CN117777150A - Alkaloid compound and preparation method and application thereof - Google Patents
Alkaloid compound and preparation method and application thereof Download PDFInfo
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- CN117777150A CN117777150A CN202211153365.9A CN202211153365A CN117777150A CN 117777150 A CN117777150 A CN 117777150A CN 202211153365 A CN202211153365 A CN 202211153365A CN 117777150 A CN117777150 A CN 117777150A
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- ethyl acetate
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 25
- -1 Alkaloid compound Chemical class 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 19
- 206010039073 rheumatoid arthritis Diseases 0.000 claims abstract description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
- 239000012046 mixed solvent Substances 0.000 claims description 19
- 239000000284 extract Substances 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 14
- 239000003208 petroleum Substances 0.000 claims description 14
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 11
- 150000001875 compounds Chemical class 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 238000004809 thin layer chromatography Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000002024 ethyl acetate extract Substances 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 5
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 5
- 239000002808 molecular sieve Substances 0.000 claims description 5
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 3
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical compound OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000049 pigment Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 5
- 229940079593 drug Drugs 0.000 abstract description 16
- 210000002437 synoviocyte Anatomy 0.000 abstract description 12
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 12
- 229960000485 methotrexate Drugs 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 7
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 239000003513 alkali Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 235000020971 citrus fruits Nutrition 0.000 description 3
- 239000000287 crude extract Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 241001081440 Annonaceae Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- 230000003356 anti-rheumatic effect Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001647 drug administration Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LTGIHGCXTGSMTA-UHFFFAOYSA-N 3-(4,5-dimethyl-1h-pyrrol-2-yl)-2,5-diphenyl-1h-tetrazol-1-ium;bromide Chemical compound [Br-].N1C(C)=C(C)C=C1N1N(C=2C=CC=CC=2)[NH2+]C(C=2C=CC=CC=2)=N1 LTGIHGCXTGSMTA-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001189830 Fissistigma Species 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 238000010262 high-speed countercurrent chromatography Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an alkaloid compound, a preparation method and application thereof, and the alkaloid compound is extracted from radix fissistigmae; the novel alkaloid compound is extracted from the radix fissistigmae, has a good inhibition effect on synovial cells, has a good effect of resisting rheumatoid arthritis, and can be used for preparing medicines for resisting the rheumatoid arthritis.
Description
Technical Field
The invention relates to the field of phytochemistry, in particular to an alkaloid compound, and a preparation method and application thereof.
Background
Radix seu folium Fissistigmatis OldhamiiFissistigma oldhamii (hemsl.) merr.) is an Annonaceae (Annonaceae) plant of the genus Fissistigma. The plant rhizome is a famous Chinese medicinal material "Zuanshanfeng" and can treat rheumatic osteodynia, numbness of hands and feet, etc.
Chinese patent document CN (application number 201510763189.4) discloses a fragrant-leaf-herb extract, a preparation method and application thereof, wherein the fragrant-leaf-herb extract is prepared by the following method: drying and crushing the raw materials of the fragrant citrus fruits, heating and reflux-extracting the raw materials by using alcohol, filtering the raw materials to obtain an extracting solution, concentrating the extracting solution under reduced pressure to obtain an alcohol crude extract, diluting the alcohol crude extract by adding water, extracting the alcohol crude extract by using an organic solvent, concentrating the extracting solution or the residual water solution after the extraction under reduced pressure to obtain extractum respectively, and vacuum-drying the extractum to obtain powder, namely the organic solvent extract of the fragrant citrus fruits or the water extract of the fragrant citrus fruits; wherein the organic solvent is any one or the combination of more of petroleum ether, chloroform and ethyl acetate; and describes the application of the radix Fissistigmatis Oldhamii extract in preparing medicines for treating rheumatoid arthritis. The petroleum ether extraction site, chloroform extraction site and aqueous phase site of this patent have good efficacy and can be used for pharmaceutical purposes, but the specific chemical components of these petroleum ether extraction site, chloroform extraction site and aqueous phase site are not known in the art.
Chinese patent document CN102241682A (application No. 201110117935.4) discloses a preparation method of the radix Fissistigmatis Oldhamii, which comprises (1) crushing radix Fissistigmatis Oldhamii, reflux-extracting with ethanol or percolating, concentrating the extractive solution to small volume, adding hydrochloric acid to adjust to acidity, filtering to remove insoluble substances, adjusting acid liquor to alkali, extracting with dichloromethane, and recovering reagent from the extractive solution to obtain radix Fissistigmatis Oldhamii total alkali; (2) Separating and purifying the total alkali of the radix Fissistigmatis Oldhamii by high-speed countercurrent chromatography to obtain radix Fissistigmatis Oldhamii alkali A.
Disclosure of Invention
The invention aims to provide an alkaloid compound extracted from radix fissistigmae, and a preparation method and application thereof.
The technical scheme for realizing the first purpose of the invention is an alkaloid compound with the following structural formula:
。
the technical scheme for realizing the second purpose of the invention is that the preparation method of the alkaloid compound comprises the following steps:
(1) extracting dried stems of radix Fissistigmatis Oldhamii with ethanol to obtain extractive solution, and concentrating under reduced pressure to obtain radix Fissistigmatis Oldhamii extract.
(2) Diluting the radix Fissistigmatis Oldhamii extract obtained in the step (1) with water to obtain suspension, sequentially extracting with petroleum ether and ethyl acetate, mixing the ethyl acetate extracts, and concentrating to obtain extract.
(3) Subjecting the ethyl acetate extract obtained in the step (2) to silica gel column chromatography, subjecting to petroleum ether-ethyl acetate mixed solvent (100:0-0:100, V/V) and ethyl acetate-methanol (100:0-0:100, V/V) to silica gel column chromatography according to increasing polarity, collecting fractions about 1000 mL each time, and combining similar fractions into 8 components by TLC detection, wherein the components are Fr. -8.
(4) Eluting the component Fr.3 obtained in the step (3) by using a 300-400 mesh silica gel column and an ethyl acetate-petroleum ether mixed solvent as an eluent; carrying out thin layer chromatography with methanol-chloroform mixed solvent as developing agent, and mixing the fractions according to the chromatography effect to obtain 3 components, which are Fr.3-1, fr.3-2 and Fr.3-3 respectively.
(5) And (3) performing molecular sieve Sephadex LH-20 column chromatography with chloroform-methanol mixed solvent as eluent on the Fr.3-2 obtained in the step (4), removing pigment to obtain a component Fr.3-2-1, and purifying Fr.3-2-1 by high performance liquid chromatography to obtain the alkaloid compound of claim 1.
And (3) concentrating under reduced pressure, wherein the temperature is 30-70 ℃ and the pressure is-0.06-0.15 MPa.
And (2) concentrating at the temperature of 30-70 ℃ and the pressure of-0.06 to-0.15 MPa.
In the step (4), the ethyl acetate-petroleum ether mixed solvent with the volume percent of 20 percent is used as an eluent for elution, and the methanol-chloroform mixed solvent with the volume percent of 5 percent is used as a developing agent for thin layer chromatography.
In the step (5), a chloroform-methanol mixed solvent with the volume percent of chloroform being 50 percent is used as an eluent to carry out molecular sieve Sephadex LH-20 column chromatography.
In the step (5), when Fr.3-2-1 is analyzed and purified by high performance liquid chromatography, the eluent is acetonitrile and water, and the volume ratio is 65:35.
The technical scheme for realizing the third purpose of the invention is the application of the compound in preparing medicines for resisting rheumatoid arthritis.
The invention has the positive effects that: the novel alkaloid compound is extracted from the radix fissistigmae, has a good inhibition effect on synovial cells, has a good effect of resisting rheumatoid arthritis, and can be used for preparing medicines for resisting the rheumatoid arthritis.
Drawings
FIG. 1 is a nuclear magnetic resonance H-spectrum of the compound extracted in example 1.
FIG. 2 is a nuclear magnetic resonance C-spectrum of the compound extracted in example 1.
FIG. 3 is an HMBC spectrum of the compound extracted in example 1.
Detailed Description
Example 1
The alkaloid compound of the embodiment is extracted from the radix fissistigmatis Oldhamii, and the extraction process is as follows:
(1) 8kg of dried stems of the radix Fissistigmatis Oldhamii are leached with 30-95% v/v (75% v/v in this example) ethanol for 3 times and 7 days each time to obtain an extract, and the extract is concentrated under reduced pressure to obtain 1000g of radix Fissistigmatis Oldhamii extract.
The temperature is 30-70 ℃ and the pressure is-0.06 to-0.15 MPa during the reduced pressure concentration; in this example, the temperature was 45℃and the pressure was-0.095 MPa.
(2) Diluting the radix Fissistigmatis Oldhamii extract obtained in the step (1) with 3L distilled water to obtain suspension, sequentially extracting with petroleum ether (3 L×3 times, 3L each time), and ethyl acetate (3 L×3 times, 3L each time), mixing the ethyl acetate extracts, and concentrating to obtain extract of about 256g.
The temperature is 30-70 ℃ and the pressure is-0.06 to-0.15 MPa during concentration; in this example, the temperature was 45℃and the pressure was-0.095 MPa.
(3) And (3) performing silica gel column chromatography on the ethyl acetate extract obtained in the step (2), performing silica gel column chromatography on the ethyl acetate extract with 200-300 meshes by using a petroleum ether-ethyl acetate mixed solvent (100:0-0:100, V/V) and ethyl acetate-methanol (100:0-0:100, V/V) according to polarity increment, and collecting fractions according to about 1000 mL each time.
The combined similar fractions were separated into 8 fractions as measured by TLC, fr. 1-8. Thin layer chromatography is carried out by taking a methanol-chloroform mixed solvent with the volume percent of 5 percent of methanol as developing agent.
(4) And (3) eluting the component Fr.3 obtained in the step (3) by using a 300-400 mesh silica gel column, eluting by using an ethyl acetate-petroleum ether mixed solvent with the volume percent of 20% as an eluent, and performing thin-layer chromatography by using a methanol-chloroform mixed solvent with the volume percent of 5% as a developing agent, wherein the components Fr.3-1, fr.3-2 and Fr.3-3 are obtained by combining the components according to the chromatography effect.
(5) And (3) performing molecular sieve Sephadex LH-20 column chromatography on Fr.3-2 obtained in the step (4) by taking chloroform-methanol mixed solvent with the volume percentage of chloroform as an eluent, removing pigment to obtain a component Fr.3-2-1, performing analysis and purification on Fr.3-2-1 by using high performance liquid chromatography, wherein the eluent of the high performance liquid chromatography is acetonitrile and water (the volume ratio of the eluent is 65:35), and obtaining a novel alkaloid compound which is named as stephorine A. The alkaloid compound obtained in this example was white powder.
The nuclear magnetic resonance H spectrum of the compound is shown in figure 1, the nuclear magnetic resonance C spectrum is shown in figure 2, and the HMBC spectrum is shown in figure 3.
High resolution Mass Spectrometry display [ M+H ]] + 282.1128 with molecular formula C 17 H 15 NO 3 。
1 H NMR (400 MHz, MeOD-d 4 ) δ7.20, 7.17, 7.05, 7.03, 6.68, 6.41, 6.39, 6.29, 6.27, 6.14, 4.56, 4.54, 4.52, 3.81, 3.61, 3.59, 3.57, 3.56, 3.30, 3.28, 3.26, 3.24, 2.93, 2.92, 2.48, 2.46, 2.45, 2.43, 2.42, 2.40, 2.37, 2.34。
13 C NMR (101 MHz, MeOD-d4)) δ187.94, 155.48, 152.07, 146.93, 145.29, 133.01, 128.57, 128.40, 128.34, 127.52, 113.07, 100.92, 57.63, 52.14, 46.72, 44.75, 25.24。
According to 1 HNMR， 13 CNMR shows that the compound has skeleton of stephorine alkaloid, and has-OCH in HMBC spectrum 2 O is related to the carbon at the 1,2 position and is consistent with the known compound stephorine data except that two methoxy groups at the 1,2 positions are reduced. The structure of this compound can be determined as follows and is designated stephorine A.
The structural identification results of stephorine a are as follows:
。
(Experimental example)
1. Experimental materials and instruments
And (3) cells: synovial cells (primary rat synovial cells).
Cell culture fluid: primary rat synovial cell culture medium.
Reagent: thiazole blue (3- (4, 5-dimethylazol-2-yl) -2,5-diphenyl tetrazolium bromide, MTT, manufactured by Sigma). Lactate dehydrogenase (lactate dehydrogenase, LDH) kit (from bi yun tian). Methotrexate (methot rexate, MTX, shanghai pharmaceutical Co., ltd., lot 20140307); stephorine a extracted in example 1.
Instrument: 96-well cell culture plates; an infinite 200Pro multifunctional enzyme labeling instrument.
2. Experimental method
(1) MTT method for determining inhibition of medicine on synovial cells
Synovial cells were seeded into 96-well plates (1X 10) 4 Individual cells/well), set as blank (no drug administered), methotrexate (1 μg/mL methotrexate administered), dosing (10, 50, 100 μg/mL drug administered), and incubation overnight for the experiment. After cells were given different concentrations of drug culture 48 h, 10 μl of 5 mg/mL MTT was added to each well, and after further culturing 4 h, culture supernatant was carefully aspirated and discarded, 100 μl of DMSO was added to each well to dissolve formazan crystals, shaking at room temperature for 5 min, and after complete dissolution, optical density (OD value) was detected at 570 nm with an enzyme-labeled instrument. Cell viability was calculated from OD values:
。
(2) LDH activity assay to determine the effect of drugs on synoviocyte viability
Slide for taking log phaseMembrane cells were seeded into 96-well plates (1X 10) 4 Individual cells/well), set as blank group (no drug administration), methotrexate group (1 μg/mL of methotrexate administration), dosing group (10, 50, 100 μg/mL of drug administration), culture and incubation overnight, after the end of each cell culture, cell supernatant was taken and LDH activity was detected by LDH detection kit. The viability of LDH was calculated according to the following formula:
。
3. statistical method
Experimental data were analyzed using SPSS 22.0 statistical software. The result is expressed by mean ± standard deviation%) The differences between groups are analyzed using one-way ANOVA. To be used forP<0.05 was considered to have statistical differences.
4. Experimental results
(1) MTT method for determining inhibition of medicine on synovial cells
Cell viability was 97.18%, 31.13%, 48.12%, 41.33%, 34.25% for the blank group (no drug), the methotrexate group (1 μg/mL of methotrexate), the dosed group (10, 50, 100 μg/mL of drug).
IC of methotrexate, stephorine A 50 The values are respectively: 1.2 And [ mu ] M and 9.8 [ mu ] M.
The result shows that the compound stephorine A has good inhibition effect on synovial cells.
(2) LDH activity assay to determine the effect of drugs on synoviocyte viability
After 48 and h cultures with different concentrations of drug, cell viability was measured using the LDH activity assay.
The effect rates of LDH on cell activity were 98.2%, 94.16%, 95.38%, 91.12%, 89.28% for the blank group (no drug), the methotrexate group (1 μg/mL of methotrexate), and the dosing group (10, 50, 100 μg/mL of drug).
The results showed that the LDH activity in the cell culture fluid was not greatly biased after the treatment with different concentrations of the drug.
The results show that the alkaloid compound stephorine A extracted by the invention has good inhibition effect on synovial cells and has small influence on LDH activity in cell culture solution. Experiments show that the alkaloid compound has good anti-rheumatoid arthritis drug effect and can be used for preparing anti-rheumatoid arthritis drugs.
Claims (8)
1. An alkaloid compound has the structural formula as follows:
。
2. a process for the preparation of the alkaloid compounds of claim 1, characterized by comprising the steps of:
(1) extracting dried stems of radix Fissistigmatis Oldhamii with ethanol to obtain extractive solution, concentrating under reduced pressure to obtain radix Fissistigmatis Oldhamii extract;
(2) diluting the radix Fissistigmatis Oldhamii extract obtained in the step (1) with water to obtain suspension, sequentially extracting with petroleum ether and ethyl acetate, mixing the ethyl acetate extracts, and concentrating to obtain extract;
(3) subjecting the ethyl acetate extract obtained in the step (2) to silica gel column chromatography, subjecting the ethyl acetate extract to silica gel column chromatography with petroleum ether-ethyl acetate mixed solvent (100:0-0:100, V/V) and ethyl acetate-methanol (100:0-0:100, V/V) according to increasing polarity, collecting fractions about 1000 mL each time, and combining similar fractions into 8 components by TLC detection, wherein the components are Fr. -8;
(4) eluting the component Fr.3 obtained in the step (3) by using a 300-400 mesh silica gel column and an ethyl acetate-petroleum ether mixed solvent as an eluent; carrying out thin layer chromatography by using methanol-chloroform mixed solvent as developing agent, and combining the fractions according to the chromatography effect to obtain 3 components, which are Fr.3-1, fr.3-2 and Fr.3-3 respectively;
(5) and (3) performing molecular sieve Sephadex LH-20 column chromatography with chloroform-methanol mixed solvent as eluent on the Fr.3-2 obtained in the step (4), removing pigment to obtain a component Fr.3-2-1, and purifying Fr.3-2-1 by high performance liquid chromatography to obtain the alkaloid compound of claim 1.
3. The method for producing an alkaloid compound according to claim 2, characterized in that: and (3) concentrating under reduced pressure, wherein the temperature is 30-70 ℃ and the pressure is-0.06-0.15 MPa.
4. The method for producing an alkaloid compound according to claim 2, characterized in that: and (2) concentrating at the temperature of 30-70 ℃ and the pressure of-0.06 to-0.15 MPa.
5. The method for producing an alkaloid compound according to claim 2, characterized in that: in the step (4), the ethyl acetate-petroleum ether mixed solvent with the volume percent of 20 percent is used as an eluent for elution, and the methanol-chloroform mixed solvent with the volume percent of 5 percent is used as a developing agent for thin layer chromatography.
6. The method for producing an alkaloid compound according to claim 2, characterized in that: in the step (5), a chloroform-methanol mixed solvent with the volume percent of chloroform being 50 percent is used as an eluent to carry out molecular sieve Sephadex LH-20 column chromatography.
7. The method for producing an alkaloid compound according to claim 6, wherein: in the step (5), when Fr.3-2-1 is analyzed and purified by high performance liquid chromatography, the eluent is acetonitrile and water, and the volume ratio is 65:35.
8. Use of a compound according to claim 1 for the preparation of a medicament for the treatment of rheumatoid arthritis.
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| CN117777150A true CN117777150A (en) | 2024-03-29 |
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