CN108690023B - Diterpene alkaloid compound in Setatarian delphinium flower and preparation method and application thereof - Google Patents
Diterpene alkaloid compound in Setatarian delphinium flower and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to diterpene alkaloid compound in setaria segetalis, a preparation method and application thereof, and the application is realized by using the setaria segetalis (L.) (Delphinium pseudoaemulans C, Y, Yang et B, Wang) as raw material, extracting with solvent, treating with acid and alkali, extracting with solvent, separating by two, three or four ways of silica gel column chromatography, preparative thin layer chromatography, Sephadex LH-20 column chromatography or high performance liquid chromatography, and detecting and analyzing by thin layer chromatography or high performance liquid chromatography to obtain the new diterpene alkaloid compounds. And the compound is subjected to in vitro tumor cytotoxic activity determination, and the experimental result shows that: diterpenoid alkaloid compounds separated from Setaria delphinium show antitumor activities of different degrees on human lung cancer A549 cells and human cervical cancer Hela cells, and can be used for preparing antitumor drugs.
Description
Technical Field
The invention relates to the field of phytochemistry, in particular to diterpene alkaloid compounds in plant medicinal materials from Delphinium stauntonii as well as a preparation method and application thereof.
Background
Delphinium (Delphinium) belongs to Ranunculaceae (Ranunculus) plants, and has more than 300 varieties all over the world, is widely distributed in northern temperate, has about 140 varieties in China, is distributed in China except Taiwan and Hainan provinces, is a main distribution area in southwest and northwest, and has 26 varieties and 3 varieties in Xinjiang. 18 Chinese medicinal plants are used for folk medicine and are used for treating traumatic injury, rheumatism, toothache, enteritis and the like. And 4 other pesticides are used for killing louse, mosquito and fly larvae.
The qualitative chemical components in the plants include diterpene alkaloids, flavonoids, sterols, fatty acids, etc. The main component is diterpenoid alkaloid which is not only considered as an effective component and a toxic component, but also is a characteristic chemical component, has wide biological activity, and particularly has obvious effects on anti-inflammation, analgesia, arrhythmia resistance, tumor resistance and the like.
The Setatachalata is grown in Saururi mountain, mountain slope grassland with the altitude of 1900 m, bush and valley tidal wetland, produced in Buckessel county, belongs to a special plant in Xinjiang, and has obvious regional specificity, but the chemical components of the Setatachalata are not reported. Therefore, the systematic and deep research on diterpenoid alkaloid components in the flowers of the Delphinium genus is carried out, the pharmacological active substance basis is clarified, and the discovery of a new diterpenoid alkaloid compound with specific activity and a high-efficiency low-toxicity drug lead compound is of great significance.
Disclosure of Invention
The invention aims to provide diterpenoid alkaloid compounds in the Delphinium staphinium flower as well as a preparation method and application thereof, wherein the Delphinium staphinium flower (Delphinium pseudo aemulans C.Y. Yang et B.Wang) is used as a raw material, and is separated by two, three or four modes of silica gel column chromatography, preparative thin layer chromatography, sephadex LH-20 column chromatography or high performance liquid chromatography (pHPLC), and is detected and analyzed by adopting the thin layer chromatography or the High Performance Liquid Chromatography (HPLC), so that the novel diterpenoid alkaloid compounds are obtained, and the in vitro tumor cell toxicity activity of the compounds is determined, and the experimental result shows that: diterpenoid alkaloid compounds separated from Setaria delphinium show antitumor activities of different degrees on human lung cancer A549 cells and human cervical cancer Hela cells, and can be used for preparing antitumor drugs.
The invention relates to a diterpene alkaloid compound in Setaria viridis, which has the structural formula as follows:
wherein:
compound 2 is named as saturnine b (pseudomorphine b);
compound 3 is named as sertraline c (pseudomorphine c);
the name of the compound 5a is sertraline A (pseudorene A);
the name of the compound 5b is sertraline B (pseudorene B);
compound 6 is named setadine a (pseudoonidine a);
compound 7 is named setadine b (pseudoonidine b);
compound 8 is named setadine c (pseudoonidine c);
compounds 5a and 5b are a pair of regioisomeric mixtures.
The preparation method of the diterpene alkaloid compound in the Setaria delphinii flower comprises the following steps:
a. pulverizing whole plant of Setaria viridis, extracting with 10-95% ethanol water solution, methanol or chloroform at room temperature by cold soaking or percolation method, heating under reflux or ultrasonic extraction, and concentrating under reduced pressure to recover solvent to obtain extract;
b. dispersing the extract obtained in the step a by using sulfuric acid with the mass fraction of 1-5%, anhydrous sodium carbonate or ammonia water to obtain an acid water layer, and adding Na2CO3Adjusting pH to 10, extracting with organic solvent such as chloroform, ethyl acetate, diethyl ether or n-butanol, and concentrating under reduced pressure to recover organic solvent to obtain total alkaloids;
c. separating the total alkaloids obtained in step b by silica gel column chromatography, preparative thin layer chromatography, Sephadex LH-20 column chromatography or preparative high performance liquid chromatography;
d. detecting and analyzing by thin layer chromatography or high performance liquid chromatography to obtain new diterpene alkaloid compounds.
The silica gel column chromatography in the step c is normal pressure or pressurized column chromatography, the used filler is forward silica gel or reverse silica gel, and chloroform and methanol are used in the volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0: 1; 0.2 percent of petroleum ether, acetone and diethylamine in the volume ratio of 20:1,10:1,5:1,2:1,1:1 and 0: 1; or methanol and water in a volume ratio of 1:9-1:0 are used as eluent, and isocratic or gradient elution is adopted.
And c, performing normal-pressure chromatography by using the thin-layer chromatography in the step c, wherein a developing system comprises a mixture of chloroform and methanol, a mixture of chloroform, methanol and water and a mixture of petroleum ether and acetone saturated by ammonia water.
And c, eluting the sephadex LH-20 column chromatography by using chloroform-methanol with the volume ratio of 10:1, and performing isocratic elution.
And c, eluting the eluent of the high performance liquid chromatography in the step c by using acetonitrile-formic acid aqueous solution with the volume ratio of 10:90, and performing isocratic or gradient elution.
The application of diterpene alkaloid compounds in the Setatariella asiatica in preparing anti-tumor drugs is provided.
The diterpene alkaloid compound in the Setaria viridis disclosed by the invention is subjected to in vitro tumor cell toxicity activity determination on the obtained new diterpene alkaloid compound, and the experimental result shows that: diterpene alkaloid compounds separated from Setaria viridis show antitumor activities of different degrees on human lung cancer A549 cells and human cervical cancer Hela cells, and can be used for preparing antitumor drugs.
The diterpene alkaloid compound in the Setatariella asiatica is obtained by separating and purifying from plants, wherein:
Compound 2 is cetuxine B, a white amorphous powderHRESI (+) MS (M/z 452.2632[ M ]]+Theoretical value 452.2643) determined to have the molecular formula C24H38NO7 +(ii) a According to1H，13C and two-dimensional nuclear magnetic resonance data, and the structure is named as pseudomorphine B1H and13c NMR data are shown in Table 1[600MHz ] (1H)，150MHz(13C) The solvent: CDCl3]。
Compound 3, named as Setarine C, is a white amorphous powder prepared by HRESI (+) MS (M/z 492.2959[ M ]]+Theoretical value 492.2956) determined to have the molecular formula C27H42NO7 +(ii) a According to1H，13C and two-dimensional nuclear magnetic resonance data to determine the structure, and the name is pseudomorphine C1H and13c NMR data are shown in Table 1[600MHz ] (1H)，150MHz(13C) The solvent: CDCl3]。
Compound 5a named Saetanin A and compound 5B named Saetanin B (regioisomeric mixture) were white amorphous powders obtained by HRESI (+) MS (M/z 725.3638[ M)]+Theoretical value 725.3644) determined to have the molecular formula C39H53N2O11 +(ii) a According to1H，13C and two-dimensional nuclear magnetic resonance data, and the structure is named as pseudorene A-B1H and13CNMR data are ascribed to Table 2[600 MHz: (C:)1H)，150MHz(13C) The solvent: CDCl3]。
Compound 6, named Setadine A, is a white amorphous powder obtained by HRESI (+) MS (M/z 464.2645[ M + H ]]+Theoretical value 464.2643) determined to have the molecular formula C25H37NO7(ii) a According to1H，13C and two-dimensional NMR data to determine its structure, named pseudoonidine A, which1H and13c NMR data are shown in Table 3[600MHz ] (1H)，150MHz(13C) The solvent: CDCl3]。
Compound 7, named Satadine B, is a white amorphous powder obtained by HRESI (+) MS (M/z 450.2489[ M + H ]]+Theoretical value 450.2486) determined to have the molecular formula C24H35NO7(ii) a According to1H，13C and two-dimensional nuclear magnetic resonance data to determine its structure, which was named pseudoonidine B. It is composed of1H and13c NMR data are shown in Table 3[600MHz ] (1H)，150MHz(13C) The solvent: CDCl3]。
Compound 8, named Satadine C, is a white amorphous powder obtained by HRESI (+) MS (M/z 536.3223[ M + H ]]+Theoretical value 536.3218) determined to have the molecular formula C29H45NO8(ii) a According to1H，13C and two-dimensional nuclear magnetic resonance data determined its structure, which was named pseudoonidine C. It is composed of1H and13c NMR data are shown in Table 3[600MHz ] (1H)，150MHz(13C) The solvent: CDCl3]。
The invention relates to a structural identification of diterpenoid alkaloid compounds in Setaria viridis flower:
of compounds 1 to 3 of Table 11H (600MHz) and13c (150MHz) NMR data [ (ppm), J (Hz)]
TABLE 2 preparation of compounds 4 to 51H (600MHz) and13c (150MHz) NMR data [ (ppm), J (Hz)]
TABLE 3 preparation of compounds 6 to 81H (600MHz) and13c (150MHz) NMR data [ (ppm), J (Hz)]
Drawings
FIG. 1 shows the preparation of Compound 1 (Setarine A) of the present invention1H NMR chart;
FIG. 2 shows the preparation of Compound 1 (Setarine A) according to the present invention13C NMR chart;
FIG. 3 shows the preparation of Compound 2 (Setarine B) according to the present invention1H NMR chart;
FIG. 4 shows the preparation of Compound 2 (Setarine B) of the present invention13C NMR chart;
FIG. 5 shows preparation of Compound 3 (Setarine C) of the present invention1H NMR chart;
FIG. 6 shows preparation of Compound 3 (Setarine C) of the present invention13C NMR chart;
FIG. 7 shows preparation of Compound 4 (Setarine D) of the present invention1H NMR chart;
FIG. 8 shows preparation of Compound 4 (Setarine D) of the present invention13C NMR chart;
FIG. 9 shows Compounds 5a and 5b (cetearenin A) of the present invention&B) Is/are as follows1H NMR chart;
FIG. 10 shows Compounds 5a and 5b (cetearenin A) of the present invention&B) Is/are as follows13C NMR chart;
FIG. 11 shows preparation of Compound 6 (Setadine A) of the present invention1H NMR chart;
FIG. 12 shows the preparation of Compound 6 (sertraline A) according to the invention13C NMR chart;
FIG. 13 shows preparation of Compound 7 (Setadine B) of the present invention1H NMR chart;
FIG. 14 shows the preparation of Compound 7 (sertraline B) according to the invention13C NMR chart;
FIG. 15 shows preparation of Compound 8 (sertraline C) according to the invention1H NMR chart;
FIG. 16 shows Compound 8 (Setadine C) of the present invention13C NMR chart.
Detailed Description
All reagents used were analytical grade, and acetonitrile was HPLC grade in HPLC (Merk, usa). Column chromatography silica gel (100 meshes 200 meshes, 200 meshes 300 meshes): marine industrial production of Qingdao; thin layer chromatography of silica gel to GF254Production of yellow silica gel development test factory in cigarette platform market; sephadex LH-20 gel: manufactured by the Pharmacia Company. High performance liquid chromatography (DIONEX corporation, usa): p680HPLC pump, ASI-100 autosampler, TCC-100 column oven, UVD170U uv detector (four wavelength), quaternary eluent, online degasser, Chromeleon chromatography workstation. Preparative high performance liquid chromatography (DIONEX, usa): p680HPLC pump, UVD170U uv detector (four wavelengths), quaternary eluent, online degasser, Chromeleon chromatography station. Mass spectra were measured on a QSTAR Elite mass spectrometer (Applied Biosystems/MDS Sciex Co.); the NMR was measured by a Varian Vnmrs 600/400 NMR spectrometer (Varian Corp.).
The Setaria delphinii flowers were collected from Tacheng area of Uygur autonomous area of Xinjiang and Buckcel county, and were identified as Delphinium pseudo aemulans C.Y. Yang et B.Wang by Von tassel researchers, institute of ecology and geography, Sinkiang, academy of China.
Example 1
Taking 10.0kg of the whole plant of the Setaria delphinii flower, crushing, extracting by a 50L ethanol percolation method with the volume fraction of 80% at room temperature, and evaporating the solvent to dryness under reduced pressure to obtain a crude extract of the Setaria delphinii flower;
mixing the obtained crude extract with 2 vol% H2SO4The dispersion is carried out, and the dispersion,the acid aqueous layer obtained was washed with Na2CO3Adjusting pH to 10, extracting with chloroform, and evaporating chloroform under reduced pressure to obtain total alkaloid extract;
separating the obtained total alkaloid extract with silica gel column chromatography with normal phase silica gel as filler, gradient eluting with chloroform and methanol at volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1 as eluent, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 6 components A-F;
separating the obtained component B by normal phase silica gel column, and performing gradient elution by using petroleum ether-acetone + 0.2% diethylamine as eluent at the volume ratio of 20:1,10:1,5:1,2:1,1:1 and 0:1 to obtain 3 components B1-B3; separating the component B1 with normal phase silica gel column, and gradient eluting with chloroform-methanol at volume ratio of 40:1-0:1 as eluent to obtain compound cetuxine C; separating the component B2 with normal phase silica gel column, isocratic eluting with chloroform-methanol as eluent at volume ratio of 40:1, and further isocratic eluting with methanol-0.1% formic acid water at volume ratio of 35:65 with high performance liquid phase pHPLC to obtain compound cetradine A; separating the component B3 by normal phase silica gel column, performing gradient elution by using chloroform-methanol and 0.2% diethylamine as eluent in a volume ratio of 100:1-1:1 to obtain a compound sertraline A, a compound sertraline B and two components B3-2 and B3-3, preparing the compound sertraline C by using chloroform-methanol in a volume ratio of 10:1 as a developing agent through the component B3-2 by using a thin layer chromatography method, and obtaining the compound sertraline B by using chloroform-methanol in a volume ratio of 10:1 as a developing agent through the component B3-3 by using a thin layer chromatography method;
separating the obtained C component with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 60:1-1:1 to obtain 4 components C1-C4, and performing preparative thin layer chromatography on the component C-2 with chloroform-methanol-water as developing agent at volume ratio of 82:16:2 to obtain compound saturnine A;
separating the obtained E component and F component with normal phase silica gel column, and gradient eluting with chloroform-methanol at volume ratio of 60:1-1:1 as eluent to obtain components E1-E6 and F1-F10; separating the components E5 and F9 by normal phase silica gel column respectively, eluting with 5:1 petroleum ether-acetone + 0.2% diethylamine as eluent at equal volume ratio, further separating by thin layer chromatography, and obtaining compound Setarine D and Setarine B by chloroform-methanol-water as developing agent at 82:16:2 volume ratio; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures.
Example 2
Taking 10.0kg of the whole plant of the Setaria delphinii flower, crushing, performing reflux extraction at room temperature by using 50L of 95% ethanol, and evaporating the solvent to dryness under reduced pressure to obtain a crude extract of the Setaria delphinii flower;
the obtained crude extract is treated with 3 vol% H2SO4Dispersing to obtain acid water layer, adjusting pH to 10 with ammonia water, extracting with diethyl ether, and evaporating diethyl ether under reduced pressure to obtain total alkaloid extract;
separating the obtained total alkaloid extract with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 6 components A-F;
separating the obtained component B by normal phase silica gel column, and performing gradient elution by using petroleum ether-acetone + 0.2% diethylamine as eluent at the volume ratio of 20:1,10:1,5:1,2:1,1:1 and 0:1 to obtain 3 segments of components B1-B3; subjecting the component B1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain compound Setarine C; subjecting the component B2 to Sephadex LH-20 column chromatography, isocratically eluting with chloroform-methanol at volume ratio of 1:1, and isocratically eluting the obtained component with methanol-0.1% formic acid water at volume ratio of 35:65 by high performance liquid chromatography (pHPLC) to obtain compound setadin A; subjecting the component B3 to Sephadex LH-20 column chromatography, isocratic eluting with chloroform-methanol at a volume ratio of 1:2 to obtain compounds of cetosterin A and cetosterin B and two components B3-2 and B3-3, subjecting the component B3-2 to thin layer chromatography with chloroform-methanol at a volume ratio of 10:1 as a developing agent to obtain a compound of cetosterin C, subjecting the component B3-3 to thin layer chromatography with chloroform-methanol at a volume ratio of 10:1 as a developing agent to obtain a compound of cetosterin B;
separating the obtained C component with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 60:1-1:1 to obtain 4 components C1-C4, separating component C-2 with Sephadex LH-20 column, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain compound cetuxine A;
separating the obtained E component and F component with normal phase silica gel column, and gradient eluting with chloroform-methanol at volume ratio of 60:1-1:1 as eluent to obtain components E1-E6 and F1-F10; subjecting the components E5 and F9 to Sephadex LH-20 column chromatography, eluting with chloroform-methanol at a volume ratio of 1:2, performing thin layer chromatography, and separating to obtain compound Setarine D and Setarine B with chloroform-methanol-water at a volume ratio of 82:16:2 as developing agent; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures.
Example 3
Taking 10.0kg of the whole plant of the Setaria delphinii flower, crushing, extracting by a 50L ethanol percolation method with the concentration of 40% at room temperature, and evaporating the solvent to dryness under reduced pressure to obtain a crude extract of the Setaria delphinii flower;
the obtained crude extract is treated with 5% H2SO4Dispersing the obtained acid water layer with Na2CO3Adjusting pH to 10, extracting with ethyl acetate, and evaporating ethyl acetate under reduced pressure to obtain total alkaloid extract;
separating the obtained total alkaloid extract with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 6 components A-F;
subjecting the obtained component B to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain 3 components B1-B3; separating the component B1 with normal phase silica gel column, and performing gradient elution with chloroform-methanol as eluent at volume ratio of 40:1-0:1 to obtain compound Setarine C; separating the component B2 with normal phase silica gel column, isocratic eluting with chloroform-methanol as eluent at volume ratio of 40:1, and further isocratic eluting with high performance liquid phase pHPLC and methanol-0.1% formic acid water at volume ratio of 35:65 to obtain compound cetradine B; separating the component B3 by normal phase silica gel column, performing gradient elution with chloroform-methanol and 0.2% diethylamine as eluent at a volume ratio of 100:1-1:1 to obtain compound sertraline A, sertraline B and two components B3-2 and B3-3, performing preparative thin layer chromatography on the component B3-2, preparing compound sertidine C with chloroform-methanol at a volume ratio of 10:1 as developing agent, and performing preparative thin layer chromatography on the component B3-3, and obtaining compound sertidine B with chloroform-methanol at a volume ratio of 10:1 as developing agent;
subjecting the obtained C component to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain 4 components C-1 to C-4; preparing the component C-2 into a compound cetuxine A by a preparative thin layer chromatography method and taking chloroform-methanol-water with the volume ratio of 82:16:2 as a developing agent;
subjecting the component E and the component F to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:2 to obtain components E1-E6 and F1-F10; separating the components E5 and F9 by normal phase silica gel column respectively, performing gradient elution by using chloroform-methanol as eluent with volume ratio of 60:1-1:1 to obtain components E5-1 to E5-3 and F9-1 to F9-10, further separating the components E5-3 and F9-10 by thin layer chromatography respectively, and obtaining compound cetuxine D and compound cetuxine B by using chloroform-methanol-water as developing agent with volume ratio of 82:16: 2; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures.
Example 4
Taking 10.0kg of the whole plant of the Setaria delphinii flower, crushing, performing ultrasonic extraction with 50L of chloroform at room temperature, and evaporating the solvent to dryness under reduced pressure to obtain a crude extract of the Setaria delphinii flower;
mixing the obtained crude extract with 1% H2SO4Dispersing to obtain acid water layer, adjusting pH to 10 with ammonia water, extracting with n-butanol, and evaporating n-butanol under reduced pressure to obtain total alkaloid extract;
separating the obtained total alkaloid extract with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 6 components A-F;
performing ODS reverse silica gel column chromatography on the obtained component B, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain 3 sections of components B1-B3; separating the component B1 with normal phase silica gel column, and performing gradient elution with chloroform-methanol as eluent at volume ratio of 40:1-0:1 to obtain compound Setarine C; separating the component B2 with normal phase silica gel column, isocratic eluting with chloroform-methanol as eluent at volume ratio of 40:1, and isocratic eluting with methanol-0.1% formic acid water at volume ratio of 35:65 with high performance liquid phase pHPLC to obtain compound cetradine A; separating the component B3 by normal phase silica gel column, performing gradient elution by using chloroform-methanol and 0.2% diethylamine as eluent in a volume ratio of 100:1-1:1 to obtain a compound sertraline A, a compound sertraline B and two components B3-2 and B3-3, preparing the component B3-2 into a thin layer, preparing the compound sertraline C by using chloroform-methanol in a volume ratio of 10:1 as a developing agent, preparing the component B3-3 into a thin layer, and preparing the compound sertraline B by using chloroform-methanol in a volume ratio of 10:1 as a developing agent;
performing ODS reversed phase silica gel column chromatography on the obtained component C, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain 4 sections of components C-1 to C-4; preparing the component C-2 into a compound cetuxine A by using chloroform-methanol-water as a developing agent according to a volume ratio of 82:16:2 through a thin layer chromatography;
performing ODS reverse silica gel column chromatography on the obtained component E and component F respectively, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain components E1-E6 and F1-F10 respectively; separating the components E5 and F9 by normal phase silica gel column respectively, eluting with petroleum ether-acetone + 0.2% diethylamine at a volume ratio of 5:1 as eluent at equal speed, further separating by thin layer chromatography, and obtaining compound saturnine D and compound saturnine B by chloroform-methanol-water at a volume ratio of 82:16:2 as developing agents; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures.
Example 5
Taking 10.0kg of the whole plant of the Setaria delphinii flower, crushing, performing ultrasonic extraction with 50L of methanol at room temperature, and evaporating the solvent to dryness under reduced pressure to obtain a crude extract of the Setaria delphinii flower;
mixing the obtained crude extract with 1% H2SO4Dispersing to obtain acid water layer, adjusting pH to 10 with ammonia water, extracting with n-butanol, and evaporating n-butanol under reduced pressure to obtain total alkaloid extract;
separating the obtained total alkaloid extract with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 6 components A-F;
performing ODS reverse silica gel column chromatography on the obtained component B, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain 3 sections of components B1-B3; subjecting the component B1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain compound Setarine C; subjecting the component B2 to Sephadex LH-20 column chromatography, isocratically eluting with chloroform-methanol at a volume ratio of 1:1, and isocratically eluting the obtained component with methanol-0.1% formic acid water at a volume ratio of 35:65 by high performance liquid chromatography (pHPLC) to obtain compound setadin A; subjecting B3 to Sephadex LH-20 column chromatography, isocratic eluting with chloroform-methanol at volume ratio of 1:2 to obtain compound Setanine A, Setanine B and two components B3-2 and B3-3, subjecting B3-2 to thin layer chromatography with chloroform-methanol at volume ratio of 10:1 as developing agent to obtain compound Setanine C, subjecting component B3-3 to thin layer chromatography with chloroform-methanol at volume ratio of 10:1 as developing agent to obtain compound Setanine B;
performing ODS reversed phase silica gel column chromatography on the obtained component C, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain 4 segments of components C3-5-1 to C3-5-4; subjecting the component C3-5-1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:2 to obtain compound Setarine A;
performing ODS reverse silica gel column chromatography on the obtained component E and component F respectively, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain components E1-E6 and F1-F10 respectively; subjecting the components E5 and F9 to Sephadex LH-20 column chromatography, eluting with chloroform-methanol at a volume ratio of 1:2, performing thin layer chromatography, and separating to obtain compound Setarine D and compound Setarine B with chloroform-methanol-water at a volume ratio of 82:16:2 as developing agent; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures.
Example 6
Taking 10.0kg of the whole plant of the Setaria delphinii flower, crushing, performing ultrasonic extraction with 50L of chloroform at room temperature, and evaporating the solvent to dryness under reduced pressure to obtain a crude extract of the Setaria delphinii flower;
mixing the obtained crude extract with 1% H2SO4Dispersing to obtain acid water layer, adjusting pH to 10 with ammonia water, extracting with n-butanol, and evaporating n-butanol under reduced pressure to obtain total alkaloid extract;
separating the obtained total alkaloid extract with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing the fractions by silica gel Thin Layer Chromatography (TLC), and mixing the same fractions to obtain 6 components A-F;
separating the obtained component B by normal phase silica gel column, and performing gradient elution by using petroleum ether-acetone + 0.2% diethylamine as eluent at the volume ratio of 20:1,10:1,5:1,2:1,1:1 and 0:1 to obtain 3 segments of components B1-B3; subjecting the component B1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain compound Setarine C; subjecting the component B2 to Sephadex LH-20 column chromatography, isocratically eluting with chloroform-methanol at volume ratio of 1:1, and isocratically eluting the obtained component with methanol-0.1% formic acid water at volume ratio of 35:65 by high performance liquid chromatography (pHPLC) to obtain compound setadin B; subjecting B3 to Sephadex LH-20 column chromatography, isocratic eluting with chloroform-methanol at volume ratio of 1:2 to obtain compound Setanine A, Setanine B and two components B3-2 and B3-3, subjecting B3-2 to thin layer chromatography with chloroform-methanol at volume ratio of 10:1 as developing agent to obtain compound Setanine C, subjecting component B3-3 to thin layer chromatography with chloroform-methanol at volume ratio of 10:1 as developing agent to obtain compound Setanine B;
subjecting the obtained C component to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain 4 components C-1 to C-4; preparing the component C-2 into a compound cetuxine A by using chloroform-methanol-water as a developing agent according to a volume ratio of 82:16:2 through a thin layer chromatography;
performing ODS reverse silica gel column chromatography on the obtained component E and the component F respectively, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain components E1-E6 and F1-F10 respectively; subjecting the components E5 and F9 to Sephadex LH-20 column chromatography, eluting with chloroform-methanol at a volume ratio of 1:2, performing thin layer chromatography, and separating to obtain compound Setarine D and compound Setarine B with chloroform-methanol-water at a volume ratio of 82:16:2 as developing agent; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures.
Example 7
The application of diterpenoid alkaloid compounds in the Setatarian delphinium in the preparation of antitumor drugs is shown by taking human lung cancer cells A549 and human cervical cancer cells Hela as examples:
screening the tumor cell toxicity activity of the obtained diterpene alkaloid compounds:
experimental cells:
human lung adenocarcinoma cell a549 and human cervical carcinoma cell Hela, manufacturers: cell bank of Shanghai Life sciences college of Chinese academy of sciences
An experimental instrument:
carbon dioxide incubator, manufacturer: german Binder;
spectra Max M5 multifunctional microplate reader, manufacturer: molecular Devices, Inc. of USA;
inverted phase contrast microscope, manufacturer: leica, Germany;
constant temperature water tank, model: model LH586-1, manufacturer: shanghai Kelerian mechanical works;
electronic balance, model: sartorius BS 110S, manufacturer: beijing Sidolis balance, Inc.;
precision balance, type: PGL, manufacturer: edm weighing apparatus (wuhan) ltd;
experimental reagent:
dimethyl sulfoxide; specification: 500 mL; the characteristics are as follows: a colorless liquid; the manufacturer: shanghai Baisai Biotechnology Ltd; batch number: 1963C 070;
the experimental contents are as follows:
preparing a test sample: preparing diterpene alkaloid separated from Setaria viridis flower into stock solution with certain concentration by dimethyl sulfoxide (DMSO), and diluting before use;
detecting the growth inhibition effect of diterpene alkaloid compounds with different concentrations and gradients on human lung cancer A549 cells and human cervical carcinoma Hela cells by using a 3- (4, 5-dimethylthiazole-2, 5-diphenyl tetrazole bromide (MTT) colorimetric method, taking A549 and Hela cells in a logarithmic growth phase, digesting the A549 and Hela cells by using 0.25 percent pancreatin protease, preparing the cells into a suspension by using a high pond culture medium containing 10 percent fetal calf serum, counting the cells, inoculating the suspension into a 96-well plate at the temperature of 37 ℃ and the concentration of 5 percent CO in a volume of 100 mu L in each well, and inoculating the suspension into the 96-well plate at the temperature of 37 ℃ and the concentration of 5 percent CO in a volume2Culturing for 24h under the condition, discarding supernatant after 24h, adding medicine to final concentration of 10, 25, 50, 75 and 100 μ g/ml, setting 5 repeat wells for each medicine concentration, and setting zeroing well, control well and anti-tumor positive medicine (adriamycin, DOX); continuously culturing cells for 48h at 37 ℃ in carbon dioxide with the concentration of 5%, discarding supernatant after 48h, adding 10 mu L of MTT solution with the final concentration of 5mg/mL, continuously culturing for 4h at 37 ℃, discarding supernatant, adding 150 mu L of dimethyl sulfoxide (DMSO) solution, vibrating for 10s, measuring OD value at 570nm to indirectly react with the anticancer activity of the compound, calculating the inhibition rate of the compound on A549 and Hela cells, and calculating the IC of the compound by SPSS software50A value;
cell growth inhibition (%) - (control group OD value-addition group OD value)/(control group OD value-blank group OD value)
Experimental results table 4 shows:
TABLE 4 cytotoxic Activity of novel diterpenoid alkaloid Compounds in Delphinium Setataricum
The results show that: the diterpenoid alkaloid compound in the Setatarian delphinium exhibits cytotoxic activities of different degrees on human lung cancer A549 cells and human cervical carcinoma Hela cells.
Claims (5)
1. A diterpene alkaloid compound separated from Setaria viridis is characterized in that the compound has a structural formula as follows:
wherein:
compound 1, sertraline A (pseudomorphine A)
Compound 2, named Saitacuisine B (pseudomorphine B)
Compound 3 named Saitacuisine C (pseudomorphine C)
Compound 4, Setarine D (pseudomorphine D)
Compound 5a is named as sertaline A (pseudorene A)
Compound 5b is named Saetanin B (pseudomonene B)
Compound 7 named Satadine B (pseudoonidine B)
Compounds 5a and 5b are a pair of regioisomeric mixtures.
2. The process for the preparation of diterpene alkaloid compounds in cytarabia sieboldii according to claim 1, characterized by comprising the steps of:
a. pulverizing whole plant of Setaria viridis, extracting with 10-95% ethanol water solution, methanol or chloroform at room temperature by cold soaking or percolation method, heating under reflux or ultrasonic extraction, and concentrating under reduced pressure to recover solvent to obtain extract;
b. dispersing the extract obtained in step a with 1-5% sulfuric acid, anhydrous sodium carbonate or ammonia water to obtain acid water layer, and adding Na2CO3Adjusting pH to 10, extracting with organic solvent such as chloroform, ethyl acetate, diethyl ether or n-butanol, concentrating under reduced pressure, and recovering organic solvent to obtain total crude productA base;
c. b, separating the total alkaloids obtained in the step b by a forward silica gel column, performing gradient elution by using chloroform-methanol as an eluent in a volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing fractions by silica gel thin layer chromatography, and combining the same fractions to obtain 6 components A-F;
separating the obtained component B by normal phase silica gel column, and performing gradient elution by using petroleum ether-acetone + 0.2% diethylamine as eluent at the volume ratio of 20:1,10:1,5:1,2:1,1:1 and 0:1 to obtain 3 components B1-B3;
separating component B1 with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 40:1-0:1, or subjecting component B1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1: 1; obtaining compound saturnine C;
separating component B3 with normal phase silica gel column, performing gradient elution with chloroform-methanol and 0.2% diethylamine as eluent at volume ratio of 100:1-1:1, or subjecting component B3 to Sephadex LH-20 column chromatography, and isocratic elution with chloroform-methanol at volume ratio of 1: 2; obtaining a compound of cetanine A, cetanine B and two components B3-2 and B3-3;
preparing the component B3-2 into a compound cetraridine C by thin layer chromatography with chloroform-methanol as a developing agent in a volume ratio of 10: 1;
the component B3-3 is subjected to thin layer chromatography, and chloroform-methanol with the volume ratio of 10:1 is used as a developing agent to obtain a compound cetradine B;
separating the obtained C component with normal phase silica gel column, and performing gradient elution with chloroform-methanol as eluent at volume ratio of 60:1-1:1 to obtain 4 components C1-C4;
subjecting component C2 to thin layer chromatography with chloroform-methanol-water as developing agent at volume ratio of 82:16:2, or subjecting component C2 to Sephadex LH-20 column separation, and isocratically eluting with chloroform-methanol at volume ratio of 1: 1; preparing compound saturnine A;
separating the obtained component E and component F with normal phase silica gel column, performing gradient elution with chloroform-methanol as eluent at volume ratio of 60:1-1:1, or performing Sephadex LH-20 column chromatography with chloroform-methanol at volume ratio of 1: 2; respectively obtaining components E1-E6 and F1-F10;
separating the components E5 and F9 with normal phase silica gel column, eluting with 5:1 petroleum ether-acetone + 0.2% diethylamine as eluent at equal ratio, or separating the components E5 and F9 with Sephadex LH-20 column chromatography, eluting with 1:2 chloroform-methanol at equal ratio; further separating by thin layer chromatography, and respectively obtaining compounds of staurodine D and staurodine B by using chloroform-methanol-water as a developing agent in a volume ratio of 82:16: 2; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures;
d. detecting and analyzing by thin layer chromatography or high performance liquid chromatography to obtain 8 diterpene alkaloid compounds.
3. The process for the preparation of diterpene alkaloid compounds in cytarabia sieboldii according to claim 1, characterized by comprising the steps of:
a. pulverizing whole plant of Setaria viridis, extracting with 10-95% ethanol water solution, methanol or chloroform at room temperature by cold soaking or percolation method, heating under reflux or ultrasonic extraction, and concentrating under reduced pressure to recover solvent to obtain extract;
b. dispersing the extract obtained in step a with 1-5% sulfuric acid, anhydrous sodium carbonate or ammonia water to obtain acid water layer, and adding Na2CO3Adjusting pH to 10, extracting with organic solvent such as chloroform, ethyl acetate, diethyl ether or n-butanol, and concentrating under reduced pressure to recover organic solvent to obtain total alkaloids;
c. b, separating the total alkaloids obtained in the step b by a forward silica gel column, performing gradient elution by using chloroform-methanol as an eluent in a volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing fractions by silica gel thin layer chromatography, and combining the same fractions to obtain 6 components A-F;
subjecting the obtained component B to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain 3 components B1-B3; separating the component B1 with normal phase silica gel column, and performing gradient elution with chloroform-methanol as eluent at volume ratio of 40:1-0:1 to obtain compound Setarine C; separating the component B2 with normal phase silica gel column, isocratic eluting with chloroform-methanol as eluent at volume ratio of 40:1, and further isocratic eluting with high performance liquid phase pHPLC and methanol-0.1% formic acid water at volume ratio of 35:65 to obtain compound cetradine B; separating the component B3 by normal phase silica gel column, performing gradient elution with chloroform-methanol and 0.2% diethylamine as eluent at a volume ratio of 100:1-1:1 to obtain compound sertraline A, sertraline B and two components B3-2 and B3-3, performing preparative thin layer chromatography on the component B3-2, preparing compound sertidine C with chloroform-methanol at a volume ratio of 10:1 as developing agent, and performing preparative thin layer chromatography on the component B3-3, and obtaining compound sertidine B with chloroform-methanol at a volume ratio of 10:1 as developing agent;
or separating the obtained component B by normal phase silica gel column, and performing gradient elution by using petroleum ether-acetone + 0.2% diethylamine as eluent at the volume ratio of 20:1,10:1,5:1,2:1,1:1 and 0:1 to obtain 3 segments of components B1-B3; subjecting the component B1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain compound Setarine C; subjecting the component B2 to Sephadex LH-20 column chromatography, isocratically eluting with chloroform-methanol at volume ratio of 1:1, and isocratically eluting the obtained component with methanol-0.1% formic acid water at volume ratio of 35:65 by high performance liquid chromatography (pHPLC) to obtain compound setadin B; subjecting B3 to Sephadex LH-20 column chromatography, isocratic eluting with chloroform-methanol at volume ratio of 1:2 to obtain compound Setanine A, Setanine B and two components B3-2 and B3-3, subjecting B3-2 to thin layer chromatography with chloroform-methanol at volume ratio of 10:1 as developing agent to obtain compound Setanine C, subjecting component B3-3 to thin layer chromatography with chloroform-methanol at volume ratio of 10:1 as developing agent to obtain compound Setanine B;
subjecting the obtained C component to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:1 to obtain 4 components C-1 to C-4; preparing the component C-2 into a compound cetuxine A by a preparative thin layer chromatography method and taking chloroform-methanol-water with the volume ratio of 82:16:2 as a developing agent;
subjecting the component E and the component F to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:2 to obtain components E1-E6 and F1-F10; separating the components E5 and F9 by normal phase silica gel column respectively, performing gradient elution by using chloroform-methanol as eluent with volume ratio of 60:1-1:1 to obtain components E5-1 to E5-3 and F9-1 to F9-10, further separating the components E5-3 and F9-10 by thin layer chromatography respectively, and obtaining compound cetuxine D and compound cetuxine B by using chloroform-methanol-water as developing agent with volume ratio of 82:16: 2;
or subjecting the obtained component E and component F to ODS reverse silica gel column chromatography, and performing gradient elution with methanol-water as eluent system at volume ratio of 1:9-1:0 to obtain components E1-E6 and F1-F10; subjecting the components E5 and F9 to Sephadex LH-20 column chromatography, eluting with chloroform-methanol at a volume ratio of 1:2, performing thin layer chromatography, and separating to obtain compound Setarine D and compound Setarine B with chloroform-methanol-water at a volume ratio of 82:16:2 as developing agent;
wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures;
d. detecting and analyzing by thin layer chromatography or high performance liquid chromatography to obtain 8 diterpene alkaloid compounds.
4. The process for the preparation of diterpene alkaloid compounds in cytarabia sieboldii according to claim 1, characterized by comprising the steps of:
a. pulverizing whole plant of Setaria viridis, extracting with 10-95% ethanol water solution, methanol or chloroform at room temperature by cold soaking or percolation method, heating under reflux or ultrasonic extraction, and concentrating under reduced pressure to recover solvent to obtain extract;
b. dispersing the extract obtained in step a with 1-5% sulfuric acid, anhydrous sodium carbonate or ammonia water to obtain acid water layer, and adding Na2CO3Adjusting pH to 10, extracting with organic solvent such as chloroform, ethyl acetate, diethyl ether or n-butanol, and concentrating under reduced pressure to recover organic solvent to obtain total alkaloids;
c. b, separating the total alkaloids obtained in the step b by a forward silica gel column, performing gradient elution by using chloroform-methanol as an eluent in a volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1,10:1,5:1,2:1,1:1 and 0:1, analyzing fractions by silica gel Thin Layer Chromatography (TLC), and combining the same fractions to obtain 6 components A-F;
performing ODS reverse silica gel column chromatography on the obtained component B, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain 3 sections of components B1-B3;
separating component B1 with normal phase silica gel column, performing gradient elution with chloroform-methanol at volume ratio of 40:1-0:1 as eluent, or subjecting component B1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1: 1; obtaining compound saturnine C;
separating component B2 with normal phase silica gel column, isocratic eluting with chloroform-methanol at volume ratio of 40:1 as eluent, or subjecting component B2 to Sephadex LH-20 column chromatography, isocratic eluting with chloroform-methanol at volume ratio of 1: 1; further carrying out isocratic elution by using high performance liquid phase pHPLC and methanol-0.1% formic acid water with the volume ratio of 35:65 to obtain a compound sertraline A;
separating the component B3 with normal phase silica gel column, and performing gradient elution with chloroform-methanol and 0.2% diethylamine as eluent at volume ratio of 100:1-1:1 to obtain compound Setanine A, Setanine B and two components B3-2 and B3-3; preparing a thin layer of the component B3-2, preparing a compound cetraridine C by using chloroform-methanol in a volume ratio of 10:1 as a developing agent, and preparing a thin layer of the component B3-3, and preparing a compound cetraridine B by using chloroform-methanol in a volume ratio of 10:1 as a developing agent;
or subjecting component B3 to Sephadex LH-20 column chromatography, isocratic eluting with chloroform-methanol at volume ratio of 1:2 to obtain compound Setanine A, Setanine B and two components B3-2 and B3-3; performing thin layer chromatography on the component B3-2, and using chloroform-methanol with the volume ratio of 10:1 as a developing agent to prepare a compound cetradine C, and performing thin layer chromatography on the component B3-3, and using chloroform-methanol with the volume ratio of 10:1 as a developing agent to obtain a compound cetradine B;
performing ODS reversed phase silica gel column chromatography on the obtained component C, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain 4 sections of components C-1 to C-4; preparing the component C-2 into a compound cetuxine A by using chloroform-methanol-water as a developing agent according to a volume ratio of 82:16:2 through a thin layer chromatography; or subjecting the component C to ODS reversed phase silica gel column chromatography, and performing gradient elution with methanol-water as eluent system at volume ratio of 1:9-1:0 to obtain 4 segments of components C3-5-1 to C3-5-4; subjecting the component C3-5-1 to Sephadex LH-20 column chromatography, and isocratically eluting with chloroform-methanol at volume ratio of 1:2 to obtain compound Setarine A;
performing ODS reverse silica gel column chromatography on the obtained component E and component F respectively, and performing gradient elution by using methanol-water as an eluent system with the volume ratio of 1:9-1:0 to obtain components E1-E6 and F1-F10 respectively; separating the components E5 and F9 with normal phase silica gel column, eluting with petroleum ether-acetone + 0.2% diethylamine at volume ratio of 5:1, or subjecting the components E5 and F9 to Sephadex LH-20 column chromatography, and eluting with chloroform-methanol at volume ratio of 1: 2; further separating by thin layer chromatography, and obtaining a compound stauntonine D and a compound stauntonine B by using chloroform-methanol-water as a developing agent in a volume ratio of 82:16: 2; wherein the compounds sertraline A and sertraline B are a pair of regioisomeric mixtures;
d. detecting and analyzing by thin layer chromatography or high performance liquid chromatography to obtain 8 diterpene alkaloid compounds.
5. Use of the diterpene alkaloid compounds isolated from the delphinium stauntoni according to claim 1 in the preparation of antitumor drugs.
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| US3415830A (en) * | 1964-01-27 | 1968-12-10 | Shionogi & Co | Process for preparing intermediates of garrya alkaloids |
| CN102274302A (en) * | 2011-06-07 | 2011-12-14 | 南京泽朗农业发展有限公司 | Preparation method of delphinium total diterpenoid alkaloids |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3415830A (en) * | 1964-01-27 | 1968-12-10 | Shionogi & Co | Process for preparing intermediates of garrya alkaloids |
| CN102274302A (en) * | 2011-06-07 | 2011-12-14 | 南京泽朗农业发展有限公司 | Preparation method of delphinium total diterpenoid alkaloids |
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| Five New C 19 -Diterpenoid Alkaloids from Delphinium tianshanicum W.T.W ANG;Ji-Fa Zhang等;《Chem.Biodiversity》;20171231;第14卷;第2页图1，第7页左栏倒数第1段 * |
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