarian cancer cells Ho-8910 and human oral epidermoid cancer cells KB, and the inhibitory rate is obviously increased along with the increase of the drug concentration, namely the inhibitory rate is concentration dependent. The novel compound of the invention is used for treating the eight tumor cells IC₅₀The values are shown in Table 5.

Table 2 inhibitory effect of the novel compounds of the present invention on tumor cells.

Neuroprotective effects of the novel compounds of the present invention.

1 main material.

1.1 drugs and reagents: the new alkaloid compound used in the experiment is prepared by the method, the purity of the compound is 90-99%, the compound is precisely weighed and diluted by DMSO to be a solution required by each dosage group. DMEM high-glucose medium, fetal bovine serum (Hyclone, usa); penicillin, streptomycin (Hangzhou Sijiqing Co.), Phosphate Buffered Saline (PBS), (Wuhan Boshi De Co., Ltd.), ROS detection kit (Haimebi Yuntian reagent Co., Ltd.)

1.2 cell lines: human neuroblastoma cell line (SH-SY5Y, IMR-32) (Shanghai cell of Chinese academy of sciences)

1.3 grouping: divided into control group, H₂O₂Injury model group and experimental group.

2 experimental methods.

2.2MTT colorimetric method for determining cell viability, the three groups are respectively inoculated in a 96-hole culture plate by SH-SY5Y cells and IMR-32 cells in logarithmic growth phase, and the cell density is 1 multiplied by 10⁴one/mL, 100. mu.L per well, temperature 37 ℃, 5% CO₂After overnight incubation under conditions, the experimental groups were added with different concentrations of the novel alkaloid compounds of the invention (5-40. mu.M), incubated for 1H and then incubated for H₂O₂Group and experimental group were each added with H at a final concentration of 800. mu.M/L₂O₂In addition, a zero-adjusting group (containing DMSO solvent)The culture solution) of (4), 3 multiple wells were set for each group, and the effect on cells after addition of the drug was examined. After culturing the above groups of cells for 24h, 20 μ L of 5mg/mLMTT was added to each well of cells at 37 deg.C with 5% CO₂After further incubation for 4h under the conditions, the culture was terminated, the liquid in the wells was aspirated, 100. mu.L of dimethyl sulfoxide (DMSO) was added to each well, shaking was carried out for 10min to dissolve the intracellular crystals sufficiently, the absorbance (A) value of each well was measured at a wavelength of 450nm with a microplate reader, the cell survival rate was calculated, and the cell survival rate was (AH ═ cell survival rate)₂O₂Lesion-a blank)/(a control-a blank).

2.3 detecting ROS in SH-SY5Y cells and IMR-32 cells by DCFH-DA method, incubating each group of cells for 24h after corresponding substances are given, incubating for 30min before incubation is finished, adding DCFH-DA into each hole to enable the final concentration to be 10 mu mol/L, continuing incubating for 30min at 37 ℃, collecting cells, washing for 2 times by PBS, counting the cells, and preparing each group of cells into cell suspension with the same concentration. And (3) taking 100 mu L of cell suspension to detect the fluorescence intensity, wherein the excitation wavelength is 485nm, and the emission wavelength is 538 nm. The change in intracellular ROS was calculated by comparing the fluorescence intensity of the control group to 100% and the fluorescence intensity of the remaining groups to that of the control group.

2.4 measurement of LDH Release amount by INT color reaction method, except for the control group and H₂O₂And (3) additionally setting a blank control group (the blank control group is not inoculated with cells) outside the damage model group and the experimental group, adding corresponding substances into the cells of each group for culturing for 24h, taking 120 mu L of supernatant of each well to a new 96-well plate, adding 60 mu L of prepared LDH detection working solution, incubating for 30min at the dark room temperature, measuring the A value by using a multifunctional microplate reader at 490nm, and calculating the percentage of LDH release amount relative to a control tube. LDH release rate ═ (a dose-a blank)/(a control-a blank)

3, experimental results.

The results of the cell relative survival experiments are shown in table 3.

TABLE 3 Effect of the present invention on the relative survival rates of human neuroblastoma cell lines SH-SY5Y and IMR-32

Note:^*P<0.05 and H₂O₂And comparing the damage model groups.

The results of ROS measurement in SH-SY5Y cells and IMR-32 cells are shown in Table 4.

TABLE 4 Effect of the present invention on the intracellular ROS levels of human neuroblastoma cell lines SH-SY5Y and IMR-32

Note:^*P<0.05 compared with the control group,^#P<0.05 and H₂O₂The results of the damage model group comparing the effects of LDH release in SH-SY5Y cells and IMR-32 cells are shown in Table 5.

TABLE 5 Effect of the present invention on the intracellular LDH Release from human neuroblastoma cell lines SH-SY5Y and IMR-32

Note:^*P<0.05 compared with the control group,^#P<0.05 and H₂O₂And comparing damage model groups.

In conclusion, the invention provides an alkaloid compound and an extraction and separation method thereof, which are characterized in that a new alkaloid compound is successfully separated and obtained by sequentially adopting water reflux extraction, silica gel column chromatography, polyamide column chromatography, ODS medium-pressure column chromatography, sephadex column chromatography separation and purification and HPLC separation preparation.

Claims

1. An indoline carboxylic acid alkaloid compound separated from a purslane medicinal material is characterized in that the molecular formula is as follows: c₉H₉NO₄The chemical name is 4, 5-dihydroindoline-1-carboxylic acid, and the chemical structural formula is as follows:

。

2. the method for extracting and separating alkaloid compounds according to claim 1, comprising the following steps:

step 1, taking dry purslane medicinal materials, decocting and extracting the medicinal materials by adopting water, and cooling the medicinal materials to room temperature to obtain liquid medicine for later use;

step 2, evaporating the liquid medicine obtained in the step 1 to dryness, then putting the liquid medicine into a silica gel column, eluting the liquid medicine by using ethyl acetate, and recovering the ethyl acetate under reduced pressure to obtain an extract, thus obtaining an ethyl acetate extract;

step 4, separating the product obtained in the step 3 by a polyamide column, performing gradient elution by adopting ethanol-water, and evaporating 30% ethanol partially for later use;

step 5, separating the product obtained in the step 4 by pretreated ODS column chromatography, performing gradient elution by using methanol-water to obtain a plurality of elution parts, detecting by using thin-layer chromatography, developing, and concentrating the developed elution parts under reduced pressure to dryness to obtain a concentrate for later use;

step 6, subjecting the product obtained in the step 5 to chromatography separation by a pretreated sephadex column, eluting by methanol to obtain a plurality of elution parts, detecting by a thin-layer chromatography, developing, combining the developed elution parts, and concentrating the combined elution parts under reduced pressure to dryness for later use;

and 7, performing HPLC separation preparation on the concentrate obtained in the step 6, wherein the volume ratio of acetonitrile to 0.1% formic acid is 13: 87 as mobile phase, and preparing the indoline carboxylic acid alkaloid compound in the purslane.

3. The extraction and separation method of claim 2, wherein in the step 1, the water is decocted and extracted for 2 times, each time for 2 hours, and the water amount is 8-16 times of that of the medicinal materials.

4. The extraction separation method according to claim 2, wherein said ODS and sephadex pretreatment is performed by soaking in methanol for 24 hours, loading on a column, washing with methanol until no turbidity is observed in the dropping water, and then equilibrating with an initial mobile phase.

5. The extraction separation method according to claim 2, wherein the mobile phase elution procedure used in step 2 is isocratic elution.

6. The extraction separation method according to claim 2, wherein the volume ratio of water to ethanol in the steps 3 and 4 is 100: 0,70: 30, 50: 50, 30: 70 and 0: elution with 100 gradient; the volume ratio of the ethyl acetate to the methanol in the step 3 is 5:1,2: 1 and 1:2, gradient elution; in the step 5, the volume ratio of methanol to water is 30: 70, 40: 60, 50: 50, 60: 40, 70: 30 and 100: elution was performed with a gradient of 0.

7. The extraction separation method according to claim 2, wherein the methanol elution procedure in step 6 is isocratic elution.

8. Use of a compound according to claim 1 for the preparation of an antitumor medicament.

9. Use of a compound according to claim 1 for the preparation of a neuroprotective medicament.