CN116444475A - Sesquiterpenoids and preparation method and application thereof - Google Patents
Sesquiterpenoids and preparation method and application thereof Download PDFInfo
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- CN116444475A CN116444475A CN202310312563.3A CN202310312563A CN116444475A CN 116444475 A CN116444475 A CN 116444475A CN 202310312563 A CN202310312563 A CN 202310312563A CN 116444475 A CN116444475 A CN 116444475A
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- petroleum ether
- fermentation
- sesquiterpenoids
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- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- -1 sesquiterpene compound Chemical class 0.000 claims abstract description 65
- 229930004725 sesquiterpene Natural products 0.000 claims abstract description 60
- 239000003814 drug Substances 0.000 claims abstract description 15
- 229940079593 drug Drugs 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- 238000000855 fermentation Methods 0.000 claims description 44
- 230000004151 fermentation Effects 0.000 claims description 44
- 239000003480 eluent Substances 0.000 claims description 40
- 244000052363 Cynodon dactylon Species 0.000 claims description 34
- 239000003208 petroleum Substances 0.000 claims description 32
- 241000233866 Fungi Species 0.000 claims description 31
- 239000002904 solvent Substances 0.000 claims description 27
- 238000004440 column chromatography Methods 0.000 claims description 26
- 239000000287 crude extract Substances 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims description 20
- 238000000926 separation method Methods 0.000 claims description 20
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 238000005227 gel permeation chromatography Methods 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000000741 silica gel Substances 0.000 claims description 13
- 229910002027 silica gel Inorganic materials 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 238000013375 chromatographic separation Methods 0.000 claims description 12
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- 244000061456 Solanum tuberosum Species 0.000 claims description 8
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 8
- 238000002137 ultrasound extraction Methods 0.000 claims description 7
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 6
- 235000007164 Oryza sativa Nutrition 0.000 claims description 5
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 5
- 235000009566 rice Nutrition 0.000 claims description 5
- 210000001113 umbilicus Anatomy 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 235000012015 potatoes Nutrition 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 abstract description 11
- 210000000952 spleen Anatomy 0.000 abstract description 8
- 230000002708 enhancing effect Effects 0.000 abstract description 7
- 210000004698 lymphocyte Anatomy 0.000 abstract description 6
- 230000001737 promoting effect Effects 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 4
- 241000699670 Mus sp. Species 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 description 26
- 230000002434 immunopotentiative effect Effects 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 229940125904 compound 1 Drugs 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 239000002158 endotoxin Substances 0.000 description 11
- 229920006008 lipopolysaccharide Polymers 0.000 description 11
- 238000004821 distillation Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 9
- 229940125782 compound 2 Drugs 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 238000012258 culturing Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 6
- 238000010898 silica gel chromatography Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000005100 correlation spectroscopy Methods 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 150000003505 terpenes Chemical class 0.000 description 5
- 241001465178 Bipolaris Species 0.000 description 4
- 241000209094 Oryza Species 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 4
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 4
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 3
- 238000003775 Density Functional Theory Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 235000007586 terpenes Nutrition 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 2
- 241000227129 Aconitum Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000004057 DFT-B3LYP calculation Methods 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 150000002596 lactones Chemical group 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000002436 one-dimensional nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 229940124531 pharmaceutical excipient Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000002495 two-dimensional nuclear magnetic resonance spectrum Methods 0.000 description 2
- 241000220451 Canavalia Species 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 108010034145 Helminth Proteins Proteins 0.000 description 1
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical group CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 1
- 238000000719 MTS assay Methods 0.000 description 1
- 231100000070 MTS assay Toxicity 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- ZEEBGORNQSEQBE-UHFFFAOYSA-N [2-(3-phenylphenoxy)-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound C1(=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)C1=CC=CC=C1 ZEEBGORNQSEQBE-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- SIKJAQJRHWYJAI-UHFFFAOYSA-N benzopyrrole Natural products C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000002518 distortionless enhancement with polarization transfer Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 244000000013 helminth Species 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000012585 nuclear overhauser effect spectroscopy experiment Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a sesquiterpene compound and a preparation method and application thereof. The invention provides a sesquiterpene compound, which has a structure shown in a formula 1 or a formula 2. The sesquiterpene compound with the structure shown in the formula 1 or the formula 2 provided by the invention is a sesquiterpene compound containing a 2- (dihydro-2H-pyran-2-yl) propanol unit, so that the diversity of the sesquiterpene compound is enriched, and meanwhile, the immune promoting activity screening test of the sesquiterpene compound provided by the invention shows that the sesquiterpene compound provided by the invention is specific to concanavalin (ConA) or lipodolSugar (LPS) induces proliferation of spleen lymphocytes of mice, shows a certain immunity enhancing effect, and can be applied to preparation of immunity enhancing medicaments.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a sesquiterpene compound and a preparation method and application thereof.
Background
Sesquiterpenes (sesquiterpenes) are natural terpenes containing 15 carbon atoms in one molecule, and are an important group of natural products containing three isoprene units and having structural diversity.
The literature reports that a variety of sesquiterpenoids with different backbones have stimulated the study of pharmaceutical analogs. Among these, the reported sesquiterpene units containing 2- (dihydro-2H-pyran-2-yl) propanol (2- (tetrahydro-2H-pyran-2-yl) pro-pan-2-ol) are one important subunit of hexapeptide-terpenoids, as well as indole diterpenoids. However, sesquiterpenes having a 2- (dihydro-2H-pyran-2 yl) propanol unit remain an extremely rare class of terpenoids. To date, in the studies of structural assays and biosynthesis of hexakenone (terpene) terpenes (Terpenoids) in 1971 and 1973, only three artifacts containing lactone or lactone ring-opening units were reported, and no nuclear magnetic data and activity assessment was available.
Disclosure of Invention
The invention aims to provide a sesquiterpene compound, a preparation method and application thereof, and the sesquiterpene compound provided by the invention has immune promoting activity, can be applied to preparation of immune promoting medicines, and enriches the diversity of the sesquiterpene compound.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a sesquiterpene compound, which has a structure shown in a formula 1 or a formula 2:
the invention provides a preparation method of the sesquiterpene compound, which comprises the following steps:
inoculating the bermudagrass fungus to a fermentation medium for fermentation to obtain a bermudagrass fungus fermentation product;
mixing the bermuda grass umbilicus vermiculosus fungus ferment with an alcohol solvent, and performing ultrasonic extraction to obtain a crude extract of sesquiterpenes compound;
mixing the crude extract of the sesquiterpenoids with silica gel, filling the obtained mixture into a column, and performing first column chromatography separation, wherein the first column chromatography separation is performed by gradient elution according to the volume ratio of chloroform to methanol of a chloroform-methanol system of which the volume ratio is 100:0-10:1, and merging the same fractions to obtain five sections of fractions which are respectively named as Fr.1-Fr.5 fractions;
subjecting the Fr.2 fraction to second column chromatography to obtain eluent containing sesquiterpenoids with structure shown in formula 1; the eluent for the second column chromatographic separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system;
subjecting the Fr.4 fraction to third column chromatography to obtain eluent containing sesquiterpenoids with structure shown in formula 2; the eluent for the third column chromatographic separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system;
purifying the eluent containing the sesquiterpene compound with the structure shown in the formula 1 by first gel chromatography to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 1;
and (3) performing second gel chromatography purification on the eluent containing the sesquiterpene compound with the structure shown in the formula 2 to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 2.
Preferably, the raw materials of the fermentation medium comprise potatoes and/or rice; the fermentation is solid fermentation.
Preferably, the temperature of the fermentation is 20-25 ℃, and the time of the fermentation is 30-35 d.
Preferably, the alcoholic solvent comprises one or more of methanol, ethanol, propanol and isopropanol; the ratio of the mass of the bermuda grass vermicular fungus ferment to the volume of the alcohol solvent is (50-70) g (80-130) mL.
Preferably, the first column chromatography separation is: gradient elution was performed sequentially with chloroform-methanol systems at volume ratios of 100:0, 100:1, 50:1, 30:1, and 10:1.
Preferably, the weight ratio of the dry weight of the crude extract of the sesquiterpenoids to the mass ratio of the silica gel is 1 (1-1.2);
the mesh number of the silica gel is 300-400 mesh.
Preferably, the eluent for the second column chromatographic separation is a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone is 30:1.
Preferably, the eluent for the third column chromatographic separation is a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone is 15:1.
The invention provides application of the sesquiterpene compound prepared by the technical scheme or the preparation method of the sesquiterpene compound in preparation of immunity-promoting medicaments.
The invention provides a sesquiterpene compound, which has a structure shown in a formula 1 or a formula 2. The sesquiterpene compound with the structure shown in the formula 1 or the formula 2 provided by the invention is a sesquiterpene compound containing 2- (dihydro-2H-pyran-2-yl) propanol (2- (tetrahydro-2H-pyran-2-yl) pro-pan-2-ol) unit, so that the diversity of the sesquiterpene compound is enriched, and meanwhile, the immune promotion activity screening test of the sesquiterpene compound provided by the invention shows that the sesquiterpene compound provided by the invention has a certain immune promotion effect on induction of mouse spleen lymphocyte proliferation in concanavalin (ConA) or Lipopolysaccharide (LPS), and can be applied to preparation of immune promotion drugs.
The invention provides a preparation method of the sesquiterpene compound. The preparation method provided by the invention is based on microbial fermentation preparation, and has the advantages of short preparation period, mild culture condition, few byproducts and low cost, thereby meeting the requirements of environmental protection and low carbon and being easy to realize industrialization.
Drawings
FIG. 1 shows the immunopotentiating compound bipodonine E of the present invention 1 H-NMR spectrum;
FIG. 2 shows the immunopotentiating compound bipodonine E of the present invention 13 C-NMR and DEPT spectra;
FIG. 3 shows the immunopotentiating compound bipodonine E of the present invention 1 H- 1 H COSY profile;
FIG. 4 is a chart showing the HMBC pattern of the immunopotentiating active compound bipodonine E of the present invention;
FIG. 5 is a spectrum of HSQC of bipodonine E, an immune enhancing active compound of the present invention;
FIG. 6 is a NOESY spectrum of bipodonine E, an immune enhancing active compound of the present invention;
FIG. 7 is a chart showing the HR-ESI-MS spectrum of bipodonine E, an immunopotentiating active compound of the present invention;
FIG. 8 is an ECD experimental and computational profile of the immunopotentiating active compound bipodonine E of the present invention;
FIG. 9 shows the immunopotentiating compound bipodonine G of the present invention 1 H-NMR spectrum;
FIG. 10 shows the immunopotentiating compound bipodonine G of the present invention 13 C-NMR and DEPT spectra;
FIG. 11 shows the immunopotentiating compound bipodonine G of the present invention 1 H- 1 H COSY profile;
FIG. 12 is a chart showing the HMBC pattern of the immunopotentiating active compound bipodonine G of the present invention;
FIG. 13 is a spectrum of HSQC of bipodonine G, an immune enhancing active compound of the present invention;
FIG. 14 is a ROESY spectrum of bipodonine G, an immune enhancing active compound of the present invention;
FIG. 15 is a chart showing the HR-ESI-MS spectrum of bipodonine G, an immunopotentiating active compound of the present invention;
FIG. 16 is an ECD experimental and computational graph of the immunopotentiating active compound bipodonine G of the present invention.
Detailed Description
The invention provides a sesquiterpene compound, which has a structure shown in a formula 1 or a formula 2:
in the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The invention identifies the structure of the sesquiterpenoids shown in the formulas 1 and 2 by combining one-dimensional nuclear magnetic resonance spectrum and two-dimensional nuclear magnetic resonance spectrum (1D/2D NMR), HR-ESI-MS and quantum chemistry ECD calculation, 1 H、 13 C、 1 H- 1 the H COSY and HMBC, HSQC, ROESY nuclear magnetic data, HR-ESI-MS and ECD calculation patterns are shown in the accompanying drawings 1-16 of the specification.
The invention provides a preparation method of the sesquiterpene compound, which comprises the following steps:
inoculating the bermudagrass fungus to a fermentation medium for fermentation to obtain a bermudagrass fungus fermentation product;
mixing the bermuda grass umbilicus vermiculosus fungus ferment with an alcohol solvent, and performing ultrasonic extraction to obtain a crude extract of sesquiterpenes compound;
mixing the crude extract of the sesquiterpenoids with silica gel, filling the obtained mixture into a column, and performing first column chromatography separation, wherein the first column chromatography separation is performed by gradient elution according to the volume ratio of chloroform to methanol of a chloroform-methanol system of which the volume ratio is 100:0-10:1, and merging the same fractions to obtain five sections of fractions which are respectively named as Fr.1-Fr.5 fractions;
subjecting the Fr.2 fraction to second column chromatography to obtain eluent containing sesquiterpenoids with structure shown in formula 1; the eluent separated by the second column chromatography is a first petroleum ether-acetone system;
subjecting the Fr.4 fraction to third column chromatography to obtain eluent containing sesquiterpenoids with structure shown in formula 2; the eluent of the third column chromatographic separation is a second petroleum ether-acetone system;
purifying the eluent containing the sesquiterpene compound with the structure shown in the formula 1 by first gel chromatography to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 1;
and (3) performing second gel chromatography purification on the eluent containing the sesquiterpene compound with the structure shown in the formula 2 to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 2.
Inoculating the bermudagrass fungus into a fermentation medium for fermentation to obtain a bermudagrass fungus fermentation product.
In the invention, the bermuda grass bipolaris fungus is endophytic fungus of aconitum brachypomum, preferably separated from the root of aconitum brachypomum. The method has no special requirement on the separation method, and adopts the conventional technical means in the field. The invention preferably activates the bermuda grass bipolaris fungus before the fermentation is carried out; the activation is preferably: inoculating the bermudagrass fungus to a PDA slant culture medium, and culturing at constant temperature to obtain the activated bermudagrass fungus. In the present invention, the temperature of the constant temperature culture is preferably 25 to 30 ℃, more preferably 28 ℃, and the time is preferably 3 to 7 days, more preferably 5 days. The invention preferably stores the obtained activated bermuda grass vermicular fungus in an environment of 5 ℃ for standby.
In the present invention, the fermentation is preferably a solid fermentation.
In the present invention, the raw material of the fermentation medium preferably includes potato and/or rice. In the present invention, the preparation method of the fermentation medium used for the fermentation preferably comprises the steps of:
and sequentially sterilizing and cooling the raw materials of the culture medium at high temperature to obtain the fermentation culture medium.
In the present invention, when the raw material of the culture medium is preferably potato, the present invention preferably peels and crushes the potato before the high temperature sterilization, the crushing preferably cuts the potato into pieces, and the volume of the pieces obtained by the cutting is preferably 1.0cm 3 . In the present invention, the preparation process of the fermentation medium is preferably performed in a tissue culture flask; the ratio of the mass of the raw material of the fermentation medium to the volume of the tissue culture flask is preferably 45 to 55g:75 to 85mL, more preferably 50g:80mL. In the present inventionThe temperature of the high-temperature sterilization is preferably 120-150 ℃, more preferably 121-130 ℃; the time is preferably 30 to 50 minutes, more preferably 35 to 40 minutes. In the invention, the tissue culture flask is preferably capped when the high-temperature sterilization is performed. The cooling mode is not particularly limited in the present invention, as long as it can cool to room temperature.
In the present invention, the fermentation is preferably: the activated bermuda grass bipolaris fungus is inoculated in a fermentation medium, and the inoculation amount (the mass ratio of the bermuda grass bipolaris fungus to the fermentation medium) is preferably 0.5%. The invention has no special requirements on the inoculation conditions, and the inoculation can be carried out by adopting a mode well known to a person skilled in the art. In the present invention, the temperature of the fermentation is preferably 20 to 30 ℃, more preferably 20 to 25 ℃; the time is preferably 30 to 40 days, more preferably 35 days. The invention adopts a solid fermentation mode, so that the fermentation time can be effectively shortened.
After obtaining the bermuda grass umbilicus vermiculus fungus ferment, the invention mixes the bermuda grass umbilicus vermiculus fungus ferment with alcohol solvent, and carries out ultrasonic extraction to obtain the crude extract of sesquiterpenes compound.
In the present invention, the alcoholic solvent preferably includes one or more of methanol, ethanol, propanol and isopropanol, more preferably includes methanol, ethanol, propanol or isopropanol, and still more preferably methanol. In the invention, the ratio of the mass of the bermuda grass vermicular fungus ferment to the volume of the alcohol solvent is preferably (50-70) g (80-130) mL, more preferably 60g:120mL. In the invention, the alcohol solvent has good solubility for the sesquiterpenoids with the structures shown in the formulas 1 and 2, and can well separate the sesquiterpenoids with the structures shown in the formulas 1 and 2 from the fermentation product of the bermuda grass helminth fungus. The invention has no special requirement on the mixing mode, and can be uniformly mixed.
In the present invention, the power of the ultrasonic extraction is preferably 250 to 350W, more preferably 300W, and the time is preferably 20 to 40min, more preferably 30min.
In the present invention, after the ultrasonic extraction, the present invention preferably further comprises filtering the system after the ultrasonic extraction; the invention is not particularly limited to the filtration, and a conventional filtration mode is adopted. After the filtration, the present invention preferably carries out reduced pressure distillation on the obtained filtrate to remove the solvent in the filtrate, thereby obtaining a crude extract of sesquiterpenoids. In the present invention, the vacuum degree of the reduced pressure distillation is preferably 10 to 15kPa, more preferably 12kPa; the temperature of the reduced pressure distillation is preferably 45 to 55 ℃, more preferably 50 ℃. The time of the reduced pressure distillation is not particularly limited as long as the solvent in the filtrate can be removed.
After obtaining a crude extract of the sesquiterpenoids, mixing the crude extract of the sesquiterpenoids with silica gel, loading the obtained mixture into a column, and carrying out first column chromatography separation, wherein the first column chromatography separation adopts a chloroform-methanol system with the volume ratio of chloroform to methanol of 100:0-10:1 for gradient elution according to the volume ratio, and merging the same fractions to obtain five sections of fractions which are respectively named as Fr.1-Fr.5 fractions.
In the present invention, the crude extract of sesquiterpenoids is preferably mixed with the silica gel in the form of a crude extract solution. In the present invention, the solvent in the crude extract solution is preferably methylene chloride. The invention preferably uses dichloromethane to obtain the crude extract of sesquiterpenes compound.
In the present invention, the ratio by mass of the dry weight of the crude extract of sesquiterpenoids to the silica gel is preferably 1 (1 to 1.2). In the present invention, the mesh number of the silica gel is 300 to 400 mesh. The invention preferably uses the mixture obtained after the solvent is removed, and the mixture is packed into a column.
In the present invention, the first column chromatography separation is preferably: gradient elution was performed sequentially with chloroform-methanol systems at volume ratios of 100:0, 100:1, 50:1, 30:1, and 10:1. In the present invention, 3 column volumes were eluted separately for each ratio, and then the eluents for each ratio were combined. Subjecting the obtained eluate to Thin Layer Chromatography (TLC), and mixing the same fractions to obtain five fractions, which are named fraction Fr.1, fraction Fr.2, fraction Fr.3, fraction Fr.4 and fraction Fr.5; the first column chromatography separation is preferably performed at room temperature.
The invention carries out second column chromatography separation on the Fr.2 fraction to obtain the eluent containing sesquiterpenoids with the structure shown in the formula 1. In the present invention, the second column chromatography separation is preferably a silica gel column chromatography separation; the eluent for the second column chromatographic separation is preferably a petroleum ether-acetone system or a petroleum ether-ethyl acetate system, and more preferably a petroleum ether-acetone system. In the invention, in the petroleum ether-acetone system used for the second column chromatographic separation, the volume ratio of petroleum ether to acetone is preferably 30:1. In the present invention, the flow rate of the eluent for the second column chromatography is preferably 2 to 5mL/min, more preferably 3.5mL/min.
The invention carries out third column chromatography separation on the Fr.4 fraction to obtain the eluent containing sesquiterpenoids with the structure shown in the formula 2. In the present invention, the third column chromatography separation is preferably a silica gel column chromatography separation; the eluent for the third column chromatographic separation is preferably a petroleum ether-acetone system or a petroleum ether-ethyl acetate system, more preferably a petroleum ether-acetone system. In the invention, in the petroleum ether-acetone system used for the second column chromatographic separation, the volume ratio of petroleum ether to acetone is preferably 15:1. In the present invention, the flow rate of the eluent for the third column chromatography is preferably 2 to 5mL/min, more preferably 3.5mL/min.
The eluent containing the sesquiterpene compound with the structure shown in the formula 1 is subjected to first gel chromatography purification to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 1. In the present invention, the first gel-chromatography purified packing is preferably a sephadex, and the first gel-chromatography purified solvent is preferably methanol or a methanol-chloroform system, more preferably methanol; in the methanol-chloroform system, the volume ratio of methanol to chloroform is preferably 2:1. The flow rate of the solvent for the first gel chromatography purification is preferably 0.5 to 0.7mL/min, more preferably 0.6mL/min. In the present invention, the first gel chromatography is preferably performed to obtain a first purified solution, and the solvent of the first purified solution is preferably removed to obtain a pure sesquiterpene compound having a structure represented by formula 1. In the present invention, the method for removing the solvent is preferably distillation under reduced pressure; the temperature of the reduced pressure distillation is preferably 48 to 55 ℃, more preferably 50 ℃.
The eluent containing the sesquiterpene compound with the structure shown in the formula 2 is subjected to second gel chromatography purification to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 2. In the present invention, the packing material of the second gel chromatography purification is preferably a sephadex, and the solvent of the second gel chromatography purification is preferably methanol or a methanol-chloroform system, more preferably methanol; in the methanol-chloroform system, the volume ratio of methanol to chloroform is preferably 2:1. The flow rate of the solvent for the second gel chromatography purification is preferably 0.5 to 0.7mL/min, more preferably 0.6mL/min. In the present invention, the second gel chromatography is preferably performed to obtain a second purified solution, and the second purified solution is preferably subjected to solvent removal to obtain a pure sesquiterpene compound having a structure represented by formula 2. In the present invention, the method for removing the solvent is preferably distillation under reduced pressure; the temperature of the reduced pressure distillation is preferably 48 to 55 ℃, more preferably 50 ℃.
The invention provides application of the sesquiterpene compound prepared by the technical scheme or the preparation method of the sesquiterpene compound in preparation of immunity-promoting medicaments.
The invention provides an immunity-promoting medicament, which comprises a sesquiterpene compound in the technical scheme or the sesquiterpene compound prepared by the preparation method in the technical scheme and a medicament auxiliary material. In the immune promoting medicine, the mass percentage of the sesquiterpene compound prepared by the technical scheme or the preparation method is preferably 1-99%, more preferably 55-90%. The pharmaceutical excipients are not particularly limited, and the pharmaceutical excipients conventional in the art are selected. The preparation formulation and the preparation method of the immunity-promoting medicament are not particularly limited, and the medicament formulation such as tablets, granules or injections can be prepared by adopting the preparation method well known in the art.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Inoculating the bermudagrass fungus to a PDA slant culture medium, culturing at constant temperature at 28 ℃ for 5 days to obtain activated bermudagrass fungus, and storing in an environment at 5 ℃ for later use;
peeling, cleaning and cutting 5.0kg potato into 1cm volume 3 Sub-packaging the potato blocks in 100 tissue culture bottles (50 g/bottle) with the volume of 350mL, capping the tissue culture bottles, sterilizing at 121 ℃ for 30min, and cooling to obtain a fermentation culture medium;
inoculating the activated bermudagrass fungus to the fermentation medium according to the inoculation amount of 0.5%, and culturing for 35 days at normal temperature after capping to obtain a bermudagrass fungus ferment;
mixing the obtained bermuda grass vermicular fungus ferment with 120mL of methanol according to the mass volume ratio (50 g/120 mL), carrying out ultrasonic treatment for 30min under the condition of 40kHz after mixing, filtering, and carrying out reduced pressure distillation (the vacuum degree is 12kPa,50 ℃) on the filtrate until no alcohol smell exists, thus obtaining 83.0g of crude extract;
dissolving 83.0g of the crude extract in 100mL of dichloromethane, mixing with 83.0g of silica gel (300-400 meshes), concentrating under reduced pressure (the vacuum degree is 12kPa,50 ℃) to remove the solvent, and loading the mixture into a column; carrying out gradient elution by using a solution with the volume ratio of chloroform to methanol of 100:0, 100:1, 50:1, 30:1 and 10:1 to respectively obtain five fractions Fr.1, fr.2, fr.3, fr.4 and F.r5; performing silica gel column chromatography eluting on the Fr.3 fraction by using petroleum ether-acetone with the volume ratio of 30:1 as an eluent to obtain an eluent of a sesquiterpene compound (marked as bipodonine E) with the structure shown in the formula 1, and performing silica gel column chromatography eluting on the Fr.4 fraction by using petroleum ether-acetone with the volume ratio of 15:1 as an eluent to obtain an eluent of a sesquiterpene compound (marked as bipodonine G) with the structure shown in the formula 1;
purifying the eluate of bipodonine E and the eluate of bipodonine G respectively by using sephadex and methanol as solvent to obtain the pure product of bipodonine E and the pure product of bipodonine G.
Example 2 identification of Structure
The bipodonine E and bipodonine G prepared in example 1 were detected by nuclear magnetic resonance spectroscopy to give 1D/2D NMR (one-dimensional and two-dimensional), HR-ESI-MS (high resolution electrospray ionization mass spectrometry) and the compounds bipodonine E and bipodonine G identified by quantum chemical ECD calculations (FIGS. 1 to 16) and their structures.
The chemical shifts delta ascribed to H and C attached thereto of the compounds bipodonine E and bipodonine G obtained from the HSQC spectrum in combination with the carbon spectrum are shown in Table 1.
TABLE 1 bipodonine E and bipodonine G 13 C and C 1 H NMR data.
| No. | 1 a | 2 b | ||
| δ H (JinHz) | δ C | δ H (JinHz) | δ C | |
| 1 | 1.76dd(6.9,1.2) | 44.6 | 10.01d | 207.2 |
| 1.10overlap | ||||
| 2 | 4.14td(11.2,4.7) | 66.5 | 2.07s | 64.8 |
| 3 | 211.5 | 71.4 | ||
| 4 | 2.43m | 57.3 | 1.41m | 39.1 |
| 1.75dt(14.3,3.1) | ||||
| 5 | 1.50m | 29.0 | 1.45m | 21.4 |
| 1.96dt(12.7,4.1) | 1.66m | |||
| 6 | 3.20overlap | 82.1 | 3.16dd(11.9,3.5) | 83.7 |
| 7 | 33.7 | 37.6 | ||
| 8 | 1.31m | 38.5 | 1.57dd(12.6,3.9) | 37.6 |
| 1.60m | 1.91dt(11.5,3.8) | |||
| 9 | 1.47m | 21.6 | 1.50m | 24.0 |
| 1.60m | 1.84m | |||
| 10 | 3.23oerlap | 85.4 | 3.25dd(11.7,2.8) | 85.1 |
| 11 | 72.0 | 72.0 | ||
| 12 | 1.16s3H | 26.1 | 1.18s3H | 26.1 |
| 13 | 1.14s3H | 23.9 | 1.17s3H | 24.0 |
| 14 | 0.91s3H | 16.5 | 1.23s3H | 13.8 |
| 15 | 2.19s3H | 29.1 | 1.21s3H | 31.1 |
a Measured in CDCl 3 (600 MHz for 1 H NMR and 150 MHz for 13 C NMR)
b Measured in CDCl 3 (400 MHz for 1 H NMR and 100 MHz for 13 C NMR)
Compound 1 was a white powder. M/z 293.1723[ M+Na ] of High resolution Mass Spectrometry (High-resolution electrospray ionization mass spectrum, HR-ESI-MS) of Compound 1] + (calcd.for C 15 H 26 O 4 Na + 293.1723) shows that its molecular formula is C 15 H 26 O 4 The unsaturation was 3. One of the carbonyl groups (-CO-) occupies one unsaturation, the remaining 2 indicating that compound 1 has a two-ring backbone. Comprehensive analysis of chemical compoundsObject 1 1 H， 13 C, DEPT and HSQC NMR spectra show that Compound 1 shows 15 carbons, including three methyl [ delta ] H 1.16(H 3 -12),1.14(H 3 -13),0.91(H 3 -14),2.19(H 3 -15)；δ C 26.1(C-12),23.9(C-13),16.5(C-14),29.1(C-15)]Four methylene [ delta ] H 1.76(H-1a),1.10(H-1b),1.50(H-5a),1.96(H-5b),1.31(H-8a),1.60(H-8b),1.47(H-9a),1.60(H-9b)；δ C 44.6(C-1),29.0(C-5),38.5(C-8),21.6(C-9)]Four methines [ delta ] H 4.14(H-2,oxygenated),2.43(H-4),3.20(H-6,oxygenated),3.23(H-10,oxygenated)；δ C 66.5(C-2,oxygenated),57.3(C-4),82.1(C-6,oxygenated),85.4(C-10,oxygenated)]Three quaternary carbons [ delta ] C 33.7(C-7),72.0(C-11,oxygenated)]And a carbonyl [ delta ] C 211.5(C-3)]。
H-10 and H were observed from HMBC spectra in combination with 1D and 2D NMR spectra analysis of Compound 1 2 -8 is related to C-6, H 2 -8 and H 2 -9 is associated with C-7 and is in combination with H-8/H-9/H-10 1 H- 1 H COSY and CH-6 (delta) H 3.20,δ C 82.1 And CH-10 (delta) H 3.23,δ C 85.4 Chemical shift of the (a) ring indicates the presence of the (a) ring of pentane oxide. As can be seen from the HMBC pattern, H 3 -12 is associated with C-13, C-10 and C-11, respectively; h 3 -13 is associated with C-12, C-11 and C-10, respectively; the chemical shift in combination with them indicates that 2-propanol of Compound 1 passes through C 10 -C 11 The bond is located at C-10, thereby constructing 2- (dihydro-2H-pyran-2-yl) propanol (2- (tetrahydro-2H-pyran-2-yl) pro-n-2-ol)]) A unit. Cyclohexane (B-ring) is present by H 2 -4/H 2 -5/H-6 1 H- 1 The H COSY correlation and the HMBC correlation of H-2 and C-7 prove to be obtained. According to CH-2 (delta) H 4.14,δ C 66.5 Chemical shift value indicates that the hydroxyl group (-OH) is located at C-2. According to H in HMBC spectra 3 -15(δ H 2.19 With respect to C-3 and C-4, respectively, indicating that the methyl ketone is located at the C-4 position.
The relative configuration of Compound 1 was determined by NOESY experiments, H 3 -14(δ H 0.91 Respectively with H-8β (delta) H 1.60 And H-2 (delta) H 4.14 NOE correlation, confirming the relative configuration of C-7 as R. H-8α (δ) H 1.31 Respectively with H-6 and H-10 (delta) H 3.21 NOE-related, and H-6 is related to NOE of H-4, and H 3 -14(δ H 0.91 And H-4 (delta) H 4.14 NOE-related deletions, indicating the relative configuration of compound 1 as 2S,4S,6R,7R, 10R. The absolute configuration of Compound 1 was determined to be 2S,4S,6R,7R,10R by comparison with the ECD experimental plot using time-Density functional theory calculation (TDDFT) at the level of B3LYP/6-311+G (d, p)/B3 LYP/6-31G (d, p) and was designated as bipodonine E.
Compound 2, pale yellow powder. By analysis of the HR-ESI-MS m/z 293.1720[ M+Na ]] + (calcd for C 15 H 26 O 4 Na + 293.1723) 13 The C and DEPTNMR data show that the molecular formula is C 15 H 26 O 4 Corresponding to 3 unsaturations. Careful comparison of this with compound 1 revealed that compound 2 had similar a-ring-pentane oxide ring and B-ring-cyclohexane to 1. Two unsaturations were removed and the other was aldehyde (-CHO). Chemical shift of binding C-3 to delta C 71.4ppm and by H 3 -15(δ H 1.21 With regard to HMBC of C-2, C-3, C-4, respectively, it is presumed that a methyl group is present in addition to the hydroxyl (-OH) substitution at the C-3 position. H-1 (delta) H 10.01 And C-2 (delta) C 64.8 With the aldehyde group at the C-2 position.
Determining the relative configuration of the compound according to the NOESY experimental map of the compound 2, H 3 -14 and H 3 -15 and H-8β (delta) H 1.91 NOE correlation, presumption H 3 -15 is in the beta-configuration. H-6 and H-8α (delta) H 1.57 And H-2, and H-8α and H-10 (δ) H 3.25 NOE, all presumably in the alpha-configuration. ECD calculations were performed on compound 2 ((2R, 3R,6R,7R, 10R) -2) using Gauss 09 at the level of B3LYP/6-311+G (d, p)/B3 LYP/6-31G (d, p) using Density Functional Theory (DFT), confirming that the configuration of compound 2 is 2R,3R,6R,7R,10R and is designated bipodonine G.
In summary, it can be determined that the compound bipodonine E (compound 1) prepared in example 1 is a sesquiterpene compound having a structure represented by formula 1, and the compound bipodonine G (compound 2) is a sesquiterpene compound having a structure represented by formula 2:
EXAMPLE 3 selection of immunopotentiating Activity of Compounds bipodonine E and bipodonine G
Preparation of a mouse spleen lymphocyte suspension: ICR mice were sacrificed by cervical dislocation, immersed in 75% ethanol for 5min, removed and placed on an ultra-clean bench to perform aseptic spleen separation, phosphate Buffer (PBS) rinse, and connective tissue and fat components around the mice were stripped. Spleen tissue was placed on a stainless steel screen (200 mesh), lightly ground with a glass syringe needle, rinsed with 3mL of PBS, transferred to a centrifuge tube, centrifuged at 4 ℃ for 15min (1000 rpm/min), and the supernatant discarded. Adding 3mL erythrocyte lysate, standing at normal temperature for 2min, balancing the osmotic pressure of spleen cells with 7mL PBS, repeatedly adding 7mL PBS, centrifuging and washing twice (1000 rpm/min,5 min), discarding supernatant to obtain off-white spleen cell precipitate, suspending cells with RPMI-1640 medium to obtain single cell suspension with certain concentration, counting trypan blue stained cells, counting living cells with number greater than 95%, and adjusting cell concentration to 1×10 5 And each mL.
The MTS assay detects mouse spleen lymphocyte proliferation: the prepared mouse spleen lymphocyte suspension is added into a 96-well cell culture plate, 180 mu L/well. The test was divided into cell blank control groups (40. Mu. LRPMI-1640 per well); inducer ConA or LPS alone stimulated groups (10. Mu.L inducer, 10. Mu.LRPMI-1640 added per well); samples were combined with ConA or LPS stimulated groups, 10. Mu.L of sample solution (final concentration of 20. Mu.M) was added to each well, 10. Mu.L of inducer ConA or LPS (final concentration of 10. Mu.g/mL), and 3 wells were multiplexed in each group. At 37 ℃,5% CO 2 Culturing in incubator for 72 hr, adding 20 μLMTS, culturing for 3 hr, shaking for 10min, and measuring absorbance at 490 nm.
Experimental results show that the compounds bipodonine E and bipodonine G have certain immunity enhancing effect on induction of mouse spleen lymphocyte proliferation in concanavalin (ConA) or Lipopolysaccharide (LPS). (Table 2ConA/LPS is positive Control, control is blank Control). Thus, bipodonine E and bipodonine G have research value for developing immune-enhancing drugs as lead compounds. The method based on a large amount of simple immune promoting compounds bipodonine E and bipodonine G by microbial fermentation not only can be used for testing the environment-friendly low-carbon requirement, but also can realize a new mass production way and provide a new choice for developing immune promoting drugs.
TABLE 2 induced cell proliferation Rate of the compounds bipodonine E and bipodonine G on (A) Canavalia (ConA) and (B) Lipopolysaccharide (LPS)
| compd | Cell proliferation(％) | compd | Cell proliferation(％) |
| Compound 1 | - | Compound 1 | 28.8 |
| Compound 2 | 37.2 | Compound 2 | 40.3 |
| Control | 12.3 | Control | 11.0 |
| ConA | 28.9 | LPS | 21.3 |
Example 4
Inoculating the bermudagrass fungus to a PDA slant culture medium, culturing at constant temperature at 28 ℃ for 5 days to obtain activated bermudagrass fungus, and storing in an environment at 5 ℃ for later use;
soaking 50g of rice in water overnight, placing the soaked rice in a tissue culture bottle with the volume of 350mL, capping the tissue culture bottle, sterilizing at 121 ℃ for 30min, and cooling to obtain a fermentation culture medium;
inoculating the activated bermudagrass fungus to the fermentation medium according to the inoculation amount of 0.5%, and culturing for 35 days at normal temperature after capping to obtain a bermudagrass fungus ferment;
mixing the obtained bermuda grass vermicular fungus ferment with 120mL of methanol according to the mass volume ratio (50 g/120 mL), carrying out ultrasonic treatment for 30min under the condition of 40kHz after mixing, filtering, and carrying out reduced pressure distillation (the vacuum degree is 12kPa,50 ℃) on the filtrate until no alcohol smell exists, thus obtaining 14.0g of crude extract;
dissolving 14.0g of the crude extract in 40mL of dichloromethane, mixing with 14.0g of silica gel (300-400 meshes), concentrating under reduced pressure (the vacuum degree is 12kPa,50 ℃) to remove the solvent, and loading the mixture into a column; carrying out gradient elution by using a solution with the volume ratio of chloroform to methanol of 100:0, 100:1, 50:1, 30:1 and 10:1 to respectively obtain five fractions Fr.1, fr.2, fr.3, fr.4 and Fr.5; and (3) performing silica gel column chromatography elution on the Fr.3 fraction by taking petroleum ether-acetone with the volume ratio of 30:1 as an eluent to obtain a bipodonine E eluent, performing silica gel column chromatography elution on the Fr.4 fraction by taking petroleum ether-acetone with the volume ratio of 15:1 as an eluent to obtain bipodonine G eluents, and purifying the eluents by taking methanol as a solvent through sephadex to obtain the compounds bipodonine E and bipodonine G.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (10)
1. A sesquiterpene compound having a structure represented by formula 1 or formula 2:
2. the process for producing sesquiterpenoids according to claim 1, which comprises the steps of:
inoculating the bermudagrass fungus to a fermentation medium for fermentation to obtain a bermudagrass fungus fermentation product;
mixing the bermuda grass umbilicus vermiculosus fungus ferment with an alcohol solvent, and performing ultrasonic extraction to obtain a crude extract of sesquiterpenes compound;
mixing the crude extract of the sesquiterpenoids with silica gel, filling the obtained mixture into a column, and performing first column chromatography separation, wherein the first column chromatography separation is performed by gradient elution according to the volume ratio of chloroform to methanol of a chloroform-methanol system of which the volume ratio is 100:0-10:1, and merging the same fractions to obtain five sections of fractions which are respectively named as Fr.1-Fr.5 fractions;
subjecting the Fr.2 fraction to second column chromatography to obtain eluent containing sesquiterpenoids with structure shown in formula 1; the eluent for the second column chromatographic separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system;
subjecting the Fr.4 fraction to third column chromatography to obtain eluent containing sesquiterpenoids with structure shown in formula 2; the eluent for the third column chromatographic separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system;
purifying the eluent containing the sesquiterpene compound with the structure shown in the formula 1 by first gel chromatography to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 1;
and (3) performing second gel chromatography purification on the eluent containing the sesquiterpene compound with the structure shown in the formula 2 to obtain a pure product of the sesquiterpene compound with the structure shown in the formula 2.
3. The method of claim 2, wherein the feedstock for the fermentation medium comprises potatoes and/or rice; the fermentation is solid fermentation.
4. A method according to claim 2 or 3, wherein the fermentation temperature is 20-25 ℃ and the fermentation time is 30-35 d.
5. The method of claim 2, wherein the alcoholic solvent comprises one or more of methanol, ethanol, propanol, and isopropanol; the ratio of the mass of the bermuda grass vermicular fungus ferment to the volume of the alcohol solvent is (50-70) g (80-130) mL.
6. The method of claim 2, wherein the first column chromatography separation is: gradient elution was performed sequentially with chloroform-methanol systems at volume ratios of 100:0, 100:1, 50:1, 30:1, and 10:1.
7. The preparation method according to claim 2 or 6, wherein the mass ratio of the dry weight of the crude extract of the sesquiterpenoids to the silica gel is 1 (1-1.2);
the mesh number of the silica gel is 300-400 mesh.
8. The method of claim 2, wherein the eluent from the second column chromatography is a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone is 30:1.
9. The method according to claim 2, wherein the eluent of the third column chromatographic separation is a petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone is 15:1.
10. The sesquiterpenoids of claim 1 or the sesquiterpenoids prepared by the preparation method of claims 2-9 are applied to the preparation of immunity-promoting drugs.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023232125A1 (en) * | 2022-06-02 | 2023-12-07 | 暨南大学 | Sesquiterpene polyketone compound, pharmaceutical composition and use |
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