CN109761994A - Carbon drop class guainane type Sesquiterpene lactones compound and preparation method thereof - Google Patents
Carbon drop class guainane type Sesquiterpene lactones compound and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of carbon drop class guainane type Sesquiterpene lactones compound and preparation method thereof, the carbon drop class guainane type Sesquiterpene lactones compound be from Achillea millefolium (Achillea millefolium L. it is tested and analyzed in herb) using thin layer chromatography and analytic type high performance liquid chromatography, it is extracted with organic solvent, then pass through solvent extraction, normal-phase silica gel column chromatography method, aperture resin column chromatography, Sephadex LH-20 gel filtration chromatography method, four kinds to five kinds methods in semi-preparative high performance liquid chromatography are separated, obtain three new carbon drop class guainane type Sesquiterpene lactones monomeric compounds, determine that three monomeric compounds are new carbon drop class guainane type Sesquiterpene lactones compound by the methods of high resolution mass spectrum and NMR spectrum, and Structural Identification is carried out to it.And anti-inflammatory activity measurement is carried out, the results showed that described three new carbon drop class guainane type Sesquiterpene lactones compounds have different degrees of anti-inflammatory activity, can be used for preparing anti-inflammatory drug.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to carbon drop class guainane type Sesquiterpene lactones compound and its
Preparation method.
Background technique
Terpenoid is an important component in natural organic-compound, it is end to end by several iso-amylene
It forms, wherein being constituted with three iso-amylene is sequiterpene.And it is three isoprenyl units that sesquiterpene lactone, which is then basic framework,
The lactone compound of composition.Guainane type Sesquiterpene lactones compound is the very important sesquialter of one kind existing for nature
Terpene, molecular skeleton is by one typical [5,7] and ring structure forms, and has a multiple biological activities, including anti-inflammatory, antibacterial, anti-
Tumour etc..The guainane type sequiterpene of separated identification has hundreds of in nature at present, and why structure is enriched, and is
Because often with having various substituent groups, such as hydroxyl, acetoxyl group, propionyloxy, isobutyl acyloxy on its skeleton.These substituent groups
Position on ring is different, and number is different, and configuration is different, forms the rich and changeful property on this kind of compound structure.At present from
The compound reported in Achillea millefolium has more than 160, mainly include phenols, flavones and its glycosides, sterol, monoterpene, sequiterpene,
Triterpene etc., wherein guainane type Sesquiterpene lactones report 14 altogether.Three new compounds in this research are thousand
The new guainane type Sesquiterpene lactones compound found in leaf alpine yarrow, structure feature are original guainane type sequiterpene
3 carbon of lactone compound are substituted with an oxygen, and have cyclopentenone ester structure, with original guainane type Sesquiterpene lactones
Compound has certain difference on skeleton.Research of this research to Achillea millefolium chemical component can be secondary generation in clear Achillea millefolium
It thanks to product and reference is provided, also provide scientific basis to disclose the material base of its drug action.
It is now recognized that macrophage is the main source of mammal nitrate.NO is by NOS approach in macrophage
Synthesis, synthesis capability are closely related with anti-inflammatory effect.The synthesis of NO can be induced by interferon, however the Induction Process is usual
Need the presence of second signal (such as lipopolysaccharides LPS).When interferon or second signal independent role, inducing macrophage synthesis is low
The NO of amount and in most cases, without direct cytotoxic effect, but interferon and second signal collective effect can be significant
The generation of NO is improved, and increases the cytotoxic effect of macrophage.In the cell of synthesis NO, with L-arginine (L-Arg)
It is substrate with molecular oxygen, under the conditions of nitricoxide synthase (NOS) and existing reduced coenzyme (NADPH), generates intermediate
To hydroxyl L-Arg, the latter and O2It reacts, the NO and L-citrulline of the gram-molecular weights such as generation, wherein NOS is that NO is generated most
Main rate-limiting factor, NADPH, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (FAD) be important auxiliary because
Son.NO is a kind of hydroxyl radical gas, and chemical property is very active, can rapidly with molecular oxygen, superoxide anion and iron, copper,
Magnesium etc. reacts, and oxidation generates terminal metabolin nitrate (NO) and nitrite (NO) and inactivates.NOS in macrophage
Mainly iNOS shows that iNOS is not present in inactive cell to the RAW264.7 cell research of LPS/IFN- γ activation,
Its activity is cytoplasm, needs L-Arg and NADPH.
The premise for being the discovery that innovation drug research of lead compound, and influence the original new drug period it is decisive because
Element.There are many approach in the source of lead compound, and active compound is found from natural products and makees primer Development of New Drugs
It is one of the effective way that world pharmaceutical worker generally acknowledges.This research is intended to carry out the sesquiterpene lactone constituents in Achillea millefolium
Further Research on Mining, to find structure novel and anti-inflammatory activity Sesquiterpene lactones compound outstanding, naturally to produce
Building provides largely using natural products as the compound library of template for high-flux medicaments sifting based on object molecular structure abundant
Compound containing abundant structure diversity.Based on a large amount of structure-activity relationship information provided by the compound library, researcher
Can pharmacological activity to drug and pharmacokinetic property study, structure is modified or is optimized, providing simplicity has
The synthetic method of effect to obtain lead compound even drug itself, and then provides reference for the exploitation of anti-inflammatory drug.
Summary of the invention
Present invention aims at provide a kind of carbon drop class guainane type Sesquiterpene lactones compound and its preparation side
Method, the carbon drop class guainane type Sesquiterpene lactones compound are from Achillea millefolium (Achillea millefolium L.)
Herb in tested and analyzed using thin layer chromatography and analytic type high performance liquid chromatography, extracted with organic solvent, then passed through
Solvent extraction, normal-phase silica gel column chromatography method, aperture resin column chromatography, Sephadex LH-20 gel filtration chromatography method, half make
Four kinds to five kinds methods in standby high performance liquid chromatography are separated, and three new carbon drop class guainane type sequiterpenes are obtained
Lactone monomeric compound determines that three monomeric compounds are new drop by the methods of high resolution mass spectrum and NMR spectrum
Carbons guainane type Sesquiterpene lactones compound, and Structural Identification has been carried out to it, the results showed that three new drops
Carbons guainane type Sesquiterpene lactones compound has different degrees of anti-inflammatory activity, can be used for preparing anti-inflammatory drug.
A kind of carbon drop class guainane type Sesquiterpene lactones compound of the present invention, the structural formula of the compound
Are as follows:
Wherein:
Formula (I) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl -2-
Butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；
Formula (II) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8-
[(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；
Formula (III) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- first
Base -2- butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
A kind of preparation method of the carbon drop class guainane type Sesquiterpene lactones compound, follow these steps into
Row:
A, taking Achillea millefolium herb is raw material, measures ethanol water, the nothing that volumetric concentration is 50-99% with 5-10 times after crushing
Diacolation, the cold soaking that water-ethanol, pure acetone, the methanol aqueous solution that volumetric concentration is 50-99% or anhydrous methanol carry out at room temperature mention
It takes or heating and refluxing extraction, is concentrated to get the crude extract of Achillea millefolium；
B, the crude extract obtained step a is water-dispersible, sequentially adds petroleum ether, n-hexane or hexamethylene, ethyl acetate,
Ethyl acetate, dichloromethane or chloroform extract liquor are concentrated, obtain by dichloromethane or chloroform, extracting n-butyl alcohol 3-5 times
To ethyl acetate, dichloromethane or chloroform extract medicinal extract；
C, the ethyl acetate that obtains step b, dichloromethane or chloroform extract medicinal extract through normal-phase silica gel column chromatography,
Reversed-phase silica gel column chromatography, aperture resin column chromatography, in Sephadex LH-20 gel filtration chromatography, preparative high performance liquid chromatography
It is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl that two or three, which is separated to get formula (I) compound is arrived,
Base -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；Formula (II) compound be (4R, 6S, 7R,
8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1-
Alkene -12,6- lactone and formula (III) compound are (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8-
[(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
Normal-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography in step c, and filler used is purification on normal-phase silica gel, used
Eluant, eluent is petroleum ether, hexamethylene or n-hexane, acetone, chloroform, methylene chloride or ethyl acetate, at least two in methanol
The mixture of solvent, using isocratic elution or gradient elution.
Reversed-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography in step c, and eluant, eluent is that volumetric concentration is 40-
99% methanol aqueous solution or the acetonitrile solution of 30-99%, using isocratic elution or gradient elution.
Sephadex LH-20 gel filtration chromatography method used is normal pressure column chromatography in step c, and eluant, eluent is methanol, dichloromethane
The mixture of alkane, chloroform or at least two solvents, using isocratic elution or gradient elution.
Aperture resin chromatography method used is normal pressure or pressurized column chromatography in step c, and eluant, eluent is volumetric concentration 40-99%'s
The acetonitrile solution of methanol aqueous solution or 30-99%, using isocratic elution or gradient elution.
Preparative high performance liquid chromatography used is pressurized column chromatography in step c, and eluant, eluent is the first of volumetric concentration 40-99%
The acetonitrile solution of alcohol solution or 30-99%, using isocratic elution or gradient elution.
A kind of carbon drop class guainane type Sesquiterpene lactones compound of the present invention, can be by separating from plant
Purifying obtains, and can also synthesize and obtain through chemical modification method well known to those skilled in the art.
A kind of carbon drop class guainane type Sesquiterpene lactones compound of the present invention, using high resolution mass spectrum, one
The Modern spectroscopies means such as peacekeeping ID NMR speetna determine that its structure, Structural Identification process are as follows:
Formula (I) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl -2-
Butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone, white amorphous powder,-13.0(c 0.1,MeOH)；UV
(MeOH)216nm；ECD (MeOH) 201 (Δ ε+18.02), 224 (Δ ε -19.80) nm；Pass through quasi-molecule in its high resolution mass spectrum
Quasi-molecular ions [M-H]-M/z 363.1444 (calculated value 363.1449) determines that its molecular formula is C19H23O7.According to1H,13C NMR
And two dimensional NMR data determine that its structural framework type is carbon drop class guainane type sesquiterpene lactone
(Guaianesesquiterpenes) class compound is named as (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl
Base -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone,1H and13C NMR ownership is shown in Table 1
[600MHz(1H), 150MHz (13C), CDCl3]。
Formula (II) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8-
[(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone, white amorphous powder,-5.0(c
0.1,MeOH)；UV(MeOH)217nm；ECD (MeOH) 210 (Δ ε+26.70), 232 (Δ ε -14.18), 259 (Δ ε+3.80)
nm；Pass through quasi-molecular ion peak in high resolution mass spectrum [M-H]-M/z 393.1554 (calculated value 393.1554) determines its molecule
Formula is C20H26O8；According to1H,13C and two dimensional NMR data determine that its structure, framework types are carbon drop class guainane type
Sesquiterpene lactone (Guaiane sesquiterpenes) class compound, is named as (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -
3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；Its1H
With13CNMR ownership be shown in Table 1 [400MHz (1H), 100MHz (13C), CDCl3]。
Formula (III) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- first
Base -2- butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone, white amorphous powder, [α]D 20-34.0(c 0.1,MeOH)；
UV(MeOH)217nm；ECD (MeOH) 202 (Δ ε+19.33), 221 (Δ ε -22.02)；Pass through quasi-molecule in its high resolution mass spectrum
Quasi-molecular ions [M-H]-379.1393 (calculated values 379.1398) determine that its molecular formula is C19H24O8；According to1H,13C NMR and
Two dimensional NMR data determine that its structure, framework types are carbon drop class guainane type sesquiterpene lactone
(Guaianesesquiterpenes) class compound is named as (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- bis-
Hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；Its1H and13C NMR ownership is shown in Table 1
[400MHz(1H), 100MHz (13C), DMSO-d6]。
1 formula of table (I), formula (II), formula (III) compound1H and13C NMR data [δ (ppm), J (Hz)]
Detailed description of the invention
Fig. 1 is formula (I) compound1H NMR(600MHz,CDCl3) spectrogram；
Fig. 2 is formula (I) compound13C NMR(150MHz,CDCl3) spectrogram；
Fig. 3 is formula (II) compound1H NMR(400MHz,CDCl3) spectrogram；
Fig. 4 is formula (II) compound13C NMR(100MHz,CDCl3) spectrogram；
Fig. 5 is formula (III) compound1H NMR(400MHz,DMSO-d6) spectrogram；
Fig. 6 is formula (III) compound13C NMR(100MHz,DMSO-d6) spectrogram.
Specific embodiment
Agents useful for same is that analysis is pure, and acetonitrile is that (Merck KGaA is not public for high-efficiency liquid chromatographic-grade in high performance liquid chromatography
Department).Column chromatographs purification on normal-phase silica gel (100-200 mesh, 200-300 mesh): Haiyang Chemical Plant, Qingdao's production；Tlc silica gel is
HSGF254: Yantai City's Huang business silica gel development experiments factory production；Sephadex LH-20 gel: General Electric's Medical Group
Production；Aperture resin aperture resin gel MCI (CHP 20/120): Mitsubishi Co., Ltd. production；Reverse phase silica gel ODS: moral
The production of Merck & Co., Inc., state；High performance liquid chromatography (Dai An company, the U.S.) configuration is as follows: P680HPLC pump, ASI-100 automatic sampling
Device, TCC-100 column oven, UVD170U UV detector (four wavelength), quaternary solvent system, on-line degassing machine, chameleon chromatography
Work station.Preparative high-performance liquid chromatographic (Dai An company, the U.S.) configuration is as follows: P680HPLC pump, UVD170U UV detector (four
Wavelength), quaternary solvent system, on-line degassing machine, chameleon chromatographic work station.Mass spectrum hybridizes mass spectrum with quadrupole rod-flight time
Instrument (Applied biosystems) measurement；The nuclear magnetic resonance 600/400 type Nuclear Magnetic Resonance (U.S. watt Varian Vnmrs
Li An company) measurement.
Achillea millefolium herb picks up from Hami Prefecture, Xinjiang Uygur Autonomous Regions, by Xinjiang ecogeography research institute, the Chinese Academy of Sciences
Specimen museum Feng Ying assistant researcher is accredited as Achillea millefolium L..
Embodiment 1
A, Achillea millefolium herb 5kg is taken, cold soaking extracts the ethanol-water solution for being 50% with the concentration of 50L after crushing at room temperature,
Evaporated under reduced pressure solvent obtains Achillea millefolium herb crude extract；
B, the crude extract obtained step a is water-dispersible, and petroleum ether, ethyl acetate, n-butanol is added and successively carries out extraction 3
Secondary, simultaneously evaporated under reduced pressure obtains ethyl acetate layer extract medicinal extract to combined ethyl acetate layer；
C, the ethyl acetate layer extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 50:1-0:1
Petroleum ether-ethyl acetate carry out gradient elution, flow point analyzes through silica gel thin-layer chromatography, merges identical flow point, obtain 9 components
F1-F9；By component F4 through Sephadex LH-20 gel post separation, is eluted, obtained with chloroform-methanol that volume ratio is 1:1
To component F4A-F4E；It is the methanol-water solution gradient elution of 40%-100% with concentration by component F4C through MCI post separation, receives
Collect 60% methanol-water solution F4C3, evaporated under reduced pressure uses F4C3 sections and prepares reversed-phase column C18, 5 μm, 10 × 150mm separation, with
The acetonitrile-aqueous solution isocratic elution that concentration is 50%, obtaining formula (I) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -
3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone and formula (II) compound
For (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl-2-butene acyl-oxygen
Base]-guaiaci lignum -1- alkene -12,6- lactone；It is 1:1's with volume ratio by component F5 through Sephadex LH-20 gel post separation
Chloroform-methanol elution, obtains component F5A-F5F；It is the first of 50%-100% with concentration by component F5B through MCI post separation
Alcohol-water solution gradient elution, collects 60% methanol-water solution F5B2, and evaporated under reduced pressure uses F5B2 sections and prepares reversed-phase column C18, 5
μm, 10 × 150mm separation, with concentration be 60% acetonitrile-aqueous solution isocratic elution, obtain formula (III) compound be (4R, 6S,
7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -
12,6- lactone.
Embodiment 2
A, Achillea millefolium herb 5kg is taken, is mentioned after crushing with flowing back at 80 DEG C of ethanol-water solution temperature that 40L concentration is 95%
It takes, evaporated under reduced pressure solvent obtains Achillea millefolium herb crude extract；
B, the crude extract obtained step a is water-dispersible, sequentially adds n-hexane, methylene chloride and n-butanol and is extracted
4 times, merge dichloromethane layer, and evaporated under reduced pressure obtains dichloromethane extract medicinal extract；
C, the dichloromethane extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1's
N-hexane-acetone carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography, merges identical flow point, obtain 9 component F1-F9；
By component F4 through Sephadex LH-20 gel post separation, is eluted with chloroform-methanol that volume ratio is 1:1, obtain component
F4A-F4E；It is the methanol-water solution gradient elution of 50%-100% with concentration by component F4C through ODS reverse phase post separation, collects
60% methanol-water solution F4C2, evaporated under reduced pressure, F4C2 sections use purification on normal-phase silica gel post separation, use volume ratio for 20:1-0:1's
N-hexane-ethyl acetate carries out gradient elution, obtains component F4C2A-F4C2H, uses to F4C2E sections and prepares reversed-phase column C18,5μ
M, 10 × 150mm separation, with concentration be 65% methanol-water solution isocratic elution, obtain formula (I) compound be (4R, 6S, 7R,
8S, 10S, 11S) in -2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6-
Ester and formula (II) compound are (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2-
Methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；By component F5 through Sephadex LH-20 gel post separation,
Chloroform-methanol the elution for being 1:1 with volume ratio, obtains component F5A-F5F；By component F5B through RP-18 reversed-phase column column point
From, it is the methanol-water solution gradient elution of 40%-100% with concentration, collects 60% methanol-water solution (F5B3), decompression steaming
It is dry, F5B3 sections are used and prepares reversed-phase column C18, 5 μm, 10 × 150mm separation, the acetonitrile-aqueous solution for being 60% with concentration is isocratic to be washed
De-, obtaining formula (III) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- first
Base -2- butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
Embodiment 3
A, Achillea millefolium herb 5kg is taken, seepage pressure effects, evaporated under reduced pressure solvent obtain at room temperature with the dehydrated alcohol of 40L after crushing
To Achillea millefolium herb crude extract；
B, the crude extract obtained step a is water-dispersible, sequentially adds hexamethylene, ethyl acetate, n-butanol and carries out extraction 5
Secondary, simultaneously evaporated under reduced pressure obtains acetic acid ethyl ester extract medicinal extract to combined ethyl acetate layer；
C, the acetic acid ethyl ester extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 50:1-0:1's
Cyclohexane-acetone carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography, merges identical flow point, obtain 9 component F1-F9；
It is the methanol-water solution gradient elution of 40%-100% with concentration by component F4 through ODS reverse phase post separation, collects 60% methanol-
Aqueous solution F4B, evaporated under reduced pressure, F4B sections use purification on normal-phase silica gel post separation, use volume ratio for petroleum ether-acetic acid of 20:1-0:1
Ethyl ester carries out gradient elution, obtains component F4B1-F4B8, uses to F4B4 sections and prepares reversed-phase column C18, 5 μm, 10 × 250mm points
From the acetonitrile-aqueous solution isocratic elution for being 55% with concentration, obtaining formula (I) compound is (4R, 6S, 7R, 8S, 10S, 11S)-
2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone and formula (II) are changed
Conjunction object is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl-2-butene acyl
Oxygroup]-guaiaci lignum -1- alkene -12,6- lactone；Component F5 is subjected to purification on normal-phase silica gel post separation, is 100:1-0:1's with volume ratio
Petroleum ether-ethyl acetate carries out gradient elution, obtains component F5A-F5D, by component F5C through Sephadex LH-20 gel column point
From the chloroform-methanol elution for being 1:1 with volume ratio, obtaining formula (III) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -
3- oxygen -4,10- dihydroxy -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
Embodiment 4
A, Achillea millefolium herb 5kg is taken, 40L pure acetone seepage pressure effects at room temperature, evaporated under reduced pressure solvent Achillea millefolium are used after crushing
Herb crude extract；
B, the crude extract obtained step a is water-dispersible, sequentially adds hexamethylene, methylene chloride, extracting n-butyl alcohol 3 times,
Merge dichloromethane layer and evaporated under reduced pressure obtains dichloromethane extract medicinal extract；
C, the dichloromethane extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1's
Petroleum ether-ethyl acetate carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography, merges identical flow point, obtain 9 components
F1-F9；Component F4 is subjected to purification on normal-phase silica gel post separation, the chloroform-methanol with volume ratio for 100:0-0:100 carries out gradient and washes
It is de-, obtain component F4A-F4F；It is the acetonitrile-aqueous solution ladder of 40%-100% with concentration by component F4C through ODS reverse phase post separation
Degree elution, collects 50% acetonitrile-aqueous solution F4C2, evaporated under reduced pressure, and F4C2 sections of uses prepare reversed-phase column C18, 5 μm, 10 × 250mm
Separation, with concentration be 50% methanol-water solution isocratic elution, obtain formula (I) compound be (4R, 6S, 7R, 8S, 10S,
11S) -2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone and formula
(II) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl -2-
Butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；Component F5 is subjected to purification on normal-phase silica gel post separation, is 100:0- with volume ratio
The n-hexane-ethyl acetate of 0:100 carries out gradient elution, component F5A-F5E is obtained, by component F5C through Sephadex LH-20
Gel post separation, with volume ratio be 1:1 methylene chloride-methanol elute, obtain formula (III) compound be (4R, 6S, 7R, 8S,
10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6-
Lactone.
Embodiment 5
A, Achillea millefolium herb 5kg is taken, is flowed back after crushing at 80 DEG C of methanol-water solution temperature that the concentration of 50L is 50%
It extracts, evaporated under reduced pressure solvent obtains Achillea millefolium herb crude extract；
B, the crude extract obtained step a is water-dispersible, sequentially adds n-hexane, methylene chloride, extracting n-butyl alcohol 4 times,
Merge dichloromethane layer and evaporated under reduced pressure obtains dichloromethane extract medicinal extract；
C, the dichloromethane extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1's
N-hexane-ethyl acetate carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography, merges identical flow point, obtain 9 components
F1-F9；Component F4 is subjected to MCI post separation, is the acetonitrile-aqueous solution gradient elution of 30%-100% with concentration, collects 40%
Acetonitrile-aqueous solution F4B, evaporated under reduced pressure, F4B sections through Sephadex LH-20 gel post separation, the trichlorine for being 1:1 with volume ratio
Methane-methanol elution, obtains component F4B1-F4B6, uses to F4B3 sections and prepares reversed-phase column C18, 5 μm, 10 × 250mm separation, with
The acetonitrile-aqueous solution isocratic elution that concentration is 45%, obtaining formula (I) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -
3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone and formula (II) compound
For (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl-2-butene acyl-oxygen
Base]-guaiaci lignum -1- alkene -12,6- lactone；Component F5 is subjected to purification on normal-phase silica gel post separation, is 100:0-0:100's with volume ratio
N-hexane-acetone carries out gradient elution, obtains component F5A-F5E, by component F5C through Sephadex LH-20 gel post separation,
It is eluted with anhydrous methanol, obtaining formula (III) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -
8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
Embodiment 6
A, Achillea millefolium herb 5kg is taken, seepage pressure effects, evaporated under reduced pressure solvent obtain at room temperature with the anhydrous methanol of 50L after crushing
To Achillea millefolium herb crude extract；
B, the crude extract obtained step a is water-dispersible, sequentially adds n-hexane, ethyl acetate, extracting n-butyl alcohol 5 times,
Simultaneously evaporated under reduced pressure obtains acetic acid ethyl ester extract medicinal extract to combined ethyl acetate layer；
C, the acetic acid ethyl ester extract medicinal extract purification on normal-phase silica gel post separation for obtaining step b, with volume ratio 100:1-0:1's
Cyclohexane-ethyl acetate carries out gradient elution, and flow point is analyzed through silica gel thin-layer chromatography, merges identical flow point, obtain 9 components
F1-F9；Component F4 washed through Sephadex LH-20 gel post separation with chloroform-methanol that volume ratio is 1:1
It is de-, obtain component F4A-F4E；It is the acetonitrile-aqueous solution ladder of 40%-100% with concentration by component F4B through ODS reverse phase post separation
Degree elution, collects 50% acetonitrile-aqueous solution F4B2, and evaporated under reduced pressure uses F42K sections and prepares reversed-phase column C18, 5 μm, 10 ×
250mm separation, with concentration be 55% methanol-water solution isocratic elution, obtain formula (I) compound be ((4R, 6S, 7R, 8S,
10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone
It is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- first with formula (II) compound
Base -2- butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；Component F5 is subjected to purification on normal-phase silica gel post separation, is with volume ratio
The petroleum ether-ethyl acetate of 100:0-0:100 carries out gradient elution, component F5A-F5D is obtained, by component F5B through Sephadex
LH-20 gel post separation, with volume ratio be 1:1 chloroform-methanol elute, obtain formula (III) compound be (4R, 6S, 7R,
8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- methyl-2-butene acyloxy]-alkene -12 guaiaci lignum -1-,
6- lactone.
Embodiment 7
The Sesquiterpene lactones compound of the present invention separated from Achillea millefolium herb is in preparing anti-inflammatory drugs
Purposes, by taking macrophage strain (RAW264.7 cell) as an example；
The test of formula (I)-formula (III) compound cell toxicant:
1. screening model:
Title: NFkB pathway (inhibitor)；
Full name in English: NFkB pathway；
Chinese name: the screening of NFkB signal pathway inhibitor；
Brief introduction: NF- κ B is a kind of transcription factor, by the expression in conjunction with target DNA, regulating and controlling several genes.It is demonstrated experimentally that
NF- κ B is high expression in kinds cancer cell.Its high expression and tumour occurs, and angiogenesis, the processes such as apoptosis have
Close ties；On the other hand, NF- κ B is proved in oncotherapy be an important factor for generating antineoplastic resistance.Therefore, it selects
Selecting property, the NF- kB inhibitor of high activity has potential value in terms for the treatment of of cancer；
2. screening technique:
Protocol name:NFkB pathway luciferase assay (inhibitor)；
Screen instrument: EnVisionTM, Biomek®；FX；
Screening material: TNFa, 96 hole blanks (PE)；
Screening process: the cell liquid after 50 μ L are mixed is added to 96 hole screen plates, and 10000, every hole is incubated overnight, and is added
20ng/mL TNF α 50 μ L, final concentration 10ng/mL；1 μ L is derived from the compound of 96 hole compound motherboards and control compound motherboard
96 hole screen plates are added；After 6 hours, 80 μ L liquid are taken away from 96 hole screen plates, add 20 μ L substrates, be protected from light 30 points of incubation
Zhong Hou, EnVisionTM detect luciferase signal strength；
3. sample treatment:
Sample is dissolved with DMSO, cryo-conservation, DMSO in final system concentration control do not influence to detect it is active
Within the scope of；
4. data processing and result explanation:
Primary dcreening operation selects under single concentration conditions, such as 20 μ g/mL, tests the activity of sample；For in certain condition
Under show active sample, such as inhibiting rate %Inhibition is greater than 50, test agents amount dependence, i.e. IC50/
EC50 value carries out Nonlinear Quasi to sample concentration by sample activity and obtains, and calculating software used is Graphpad Prism
4, being fitted used model is sigmoidaldose-response (varible slope), and most of inhibitor are sieved
Matched curve bottom and top are set as 0 and 100 by modeling type；Under normal circumstances, each sample is respectively provided with multiple holes in testing
(n >=2), in the result with standard deviation (Standard Deviation, SD) or standard error (Standard Error,
SE it) indicates；
2 reference compound of table and formula (I)-formula (III) Compound cellular poison test data
| Sample ID | Concentration | Type | Unit | As a result | Error | Remarks |
| PS-341 | IC50 | μM | 0.072 | 0.013 | ||
| Formula (I) | 20μg/mL | %Activity | percent | 85.872 | 3.590 | It is non-toxic |
| Formula (II) | 20μg/mL | %Activity | percent | 67.607 | 6.241 | It is non-toxic |
| Formula (III) | 20μg/mL | %Activity | percent | 77.198 | 8.723 | It is non-toxic |
5. conclusion: one, which shares 0 compound, shows activity to a certain degree；
Formula (I)-formula (III) compound anti-inflammatory activity test:
Experimental material:
Preparation of samples:
Formula (I)-formula (III) compound initial concentration is 10mM, successively asks for liquid 0.5 μ L, 2.5 μ L, 5 μ L, is diluted
1000,200,100 times, concentration is followed successively by 10 μM, 50 μM, 100 μM after dilution；
Test consumptive material:
Material and reagent: 15mL centrifuge tube is purchased from Thermo company (product batch number: 339605)；24 orifice plates are purchased from
CORNING company (product batch number: 3524)；NO detection kit is purchased from green skies company (product batch number: S0023)；
Experiment cell: RAW264.7 cell, cell type are attached cell, and culture medium is HG-DMEM (adding ingredient
10% serum+1%P/S+1mM N-pyruvate).
Experimental procedure:
RAW264.7 cell is cultivated, culture medium HG-DMEM, 1:5 are passed on and counted 1 × 105Cells/mL is inoculated with 24 holes
Plate；
After adherent 6h, compound pretreatment cell 1h is added, collects 60 μ of supernatant after LPS (1mg/mL) stimulation 18h is added afterwards
L；
Following reagent is sequentially added according to the requirement in green skies Griess kit:
Table 3
Standard curve is made with absorbance value and calculates the inhibiting value that compound discharges NO；
Experimental result:
4 formula of table (I)-formula (III) compound anti-inflammatory activity test
| Compound | Nitric oxide | Blank group | LPS(1μg/mL) | 10μM | 50μM | 100μM |
| Number | Content | (Blank) | Inflammation group (Control) | Medicine group 1 | Medicine group 2 | Medicine group 3 |
| Formula (I) | NO(μmol/L) | 1.12±0.037 | 9.29±0.043 | 8.13±0.223 | 6.52±0.388 | 5.95±0.261 |
| Formula (II) | NO(μmol/L) | 1.12±0.037 | 9.29±0.043 | 7.17±0.633 | 5.15±0.239 | 5.11±0.872 |
| Formula (III) | NO(μmol/L) | 1.12±0.037 | 9.29±0.043 | 7.50±0.138 | 5.07±0.037 | 4.86±0.457 |
Conclusion:
3 compounds of the present invention can inhibit the NO burst size of mouse macrophage, with anti-inflammatory activity, and with
Concentration increase present gradient dependence decreasing trend；
The concentration dependent of formula (I) compound is more preferable, and the anti-NO generative capacity of formula (II) and formula (III) compound is stronger, and 50
μM can by NO burst size reduce by 50% or so.
Claims (7)
1. a kind of carbon drop class guainane type Sesquiterpene lactones compound, it is characterised in that the structural formula of the compound are as follows:
Wherein:
Formula (I) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -8- [(E) -2- methyl-2-butene
Acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；
Formula (II) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2-
Methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；
Formula (III) compound is (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -2- methyl -
2- butylene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
2. a kind of preparation method of carbon drop class guainane type Sesquiterpene lactones compound according to claim 1,
It is characterized in that following these steps to carry out:
A, taking Achillea millefolium herb is raw material, measures the ethanol water, anhydrous second that volumetric concentration is 50-99% with 5-10 times after crushing
Alcohol, pure acetone, the methanol aqueous solution that volumetric concentration is 50-99% or anhydrous methanol carry out diacolation at room temperature, cold soaking extracts or
Heating and refluxing extraction is concentrated to get the crude extract of Achillea millefolium；
B, the crude extract obtained step a is water-dispersible, sequentially adds petroleum ether, n-hexane or hexamethylene, ethyl acetate, dichloro
Ethyl acetate, dichloromethane or chloroform extract liquor are concentrated, obtain second by methane or chloroform, extracting n-butyl alcohol 3-5 times
Acetoacetic ester, dichloromethane or chloroform extract medicinal extract；
C, the ethyl acetate that obtains step b, dichloromethane or chloroform extract medicinal extract are through normal-phase silica gel column chromatography, reverse phase
Silica gel column chromatography, aperture resin chromatography, Sephadex LH-20 gel filtration chromatography, two kinds in preparative high performance liquid chromatography or
Three kinds are separated to get being (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -8- to formula (I) compound
[(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone；Formula (II) compound be (4R, 6S, 7R, 8S,
10S, 11S) -2- ketone -3- oxygen -10- hydroxyl -4- methoxyl group -8- [(E) -2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -
12,6- lactone and formula (III) compound are (4R, 6S, 7R, 8S, 10S, 11S) -2- ketone -3- oxygen -4,10- dihydroxy -8- [(E) -
2- methyl-2-butene acyloxy]-guaiaci lignum -1- alkene -12,6- lactone.
3. the preparation method of carbon drop class guainane type Sesquiterpene lactones compound according to claim 2, feature
It is in step c that normal-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography, filler used is purification on normal-phase silica gel, elution used
Agent is petroleum ether, hexamethylene or n-hexane, acetone, chloroform, methylene chloride or ethyl acetate, at least two solvents in methanol
Mixture, using isocratic elution or gradient elution.
4. the preparation method of carbon drop class guainane type Sesquiterpene lactones compound according to claim 2, feature
It is in step c that reversed-phase silica gel column chromatography method used is normal pressure or pressurized column chromatography, eluant, eluent is that volumetric concentration is 40-99%'s
The acetonitrile solution of methanol aqueous solution or 30-99%, using isocratic elution or gradient elution.
5. the preparation method of carbon drop class guainane type Sesquiterpene lactones compound according to claim 2, feature
Be in step c that Sephadex LH-20 gel filtration chromatography method used is normal pressure column chromatography, eluant, eluent be methanol, methylene chloride,
The mixture of chloroform or at least two solvents, using isocratic elution or gradient elution.
6. the preparation method of carbon drop class guainane type Sesquiterpene lactones compound according to claim 2, feature
It is in step c that aperture resin chromatography method used is normal pressure or pressurized column chromatography, eluant, eluent is the methanol of volumetric concentration 40-99%
The acetonitrile solution of aqueous solution or 30-99%, using isocratic elution or gradient elution.
7. the preparation method of carbon drop class guainane type Sesquiterpene lactones compound according to claim 2, feature
It is in step c that preparative high performance liquid chromatography used is pressurized column chromatography, eluant, eluent is the methanol-water of volumetric concentration 40-99%
The acetonitrile solution of solution or 30-99%, using isocratic elution or gradient elution.
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