CN115583953A - Quinazolinone alkaloid compound and preparation method and application thereof - Google Patents
Quinazolinone alkaloid compound and preparation method and application thereof Download PDFInfo
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- -1 Quinazolinone alkaloid compound Chemical class 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 44
- 241000203233 Aspergillus versicolor Species 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 22
- 230000004151 fermentation Effects 0.000 claims abstract description 22
- 241000723873 Tobacco mosaic virus Species 0.000 claims abstract description 14
- 241000208125 Nicotiana Species 0.000 claims abstract description 12
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 12
- 229940125904 compound 1 Drugs 0.000 claims abstract description 10
- 229940125782 compound 2 Drugs 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 39
- 239000003480 eluent Substances 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
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- PZZXYDQKZIGACT-UHFFFAOYSA-N 5-hydroxyskytanthine Natural products C1N(C)CC(C)C2(O)C1C(C)CC2 PZZXYDQKZIGACT-UHFFFAOYSA-N 0.000 description 3
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- ABRVLXLNVJHDRQ-UHFFFAOYSA-N [2-pyridin-3-yl-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound FC(C1=CC(=CC(=N1)C=1C=NC=CC=1)CN)(F)F ABRVLXLNVJHDRQ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- 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
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- 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
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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Abstract
The invention relates to an alkaloid in a tobacco endophytic aspergillus versicolor fungus fermentation product, in particular to a quinazolinone alkaloid compound and a preparation method and application thereof, belonging to the technical field of natural product chemistry. The molecular formula of the quinazolinone alkaloid compound is as follows: c 22 H 21 N 3 O 2 Has a structure of formula 1 or formula 2. The compound of the invention has good activity of resisting tobacco mosaic virus. Through experiments of resisting tobacco mosaic virus, the relative inhibition rate of the compound 1 is 31.5 percent, and the relative inhibition rate of the compound 2 is 29.4 percentThe activity of the compound is similar to the relative inhibition rate (33.2%) of a reference substance ningnanmycin.
Description
Technical Field
The invention relates to an alkaloid in a tobacco endophytic aspergillus versicolor fungus fermentation product, in particular to a quinazolinone alkaloid compound and a preparation method and application thereof, belonging to the technical field of natural product chemistry.
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. According to literature reports, more than 4000 compounds are identified from tobacco, and the main components comprise diterpenoid compounds, sesquiterpene compounds, flavonoid compounds, alkaloids and coumarin. Meanwhile, researches prove that the compounds 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.
Disclosure of Invention
According to the invention, two novel quinazolinone alkaloid compounds with tobacco mosaic virus resisting activity are obtained by separating and identifying the culture solution of the aspergillus versicolor strain from tobacco, and the compounds have not been reported yet.
The invention provides a quinazolinone alkaloid compound, wherein the molecular formula of the quinazolinone alkaloid compound is as follows: c 22 H 21 N 3 O 2 Having the structure of formula 1 or formula 2:
both compounds were brown gums, designated: aspergillus versicolor alkaloid-B, english name: isoaspergilline B, aspergillus versicolor alkaloid-C, english name: isoaspergilline C.
Fungi of the genus aspergillus are widely present in nature. Wherein, aspergillus oryzae is a strain capable of generating complex enzyme, and the strain can generate various functional enzymes such as amylase, saccharifying enzyme, cellulase, phytase and the like besides protease, thereby being 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. The compound is two new quinazolinone alkaloid compounds separated from tobacco endogenous Aspergillus versicolor (Aspergillus versicolor) YATS1111 fungus fermentation products. It is worth mentioning that the relative inhibition rate of the compound 1 is 31.5% and the relative inhibition rate of the compound 2 is 29.4% through the experiment of resisting the tobacco mosaic virus, and the activities of the two compounds are similar to the relative inhibition rate (33.2%) of the reference ningnanmycin.
The second aspect of the invention provides a preparation method of the quinazolinone alkaloid compound.
The preparation method of the quinazolinone alkaloid compound comprises the following steps:
1) And extracting the extract
Performing solid fermentation on Aspergillus versicolor strain YATS1111 separated and identified from tobacco, performing ultrasonic extraction on the fermentation product by using ethanol, filtering, concentrating the filtrate, adding a mixed solution of ethyl acetate and tartaric acid, fully and uniformly stirring, standing for layering, separating out a water phase, and then using Na as the water phase 2 CO 3 Adjusting the pH value to 8.0-10.0, extracting again by using ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain an extract;
2) Silica gel column chromatography
Filling the extract obtained in the step 1) 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, mixing the parts with the same polarity, collecting eluate of each part, and concentrating, wherein the eluate with first concentration gradient is collected and called as first eluate;
continuously separating the first eluate with silica gel chromatographic column, performing gradient elution with chloroform-acetone solution, and collecting eluate with second concentration gradient, which is called second eluate;
3) Separating by high performance liquid chromatography
Introducing the second eluent finally obtained in the step 2) into a high performance liquid chromatography for separation and purification, and collecting the eluent corresponding to the chromatographic peak after each sample injection to obtain a third eluent; removing the solvent from the third eluent to obtain a crude quinazolinone alkaloid compound product;
4) And separating by gel column chromatography
And (3) carrying out gel column chromatography separation on the crude quinazolinone alkaloid compound by taking methanol as fluidity again to obtain a pure quinazolinone alkaloid compound.
Preferably, in the step 1), the concentration of the ethanol is 90-99 wt%; further, the concentration of ethanol was 95wt%.
Preferably, in the step 2), before the crude separation by silica gel column chromatography, the extract is dissolved by methanol and then mixed with 80-120 mesh silica gel with the weight 1.5-2.5 times that of the extract.
Preferably, in step 3), after the separation and purification by high performance liquid chromatography, the obtained compound is dissolved again in pure methanol, and then the solution is separated and purified by gel column chromatography using pure methanol as a mobile phase.
Preferably, in step 2), when the chloroform-methanol solution is subjected to gradient elution, the concentration gradient is set to be in a range of 20; the first eluent is an eluent when 20.
As a preferable mode of the above technical solution, in the step 2), when the chloroform-acetone solution is subjected to gradient elution, the concentration gradient is set as the following volume ratio of 9, 2, 7; the second eluent is an eluent when the eluent is chloroform-acetone solution of 8.
Preferably, in step 3), the separation and purification by high performance liquid chromatography is performed by using a zorbaxpreph t GF chromatographic column of 21.2mm × 250mm,5 μm, the flow rate is 20mL/min, the mobile phase is 52wt% methanol aqueous solution, the detection wavelength of the ultraviolet detector is 359nm, the second eluent is injected with 200 μ L each time, the eluent corresponding to the chromatographic peak after each injection is collected, the compound 1 is the eluent corresponding to the retention time of 29.6min, and the compound 2 is the eluent corresponding to the retention time of 29.8 min.
The invention further aims to provide application of the quinazolinone alkaloid compound in preparation of a drug for resisting tobacco mosaic virus.
In conclusion, the invention has the following beneficial effects:
1. the compound is separated from a fermentation product of a tobacco endophytic aspergillus versicolor fungus strain, and as the endophytic fungus is easy to realize batch fermentation production, the compound raw material is easy to obtain; the extraction method of the compound is simple, the compound is easy to separate and obtain, and the industrial preparation is easy to realize.
2. The compound of the invention has good activity of resisting tobacco mosaic virus. Through experiments of resisting tobacco mosaic virus, the relative inhibition rate of the compound 1 is 31.5%, the relative inhibition rate of the compound 2 is 29.4%, and the activities of the two compounds are similar to the relative inhibition rate (33.2%) of a control ningnanmycin. The compound has good application prospect in preparing the anti-tobacco mosaic virus medicament.
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 ideal new skeleton type drug source molecules for preventing and treating tobacco mosaic virus.
Drawings
FIG. 1 shows the NMR carbon spectrum of Compound 1.
FIG. 2 shows the NMR spectrum of Compound 1.
FIG. 3 shows the principal HMBC and HMBC of said compound 1 1 H- 1 HCOSY is related.
FIG. 4 is the ECD pattern of Compound 1.
FIG. 5 is the NMR carbon spectrum of Compound 2.
FIG. 6 shows the NMR spectrum of Compound 2.
FIG. 7 shows the principal HMBC and HMBC of said Compound 2 1 H- 1 HCOSY is related.
Detailed Description
The present invention is further illustrated by the following examples, but is not limited to these examples. The experimental methods in which specific conditions are not specified in examples are generally commercially available under the conventional conditions and the conditions described in the manual, or under the general-purpose equipment, materials, reagents and the like used under the conditions recommended by the manufacturer, unless otherwise specified.
The raw materials used in the present invention are not affected by the type of the culture medium, and the present invention is further explained by the following culture medium of the Aspergillus versicolor strain isolated and identified from Yunnan tobacco.
Isolation and identification of strains
Isolation of the endophytic fungus Aspergillus versicolor YATS1111
Putting tobacco roots and stems sterilized by 75% ethanol into a sterile mortar for grinding, transferring the ground tobacco roots and stems into a sterile plastic tube, centrifuging the tube at 1000-3000 rpm for 2-10 min, sucking 1-100 microliters of supernatant, coating the supernatant on a BL (basic BL) plate, inverting the tube in an incubator, carrying out dark culture at 25-30 ℃ for 2-10 days, repeatedly picking a single colony for culture, numbering and preserving the single colony until a single endophytic fungus colony is obtained; identified as Aspergillus sp.
Microbiological characteristics of Aspergillus versicolor YATS1111
1) The cells were cultured on PDA medium for 5 days, and the diameter of the colonies was about 2cm. The colony is small and compact, convex, white at the edge, slightly green in the middle, purple-red transparent secretion, dry and opaque, difficult to pick and slow in growth speed.
2) Developed hyphae, few branches, smooth hyphae without septation.
3) The conidiophores are fan-shaped, the conidiophores are smooth, the apical sac is oval, the small peduncles are radial, the conidiophores are in two layers and grow in 3/4 of the apical sac, and the conidiophores are spherical.
Aspergillus versicolor (Aspergillus versicolor) YATS1111 strain culture
Inoculating the aspergillus versicolor strain separated in the step on a potato glucose agar culture medium at room temperature, culturing for 7-10 days at 25-30 ℃, then inoculating in triangular flasks of 50-500 ml, wherein each triangular flask contains 10-100 ml of potato glucose culture medium, and placing at 25-30 ℃ for shake culture for 5-10 days to obtain a liquid fermentation strain; the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No.19910.
Example 1
Carrying out large-scale fermentation on the liquid fermentation seeds obtained by cultureFermenting, namely performing large-scale fermentation in 500 Von Basher bottles with 500mL, wherein each bottle contains 180g of rice and 180mL of distilled water, inoculating 2.5mL of liquid fermentation seeds obtained by culture in each bottle, and culturing 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 30min; mixing the extractive solutions, adding into a mixture of ethyl acetate and 3% tartaric acid (ethyl acetate: tartaric acid =97, mass ratio) 2 CO 3 The solution adjusted the pH of the aqueous layer to 9.0 and re-extracted with ethyl acetate; separating ethyl acetate phase, and concentrating under reduced pressure to obtain extract 580g. The extract is stirred by 1.0kg of 80-120 mesh silica gel, and is subjected to silica gel column chromatography by using a 3.0kg200 mesh silica gel loading column, chloroform-methanol gradient elution with volume ratio of 20; dissolving the crude product with pure methanol again, separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain two pure compounds.
Example 2
And (3) performing large-scale fermentation on the liquid fermentation seeds obtained by culture in 250 Von Basher bottles of 1.0L, wherein each bottle contains 360g of rice and 360mL of distilled water, inoculating 5.0mL of the liquid fermentation seeds obtained by culture in each bottle, and culturing at 25-30 ℃ for 30 days to obtain the aspergillus versicolor fermented product. Ultrasonically extracting the fermentation product with 95% ethanol for 3 times, each time for 30min; mixing the extractive solutions, adding into mixture of ethyl acetate and 3% tartaric acidEthyl ester acid: tartaric acid =97, mass ratio), standing the mixed solution for layering, separating out a water phase, and adding Na to the water phase 2 CO 3 Adjusting 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 610g extract. The extract is stirred by 1.0kg of 80-120 mesh silica gel, and is subjected to silica gel column chromatography by using a 3.2kg 200 mesh silica gel loading column, chloroform-methanol elution portions with volume ratios of 20; dissolving the crude product with pure methanol again, separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain two pure compounds.
Example 3
The structure of the quinazolinone alkaloid compound prepared by the method of example 1 was identified by the following method:
the appearance observation shows that: both compounds of the invention are brown gums.
As shown in FIGS. 1 to 4, the UV-VIS absorption spectrum of Compound No.1 shows the absorption maxima at 200, 210, 269, and 304nm, confirming the presence of an aromatic ring structure in this compound. The high resolution mass spectrum (HRESIMS) gives the peak 382.1522[ m ] +Na of the excimer ion] + It can be confirmed that the compound has the formula C 22 H 21 N 3 O 2 . Bonding of 1 H and 13 c and HSQC NMR data are similar to that of compound protuboxepin K, and C-3 and C-16 of compound 1 form a double bond and C-7 is reduced compared with that of protuboxepin KThe above may be presumed to be hydrogen 1 H– 1 H-7/H-8/H-9 and H in H COSY spectrogram 3 Correlation of-17 with HMBC at C-3, C-16, and C-18 was confirmed.
After the planar structure of the compound is determined, the corresponding configuration of the compound 1 is mainly determined by analyzing an ROESY spectrum and an ECD spectrum of the compound. NH-2 and H in the ROESY spectrum of the compound 2 The correlation of 18 shows that the orientation is the same, and indirectly proves that the olefin configuration formed by C-3 and C-16 is Z type, and the spatial configuration of the compound C-14 is obtained by TDDFT ECD calculation. The results indicate that the configuration of C-14 is 14R. Thus far, the structure of the compound of the present invention was confirmed. The compound was named: aspergillus versicolor alkaloid-B, english name: isoaspergilline B.
Compound 2, whose high resolution mass spectrum gives an excimer ion peak the same as that of compound 1, and of which 1 H and 13 the NMR data for C and HSQC are highly similar to that of Compound 1, and the two compounds were found to be E/Z isomers by carefully comparing their 1D and 2D NMR data, which is presumed to be due to the presence of NH-2 and H in the ROESY spectrum 3 -17 correlation was confirmed. Thus far, the structure of the compound of the present invention was confirmed. The compound was named: aspergillus versicolor alkaloid-C, english name: isoaspergilline C.
The following table is for compounds 1 and 2 1 H NMR and 13 c NMR data (CDCl) 3 )
Example 4
The compound prepared in example 2 was taken as a brown gum.
As shown in FIGS. 5 to 7, the measurement method was the same as in example 3; the compound prepared in example 2 was confirmed to be the quinazolinone alkaloid compound aspergillus isoloba alkaloid-B and: aspergillus versicolor alkaloid-C.
Example 5
The quinazolinone alkaloid compounds prepared in examples 1-2 were tested for activity against tobacco mosaic virus 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 20M.
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 × 10) -3 ) Uniformly smearing on the leaves scattered with carborundum, immediately placing in a culture dish containing liquid medicine for processing for 20min after all selected leaves are disinfected, taking out, sprinkling water drops and confining liquid on the leaves, recovering and arranging two half leaves, covering glass in an enamel officer spread with toilet paper for moisturizing, controlling the temperature to be (23 +/-2) DEG C, and placing in a greenhouse for natural light irradiation, wherein the withered spots can be seen after 2-3 d. The other half of the leaves were set for each treatment as a control, and 1 group of treatments with commercial ningnanmycin was set as a control, and the relative inhibition was calculated according to the following formula.
XI％＝(CK-T)/CK×100％
X: relative inhibition ratio (%), CK: number of dead spots of half-leaf inoculated in clear water, T: the number of dead spots of half leaf inoculated with virus is soaked in the liquid medicine.
The results show that the relative inhibition rate of the compound 1 is 31.5 percent, the relative inhibition rate of the compound 2 is 29.4 percent, the activity of the two compounds is similar to that of a control ningnanmycin (33.2 percent), and the compound has good anti-tobacco mosaic virus activity.
Claims (10)
1. Quinazolinone alkaloid compounds, characterized in that: the quinazolinone alkaloid compound has a structure of formula 1 or formula 2:
2. the process for preparing quinazolinone alkaloid compounds according to claim 1, comprising the following steps:
1) And extracting the extract
Performing solid fermentation on Aspergillus versicolor strain YATS1111 separated and identified from tobacco, performing ultrasonic extraction on a fermentation product by using ethanol, filtering, concentrating a filtrate, adding a mixed solution of ethyl acetate and tartaric acid, fully and uniformly stirring, standing for layering, separating a water phase, and then using Na as the water phase 2 CO 3 Adjusting the pH value to 8.0-10.0, extracting again by using ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain an extract;
2) Silica gel column chromatography
Filling the extract obtained in the step 1) 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, mixing the parts with the same polarity, collecting eluate of each part, and concentrating, wherein the eluate with first concentration gradient is collected and called as first eluate;
continuously separating the first eluate with silica gel chromatographic column, performing gradient elution with chloroform-acetone solution, and collecting eluate with second concentration gradient, which is called second eluate;
3) Separating by high performance liquid chromatography
Introducing the second eluent finally obtained in the step 2) into a high performance liquid chromatography for separation and purification, and collecting eluent corresponding to a chromatographic peak after each sample injection to obtain a third eluent; removing the solvent from the third eluent to obtain a crude product of the quinazolinone alkaloid compound;
4) And separating by gel column chromatography
And (3) carrying out gel column chromatography separation on the crude quinazolinone alkaloid compound by taking methanol as fluidity again to obtain a pure quinazolinone alkaloid compound.
3. The method for producing a quinazolinone alkaloid compound according to claim 2, characterized in that: in the step 1), the concentration of the ethanol is 90-99 wt%.
4. The method for producing a quinazolinone alkaloid compound according to claim 2, characterized in that: in step 1), the concentration of ethanol is 95wt%.
5. The method for producing a quinazolinone alkaloid compound according to claim 2, characterized in that: in the step 2), the extract is dissolved by methanol before being subjected to silica gel column chromatography and then is mixed with 80-120 mesh silica gel with the weight of 1.5-2.5 times of that of the extract.
6. The process for producing quinazolinone alkaloid compounds according to claim 2, characterized in that: in the step 3), after the separation and purification by the high performance liquid chromatography, the obtained compound is dissolved by pure methanol again, and then the pure methanol is used as a mobile phase to be separated by gel column chromatography for further separation and purification.
7. The method for producing a quinazolinone alkaloid compound according to claim 2, characterized in that: in step 2), when the chloroform-methanol solution is subjected to gradient elution, the concentration gradient is set as the following components in volume ratio of 20; the first eluent is an eluent when 20.
8. The process for producing quinazolinone alkaloid compounds according to claim 2, characterized in that: in step 2), when the chloroform-acetone solution is subjected to gradient elution, the concentration gradient is set as the following volume ratio of 9; the second eluent is an eluent when the eluent is chloroform-acetone solution of 8.
9. The method for producing a quinazolinone alkaloid compound according to claim 2, characterized in that: in the step 3), the high performance liquid chromatography is used for separation and purification, a Zorbax PherpHT GF chromatographic column with the flow rate of 20mL/min and the flow phase of 52wt% methanol aqueous solution is adopted, the detection wavelength of an ultraviolet detector is 359nm, 200 mu L of second eluent is injected each time, the eluent corresponding to the chromatographic peak after each sample injection is collected, the compound 1 is the eluent corresponding to the retention time of 29.6min, and the compound 2 is the eluent corresponding to the retention time of 29.8 min.
10. The use of quinazolinone alkaloid compounds according to claim 1 for the preparation of a medicament against tobacco mosaic virus.
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