CN115583953B - 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 20
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 241000723873 Tobacco mosaic virus Species 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 abstract description 45
- 241000203233 Aspergillus versicolor Species 0.000 abstract description 22
- 238000000855 fermentation Methods 0.000 abstract description 22
- 230000004151 fermentation Effects 0.000 abstract description 22
- 241000208125 Nicotiana Species 0.000 abstract description 13
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract description 13
- 229940125904 compound 1 Drugs 0.000 abstract description 12
- 229940125782 compound 2 Drugs 0.000 abstract description 11
- 230000005764 inhibitory process Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 10
- AMNAZJFEONUVTD-KEWDHRJRSA-N (2s,3s,4s,5r,6r)-6-(4-amino-2-oxopyrimidin-1-yl)-4,5-dihydroxy-3-[[(2s)-3-hydroxy-2-[[2-(methylamino)acetyl]amino]propanoyl]amino]oxane-2-carboxamide Chemical compound O1[C@H](C(N)=O)[C@@H](NC(=O)[C@H](CO)NC(=O)CNC)[C@H](O)[C@@H](O)[C@@H]1N1C(=O)N=C(N)C=C1 AMNAZJFEONUVTD-KEWDHRJRSA-N 0.000 abstract description 4
- FEACDOXQOYCHKU-UHFFFAOYSA-N Gougerotin Natural products CNCC(=O)NC1=NC(=O)N(C=C1)C2OC(C(O)C(NC(=O)C(N)CO)C2O)C(=O)N FEACDOXQOYCHKU-UHFFFAOYSA-N 0.000 abstract description 4
- 229930013930 alkaloid Natural products 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 229930014626 natural product Natural products 0.000 abstract description 3
- 150000003797 alkaloid derivatives Chemical class 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- 239000003480 eluent Substances 0.000 description 17
- 239000000047 product Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 8
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 239000000741 silica gel Substances 0.000 description 7
- 229910002027 silica gel Inorganic materials 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- 239000011975 tartaric acid Substances 0.000 description 5
- 235000002906 tartaric acid Nutrition 0.000 description 5
- 241001513093 Aspergillus awamori Species 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- ZTNBSFMIFOLVCM-MCTVSQGJSA-N (1s)-1-[(3s,8s,9s,10r,13s,14s,17s)-3-methoxy-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl]-n,n-dimethylethanamine Chemical compound C([C@@H]12)C[C@]3(C)[C@@H]([C@H](C)N(C)C)CC[C@H]3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC)C1 ZTNBSFMIFOLVCM-MCTVSQGJSA-N 0.000 description 3
- ZTNBSFMIFOLVCM-UHFFFAOYSA-N 20S-Dimethylamino-3alpha-methoxypregn-5-ene Natural products C12CCC3(C)C(C(C)N(C)C)CCC3C2CC=C2C1(C)CCC(OC)C2 ZTNBSFMIFOLVCM-UHFFFAOYSA-N 0.000 description 3
- PZZXYDQKZIGACT-UHFFFAOYSA-N 5-hydroxyskytanthine Natural products C1N(C)CC(C)C2(O)C1C(C)CC2 PZZXYDQKZIGACT-UHFFFAOYSA-N 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241000400611 Eucalyptus deanei Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- SZCHXNLVRKQEGO-XKDXWZMYSA-N alkaloid b Chemical compound C([C@@]12C=C([C@H]([C@H]2C2=C3)O)OC)CCN1C[C@H](O)C2=CC1=C3OCO1 SZCHXNLVRKQEGO-XKDXWZMYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 210000003298 dental enamel Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 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
- 238000012544 monitoring process Methods 0.000 description 2
- 239000001965 potato dextrose agar Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 2
- 229910010271 silicon carbide Inorganic materials 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- AVRPFRMDMNDIDH-UHFFFAOYSA-N 1h-quinazolin-2-one Chemical class C1=CC=CC2=NC(O)=NC=C21 AVRPFRMDMNDIDH-UHFFFAOYSA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 108010011619 6-Phytase Proteins 0.000 description 1
- 244000100578 Amorphophallus variabilis Species 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 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
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- FIMJSWFMQJGVAM-UHFFFAOYSA-N chloroform;hydrate Chemical compound O.ClC(Cl)Cl FIMJSWFMQJGVAM-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000002212 electronic circular dichroism spectrum Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229940085127 phytase Drugs 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- C12P17/18—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
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Dentistry (AREA)
- Biotechnology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 (C) 22 H 21 N 3 O 2 Has a structure of formula 1 or formula 2. The compound has good tobacco mosaic virus resistance. Experiments on tobacco mosaic virus resistance show that 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 that of the control Ningnanmycin (33.2%).
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 planted commercial crops worldwide. According to literature reports, the compounds identified from tobacco are up to 4000, and the main components comprise diterpenoid compounds, sesquiterpenoid compounds, flavonoid compounds, alkaloids and coumarin. Meanwhile, researches prove that the compounds have different pharmacological effects, such as antibiosis, antioxidation, anti-tumor, tobacco mosaic virus resistance and the like. Therefore, the research on the metabolic products of the endophytic fungi in tobacco is enhanced, and the method has important scientific significance for discovering the metabolic products of the new skeleton type with remarkable activity.
Disclosure of Invention
The invention separates and identifies the aspergillus versicolor strain culture solution from tobacco to obtain two new quinazolinone alkaloid compounds with anti-tobacco mosaic virus activity, and the compounds have no related report so far.
The first aspect of the invention provides a quinazolinone alkaloid compound, which has a molecular formula as follows: c (C) 22 H 21 N 3 O 2 Has the structure of formula 1 or formula 2:
both compounds were brown gum, named: aspergillus awamori alkaloid-B, english name: isoaspergilline B A.variabilis alkaloid-C, english name: isoaspergilline C.
Aspergillus fungi are widely found in nature. Wherein, aspergillus oryzae is a strain capable of producing complex enzyme, which can produce amylase, saccharifying enzyme, cellulase, phytase and other enzymes besides protease, thus being widely applied to fermentation industries such as food, feed, brewing and the like. Meanwhile, aspergillus versicolor secondary metabolites are also considered as an important resource to be developed urgently. A series of natural products with biological activity including alkaloids, polypeptides, terpenoids and polyphenols are also isolated from Aspergillus versicolor fermentation products from different sources. The compound is two novel quinazolinone alkaloid compounds separated from tobacco endogenous aspergillus versicolor (Aspergillus versicolor) YATS1111 fungus fermentation products. It is worth mentioning that, through experiments on tobacco mosaic virus resistance, the relative inhibition rate of the compound 1 is found to be 31.5%, the relative inhibition rate of the compound 2 is found to be 29.4%, and the activities of the two compounds are similar to that of the control Ningnanmycin (33.2%).
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) Extraction of extractum
Performing solid fermentation on aspergillus versicolor strain YATS1111 separated and identified in tobacco, ultrasonically extracting the fermentation product with ethanol, filtering, concentrating the filtrate, adding mixed solution of ethyl acetate and tartaric acid, stirring thoroughly, standing for layering, separating out water phase, and using Na for the water phase 2 CO 3 Regulating pH to 8.0-10.0, extracting again with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain extract;
2) Silica gel column chromatography
Loading the extract obtained in the step 1) into a column by using 200-300 mesh silica gel dry method, and performing silica gel column chromatography; gradient eluting with chloroform-methanol solution, mixing the same polarity fractions, collecting the eluate, and concentrating, wherein the eluate with first concentration gradient is called first eluate;
continuing to separate the first eluent by using a silica gel chromatographic column, performing gradient elution by using chloroform-acetone solution, and collecting eluent with a second concentration gradient, namely second eluent;
3) High performance liquid chromatography separation
Introducing the second eluent obtained in the step 2) into high performance liquid chromatography for separation and purification, and collecting eluent corresponding to chromatographic peaks after each sample injection to obtain a third eluent; removing the solvent from the third eluent to obtain a crude quinazolinone alkaloid compound;
4) Chromatographic separation by gel column
And (3) performing gel column chromatographic separation on the crude quinazolinone alkaloid compound by taking methanol as fluidity again to obtain a pure quinazolinone alkaloid compound.
As a preferable mode of the technical scheme, in the step 1), the concentration of the ethanol is 90-99 wt%; further, the concentration of ethanol was 95wt%.
As a preferable mode of the technical scheme, in the step 2), before the extract is roughly separated by silica gel column chromatography, methanol is used for dissolving, and 80-120 meshes of silica gel with the weight of 1.5-2.5 times is used for mixing samples.
In step 3), the compound obtained after separation and purification by high performance liquid chromatography is dissolved again with pure methanol, and then separated by gel column chromatography with pure methanol as mobile phase to further separate and purify.
As a preferable mode of the above technical scheme, in step 2), when the chloroform-methanol solution is subjected to gradient elution, the concentration gradient is set to be 20:1, 9:1,8:2,7:3,6:4, 5:5 by volume ratio; the first eluent is an eluent when the chloroform-methanol solution is eluted at a ratio of 20:1.
As a preferable mode of the above technical scheme, in step 2), when the chloroform-acetone solution is subjected to gradient elution, the concentration gradient is set to be 9:1,8:2,7:3,6:4, 5:5 by volume ratio; the second eluent is an eluent in the process of eluting by using a chloroform-acetone solution with the ratio of 8:2.
As a preferable mode of the technical scheme, in the step 3), the separation and purification by the high performance liquid chromatography are carried out by adopting a ZorbaxPREST GF chromatographic column with 21.2mm multiplied by 250mm and 5 mu m, the flow rate is 20mL/min, the mobile phase is 52wt% of methanol water solution, the detection wavelength of an ultraviolet detector is 359nm, 200 mu L of second eluent is injected each time, eluent corresponding to chromatographic peaks after each injection is collected, the eluent corresponding to the retention time of 29.6min is compound 1, and the eluent corresponding to the retention time of 29.8min is compound 2.
The invention also aims to provide application of the quinazolinone alkaloid compound in preparation of tobacco mosaic virus resistant medicines.
In summary, the invention has the following beneficial effects:
1. the compound is separated from fermentation products of aspergillus versicolor fungus strains in tobacco, and the endophytic fungi are easy to realize batch fermentation production, so that the raw materials of the compound are easy to obtain; the extraction method of the compound is simple, the compound is easy to separate and obtain, and the industrialized preparation is easy to realize.
2. The compound has good tobacco mosaic virus resistance. Experiments on tobacco mosaic virus resistance show that 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 that of the control Ningnanmycin (33.2%). The compound has good application prospect in preparing medicines for resisting tobacco mosaic virus.
3. The compound has simple molecular structure, is easy to realize artificial synthesis, and can be realized through artificial synthesis in the subsequent industrialization.
4. The preparation method combining the conventional column chromatography and the 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 the subsequent industrial production are ensured.
5. The compound disclosed by the invention is safe and nontoxic, shows good activity of resisting the tobacco mosaic virus, and can provide ideal new skeleton type medicine source molecules for preventing and treating the tobacco mosaic disease.
Drawings
FIG. 1 shows the nuclear magnetic resonance carbon spectrum of the compound 1.
FIG. 2 shows the nuclear magnetic resonance hydrogen spectrum of the compound 1.
FIG. 3 is the main HMBC and the compound 1 1 H- 1 HCOSY correlation.
FIG. 4 is an ECD diagram of the compound 1.
FIG. 5 is a nuclear magnetic resonance carbon spectrum of the compound 2.
FIG. 6 shows the nuclear magnetic resonance hydrogen spectrum of the compound 2.
FIG. 7 is a main HMBC and of the compound 2 1 H- 1 HCOSY correlation.
Detailed Description
The present invention will be further illustrated by the following examples, but is not limited to the examples. Experimental methods, in which specific conditions are not specified in examples, are generally available commercially according to conventional conditions as well as those described in handbooks, or according to general-purpose equipment, materials, reagents, etc. used under conditions suggested by manufacturers, unless otherwise specified.
The raw materials used in the invention are not affected by the type of the culture medium, and the invention is further described below by using a culture medium of aspergillus versicolor strain isolated and identified from tobacco derived from Yunnan.
Isolation and identification of strains
Isolation of the endophytic fungus Aspergillus versicolor (Aspergillus versicolor) YATS1111
Putting the tobacco rhizome sterilized by 75% ethanol into a sterile mortar for grinding, transferring into a sterile plastic tube after grinding, centrifuging at 1000-3000 rpm for 2-10 min, sucking 1-100 microlitres of supernatant, coating on a BL flat plate, inverting in an incubator for 2-10 days in darkness at 25-30 ℃, repeatedly picking single bacterial colony for culturing and numbering for preserving the bacterial strain until a single endophytic fungus bacterial colony is obtained; aspergillus versicolor (Aspergillus versicolor) was identified as the fungus Aspergillus.
Microbiological characteristics of Aspergillus versicolor (Aspergillus versicolor) YATS1111
1) The colonies were grown 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 and no separation.
3) Conidiophores are smooth, the apotheca is elliptic, the small peduncles are radial, and the conidiophores are in two layers and are in the shape of conidiophore spheres at 3/4 of the apotheca.
Aspergillus versicolor (Aspergillus versicolor) YATS1111 strain culture
Inoculating the aspergillus versicolor strain obtained by the separation in the steps on a potato dextrose agar culture medium at room temperature, culturing for 7-10 days at 25-30 ℃, then inoculating the aspergillus versicolor strain into 50-500 ml triangular flasks, wherein each triangular flask contains 10-100 ml of potato dextrose culture medium, and performing shake culture for 5-10 days at 25-30 ℃ to obtain liquid fermentation seeds; the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and the preservation number is: CGMCC No.19910.
Example 1
And (3) carrying out large-scale fermentation on the liquid fermentation seeds obtained by culture, wherein the large-scale fermentation is carried out in 500mL Feng Bahe bottles, each bottle contains 180g of rice and 180mL of distilled water, 2.5mL of the liquid fermentation seeds obtained by culture are inoculated in each bottle, and the aspergillus versicolor fermentation product is obtained by culturing for 30 days at the temperature of 25-30 ℃. Ultrasonic extracting the fermentation product with 95% ethanol for 3 times for 30min each time; mixing the extractive solutions, adding into a mixed solution of ethyl acetate and 3% tartaric acid (ethyl acetate: tartaric acid=97:3, mass ratio), stirring, standing for layering, separating water phase, and Na-treating the water phase 2 CO 3 The pH of the aqueous layer was adjusted to 9.0 and re-extracted with ethyl acetate; the ethyl acetate phase is separated out and concentrated into extract under reduced pressure, thus obtaining 580g of extract. The extract is stirred with 1.0kg of 80-120 mesh silica gel, silica gel column chromatography is carried out by using 3.0kg of 200 mesh silica gel column, chloroform-methanol gradient elution with the volume ratio of 20:1,8:2,7:3,6:4 and 5:5 is carried out, TLC monitoring is carried out to combine the same parts, 5 parts are obtained, wherein the chloroform-methanol elution part with the volume ratio of 7:3 is concentrated, and chloroform-water is respectively carried out according to the volume ratio of 9:1,8:2,7:3,6:4 and 1:1Eluting with acetone solution, separating by high performance liquid chromatography with An Jielun 1100, preparing with 59% methanol as mobile phase, zorbaxPREPEREPT GF column (21.225 mm,5 m) as stationary phase, measuring wavelength with ultraviolet detector 359nm at 20mL/min, sampling 200L each time, collecting eluate corresponding to retention time of 29.6min for compound 1, collecting eluate corresponding to retention time of 29.8min for compound 2, accumulating for several times, and evaporating to obtain crude compound; dissolving the crude product with pure methanol again, and separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the pure product of the two new compounds.
Example 2
The liquid fermentation seeds obtained by culture are subjected to large-scale fermentation, the large-scale fermentation is carried out in 250 Feng Bahe bottles 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 in each bottle, and the aspergillus versicolor fermentation product is obtained by culturing for 30 days at the temperature of 25-30 ℃. Ultrasonic extracting the fermentation product with 95% ethanol for 3 times for 30min each time; mixing the extractive solutions, adding into a mixed solution of ethyl acetate and 3% tartaric acid (ethyl acetate: tartaric acid=97:3, mass ratio), stirring, standing for layering, separating water phase, and Na-treating the water phase 2 CO 3 The pH of the aqueous layer was adjusted to 9.0 and re-extracted with ethyl acetate; the ethyl acetate phase is separated out and concentrated into extractum under reduced pressure to obtain 610g of extractum. Mixing the extract with 1.0kg of 80-120 mesh silica gel, performing silica gel column chromatography with 3.2kg of 200 mesh silica gel column, performing gradient elution with chloroform-methanol with volume ratio of 20:1,8:2,7:3,6:4,5:5, performing TLC monitoring, mixing the same parts to obtain 5 parts, concentrating chloroform-methanol elution part with volume ratio of 8:2, eluting with chloroform-acetone solution with volume ratio of 9:1,8:2,7:3,6:4,1:1 respectively, preparing high performance liquid chromatography with An Jielun, preparing column with 59% methanol as mobile phase, preparing column with Zorbax pH T GF column (21.2250 mm,5 m) as stationary phase, flow rate of 20mL/min, ultraviolet detector detection wavelength of 359nm, sampling 200L each time, collecting eluent corresponding to compound 1 with retention time of 29.6min, and collecting compound 2 with retention time of 29.8min, accumulating the corresponding eluents for a plurality of times, and evaporating to dryness to obtain a crude compound product; dissolving the crude product with pure methanol again, and separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the pure product of the two new compounds.
Example 3
The structure of the quinazolinone alkaloid compound prepared as described in example 1 was identified by the following method:
appearance observation finds that: both compounds of the invention were brown gum.
As shown in fig. 1 to 4, the ultraviolet-visible absorption spectrum of the compound 1 showed maximum absorption at 200, 210, 269, and 304nm, proving that an aromatic ring structure exists in the compound. High resolution mass spectrometry (hresis) gives an excimer ion peak 382.1522[ m+na ]] + The molecular formula of the compound can be determined to be C 22 H 21 N 3 O 2 . Bonding of 1 H and 13 c and HSQC NMR data are similar to those of the compound protuboxepin K, with C-3 and C-16 of compound 1 forming a double bond and C-7 being reduced to hydrogen, presumably by 1 H– 1 H-7/H-8/H-9 in H COSY spectra and H 3 The HMBC correlation of-17 with C-3, C-16, and C-18 was confirmed.
After the planar structure of the compound is determined, the corresponding configuration of compound 1 is determined mainly by analyzing the reesy spectrum and the ECD spectrum of the compound. NH-2 and H in the ROESY spectrum of the Compound 2 The correlation of-18 shows that they are oriented identically and indirectly demonstrates that the olefin formed by C-3 and C-16 is Z-shaped, the stereochemistry of this compound C-14 being obtained by TDDFT ECD calculation. The result showed that C-14 had the configuration of 14R. Thus, the structure of the compound of the present invention was confirmed. The compounds were named: aspergillus awamori alkaloid-B, english name: isoaspergilline B.
Compound 2, whose high resolution mass spectrum gives an excimer ion peak as in compound 1, and which compound 1 H and 13 c and HSQC NMR data are highly similar to those of Compound 1, by careful comparison of 1D and 2D NMR data for these two compoundsIt is now the E/Z isomer, which can be presumed by NH-2 and H in the ROESY spectrum 3 The correlation of-17 was confirmed. Thus, the structure of the compound of the present invention was confirmed. The compounds were named: aspergillus awamori alkaloid-C, english name: isoaspergilline C.
The following tables are for compounds 1 and 2 1 H NMR 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 compounds prepared in example 2 were identified as the quinazolinone alkaloids, aspergillus isobenzophenone-B and: aspergillus awamori alkaloid-C.
Example 5
The quinazolinone alkaloid compounds prepared in examples 1-2 are taken for an activity test for resisting tobacco mosaic virus, and the test conditions are as follows:
the tobacco mosaic virus resistance activity of the compound is measured by adopting a half leaf method when the mass concentration of the medicament is 20M.
Selecting leaves (leaf rows are normal, disease and insect free) suitable for testing on plants of 5-6 flue-cured tobacco plants, uniformly scattering fine silicon carbide on the leaves, and using a writing brush to make a spare tobacco mosaic virus source (3.0 multiplied by 10) -3 ) Uniformly smearing on the leaves scattered with silicon carbide, immediately placing the leaves in a culture dish containing liquid medicine for 20min after the selected leaves are subjected to poison receiving, taking out, sprinkling water drops on the leaves and the liquid, recovering and discharging the two half leaves, covering glass in enamel covered with toilet paper for moisture preservation, controlling the temperature (23+/-2) ℃, and placing the enamel in a greenhouse for natural light irradiation for 2-3 d to obtain the visible dead spots. For each treatment, the other half of the leaves were used as a control, and for the other 1 group of treatments were used as a control, and the relative inhibition was calculated according to the following formula.
XI％＝(CK-T)/CK×100％
X: relative inhibition (%), CK: the number of dead spots of half-leaf virus-inoculated leaves (a plurality of leaves) soaked in clear water, T: the number of the dead spots of the half-leaf of the virus-inoculated leaf soaked in the liquid medicine.
The result shows that 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 the control Ningnan mycin, which indicates that the compounds have good tobacco mosaic virus resistance activity.
Claims (1)
1. Application of quinazolinone alkaloid compound in preparing tobacco mosaic virus resisting medicine; the quinazolinone alkaloid compound is characterized by having a structure of formula 1 or formula 2:
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