CN114456102A - Indole alkaloid compound and preparation method and application thereof - Google Patents
Indole alkaloid compound and preparation method and application thereof Download PDFInfo
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- CN114456102A CN114456102A CN202210098567.1A CN202210098567A CN114456102A CN 114456102 A CN114456102 A CN 114456102A CN 202210098567 A CN202210098567 A CN 202210098567A CN 114456102 A CN114456102 A CN 114456102A
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- -1 Indole alkaloid compound Chemical class 0.000 title claims abstract description 36
- 229930005303 indole alkaloid Natural products 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 150000001875 compounds Chemical class 0.000 claims abstract description 56
- 238000000855 fermentation Methods 0.000 claims abstract description 33
- 230000004151 fermentation Effects 0.000 claims abstract description 33
- 241000203233 Aspergillus versicolor Species 0.000 claims abstract description 31
- 241000723873 Tobacco mosaic virus Species 0.000 claims abstract description 20
- 239000003814 drug Substances 0.000 claims abstract description 10
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Chemical class C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 60
- 239000000243 solution Substances 0.000 claims description 26
- 239000003480 eluent Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 238000000926 separation method Methods 0.000 claims description 16
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- LCYYYYGFLZJERP-UHFFFAOYSA-N alkaloid G Natural products CN1C2=CC=CC=C2C(C2OC(O)C3C22)=C1C1N2C(O)C(CC)C3C1 LCYYYYGFLZJERP-UHFFFAOYSA-N 0.000 description 4
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- 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 description 2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/80—[b, c]- or [b, d]-condensed
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
- A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention belongs to the technical field of natural product chemistry, and particularly relates to an indole alkaloid compound and a preparation method and application thereof. The compound has the molecular formula: c15H15NO2Which has the following structure:
. The invention also discloses a preparation method and application of the compound. The compound is a novel compound separated from a tobacco endogenous Aspergillus versicolor (Aspergillus versicolor) fungus fermentation productAnd the indole alkaloid compounds are identified by spectrum data such as nuclear magnetic resonance, mass spectrum, infrared and the like. And the indole alkaloid compound has good activity of resisting tobacco mosaic virus: the activity test of resisting tobacco mosaic virus shows that the relative inhibition rate of the indole alkaloid compound to the tobacco mosaic virus reaches 41.2 percent, and the activity of the indole alkaloid compound is higher than that of a reference substance ningnanmycin (32.5 percent). The compound has simple structure and good activity, can be used as a guiding compound of a tobacco mosaic virus resistant medicament, and has good application prospect in the preparation of the tobacco mosaic virus resistant medicament.
Description
Technical Field
The invention belongs to the technical field of natural product chemistry, and particularly relates to an indole alkaloid compound and a preparation method and application thereof.
Background
Tobacco is a plant of the genus nicotiana of the family solanaceae, and is one of the most widely cultivated commercial crops in the world. China is the biggest tobacco producing country and consuming country in the world, and annual output can reach 4-5 hundred million tons. However, the research on metabolites of endophytes is still relatively rare, and secondary metabolites and biological activities of endophytes are yet to be further studied. Therefore, the strengthening of the comprehensive utilization of the secondary metabolites of the tobacco endophytes has attracted the wide attention of researchers.
According to literature reports, more than 4000 compounds are identified from tobacco at present, and main components comprise diterpenoids, sesquiterpenes, flavonoids, alkaloids, coumarins and the like. Meanwhile, researches prove that the compounds separated and identified from tobacco endogenous fungi have different pharmacological effects, such as antibiosis, antioxidation, antitumor, tobacco mosaic virus resistance and the like. Therefore, the research of strengthening the tobacco endophytic fungi metabolite has important scientific significance for finding new skeleton type metabolites with remarkable activity.
Fungi of the genus aspergillus are widely present in nature. The aspergillus versicolor is a strain capable of producing complex enzyme, and the strain can produce various functional enzymes such as amylase, diastase, cellulase, phytase and the like besides protease, so that the aspergillus versicolor is widely applied to fermentation industries such as food, feed, wine brewing and the like. Meanwhile, the secondary metabolites of aspergillus versicolor are considered as an important resource to be developed urgently. A series of natural products with biological activity, including alkaloid, polypeptide, terpenoid and polyphenol compounds, are also separated from the aspergillus versicolor fermentation products of different sources.
Disclosure of Invention
The compound is a novel indole alkaloid compound separated from a tobacco endogenous Aspergillus versicolor (Aspergillus versicolor) fungus fermentation product. It is worth mentioning that the indole alkaloid compound has significant activity for resisting tobacco mosaic virus, and the relative inhibition rate of the indole alkaloid compound on tobacco mosaic virus reaches 41.2 percent, which is higher than the relative inhibition rate (32.5 percent) of a reference substance ningnanmycin.
The invention separates and identifies the culture solution of the aspergillus versicolor strain from tobacco to obtain a novel alkaloid compound with the activity of resisting tobacco mosaic virus, and the compound has not been reported so far.
All percentages used in the present invention are mass percentages unless otherwise indicated.
In a first aspect, the present invention provides an indole alkaloid compound, wherein the compound has a molecular formula: c15H15NO2It has the following structure:
this compound was a brown gum, designated: aspergillus versicolor alkaloid-G, english name: aspergilline-I.
The second aspect of the present invention provides a method for preparing an indole alkaloid compound according to the first aspect, specifically comprising the following steps:
A. extracting the extractum: fermenting the culture medium by using an aspergillus versicolor strain YATS1111, ultrasonically extracting a fermentation product by using 90-99 wt% ethanol, filtering, concentrating a filtrate, adding a mixed solution of ethyl acetate and 3 wt% tartaric acid aqueous solution, stirring, standing for layering, separating out a water phase, and using Na for the water phase2CO3Adjusting pH to 9.0, extracting with ethyl acetate again, and concentrating ethyl acetate phase under reduced pressure to obtain extract;
wherein, the aspergillus versicolor strain YATS1111 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC N. 19910; the preservation date is as follows: 6, 8 months in 2020; and (4) storage address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
In the mixed solution of ethyl acetate and 3 wt% of tartaric acid aqueous solution, the volume ratio of ethyl acetate to 3 wt% of tartaric acid aqueous solution is 1: 1-1: 2;
the culture medium comprises: the weight volume ratio of the rice to the distilled water is 50-400 g: 50-400 mL.
B. Silica gel column chromatography: b, filling the extract obtained in the step A into a column by using a 200-300-mesh silica gel dry method, and performing silica gel column chromatography; gradient eluting with chloroform-methanol solution at volume ratio of 10:0, 9:1,8:2,7:3,6:4, and 5:5 respectively, mixing the parts with the same polarity, collecting eluate of each part, and concentrating; wherein the mass ratio of the silica gel to the extract is 2-5; collecting eluate obtained by eluting with chloroform-methanol solution at volume ratio of 9:1, and making into first eluate; concentrating the first eluent, continuously separating by using a silica gel chromatographic column, performing gradient elution by using a series of chloroform-acetone solutions with volume ratios of 9:1,8:2,7:3,6:4 and 5:5 in sequence, and collecting eluent obtained when the chloroform-acetone solution with the volume ratio of 7:3 is used for elution, wherein the eluent is called as second eluent;
C. high performance liquid chromatography separation: and C, concentrating the second eluent finally obtained in the step B, replacing the solvent with methanol, and separating and purifying by using high performance liquid chromatography, wherein the separation and purification by using the high performance liquid chromatography adopt a ZorbaxPrepHT GF chromatographic column with the size of 21.2mm multiplied by 250mm and the size of 5 mu m, the flow rate is 20mL/min, the mobile phase is 52 wt% methanol aqueous solution, the detection wavelength of an ultraviolet detector is 359nm, the sample injection of the second eluent is 200 mu L each time, the eluent corresponding to the chromatographic peak retention time of 25.2min after each sample injection is collected and is called as a third eluent, and the indole alkaloid compound is obtained after the solvent is removed from the third eluent.
Preferably, in the step A, the concentration of the ethanol is 95 wt%.
Preferably, before the extract is subjected to silica gel column chromatography, the extract is dissolved by methanol, and then is mixed with 80-120 mesh silica gel with the weight ratio of 1.5-2.5 times.
Preferably, in the step C, after the separation by the high performance liquid chromatography, the obtained indole alkaloid compound is dissolved by methanol again, and then the obtained indole alkaloid compound is separated by sephadex column chromatography with methanol as a mobile phase for further separation and purification.
The third aspect of the invention provides application of the indole alkaloid compounds in tobacco mosaic virus resistance or application in preparation of tobacco mosaic virus resistance medicines.
The preparation method of the CPA type indole alkaloid compound comprises the following steps:
A. and (3) strain separation and identification:
isolation of the endophytic fungus Aspergillus versicolor: putting tobacco roots sterilized by 75% ethanol into a sterile mortar for grinding, transferring the ground tobacco roots into a sterile plastic tube, centrifuging for 2-10 min at 1000-3000 rpm, sucking 1-100 microliters of supernatant, coating the supernatant on a BL (BL) plate, inverting the BL plate in an incubator, performing dark culture at 25-30 ℃ for 2-10 days, repeatedly selecting a single colony for culturing, numbering and preserving the colony until a single endophytic fungus colony is obtained, and determining the colony to be Aspergillus versicolor (Aspergillus versicolor) by ITS sequencing (Genbank Accession number MT549144, GCGGGCTGCCTCCGGGCGCCCAACCTCCCACCCGTGAATACCTAACACTGTTGCTT CGGCGGGGAACCCCCTCGGGGGCGAGCCGCCGGGGACTACTGAACTTCATGCCTG AGAGTGATGCAGTCTGAGTCTGAATATAAAATCAGTCAAAACTTTCAACAATGGAT CTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAACTGCGATAAGTAATGTGAATT GCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCC GGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCCCGGCTTGTGTGTTG GGTCGTCGTCCCCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGTGTCC GGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCGACTAGGGCCGGCCGGGCGCC AGCCGACGTCTCCAACCATTTTTCTTCAGGTTGACCTCGGATCAGGTAGGGATACC CGCTGAACTTAAGCATATCAATAAGCGGAGGAA), wherein the picture of the Aspergillus versicolor is shown in figure 1;
B. culture of endophytic fungus Aspergillus versicolor strain
Inoculating aspergillus versicolor strains separated in the A step on a potato glucose agar culture medium at room temperature, culturing for 7-10 days at 25-30 ℃, then inoculating in 50-500 ml triangular bottles, wherein each triangular bottle contains 10-100 ml of potato glucose culture medium, and placing at 25-30 ℃ for shake culture for 5-10 days to obtain liquid fermentation strains; the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC N. 19910.
C. Amplifying fermentation of strain
And C, performing large-scale fermentation on the liquid fermentation seeds obtained by the culture in the step B, wherein the large-scale fermentation is performed in 100-1000 Von Basher bottles of 100-500 mL, each bottle contains 50-400 g of rice and 50-400 mL of distilled water, 1.0-5.0 mL of the liquid fermentation seeds obtained by the culture in the step B are inoculated into each bottle after autoclaving, and the liquid fermentation seeds are cultured for 15-45 days at 25-30 ℃ to obtain the aspergillus versicolor fermentation product.
D. Extracting the extractum:
c, ultrasonically extracting the aspergillus versicolor fermented substance obtained by fermentation in the step C with 90-99 wt% ethanol for 2-5 times, each time for 30-50min, combining the extracting solutions, filtering and concentrating to a small volume, and then adding ethyl acetate and 3 wt% wine into the concentrated solutionSufficiently stirring the mixed solution of the aqueous solution of the tartaric acid (the volume ratio of the ethyl acetate to the 3 wt% aqueous solution of the tartaric acid is 1: 1-1: 2), standing for layering, separating out an aqueous phase, and using Na for the aqueous phase2CO3Adjusting pH to 9.0, extracting with ethyl acetate again, concentrating under reduced pressure to obtain extract, and separating by column chromatography.
E. Silica gel column chromatography:
c, filling the extract obtained in the step C into a column by using a 200-300-mesh silica gel dry method, and performing silica gel column chromatography; gradient eluting with chloroform-methanol solution at volume ratio of 10:0, 9:1,8:2,7:3,6:4, and 5:5 respectively, mixing the parts with the same polarity, collecting eluate of each part, and concentrating; wherein the mass ratio of the silica gel to the extract is 2-5; collecting eluate obtained by eluting with chloroform-methanol solution at volume ratio of 9:1, and making into first eluate; concentrating the first eluent, separating with silica gel chromatographic column, gradient eluting with a series of chloroform-acetone solutions at volume ratio of 9:1,8:2,7:3,6:4, and 5:5, and collecting eluate obtained by eluting with chloroform-acetone solution at volume ratio of 7:3, and called second eluate.
F. High performance liquid chromatography separation
And E, concentrating the second eluent finally obtained in the step E, replacing the solvent with methanol, and separating and purifying by using a high performance liquid chromatography, wherein the separation and purification by using the high performance liquid chromatography adopt a ZorbaxPrepHT GF chromatographic column with the thickness of 21.2mm multiplied by 250mm and the thickness of 5 mu m, the flow rate is 20mL/min, the mobile phase is 52 wt% methanol aqueous solution, the detection wavelength of an ultraviolet detector is 359nm, the sample injection of the second eluent is 200 mu L each time, collecting the eluent corresponding to the chromatographic peak retention time of 25.2min after sample injection each time, and the eluent is called as a third eluent, and the crude indole alkaloid compound is obtained after the solvent is removed from the third eluent.
G. Purification by gel column chromatography
And (3) carrying out sephadex column chromatography separation on the alkaloid compound crude product by using a pure methanol solvent and methanol as fluidity again to obtain the alkaloid compound pure product.
The structure of the indole alkaloid compound prepared by the method is identified by the following method:
the appearance observation shows that: the compounds of the present invention are brown gums; the ultraviolet-visible absorption spectrum shows that the compound has maximum absorption at 215 nm and 358nm, and the aromatic ring structure is proved to exist in the compound; infrared spectrum (potassium bromide tablet) shows that the compound has amido (3228 cm)-1) Carbonyl group (1698 cm)-1) Aromatic rings (1615, 1468 and 1365 cm)-1) A characteristic functional group; high resolution mass spectrometry (HRESIMS) gave an excimer ion peak 264.1000[ M + Na ]]+Determining the formula of the compound as C15H15NO2The unsaturation number was 9.
Bonding with1H and13NMR data on C and HSQC show that the compound includes a 1,2,3, 4-tetrasubstituted naphthalene ring (C-3, C-4, C-8-C-15 and H-9, H-11-H-13), an isopropyl group (C-7, C-16,17, H-7, and H-13)6-16,17), one-NHCO-structural fragment (C-2-and NH-1), one hydroxymethyl (C-5 and H)2-5). In order to satisfy the 9 unsaturations of the compounds, the-NH-CO-structural fragment should form 1 benzo [ cd ] with the naphthalene ring]Indole-2 (1H) -ketone structure. This inference can be further confirmed by the correlation of NH with HMBC at C-2, C-3, C-14, C-15.
After the parent framework of the compound is determined, the remaining substituent positions can be determined by further analysis of its HMBC association. The isopropyl substitution at C-8 was determined based on the correlation of H-7 with HMBC at C-4, C-8, and C-9; according to H25 is related to HMBC at C-3, C-4 and C-8, and the substitution of hydroxymethyl at C-4 position can be determined. Thus far, the structure of the compound of the present invention was confirmed. The compound was named: aspergillus versicolor alkaloid-G, english name: aspergilline G.
TABLE-1 preparation of the compounds1H NMR and13c NMR data (CDCl)3)
Spectral data of compounds: UV (methanol), lambdamax(log ε)220(4.23), 262(3.89), 338(3.65), 359(3.56) nm; IR (Potassium bromide tablet) vmax 3394、3228、2965、2912、2846、1698、1615、1468、1365、 1087、1036、853cm-1；1H and 13C NMR data (CDCl)3500 and 125MHz), table-1; ESIMS (Positive ion mode) M/z 264[ M + Na ]]+(ii) a HRESIMS (positive ion mode) M/z 264.1006[ M + Na ]]+(calculated 264.1000, C15H15NNaO2)。
The invention has the following beneficial effects:
1. the compound is separated from a fermentation product of the tobacco endophytic aspergillus versicolor fungus strain. In particular, the aspergillus versicolor strain YATS1111 of the present invention is a novel microbial material, which is first isolated from tobacco roots. In addition, the compound of the present invention is obtained by specifically designing the culture medium of the fermentation process of the strain and by designing the separation conditions of the fermentation product.
2. The compound has good activity of resisting tobacco mosaic virus. Through screening of tobacco mosaic virus resistance, the relative inhibition rate of the indole alkaloid compound is found to reach 41.2%, and the activity of the indole alkaloid compound is higher than that of a control ningnanmycin (32.5%). The results show that the compound has good application prospect in the preparation of the anti-tobacco mosaic virus medicine.
3. The compound of the invention has simple molecular structure, is easy to realize artificial synthesis, and can realize subsequent industrialization by artificial synthesis.
4. The preparation method combining conventional column chromatography and high performance liquid chromatography is adopted, the preparation operation flow of the compound is simple, the purity of the obtained compound is high, and the quality and purity of the compound in subsequent industrial production can be guaranteed.
5. The compound is safe and nontoxic, shows good activity of resisting tobacco mosaic virus, and can provide an ideal new skeleton type drug source molecule for preventing and treating tobacco mosaic disease.
6. The aspergillus versicolor strain YATS1111 belongs to endophytic fungi, so that batch fermentation production is easy to realize. Therefore, the compound of the invention has easily obtained raw materials, simple extraction method of the compound, easy separation and easy realization of industrialized preparation.
Drawings
FIG. 1 is a photograph of Aspergillus versicolor strain YATS1111 of the present invention, a, colony morphology; b. microscopic morphology.
FIG. 2 is a nuclear magnetic resonance carbon spectrum of the indole alkaloid compounds.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the indole alkaloid compounds.
FIG. 4 shows the major HMBC and of the indole alkaloid compounds1H-1HCOSY is related.
Detailed Description
The present invention is further illustrated by the following examples, but is not limited to these examples. The experimental methods not specified in the examples are generally commercially available according to the conventional conditions and the conditions described in the manual, or according to the general-purpose equipment, materials, reagents and the like used under the conditions recommended by the manufacturer, unless otherwise specified. The starting materials required in the following examples are all commercially available.
The raw materials used in the invention are not affected by the type of the culture medium, and the invention is further explained by the culture medium of the aspergillus versicolor strain separated and identified from the tobacco from Yunnan as follows:
example 1
And (3) performing large-scale fermentation on the liquid fermentation seeds obtained by the culture in the step (B) in the invention content part, wherein the large-scale fermentation is performed in 500 Von Basher bottles with 500mL, each bottle contains 180g of rice and 180mL of distilled water, 2.5mL of liquid fermentation seeds obtained by culture are inoculated into each bottle, and the liquid fermentation seeds are cultured at 25-30 ℃ for 30 days to obtain the aspergillus versicolor fermentation product. Ultrasonically extracting the fermentation product with 95% ethanol for 3 times, each time for 30 min; mixing the extractive solutions, adding into mixture of ethyl acetate and 3 wt% tartaric acid (volume ratio of ethyl acetate to 3 wt% tartaric acid water solution is 1:1)Stirring thoroughly, standing for layering, separating water phase, and adding Na2CO3Adjusting the pH value of the water layer to 9.0 by using the solution, and extracting the solution again by using ethyl acetate; separating ethyl acetate phase, and concentrating under reduced pressure to obtain extract 580 g. Mixing the extract with 1.0kg of 80-120 mesh silica gel, loading the extract into a 200-mesh silica gel column with 3.0kg of silica gel, performing silica gel column chromatography, performing gradient elution with chloroform-methanol at volume ratio of 20:1,8:2,7:3,6:4,5:5, monitoring by TLC, combining the same parts to obtain 5 parts, concentrating the chloroform-methanol elution part at volume ratio of 9:1, performing silica gel column chromatography again, eluting with chloroform-acetone solution at volume ratio of 9:1,8:2,7:3,6:4,1:1, collecting the elution part at 7:3, concentrating, replacing solvent with methanol, preparing high performance liquid chromatography with Agilent 1100, using 52 wt% methanol water solution as mobile phase, preparing a ZorbaxPreTGF column (21.2 × 250mm,5 μm) with stationary phase at flow rate of 20mL/min and ultraviolet detector detection wavelength of 359nm, sampling 200 μ L each time, collecting chromatographic peak of 25.2min, accumulating for multiple times, and evaporating to obtain crude compound; dissolving the obtained crude product with pure methanol again, taking the pure methanol as a mobile phase, and carrying out Sephadex LH-20 gel column chromatography separation to obtain the pure product of the new compound.
Characterization of the novel compound:
the appearance observation shows that: the compounds of the present invention are brown gums. The ultraviolet-visible absorption spectrum shows that the compound has maximum absorption at 215 nm and 358nm, and the aromatic ring structure is proved to exist in the compound; infrared spectrum (potassium bromide tablet) shows that the compound has amido (3228 cm)-1) Carbonyl group (1698 cm)-1) Aromatic rings (1615, 1468 and 1365 cm)-1) A characteristic functional group; high resolution mass spectrometry (HRESIMS) gave an excimer ion peak 264.1000[ M + Na ]]+Determining the formula of the compound as C15H15NO2The unsaturation number was 9. Bonding of1H and13NMR data on C and HSQC show that the compound includes a 1,2,3, 4-tetrasubstituted naphthalene ring (C-3, C-4, C-8-C-15 and H-9, H-11-H-13), an isopropyl group (C-7, C-16,17, H-7, and H-13)6-16,17), one-NHCO-structural fragment (C-2-and NH-1), one hydroxymethylRadical (C-5 and H)2-5). In order to satisfy the 9 unsaturations of the compounds, the-NH-CO-structural fragment should form 1 benzo [ cd ] with the naphthalene ring]Indol-2 (1H) -ones. This inference can be further confirmed by HMBC correlation of C-2, C-3, C-14, C-15.
After the parent framework of the compound is determined, the remaining substituent positions can be determined by further analysis of its HMBC association. The isopropyl substitution at C-8 was determined based on the correlation of H-7 with HMBC at C-4, C-8, and C-9; according to H25 is related to HMBC at C-3, C-4 and C-8, and the substitution of hydroxymethyl at C-4 position can be determined. Thus, the structure of the compound of the present invention was confirmed. The compound was named: aspergillus versicolor alkaloid-G, english name: aspergilline G.
Example 2
And (3) performing large-scale fermentation on the liquid fermentation seeds obtained by the culture in the step (B) in the invention content part, wherein the large-scale fermentation is performed in 250 Von Bashel bottles with the volume of 1.0L, each bottle contains 360g of rice and 360mL of distilled water, 5.0mL of the liquid fermentation seeds obtained by culture are inoculated into each bottle, and the liquid fermentation seeds are cultured at the temperature of 25-30 ℃ for 30 days to obtain the aspergillus versicolor fermentation product. Ultrasonically extracting the fermentation product with 95% ethanol for 3 times, each time for 30 min; mixing the extractive solutions, adding into mixture of ethyl acetate and 3 wt% tartaric acid water solution (volume ratio of ethyl acetate to 3 wt% tartaric acid water solution is 1:2), stirring, standing for layering, separating water phase, and adding Na2CO3Adjusting the pH value of the water layer to 9.0 by using the solution, and extracting the solution again by using ethyl acetate; separating ethyl acetate phase, and concentrating under reduced pressure to obtain extract 618 g. Mixing the extract with 1.0kg of 80-120 mesh silica gel, loading 3.2kg of 200 mesh silica gel into a column, performing silica gel column chromatography, performing gradient elution with chloroform-methanol at volume ratio of 20:1,8:2,7:3,6:4,5:5, monitoring by TLC, combining the same parts to obtain 5 parts, concentrating the chloroform-methanol elution part at volume ratio of 9:1, performing silica gel column chromatography again, eluting with chloroform-acetone solution at volume ratio of 9:1,8:2,7:3,6:4,1:1, respectively, collecting the eluate at 7:3 part, concentrating and replacing solvent with methanol, preparing high performance liquid chromatography with Anlun 1100, separating with 52% methanol as mobile phase, ZorbaAn xPrepHT GF column (21.2 multiplied by 250mm,5 mu m) is prepared by taking a column as a stationary phase, the flow rate is 20mL/min, the detection wavelength of an ultraviolet detector is 359nm, 200 mu L of sample injection is carried out each time, a chromatographic peak of 25.2min is collected, and a crude compound product can be obtained by evaporation after multiple accumulation; dissolving the obtained crude product with pure methanol again, taking the pure methanol as a mobile phase, and carrying out Sephadex LH-20 gel column chromatography separation to obtain the pure product of the new compound.
The compound prepared in example 2 was taken as a brown gum. The assay method was the same as in example 1, and it was confirmed that the compound prepared in example 2 was aspergillus alkaloid-G, which is the indole alkaloid compound.
Example 3
Any indole alkaloid compound prepared in the examples 1-2 is used for an activity test of tobacco mosaic virus, and the test conditions are as follows:
the activity of the compound of the invention against tobacco mosaic virus is measured by a half-leaf method when the mass concentration of the medicament is 20 mu M. Selecting leaves suitable for testing (normal leaves, no disease and no insect) from 5-6 flue-cured tobacco plants, uniformly spraying fine carborundum on the leaves, and writing the tobacco mosaic virus source (3.0 multiplied by 10) for standby use with a writing brush-3) Uniformly smearing on blades scattered with carborundum, immediately placing in a culture dish filled with liquid medicine for processing for 20min after all selected blades are disinfected, taking out, sprinkling water drops and confining liquid on the blades, recovering and arranging two half blades, covering glass in an enamel officer paved with toilet paper for moisturizing, controlling the temperature to be (23 +/-2) DEG C, placing in a greenhouse for natural light irradiation, and enabling the officer to see withered spots after 2-3 d.
XI％＝(CK-T)/CK×100％
X: relative inhibition ratio (%), CK: the number of dead spots of half leaf after being soaked in clear water is one, and the number of dead spots of half leaf after being soaked in liquid medicine is one.
The results show that: the relative inhibition rate of the compound is 41.2 percent and is higher than the relative inhibition rate of 32.5 percent of the contrast ningnanmycin, which indicates that the compound has good activity of resisting tobacco mosaic virus.
Claims (6)
1. An indole alkaloid compound, wherein the compound has the molecular formula: c15H15NO2Which has the following structure:
2. a method for preparing the indole alkaloid compound of claim 1, comprising the steps of:
A. extracting the extractum: fermenting the culture medium by using an aspergillus versicolor strain YATS1111, ultrasonically extracting a fermentation product by using 90-99 wt% ethanol, filtering, concentrating a filtrate, adding a mixed solution of 3 wt% tartaric acid aqueous solution and ethyl acetate, stirring, standing for layering, separating out a water phase, and using Na for the water phase2CO3Adjusting pH to 9.0, extracting with ethyl acetate again, and concentrating ethyl acetate phase under reduced pressure to obtain extract;
wherein, the aspergillus versicolor strain YATS1111 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC N. 19910;
the volume ratio of the mixed solution of 3 wt% tartaric acid aqueous solution and ethyl acetate is 1: 1-2: 1;
the culture medium comprises: the weight volume ratio of the rice to the distilled water is 50-400 g: 50-400 mL.
B. Silica gel column chromatography: b, filling the extract obtained in the step A into a column by using a 200-300-mesh silica gel dry method, and performing silica gel column chromatography; gradient eluting with chloroform-methanol solution at volume ratio of 10:0, 9:1,8:2,7:3,6:4, and 5:5 respectively, mixing the parts with the same polarity, collecting eluate of each part, and concentrating; wherein the mass ratio of the silica gel to the extract is 2-5; collecting eluate obtained by eluting with chloroform-methanol solution at volume ratio of 9:1, and making into first eluate; concentrating the first eluent, continuously separating by using a silica gel chromatographic column, performing gradient elution by using a series of chloroform-acetone solutions with volume ratios of 9:1,8:2,7:3,6:4 and 5:5 in sequence, and collecting eluent obtained when the chloroform-acetone solution with the volume ratio of 7:3 is used for elution, wherein the eluent is called as second eluent;
C. high performance liquid chromatography separation: and C, concentrating the second eluent finally obtained in the step B, replacing the solvent with methanol, and separating and purifying by using high performance liquid chromatography, wherein the separation and purification by using the high performance liquid chromatography adopt a ZorbaxPrepHT GF chromatographic column with the size of 21.2mm multiplied by 250mm and the size of 5 mu m, the flow rate is 20mL/min, the mobile phase is 52 wt% methanol aqueous solution, the detection wavelength of an ultraviolet detector is 359nm, the sample injection of the second eluent is 200 mu L each time, the eluent corresponding to the chromatographic peak retention time of 25.2min after each sample injection is collected and is called as a third eluent, and the indole alkaloid compound is obtained after the solvent is removed from the third eluent.
3. The process for producing an indole alkaloid compound according to claim 2, wherein the concentration of ethanol in step A is preferably 95 wt%.
4. The preparation method of indole alkaloid compounds according to claim 2, wherein in step B, before the silica gel column chromatography, the extract is dissolved in methanol and then mixed with 80-120 mesh silica gel in a weight ratio of 1.5-2.5.
5. The process for producing an indole alkaloid compound according to claim 2, wherein in the step C, after the separation by high performance liquid chromatography, the obtained indole alkaloid compound is dissolved again in methanol, and then the resulting indole alkaloid compound is separated by sephadex column chromatography using methanol as a mobile phase for further separation and purification.
6. The use of the indole alkaloid compounds according to claim 1 in the preparation of a medicament against tobacco mosaic virus or in the preparation of a medicament against tobacco mosaic virus.
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