CN109053651A - A kind of new skeleton loop coil sesquiterpene dimers compound and preparation method thereof - Google Patents
A kind of new skeleton loop coil sesquiterpene dimers compound and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of new skeleton loop coil sesquiterpene dimers compounds and preparation method thereof.The compound includes structure shown in Formulas I, wherein R1~R18 is separately selected from hydrogen, halogen, hydroxyl, amino.In the preparation, comprising the following steps: (1) echinops grijisii Hance dry root is extracted using ethyl alcohol, obtain ethanol extract；(2) ethanol extract is used into silica gel column chromatography, gradient elution is successively carried out as eluant, eluent using petroleum ether-ethyl acetate and petroleum ether-acetone and obtains fraction；(3) it uses hydroxypropyl sephadex to elute with chloroform-methanol the fraction that step (2) obtain, obtains new skeleton loop coil sesquiterpene dimers compound.The present invention provides the compound for the first time, and finds that the compound object has in fields such as medicine, pesticides and be widely applied.
Description
Technical field
The invention belongs to technical field of pharmaceuticals more particularly to a kind of new skeleton loop coil sesquiterpene dimers compound and its systems
Preparation Method.
Background technique
Plant resources are the important source and model foundation of new drug development.On the one hand, in natural plants it is abundant chemistry at
It is the important sources it is found that active medical primer that dividing, which makes it always with the bioactivity of multiplicity,；On the other hand, plant time
Raw metabolite has many advantages, such as to be not likely to produce drug resistance, good to non-target organism safety, Environmental compatibility, has become environment
The important directions of friendly agricultural chemicals research and development.The research and product development of integration of drinking and medicinal herbs Secondary Metabolite Production in Plants are the mankind couple
Anti-disease provides powerful mean, provides green low toxicity material for plant-based pesticides development.
Echinops grijisii Hance (Echinops grijsii Hance) is composite family (Compositae) Echinops
(Echinops) plant.Its dry root is also known as radix echinopsis latifolii, promote blood circulation with the clearing heat and detoxicating, carbuncle that disappears, lower cream, relaxing muscles and tendons and other effects,
It is included in 2010 editions " Chinese Pharmacopoeias ".Have in treatment acute mastitis swelling and pain, ulcer carbuncle on the back, scrofula sore, alactation, arthritis with fixed pain caused by dampness muscular constricture
Biggish development and application prospect.
So far, the domestic and international researcher isolated various structures type chemical combination from echinops grijisii Hance dry root
Object mainly includes thiophene, Phenylpropanoid Glycosides class, terpene (monoterpene, sequiterpene, diterpene etc.), steroid, alkaloids and fatty acid
Deng.Wherein thiophene, sesquiterpenoids are many kinds of, rich content.
Spiro-compound refers to that two monocycles share the polycyclic compound of a carbon atom.Because it is with rigid structure, knot
Structure is stablized, and chiral ligand has biggish specific rotatory power, in asymmetry catalysis, luminescent material, pesticide, hospital, macromolecule bonding
Agent etc. has important application.Therefore, it separates and extracts from the dry root herb of echinops grijisii Hance containing whorled sequiterpene two
Dimeric structure compound has important practical significance.
Currently, there are no the relevant report of the compounds of this invention, more there are no the compounds of this invention is used for it is medical, agricultural
Active relevant report.
Summary of the invention
In view of the problems of prior art, the present invention provide a kind of new skeleton loop coil sesquiterpene dimers compound,
Preparation method and applications.The compound of the new skeleton structure has medicine, pesticides application value, and includes the loop coil sesquialter
The pharmaceutical composition of terpene dimer compound, to provide more drug choosings for antitumor, anti-inflammatory liver protection, killing aphids and nematode
Select approach.
The technical scheme to solve the above technical problems is that
A kind of new skeleton loop coil sesquiterpene dimers compound, including structure shown in Formulas I:
Wherein, R1~R18 is separately selected from hydrogen, halogen, hydroxyl, amino.
The present invention provides Formulas I compound represented or its crystal form or its stereoisomer or its is pharmaceutically acceptable
Salt or its solvate or its pro-drug or its metabolite.
Preferably, in above-mentioned Formulas I, R1~R18 is hydrogen.Inventor has been surprisingly found that under study for action when R1~R18 is hydrogen,
There are four highly oxidized carbon atoms for loop coil sesquiterpene dimers compound tool, and it is excellent to possess preferable fat-soluble, permeability etc.
Point, enters conducive to drug molecule and plays dependent interaction in organism.
Preferably, new skeleton loop coil sesquiterpene dimers compound, including structure shown in formula II:
Inventor has found that the compound of structure shown in formula II has preferable fat-soluble, permeability and infiltration under study for action
The advantages that property.
The present invention also provides a kind of pharmaceutical preparations, including above-mentioned new skeleton loop coil sesquiterpene dimers compound.The drug
Preparation is composition, it be above-mentioned formula I or II compound represented of formula or its crystal form or its stereoisomer or its pharmaceutically
Acceptable salt or its solvate or its pro-drug or its metabolite, in addition prepared by pharmaceutically acceptable auxiliary material
Made of preparation.
One or more compounds of the invention can be used in conjunction with one another, and also be may be selected the compound of the present invention and appointed
What other active agent is used in combination.It, can be by these compounds simultaneously, respectively or have if using one group of compound
Sequence study subject is administered.
Pharmaceutically acceptable auxiliary material of the present invention refers in addition to the active ingredient (s include substance in dosage form.
The present invention also provides the preparation methods of above-mentioned new skeleton loop coil sesquiterpene dimers compound, comprising the following steps:
(1) echinops grijisii Hance dry root is extracted using ethyl alcohol, obtains ethanol extract；
(2) ethanol extract is used into silica gel column chromatography, is successively elution with petroleum ether-ethyl acetate and petroleum ether-acetone
Agent carries out gradient elution and obtains fraction；
(3) it uses hydroxypropyl sephadex to elute with chloroform-methanol the fraction that step (2) obtain, obtains new skeleton
Loop coil sesquiterpene dimers compound.
Specifically, the concrete operation step of step (2) can be with are as follows:
(a) ethanol extract obtained by step (1) is taken, using silica gel column chromatography, with petroleum ether-ethyl acetate volume ratio (25:1)
~(0:1) is that eluant, eluent carries out gradient elution, collects eluent as fraction；
(b) fraction for collecting step (a) is again through silica gel column chromatography, the petroleum ether-for being (30:1)~(1:1) with volume ratio
Acetone solvent gradient elution collects fraction；
(c) fraction for collecting step (b) is again through silica gel column chromatography, the petroleum ether-for being (8:1)~(1:1) with volume ratio
Acetone gradient elution collects fraction.
Specifically, can carry out preparing new skeleton loop coil sesquiterpene dimers compound using following specific steps:
(1) echinops grijisii Hance dry root is taken, is crushed, is extracted with 95% alcohol reflux, second is concentrated under reduced pressure to obtain in combined extract
Alcohol medicinal extract；
(2) the following steps are included:
(a) ethanol extract obtained by step (1) is taken, using silica gel column chromatography, successively with petroleum ether-ethyl acetate volume ratio
25:1,10:1,8:1,5:1,3:1,1:1,0:1 are that eluant, eluent carries out gradient elution, are detected, are developed the color through thin-layer chromatography, closed
And identical elution position, it is spare by the elution position after merging through being concentrated to dryness.
(b) by some component in step (a) again through silica gel column chromatography, the petroleum ether-for being (30:1)~(1:1) with volume ratio
Acetone solvent gradient elution, thin-layer chromatography detection, colour developing merge identical elution position.
(c) again through silica gel column chromatography, the petroleum ether-acetone gradient elution for being (8:1)~(1:1) with volume ratio, thin layer color
Spectrum detection, colour developing merge identical elution position.
(3) by middle some fraction of gained of step (c) through Sephadex LH-20 (hydroxypropyl sephadex), with 1:1 chlorine
Imitation-carbinol affords new skeleton loop coil sesquiterpenoid.
Preferably, it in step (a), collects elute by the eluant, eluent of 5:1 with petroleum ether-ethyl acetate volume ratio and obtain
Eluent as fraction.
Preferably, in step (b), collection is carried out eluting the elution obtained by 5:1 eluant, eluent with petroleum ether-acetone volume ratio
Liquid is as fraction.
Preferably, in step (c), collection is carried out eluting the elution obtained by 3:1 eluant, eluent with petroleum ether-acetone volume ratio
Liquid is as fraction.
Inventor in the course of the study, once attempts test of many times, is found surprisingly that under study for action, using above-mentioned parameter
Beneficial effect is: obtaining the Echinops latifolius element A of higher degree and better quality.
The present invention also provides the new applications of above-mentioned new skeleton loop coil sesquiterpene dimers compound.
The present invention provides above-mentioned formula I or II compound represented of formula or its crystal forms or its stereoisomer or its medicine
The application of acceptable salt or its solvate or its pro-drug or its metabolite in preparation medicine, pesticide on.
The present invention also provides the new applications of above-mentioned new skeleton loop coil sesquiterpene dimers compound.
Above-mentioned new skeleton loop coil sesquiterpene dimers compound can be applied in the preparation of antitumor drugs, have bright
Aobvious anti-tumor activity can be further prepared into anti-tumor drug for treatment of cancer.
Above-mentioned new skeleton loop coil sesquiterpene dimers compound can be applied in preparing anti-inflammatory drug.Inventor exists
It is found surprisingly that in research process, anti-inflammatory power is better than the anti-inflammatory power of existing agent dexamethasone.
Above-mentioned new skeleton loop coil sesquiterpene dimers compound can be applied in preparing hepatic.Above-mentioned new bone
Frame loop coil sesquiterpene dimers compound can be answered in the drug that preparation inhibits key virulence factor IFN-γ expression
With.Inventor confirmed by the acute hepatic injury model that Con A is induced, above-mentioned new skeleton loop coil sesquiterpene dimers compound
Key virulence factor IFN-γ expression can be significantly inhibited, it can through above-mentioned new skeleton loop coil sesquiterpene dimers compound processing
The secretion for reducing the inflammatory mediator of ConA induction, mitigates immunological liver injury, has protection liver effect.
Above-mentioned new skeleton loop coil sesquiterpene dimers compound can be applied in preparing pesticide.
Above-mentioned new skeleton loop coil sesquiterpene dimers compound can be applied in preparing killing aphids drug.Inventor
Studies have shown that above-mentioned new skeleton loop coil sesquiterpene dimers compound is to turnip aphid, grain aphid and rhopalosiphum padi, semen viciae fabae
Four kinds of aphids of aphid all show obvious virulence, have extensive insecticidal spectrum and apparent insecticidal activity, can be used as active constituent into
One step is prepared into killing aphids botanical pesticide.
Above-mentioned new skeleton loop coil sesquiterpene dimers compound can be applied in preparation nematicidal drug.Inventor
Research shows that above-mentioned new skeleton loop coil sesquiterpene dimers compound has killing ability to Meloidogyne incognita, and unexpected
It was found that its nematicidal ability is better than existing commercially available pesticide fosthiazate, illustrate new skeleton loop coil sesquiterpene dimers compound as plant
Material resource pesticide nematicidal direction with good application prospect.
It, can be according to actual requirement, by new skeleton loop coil sesquiterpene dimers compound during application
The auxiliary material for being prepared into suitable dosage form or adding one or more of this fields routine is applied so that pharmaceutical composition is made.
Test result shows that the compounds of this invention has preferable antitumor, anti-inflammatory, killing aphids, nematocidal effect, to face
Antitumor, anti-inflammation drugs are screened and/or prepared on bed and provide a kind of new selection, kill aphid agriculturally to screen and/or preparing
Worm, nematode drug provide a kind of new selection.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made, modifies, replace
Scheme after changing or changing is also in the scope of the present invention.
Detailed description of the invention
Fig. 1 is the HRESIMS figure of compound shown in Formula II.
Fig. 2 is the infrared spectrogram (IR) of compound shown in Formula II.
Fig. 3 is compound shown in Formula II1H NMR spectra.
Fig. 4 is compound shown in Formula II13C NMR spectra.
Fig. 5 is the nuclear magnetic resonance HSQC spectrogram of compound shown in Formula II.
Fig. 6 is the nuclear magnetic resonance HMBC spectrogram of compound shown in Formula II.
Fig. 7 is COSY the and HMBC signal graph of compound shown in formula II.
Fig. 8 is the X monocrystalline figure of compound shown in formula II.
Fig. 9 is experimental group and control group serum I FN- γ expression comparison result in embodiment 4.
Specific embodiment
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
If raw material, equipment used in the specific embodiment of the invention are known product without specified otherwise, pass through purchase
Commercial product obtains.
Embodiment 1 prepares compound of the present invention
1, experimental material:
1. medicinal material
Echinops grijisii Hance root is bought in June, 2017 from Anguo medicine city of Hebei province, through inventor herein doctor Liu Tingting
It is accredited as the dry root of composite family Echinops plant echinops grijisii Hance (Echinops grijsii Hance).
2. reagent and filler
Column chromatography silica gel, 200~300 mesh (SILVER REAGENT) are purchased from Qingdao Haiyang silica-gel desiccant factory；
Tlc silica gel GF254 (chemistry is pure), is purchased from Qingdao Haiyang silica-gel desiccant factory；
Sephadex LH-20 sephadex is purchased from Amersham company of Sweden；
GF254 silica gel prepares thin layer, is purchased from Yantai Jiang You silica gel development corporation, Ltd.；
The analytical reagents such as petroleum ether, n-hexane, chloroform, ethyl acetate, acetone, methanol, are purchased from Beijing Chemical Plant.
3. laboratory apparatus
Bruker-AVIIIHD-600 Nuclear Magnetic Resonance (Switzerland Bruker)；5700 infrared spectrometer (the U.S. Nicolet
Thermo company)；341 polariscopy instrument (U.S. PerkinElmer) of Perkin-Elmer；Ten a ten thousandth electronics of BP211D
Balance (Switzerland Sartorius)；R-210 rotary evaporator (Switzerland BUCHI)；(Shanghai is gloomy for DZG-6050 type vacuum oven
Letter).
2, ingredient isolates and purifies:
1) echinops grijisii Hance dry root 5kg is taken, after crushing, is extracted 3 times with 95% alcohol reflux of volume fraction, is merged and extract
Liquid is concentrated under reduced pressure to give ethanol extract 400g；
2) gained ethanol extract in step 1) is taken, using silica gel column chromatography, successively with petroleum ether-ethyl acetate volume ratio
25:1,10:1,8:1,5:1,3:1,1:1,0:1 are that eluant, eluent carries out gradient elution, are detected, are developed the color through thin-layer chromatography, closed
And identical elution fraction, obtain 7 fractions, Fr.1-Fr.7.(Fr.1 is the fraction of petroleum ether-ethyl acetate 25:1, and Fr.2 is
The fraction of petroleum ether-ethyl acetate 10:1, Fr.3 are the fraction of petroleum ether-ethyl acetate 8:1, and Fr.4 is petroleum ether-acetic acid second
The fraction of ester 5:1, and so on, totally 7 fractions)
3) by Fr.4 in step 2) again through silica gel column chromatography, with petroleum ether-acetone ladder that volume ratio is (30:1) to (1:1)
Degree elution, thin-layer chromatography detection, 10% sulfuric acid ethyl alcohol chromogenic reagent, according to Rf value elution portion identical with colour developing color merging
Point, obtaining 7 fraction Fr.4.1-Fr.4.7, (Fr.4.1 is petroleum ether-acetone 30:1 fraction, and Fr.4.2 is with petroleum ether-the third
The fraction of ketone 15:1, Fr.4.3 are petroleum ether-acetone 8:1 fraction, and Fr.4.4 is petroleum ether-acetone 5:1 fraction, Fr.4.5
For petroleum ether-acetone 3:1 fraction, Fr.4.6 is petroleum ether-acetone 2:1 fraction, and Fr.4.7 is petroleum ether-acetone 1:1
Fraction).Fr.4.4 take volume ratio as the petroleum ether-acetone gradient elution of (8:1) to (1:1), thin layer color again through silica gel column chromatography
Spectrum detection, colour developing merge identical elution fraction, and obtaining Fr.4.4.1-Fr.4.4.4, (Fr.4.4.1 is petroleum ether-acetone 8:1
Fraction, Fr.4.4.2 are petroleum ether-acetone 5:1 fraction, and Fr.4.4.3 is petroleum ether-acetone 3:1 fraction, and Fr.4.4.4 is
Petroleum ether-acetone 1:1 fraction).
4) by the middle gained Fr.4.4.3 of step 3) through Sephadex LH-20 (hydroxypropyl sephadex), with 1:1 chloroform-
Methanol affords Echinops latifolius element A.
5) present invention carries out thin-layer chromatography color condition: observing blackening under ultraviolet lamp (254nm), sprays 10% sulfuric acid ethyl alcohol
Afterwards, it is baked to colour developing for 105 DEG C.
3, the identification of target compound:
Echinops latifolius element A, white crystal, infrared data shows the compound, and there are hydroxyl (3433cm-1) and carbonyl
(1731cm-1) signal (as shown in Figure 2).As shown in Figure 1, the structural formula that high resolution mass spectrum provides compound is ([M+Na]+m/z
489.2975, calcd 489.2981), degree of unsaturation 10.The carbon modal data (Fig. 4) of the compound shows that it has 30 carbon originals
Son, including 4 methyl, 12 methylene, 5 methines and 9 quaternary carbons, including apparent 2 carbonyl signals, 6 rings
External double bond signal and two company's oxygen carbon signals.The one-dimensional nuclear magnetic data (Fig. 3 and Fig. 4) of binding compounds and two-dimentional nuclear magnetic data
(Fig. 5, Fig. 6 and Fig. 7) can be classified as part A and part B.Part A includes 2 methyl, and (2 are believed 6 methylene for olefinic carbon
Number), 2 company's oxygen methines and 5 quaternary carbons (including 1 carbonyl signals and 2 olefinic carbon signals), our portions A of these signal prompts
Subpackage contains 2 terminal double bonds.1H-1H COSY signal can significantly see that there are H (5)-H2(6)-H(7)-H2(8)-H2(9)
It connects (Fig. 7).The further long-range correlation of HMBC gives H2- 1 and C-2, C-3 have long-range related to C-14；H2- 15 and C-3,
C-4 have to C-5 it is long-range related, these Notes of Key Datas we 2 be connected with carbonyl, 3 are connected with oxygen atom (Fig. 7).Part B and the portion A
It is point closely similar, except that is be separately connected in C-4 ' and C-15 ' is methylene and methine.Further HMBC signal
H-4 ' is long-range related to C-3's；H2The long-range correlation of -15 ' and C-3, C-4 and C-2 give the connection side of part A and part B
Formula is directly connected to by novel carbon carbon.H-4 and H2- 15 '1H-1H COSY correlation further demonstrates the connection side of the two
Formula.It is in low field since the carbon chemical shifts of connection oxygen atom are opposite, determines that hydroxyl is connected to the position C-3 '.The structural formula of compound
(Fig. 8) is further confirmed by X-ray single crystal diffraction.The compound is the eucalyptus alkane dimer chemical combination with new skeleton
Object, it has the connection type of unique C-3 and C-15 ', and has special 1-oxaspiro [4.5] decan-6-one
Ring is named as Echinops latifolius element A.Accordingly, it is determined that the chemical structure of noval chemical compound of the present invention is shown in formula I, while including exhausted
Determination has also been obtained inside to configuration, as shown in formula II:
4, nuclear magnetic resonance spectroscopy (1H-NMR): Bruker-AVIII HD-600spectrometer measurement, data are shown in Table 1.
5, carbon-13 nmr spectra (13C-NMR): Bruker-AVIII HD-600spectrometer measurement, data are shown in Table 1.
1 Echinops latifolius element A's of table1H-NMR(600MHz)、13C-NMR (150MHz) nuclear magnetic data (measurement solvent: CDCl3；δ:
ppm；J:Hz)
6, X- monocrystalline: the bis- light source X-ray single crystal diffractometer measurements of Agilent Gemini E, data are as follows:
Molecular formula: C30H42O4, M=466.31, anorthic system, α=108.971 (5) °, β=90.502 (4) °, γ=107.466 (4) °,T=106.6K, space group P1, Z=2, μ (CuK α)=0.562mm-1,19415
Reflections collected,10018 Independent reflections(Rint=0.0496) .The final wR
(F2)values were 0.1482(all data).The goodness of fit on F2 was 1.023.Flack
Parameter=-0.17 (16)
The above crystal data determines the absolute configuration of Echinops latifolius element A, as shown in formula II.
In order to illustrate beneficial effects of the present invention, the present invention further provides following embodiments.
2 anticancer experiment in vitro of embodiment
1, experimental cell
Human liver cancer cell (HepG2), human cervical carcinoma cell (Hela) and human laryngeal cancer cell (Hep2) are bought from Chinese medicine
Central laboratory of BJ Union Hospital of the academy of sciences.
2, experimental drug
The loop coil sesquiterpene dimers (Echinops latifolius element A) and camptothecine prepared in embodiment 1 (are bought from Chinese medicines group
Chemical reagent Co., Ltd) it is used as positive control medicine.The drug solution for being 5-80 μM with culture medium configuration concentration.
3, drug inhibiting tumor cell determination of activity (mtt assay)
It is utilized respectively the DMEM in high glucose culture medium culture human liver cancer cell containing mass percent for 10% fetal calf serum
(HepG2), human cervical carcinoma cell (Hela) and human laryngeal cancer cell (Hep2).Then three kinds of cells of logarithmic growth phase will be in,
According to 2 × 104The density of a/ml is inoculated in 96 orifice plate culture plates, every 100 μ L cell liquid of hole.After 24 hours, toward three kinds of tumours
It is separately added into the untested compound (1,3,6,12,24,48 μM) of various concentration in cell, continues culture 72 hours.Remove cell
Culture medium, each hole are added the MTT solution (PBS is prepared, 5mg/ml) of 20 μ L, continue to be incubated for 4 hours, 20% is added in each hole
It is stood overnight in 50 μ L, CO2 incubator of SDS solution.The absorbance under 570nm wavelength is measured using microplate reader, is calculated different dense
Degree untested compound is to the growth inhibition ratio of tumour cell and when calculating untested compound to tumour cell half growth inhibition
Drug concentration (IC50), each experiment is in triplicate.
4, experimental result
Above-mentioned Echinops latifolius element A is through anti tumor activity in vitro screening test as a result, it was confirmed that Echinops latifolius element A is to human liver cancer cell
(HepG2), three kinds of tumor cell lines of human cervical carcinoma cell (Hela) and human laryngeal cancer cell (Hep2) all show apparent inhibition
It acts on (as shown in table 2), the especially anti-tumor activity (IC to human cervical carcinoma (Hela) tumor cell line50=26.17 μM) it is better than
Existing anti-tumor drug camptothecine (IC50=31.66 μM).
Inhibiting effect (IC of the 2 Echinops latifolius element A of table to three kinds of tumor cell lines50, μM)
Test result shows
Can be seen that Echinops latifolius element A from the data of table 2 has inhibition to make tri- kinds of tumour cells of HepG2, Hela and Hep2
With, and inventor is found surprisingly that Echinops latifolius element A inhibits the effect of Hela cell better than camptothecine.
The new loop coil sesquiterpene dimers compound (Echinops latifolius element A) of one kind of the present invention has apparent antitumor
Activity can be further prepared into anti-tumor drug for treatment of cancer.
3 anti-inflammatory activity of embodiment --- LPS inducing mouse macrophages secrete inflammatory factor is influenced
1, experimental cell
Mouse macrophage strain RAW264.7 is purchased from Chinese Academy of Sciences's consonance cell resource center.
2, experimental drug and reagent
Pancreatin, dimethyl sulfoxide (DMSO) (Gibco company)；RP-MI1640 cell culture medium (Hyclone company)；One
Nitrogen oxide (NO) test kit (green skies company)；MTS reagent (Promega company)；LPS (Sigma company), other reagents
It is that commercially available analysis is pure.
3, cell culture
RAW264.7 mouse monokaryon macrophage, which is used, contains 10% fetal calf serum, penicillin (1 × 105U/L) and streptomysin
1640 culture medium of RP-MI of (100mg/L) is incubated at 37 DEG C, 5%CO2Incubator in cultivate, pass on every other day.
4, cytotoxicity experiment
The mouse RAW264.7 cell prepared is taken, with 5 × 104Cells/well is set in 96 well culture plates, in 37 DEG C,
5%CO2, after culture for 24 hours, various concentration drug is added, concurrently sets blank control group.Continue culture for 24 hours, 20 μ L are added in every hole
MTS reagent, 37 DEG C, 5%CO2, cultivate 1h.To measure absorbance at microplate reader 490nm, observation drug is made with the presence or absence of cell toxicant
With.
5, Echinops latifolius element A influences the NO of the LPS RAW264.7 cell secretion induced
The mouse RAW264.7 cell prepared is taken, with 2 × 105Cells/well is set in 96 well culture plates, is placed in 37 DEG C,
5%CO2, after culture for 24 hours, culture medium is abandoned, gives drug and LPS processing respectively.Be divided into Normal group (complete medium),
LPS model group (culture medium containing 1 μ g/mL LPS), (culture containing 1 μ g/mL LPS and various concentration Echinops latifolius element A of processing group
Base).And positive drug group (containing 1 μ g/m L, 100 μM of LPS of dexamethasone) is set, it sets after being cultivated for 24 hours in incubator, draws culture
50 μ L of liquid supernatant, is operated by NO kit specification, reads absorbance value under 540nm, and the content of each hole NO is calculated.
6, experimental result
After Echinops latifolius element A acts on RAW264.7 cell for 24 hours, compared with blank, without obvious cytotoxicity (table at 100 μM
3).After 1 μ g/mL LPS stimulation, macrophage can release a large amount of NO, and positive drug dexamethasone can reduce NO level, real
It tests the result shows that it is more than 50% (NO inhibiting rate that 25 μM of Echinops latifolius element A, which inhibit the release of the RAW264.7 cell NO of LPS induction,
55.09%), illustrate that the Echinops latifolius element A (25 μM) of low concentration is able to suppress the suppression of the RAW264.7 cell NO release of LPS induction
Rate processed is more than 50%.50 μM of Echinops latifolius element A inhibit the releasability of the RAW264.7 cell NO of LPS induction to be significantly stronger than 100 μM
Dexamethasone (such as table 4).Illustrate that Echinops latifolius element A has the anti-inflammatory power better than existing agent dexamethasone.
3 Echinops latifolius element A of table inhibits RAW264.7 cell Proliferation
4 Echinops latifolius element A of table discharges the influence of NO to LPS stimulation RAW264.7 cell
4 anti-inflammatory activity of embodiment --- ConA inducing mouse liver injury model
1, experimental animal
C57BL/6 male mice, 20-22 week old, tie up experimental animal company of tonneau China purchased from Beijing by 28-32 grams of weight.
2, experimental drug and reagent
Concanavalin A (ConcanavalinA, ConA, Sigma)；IFN-γ ELISA detection kit is purchased from R&D company.
3, it is grouped and models
Grouping and modeling mouse are randomly divided into experimental group and control group, and every group each 24, ad lib during experiment.Indigo plant thorn
Head element A is dissolved to storage concentration with DMSO, is dissolved to sterile phosphate buffer (PBS) using concentration using preceding；ConA is molten
In the sterile PBS of Xie Yu.Experimental mice is first injected intraperitoneally after 50mg/kg Echinops latifolius element A, 2h duplicate injection 1 time, through tail after
Acute hepatic injury mice model is established in intravenous injection 15mg/kgConA induction.Control group mice is first through intraperitoneal injection and experimental group
The control solvent (DMSO) of same dose duplicate injection 1 time after 2h, models after through tail vein injection 15mg/kg ConA.It builds
8h after mould, chloral hydrate anesthesia mouse, heart extracting blood separate serum, and -80 DEG C of storages are stand-by.
4, ELISA method measures serum levels IFN-γ
The ELISA micro reaction plate adsorbed by force to antibody is selected, first washed once with coating buffer, pat dry reaction plate.
Antibody is diluted to certain concentration with coating buffer by kit specification, every hole adds 250 μ L of confining liquid to carry out after adding 100 μ L
Closing.Every hole adds diluted 100 μ L of blood serum sample；Standard by specification requires dilution, and every hole adds 100 μ L；Blank well is only loaded
Product dilution.Every hole adds 100 μ L of primary antibody, after room temperature 1 hour, washs 4 times.Add 100 μ L of ELIAS secondary antibody, after room temperature 1 hour, washing
5 times.Every hole adds 100 μ L of substrate, is placed at room temperature for 10-20 minutes, until the color of gauge orifice adds terminate liquid when reaching depth appropriate
100 μ L terminate reaction, and 450nm surveys absorbance.
5, experimental result
The Con A energy intracorporal immunocyte of acute activation mouse, then releases a large amount of cell factor, inducing hepatocyte
Necrosis.IFN-γ in the hepatic injury mouse peripheral blood that Fig. 9 induces ConA for the Echinops latifolius element A of ELISA elisa kit detection
Content influences variation.As the result is shown in control group and experimental group serum the mass concentration of IFN-γ be respectively (2498.45 ±
63.67) pg/mL and (450.7 ± 37.28) pg/mL, difference have statistical significance P < 0.01.Experimental result illustrates Echinops latifolius
Plain A can significantly inhibit key virulence factor IFN-γ expression.Con A induction acute hepatic injury model confirm, Echinops latifolius
Plain A processing can reduce the secretion of the inflammatory mediator of ConA induction, mitigate immunological liver injury, and there is protection liver to make
With.
5 killing aphids activity experiment of embodiment
1, selected insect source
Turnip aphid (Lipaphis erysimi), grain aphid (Sitobion avenae), rhopalosiphum padi
(Rhopalosiphum padi), black bean aphid (Aphis craccivora), raising are ground in Chinese Academy of Agricultural Sciences's plant protection
Study carefully institute's pesticide toxicity and natural product chemistry group.
2, for reagent object
The loop coil sesquiterpene dimers (Echinops latifolius element A) prepared in embodiment 1 and 25% pymetrozine suspension agent (Beijing
Middle guarantor green agriculture science and technology Group Co., Ltd) it is used as positive control medicine.Configuration concentration is 1,3,6,12,24,48mg/L.
3, indoor toxicity test (infusion process)
Diameter 9cm culture dish is taken, ware bottom covers the wet filter paper of one layer of diameter 9cm to keep humidity after label.Select band
The blade of aphid retains aphid 30 of health, soaks 5s in the liquid medicine of each concentration respectively, and dipping is placed on culture dish
It is interior, extra medical fluid is blotted with blotting paper, after room temperature is dried, is sealed with preservative film, is pierced multiple ventholes with insect needle, is sticked
Label.Every concentration sets 3 repetitions, compares as the 0.1% Tween-80 solution containing acetone.Be placed in photoperiod L: D=14h: 10h,
Temperature is (25 ± 2) DEG C, raises in the growth cabinet that relative humidity be 48%-52%, investigates aphid death toll afterwards for 24 hours and deposits
Number living.
Test result shows above-mentioned Echinops latifolius element A to four kinds of turnip aphid, grain aphid and rhopalosiphum padi, black bean aphid
Aphid all shows obvious virulence (LC50Respectively 3.49,16.63,7.51,9.36mg/L) (as shown in table 5).Echinops latifolius element A
To turnip aphid, rhopalosiphum padi, black bean aphid LC50Value is above commercially available common pesticides pymetrozine, shows that Echinops latifolius element A has
Preferable insecticidal activity.
Indoor toxicity test (LC of the 5 Echinops latifolius element A of table to a variety of aphids50, mg/L)
The new loop coil sesquiterpene dimers compound (Echinops latifolius element A) of one kind of the present invention has extensive insecticidal spectrum
With apparent insecticidal activity, it can be used as active constituent and be further prepared into killing aphids botanical pesticide.
The experiment of 6 eelworm-killing activity of embodiment
1, selected insect source
Meloidogyne incognita (Meloidogyne incognita), Plant Protection institute, Chinese Academy of Agricultral Sciences's pesticide
Toxicity and natural product chemistry group are spare after larvae in laboratory from picking pieces of an egg in cucumber old complaint.
2, for reagent object
The loop coil sesquiterpene dimers (Echinops latifolius element A) and lythidathion prepared in embodiment 1 are as positive control drug
Object.Configuration concentration is 6,12,24,48,96mg/L.
3, prepared by Meloidogyne incognita suspension
It falls ill the pieces of an egg on heavier plant in picking root knot from root-knot nematode, hatches at normal temperature to root-knot nematode larva
Afterwards, larva is separated with tray method, dilutes nematode suspension after microscopy, made in every 1ml suspension containing 300 or so two ages childrens
Worm, room temperature storage are spare.
4, biological activity determination
Using infusion process.Drug is respectively arranged 5 concentration processing, blank controls and is arranged 1, and each processing is respectively provided with 4 weights
It is multiple.24 hole biochemical test plates are taken, respectively take 1 milliliter to be placed in aperture for reagent liquid, isometric nematode suspension is added, respectively
To the drug-treated liquid of 3,6,12,24,48mg/L, space management is added the clear water of equivalent, southern root is checked after processing 48 hours
The amount of survival and The dead quantity of tie lines worm second instar larvae calculate LD50。
Toxicity test of the 6 Echinops latifolius element A of table to Meloidogyne incognita
Indoor toxicity test is as shown in table 6, LD of the fosthiazate to Meloidogyne incognita50For 29.63mg/L, Echinops latifolius element A
To the LD of Meloidogyne incognita50For 13.25mg/L.The experimental results showed that nematicidal energy of the Echinops latifolius element A to Meloidogyne incognita
Power is better than existing commercially available pesticide fosthiazate, illustrates that Echinops latifolius element A has as botanical pesticide in nematicidal direction and preferably answers
Use prospect.
In conclusion the present invention provides a kind of new skeleton loop coil sesquiterpene dimers compound and its extraction separation method,
The compound is extracted due to its unique chemical structure, and from conventional Chinese medicine echinops grijisii Hance, the experimental results showed that its
With antitumor, liver protection, anti-inflammatory and killing aphids, eelworm-killing activity, therefore one kind as plant source natural product exploitation Chinese medicine
New drug has broad application prospects in medicine, pesticide field.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of new skeleton loop coil sesquiterpene dimers compound, which is characterized in that including structure shown in Formulas I:
Wherein, R1~R18 is separately selected from hydrogen, halogen, hydroxyl, amino.
2. new skeleton loop coil sesquiterpene dimers compound according to claim 1, which is characterized in that further include shown in Formulas I
Crystal form, stereochemical structure, its pharmaceutically acceptable salt, its solvate, its pro-drug or its metabolite of structure.
3. new skeleton loop coil sesquiterpene dimers compound according to claim 1, which is characterized in that R1~R18 is hydrogen.
4. new skeleton loop coil sesquiterpene dimers compound according to claim 1, which is characterized in that including being tied shown in formula II
Structure:
5. a kind of pharmaceutical preparation, which is characterized in that including any one of the claim 1-4 new skeleton loop coil sesquiterpene dimers
Compound.
6. a kind of preparation method of any one of claim 1-4 new skeleton loop coil sesquiterpene dimers compound, feature
It is, comprising the following steps:
(1) echinops grijisii Hance dry root is extracted using ethyl alcohol, obtains ethanol extract；
(2) ethanol extract is used into silica gel column chromatography, successively using petroleum ether-ethyl acetate and petroleum ether-acetone as eluant, eluent into
Row gradient elution obtains fraction；
(3) it uses hydroxypropyl sephadex to elute with chloroform-methanol the fraction that step (2) obtain, obtains new skeleton loop coil
Sesquiterpene dimers compound.
7. the preparation method of new skeleton loop coil sesquiterpene dimers compound described in claim 6, which is characterized in that step (2)
In, concrete operation step are as follows:
(a) take ethanol extract obtained by step (1), using silica gel column chromatography, with petroleum ether-ethyl acetate volume ratio (25:1)~
(0:1) is that eluant, eluent carries out gradient elution, collects eluent as fraction；
(b) fraction for collecting step (a) is again through silica gel column chromatography, the petroleum ether-acetone for being (30:1)~(1:1) with volume ratio
Solvent gradient elution collects fraction；
(c) fraction for collecting step (b) is again through silica gel column chromatography, the petroleum ether-acetone for being (8:1)~(1:1) with volume ratio
Gradient elution collects fraction.
8. the preparation method of new skeleton loop coil sesquiterpene dimers compound described in claim 7, which is characterized in that step (a)
In, it collects using petroleum ether-ethyl acetate volume ratio and elute the eluent obtained as fraction by the eluant, eluent of 5:1.
9. the preparation method of the new skeleton loop coil sesquiterpene dimers compound of claim 7 or 8, which is characterized in that step
(b) it in, collects using petroleum ether-acetone volume ratio and elute the eluent obtained as fraction by 5:1 eluant, eluent.
10. the preparation method of the new skeleton loop coil sesquiterpene dimers compound of claim 7 or 8, which is characterized in that step
(c) it in, collects using petroleum ether-acetone volume ratio and elute the eluent obtained as fraction by 3:1 eluant, eluent.
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