CN105294623B - A kind of Sesquiterpene lactones compound, its preparation method and application - Google Patents
A kind of Sesquiterpene lactones compound, its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of Sesquiterpene lactones compound, its preparation method and the application in treatment of cancer, shown in the structure such as formula (I) of the Sesquiterpene lactones compound.The preparation method of the Sesquiterpene lactones compound includes：A, using Extraction solvent to medicinal material extract, obtain extract solution；B, extract solution concentrated, add water suspension, is extracted respectively with the ascending organic solvent of polarity, obtains extract, and extract concentration obtains crude extract；Normal phase column is eluted with eluant, eluent on C, crude extract；Eluent is collected, is dried, the extract rich in novel sesquiterpene lactone compound is obtained；D, extract carry out anti-phase medium pressure column chromatography and preparative efficient liquid phase column chromatography for separation, obtain described novel sesquiterpene lactone compound.The preparation method is simple to operate, and production cost is low, and obtained product can be used for the preparation of cancer treatment drugs, with good DEVELOPMENT PROSPECT.
Description
Technical field
The invention belongs to biological active constituents from natural medicines technical field, it is related to and is extracted from Chinese medicine, prepares active ingredient and answer
Field, preparation method and its cancer more particularly, to a kind of one in composite family feverwort novel sesquiterpene lactone are controlled
Treat application.
Background technology
Composite family (Compositae) Eupatorium (Eupatorium L.) the plant whole world about more than 600 is planted, in being distributed mainly on
The temperate zone and torrid areas of South America.China has 14 kinds, several mutation, and the whole nation is produced in addition to Xinjiang, Tibet.Eupatorium conduct
Medicinal plant, has eight kinds such as woods Herba Lycopi using more at present.The platymiscium has long medication history.
Eupatorium lindleynun var. trifoliolatum is the drying overground part of composite family feverwort woods Herba Lycopi (Eupatorium lindleyanum DC.)
Point, it is distributed more widely in China, with medication history for many years, now recorded by 2015 editions pharmacopeia of China.Eupatorium lindleynun var. trifoliolatum, bitter is mild-natured, returns
Lung channel, with clearing heat and detoxicating, preventing phlegm from forming and stopping coughing, the effects such as relieving asthma.Eupatorium lindleynun var. trifoliolatum branches and leaves have an inducing diaphoresis clearing damp, and middle dampness elimination effect, be used for
The treatment such as epersalgia cough, haematemesis hemoptysis and stranguria with turbid discharge leukorrhea, nameless gall.
Eupatorium lindleynun var. trifoliolatum has been developed that into a variety of Chinese patent drugs such as granule, syrup and is widely used in clinic.Chasing Wildhorse syrup is
Famous-brand and high-quality Chinese patent drug, is mainly used in the treatment of respiratory disease, generates significant society and economic benefit.
Containing the sesquiterpene lactone constituents that abundant structure is novel in eupatorium lindleynun var. trifoliolatum medicinal material, the constituents generally contain " alpha-beta
Unsaturated carbonyl " construction unit, is michael reaction acceptor molecule (the Michael addition of a quasi-representative
acceptors).Michael reaction acceptor molecule has notable biological activity, particularly to containing in Cell signal propagation pathways
Albumen, the enzyme of cysteine residues, such as STAT3, CDK, Hsp90, NF- κ B have inhibitory activity, michael reaction acceptor point
Son has turned into one of focus that small molecule bio-pharmaceutical is studied.
So far, although a series of isolated Sesquiterpene lactones compounds from feverwort, but not
It is provided with a kind of technique simple, scientific and reasonable, the significant composite family Eupatorium of active ingredient stable in physicochemical property, bioactivity is planted
Thing Sesquiterpene lactones compound and preparation method thereof.
In the application aspect of the Chinese patent medicine preparations such as Chasing Wildhorse syrup, the breathing such as such preparation is used for resolving sputum, cough-relieving, relievingd asthma
The treatment of systemic disease, the index components or active ingredient of detection are based on flavones, alkaloid, and eupatorium lindleynun var. trifoliolatum and its preparation are in cancer
Application in terms for the treatment of is rarely reported.
The content of the invention
It is an object of the invention to provide a kind of novel sesquiterpene lactone, its preparation method and application, the sesquiterpene lactone
With preferable active anticancer, while the preparation method technological operation is simple, scientific and reasonable, stable in physicochemical property can be obtained,
The higher Sesquiterpene lactones compound of purity.
A kind of Sesquiterpene lactones compound, shown in structure such as formula (I)：
The structure of the compound there are no document report at present, while MTT experiments show the compound to two plants of people source breasts
Adenocarcinoma cell has after significant inhibitory action, administration 24h, to the half-inhibition concentration (IC of two plants of people source tumor cell lines50)
For：2.67~3.05 μm of ol/L.With antitumor activity.
Present invention also offers a kind of preparation method of the Sesquiterpene lactones compound, comprise the steps：
(A) extract：Medicinal material is extracted using Extraction solvent, extract solution is obtained；
Described medicinal material is the dry aerial parts and/or herb of composite family feverwort；
(B) extract：The extract solution that step (A) is obtained is concentrated, obtained concentrate adds water suspension, then using extraction
Solvent is taken to carry out that extract is obtained by extraction, extract obtains crude extract by concentration；
(C) column chromatography：Normal phase column on the crude extract that step (B) is obtained, is eluted with eluting solvent, collects eluent,
Solvent is recovered under reduced pressure, the extract rich in the sesquiterpene lactone compound is obtained；
(D) medium pressure column chromatography：The extract that step (C) is obtained carries out medium pressure column chromatography separation, obtains containing formula (1) institute
Show the crude product of compound.In described medium pressure column chromatography, chromatographic column filler is Octadecylsilane bonded phase silica gel；Medium pressure post
Chromatographic flow mutually at least includes 40~70% methanol aqueous solution；
(E) preparative efficient liquid phase column chromatography：The crude product that step (D) is obtained carries out preparative efficient liquid phase column chromatography,
Obtain the compound as shown in formula (1).In the preparative efficient liquid phase column chromatography, chromatographic column filler is Octadecylsilane bonded phase
Silica gel；The preparative efficient liquid phase column chromatography mobile phase at least includes 20~40% acetonitrile solution.
In step A, the extracting method of medicinal material is more, including percolation.Percolation extraction efficiency is high, post processing extraction solvent consumption
Few, normal temperature extraction energy consumption is low, simple and feasible.Percolation is operated at normal temperatures, does not also result in the destruction of thermal sensitivity active ingredient.
In step B, extractant can have multiple choices, but at least include ethyl acetate.Ethyl acetate polarity is more than
Petroleum ether of the prior art, ether, dichloromethane, chloroform, can by Sesquiterpene lactones in feverwort extract into
Divide extraction complete；Ethyl acetate polarity is less than n-butanol of the prior art, not only can extract sesquiterpene lactone constituents completely
Take out, additionally it is possible to the larger glycoside impurity of polarity that some are soluble in n-butanol is rejected, so that feverwort slightly carries
The content of sesquiterpene lactone active component is higher in thing, and purity is higher.
In step C, eluant, eluent, which has, compares strict requirements, and has restriction to volume ratio, and the eluant, eluent selectivity is high, washes
De- ability is strong, ensure that the extract for obtaining that physicochemical property is stable, the compound is distributed concentration.
It is 20~0 that described eluant, eluent, which is included by volume ratio,：The eluant, eluent of 1 petroleum ether and ethyl acetate composition.
In step D, described medium pressure column chromatography is reverse-phase chromatography, and chromatographic column filler is Octadecylsilane bonded phase silica gel.Institute
Stating medium pressure column chromatography mobile phase at least includes 40~70% methanol aqueous solution.
In step E, described preparative efficient liquid phase column chromatography is also reverse-phase chromatography, and chromatographic column filler is octadecyl key
Phase silica gel is closed, mobile phase at least includes 20~40% acetonitrile solution.
In order to obtain more preferable invention effect, below as the preferred of the present invention：
In step (A), the medicinal material is the drying herb of eupatorium lindleynun var. trifoliolatum (Eupatorium lindleyanum DC.), wild horse
Chase after and recorded by 2015 editions Chinese Pharmacopoeias, it is cheap and easy to get.
The Extraction solvent is one or both of water, alcohol, and the Extraction solvent is used safe, inexpensive and had preferable
Extraction efficiency.Further preferably, the Extraction solvent is the ethanol water that mass fraction containing ethanol is 90~95%.Should
The property of Extraction solvent is very suitable for extracting feverwort dry aerial parts and/or herb, can more efficiently, completely
Ground obtains the sesquiterpene lactone active component in medicinal part, and is avoided that the larger non-Sesquiterpene lactones of a large amount of polarity are miscellaneous
Matter is extracted.
The extracting method is solvent extraction method.Further preferably, the extracting method is percolation.Medicinal material beats powder, mistake
40 mesh sieves, fill post after medicinal powder wetting, plus 90~95% ethanol cold soaking 24h, diacolation, the amount ratio of Extraction solvent and medicinal material for 15~
20L ﹕ 1kg.The method extraction efficiency is high, and post processing extraction solvent consumption is few, and extraction energy consumption is low under normal temperature, and is avoided that Heat sensitive active composition
Degraded, Percolation device is simple, also can largely save production cost.
In step (B), described extractant is petroleum ether, ether, dichloromethane, chloroform, ethyl acetate, n-butanol,
It is two or more in water.Extracted successively using single solvent during extraction by polarity is ascending.Further preferably, it is described
Extractant is petroleum ether, ethyl acetate, is extracted in equal volume using single solvent successively by polarity size during extraction.According to
The similar principle that mixes, is extracted by above-mentioned extractant respectively, so that the herbal extract concentration that step A is obtained adds water after suspension
Solution according to the ascending separation of polarity, known by thin-layer chromatography (TLC), LC-MS inspections, times in feverwort extract
Sesquiterpene lactone is concentrated mainly in acetic acid ethyl acetate extract, and other extractants are mainly used in removing non-sesquialter in medicinal substances extract
Terpene lactones impurity, to obtain the crude extract rich in sesquiterpene lactone.Therefore, further preferably, it is acetic acid ethyl acetate extract is dense
Normal phase column is further purified on contracting, ethyl acetate extract, and remaining can ignore, so as to reduce production cost.
In step (C), the positive column packing is the column chromatography silica gel or aluminum oxide of 100~200 mesh.
Described eluant, eluent include by volume ratio for 20 ﹕ 1 petroleum ether and ethyl acetate constitute mixed solvent, by volume
The mixed solvent that is constituted than the petroleum ether for 15 ﹕ 1 and ethyl acetate, the petroleum ether and ethyl acetate for being 10 ﹕ 1 by volume ratio are constituted
Mixed solvent, be 6 ﹕ 1 by volume ratio petroleum ether and ethyl acetate constitute mixed solvent, be 3 ﹕ 1 by volume ratio oil
Ether and ethyl acetate composition mixed solvent, by volume ratio for 2 ﹕ 1 petroleum ether and ethyl acetate constitute mixed solvent, by body
The long-pending mixed solvent constituted than the petroleum ether and ethyl acetate that are 1 ﹕ 1, the petroleum ether and ethyl acetate for being 0 ﹕ 1 by volume ratio are constituted
Mixed solvent, and carry out gradient elution successively by polarity is ascending.The eluant, eluent constituted using petroleum ether and ethyl acetate,
And gradient elution is carried out successively by polarity is ascending, eluent system selectivity is high, and eluting power is strong, passes through TLC, LC-MS
Inspection is known, and it is 0 ﹕ 1 petroleum ether and ethyl acetate composition that novel sesquiterpene lactone almost all of the present invention, which is located at volume ratio,
In eluent after eluent so that novel sesquiterpene lactone active component content is higher in feverwort crude extract,
Distribution is more concentrated.
In step (D) (E), described middle pressure chromatogram and preparative high performance liquid chromatography filler are Octadecylsilane bonded phase
Silica gel, the filler has stronger selectivity, good separating effect to Sesquiterpene lactones compound.Medium pressure column chromatography, with upper
Sample amount is big, the advantage such as separative efficiency height.
The extract rich in novel sesquiterpene lactone that step (C) is obtained, after being dissolved with tetrahydrofuran, wet method loading, point
Do not eluted with 45%, 65% methanol aqueous solution, the mobile phase eluting power is strong, the novel sesquiterpene lactone almost all collection
In in the fractions of 65% methanol water elution.
The crude product rich in novel sesquiterpene lactone that step (D) is obtained, carries out Reverse phase preparative efficient liquid phase column chromatography,
With 20~40% acetonitrile water elution, further preferably, with 30% acetonitrile water isocratic elution, elution gained fractions steam
It is dry, that is, obtain the novel sesquiterpene lactone.The novel sesquiterpene lactone and another Sesquiterpene lactones compositional polarity are very
Close, common silica gel column chromatography and anti-phase medium pressure column chromatography are difficult to separate both materials.Preparative high performance liquid chromatography
Big with applied sample amount, post effect is high, the advantages of separative efficiency is high.In experiment, first, it is mobile phase to attempt with 40% methanol water system
Preparative separation, but fail to efficiently separate two kinds of close sesquiterpene lactones of polarity, the obtained novel sesquiterpene lactone is pure
Degree is relatively low, and separative efficiency is also very low；Then, 30% acetonitrile water is used instead for after eluent so that two kinds of polarity are very
Close Sesquiterpene lactones compound has reached good separation.
Present invention also offers a kind of application of described Sesquiterpene lactones compound in treating cancer medicine is prepared.
More specifically, the sesquiterpene lactone compound is to two kinds of people source breast cancer cells, with more significant inhibitory action.With
Mtt assay, to novel sesquiterpene lactone compound described in obtained feverwort, has carried out the tumor cell line increasing of suppression people source
Activity research is grown, is as a result shown, novel sesquiterpene lactone compound prepared by the present invention has to two plants of people source breast cancer cells
After more significant inhibitory action, administration 24h, half-inhibition concentration IC50About：2.67~3.05 μm of ol/L.Above-mentioned cell line can
Using commercially available prod, such as：American Type Culture collection warehousing ATCC (American type culture can be used
Collection various cell lines).
Novel sesquiterpene lactone compound can be combined with commercially available or conventional carrier in feverwort of the present invention, be used for
Prepare prevention either/and treatment or synergistic treatment cancer medicine.Described medicine can be fat emulsion, injection oil
The forms such as agent, powder-injection, tablet, capsule.
Compared with prior art, novel sesquiterpene lactone compound preparation method and its cancer are controlled in this feverwort
The advantage for treating application is：Compound structure is novel, and bioactivity is strong.Using systematic solvent extraction and positive, reverse-phase chromatography, in
Pressure, high pressure chromatogram, separation, purifying have obtained the novel sesquiterpene lactone compound of a structure.The solvent used is cheap and easy to get,
And toxicity is relatively low.The preparation method of novel sesquiterpene lactone is scientific and reasonable in feverwort of the present invention, simple to operate, is produced into
This is low, has a good application prospect.
Brief description of the drawings
Fig. 1 (A) is the proton nmr spectra of compound 1.
Fig. 1 (B) is the carbon-13 nmr spectra of compound 1.
Fig. 1 (C) is the DEPT spectrums of compound 1.
Fig. 1 (D) is the H-HCSOY spectrums of compound 1.
Fig. 1 (E) is the hsqc spectrum of compound 1.
Fig. 1 (F) is the HMBC spectrums of compound 1.
Fig. 1 (G) is the NOESY spectrums of compound 1.
Fig. 1 (H) is the high resolution mass spectrum of compound 1.
Embodiment
Present disclosure is described in further detail below by way of specific embodiment, but embodiment should not be understood
For limitation of the present invention.In the case where not departing from above-mentioned thought of the invention, according to ordinary skill knowledge and routine
Various replacement means or change that means are made, are all contained within the present invention.
Embodiment 1
The preparation method of novel sesquiterpene lactone comprises the steps in this feverwort：
A, extraction：Eupatorium lindleynun var. trifoliolatum medicinal material beats powder, crosses 40 mesh sieves standby.Take eupatorium lindleynun var. trifoliolatum medicinal material coarse powder 2kg, plus appropriate 95% ethanol
Post is filled after wetting, then adds seepage pressure effects after appropriate 95% ethanol cold soaking 24h, percolate is collected.Extract total solvent volume equivalent to
18 times of amounts (total spends 95% ethanol 36L) of quality of medicinal material.
B, extraction：The obtained percolates of step A are concentrated under reduced pressure into and closely done, thick medicinal extract 84.3g is obtained, adds water and be suspended to 1L,
1L petroleum ether, 1L ethyl acetate is used respectively, and each extractant is extracted three times, is merged the extract of each time, is respectively obtained
Eupatorium lindleynun var. trifoliolatum petroleum ether extraction liquid, acetic acid ethyl acetate extract is examined by TLC and known, and eupatorium lindleynun var. trifoliolatum sesquiterpene lactone is concentrated mainly on acetic acid
In ethyl ester extract, solvent is recovered under reduced pressure in acetic acid ethyl acetate extract, obtains eupatorium lindleynun var. trifoliolatum ethyl acetate extract 57.2g.
C, column chromatography：Above-mentioned eupatorium lindleynun var. trifoliolatum ethyl acetate extract is taken, the normal phase silicagel column of upper 100~200 mesh is separated,
Multiple different eluents are used respectively, and each eluant, eluent is constituted by petroleum ether and ethyl acetate, and volume is
The volume ratio of 1.5L, each eluant, eluent petrochina ether and ethyl acetate is respectively the ﹕ of 20 ﹕, 1,15 ﹕, 1,10 ﹕, 1,6 ﹕, 1,3 ﹕, 1,2 ﹕ 1,1
1,0 ﹕ 1 simultaneously carries out gradient elution successively by the ascending order of polarity.Gained flow point is examined through TLC, LC-MS to be known, rich in described
In the flow point of novel sesquiterpene lactone, the eluent for being concentrated mainly on petroleum ether and the ﹕ 1 of ethyl acetate 0 eluant, eluent, collect above-mentioned
Eluent, solvent is recovered under reduced pressure with Rotary Evaporators at 40 DEG C, obtains the extract 9.5g rich in the novel sesquiterpene lactone.
D, medium pressure column chromatography and preparative efficient liquid phase column chromatography：Said extracted thing about 4.2g is taken, medium pressure column chromatography is carried out
Separation, middle compression leg filler is Octadecylsilane bonded phase silica gel, and particle diameter is 40-60 μm, after sample tetrahydrofuran dissolves in wet method
Sample, is eluted, each gradient elution about 2L with 45%, 65% aqueous methanol gradient, collects 65% water methanol eluent correspondence
Fractions, solvent is recovered under reduced pressure, extract about 876mg is obtained, the extract about 200mg is taken, with flowing phased soln after, enter
Row Reverse phase preparative efficient liquid phase column chromatography, with 30% acetonitrile solution isocratic elution, collects respective absorption peak correspondence flow point,
Solvent is recovered under reduced pressure and obtains compound (C as shown in Equation 122H26O8) about 43.8mg.
Embodiment 2
A, extraction：Eupatorium lindleynun var. trifoliolatum medicinal material 2kg, plus 8 times of 95% ethanol of amount are taken, 85 DEG C of refluxing extractions, each extraction time is 2h,
Extract 3 times altogether, merge No. 3 extract solutions.Total spends 95% ethanol 48L.
B, extraction：The obtained extract solutions of step A are concentrated under reduced pressure into and closely done, thick medicinal extract 92.7g is obtained, adds water and be suspended to 1L,
Extracted respectively with 1L petroleum ether, 1L ethyl acetate, each extractant is extracted three times, merge the extract of each time, respectively
Eupatorium lindleynun var. trifoliolatum petroleum ether extraction liquid is obtained, acetic acid ethyl acetate extract is examined by TLC and known, and eupatorium lindleynun var. trifoliolatum sesquiterpene lactone constituents are main
Concentrate in acetic acid ethyl acetate extract, solvent is recovered under reduced pressure in acetic acid ethyl acetate extract, obtain eupatorium lindleynun var. trifoliolatum ethyl acetate extract
60.3g。
C, column chromatography：Above-mentioned eupatorium lindleynun var. trifoliolatum ethyl acetate extract is taken, the silica normal phase column of upper 100~200 mesh is separated,
Then multiple different solvent gradient elutions are used respectively, and each eluant, eluent is by petroleum ether and ethyl acetate composition and volume is equal
For 1.5L, the volume ratio of each eluant, eluent petrochina ether and ethyl acetate is respectively the ﹕ 1 of 20 ﹕, 1,15 ﹕, 1,10 ﹕, 1,6 ﹕, 1,3 ﹕ 1,2,
The ﹕ 1 of 1 ﹕ 1,0, and gradient elution is carried out successively by the ascending order of polarity.Gained flow point is examined through TLC, LC-MS to be known, described new
Clever sesquiterpene lactone is focused primarily upon in the eluent of petroleum ether and the ﹕ 1 of ethyl acetate 0 eluant, eluent, collects above-mentioned eluent, is used
Solvent is recovered under reduced pressure at 40 DEG C for Rotary Evaporators, that is, obtains the extract 11.2g rich in novel sesquiterpene lactone.
D, medium pressure column chromatography：Said extracted thing about 4.2g is taken, medium pressure column chromatography separation is carried out, middle compression leg filler is octadecane
Base bonded-phase silica, particle diameter is 40-60 μm, after sample is dissolved with 45% methanol-water, wet method loading, with 45%, 65% methanol
Aqueous solution gradient elution, each gradient elution about 2L collects the corresponding fractions of 65% water methanol eluent, is recovered under reduced pressure molten
Agent, obtains extract about 816mg, takes the extract about 200mg, after flowing phased soln, carries out Reverse phase preparative efficient liquid phase
Column chromatography, with 30% acetonitrile isocratic elution, collects respective absorption peak correspondence flow point, solvent is recovered under reduced pressure and obtains as shown in Equation 1
Compound (C22H26O8) about 35.4mg.
Embodiment 3
A, extraction：Eupatorium lindleynun var. trifoliolatum medicinal material beats powder, crosses 40 mesh sieves standby.Take eupatorium lindleynun var. trifoliolatum medicinal material coarse powder 2kg, plus appropriate 95% ethanol
Post is filled after wetting, then adds seepage pressure effects after appropriate 95% ethanol cold soaking 24h, percolate is collected.Extract total solvent volume equivalent to
18 times of amounts (total spends 95% ethanol 36L) of quality of medicinal material.
B, extraction：The obtained percolates of step A are concentrated under reduced pressure into and closely done, thick medicinal extract 82.4g is obtained, adds water and be suspended to 1L,
Extracted respectively with 1L petroleum ether, 1L ethyl acetate, each extractant is extracted three times, merge the extract of each time, respectively
Eupatorium lindleynun var. trifoliolatum petroleum ether extraction liquid is obtained, acetic acid ethyl acetate extract is examined by TLC and known, and eupatorium lindleynun var. trifoliolatum sesquiterpene lactone is concentrated mainly on
In acetic acid ethyl acetate extract, solvent is recovered under reduced pressure in acetic acid ethyl acetate extract, obtains eupatorium lindleynun var. trifoliolatum ethyl acetate extract 58.3g.
C, column chromatography：Above-mentioned eupatorium lindleynun var. trifoliolatum ethyl acetate extract is taken, the normal phase silicagel column of upper 100~200 mesh is separated,
Then multiple different eluents are used respectively, and each eluant, eluent is constituted by petroleum ether and ethyl acetate, and volume is equal
For 1.5L, the volume ratio of each eluant, eluent petrochina ether and ethyl acetate is respectively the ﹕ 1 of 20 ﹕, 1,15 ﹕, 1,10 ﹕, 1,6 ﹕, 1,3 ﹕ 1,2,
The ﹕ 1 of 1 ﹕ 1,0 simultaneously carry out gradient elution successively by the ascending order of polarity.Gained flow point is examined through TLC, LC-MS to be known, rich in institute
The flow point of novel sesquiterpene lactone is stated, in the eluent for being concentrated mainly on petroleum ether and the ﹕ 1 of ethyl acetate 0 eluant, eluent, in collection
Eluent is stated, solvent is recovered under reduced pressure at 40 DEG C with Rotary Evaporators, the extract rich in the novel sesquiterpene lactone is obtained
8.9g。
D, medium pressure column chromatography and preparative efficient liquid phase column chromatography：Said extracted thing about 4.2g is taken, medium pressure column chromatography is carried out
Separation, middle compression leg filler is Octadecylsilane bonded phase silica gel, and particle diameter is 40-60 μm, after sample tetrahydrofuran dissolves in wet method
Sample, is eluted, each gradient elution about 2L with 45%, 65% aqueous methanol gradient, collects 65% water methanol eluent correspondence
Fractions, solvent is recovered under reduced pressure, extract about 851mg is obtained, the extract about 200mg is taken, with flowing phased soln after, enter
Row Reverse phase preparative efficient liquid phase column chromatography, with 40% methanol aqueous solution isocratic elution, collects respective absorption peak correspondence flow point,
Solvent is recovered under reduced pressure and obtains compound (C as shown in Equation 122H26O8) about 22.3mg.
Compound 1 is characterized by proton nmr spectra, carbon-13 nmr spectra, ID NMR speetna, high resolution mass spectrum, tool
Just like chemical constitution shown in formula (I).Characterize data is shown in Table 1.
Table 1：Compound1H NMR(600MHz)、13C H NMR spectroscopies (150MHz) (δ, ppm, TMS, CDCl3)
6 times compared with embodiment more than 1 amounts of the Extraction solvent of embodiment 2, but the novel sesquiterpene lactone yield compared with embodiment 1
It is low, it is seen that percolation is high compared with reflux extraction to the extraction efficiency of sesquiterpene lactone.Reflux extraction is in 85 DEG C of operations, if industrial
Change big production, its energy resource consumption will also increase production cost；In addition, 85 DEG C of high temperature can may also cause in thermal sensitivity sequiterpene
The degraded of ester.The step D of embodiment 2, after 45% methanol sample dissolution, wet method loading, rich in the novel sequiterpene
The crude extract of ester is in 45% methanol aqueous solution, and solubility is poor, causes loading difficulty to increase, therefore rich in the dilute ether terpene ester of novel ring
Extract, during the separation of underway pressure column chromatography, loading is more excellent after being dissolved with tetrahydrofuran.
Embodiment 3 is compared with Example 1 compared with extraction, extraction, normal phase column chromatography, medium pressure column chromatography process conditions are complete
It is identical.Found when carrying out preparative efficient liquid phase column chromatography to the crude product rich in the novel sesquiterpene lactone, if with first
Alcohol water is (such as：40% methanol-water) system is mobile phase, the target chromatographic peak retention time of the novel sesquiterpene lactone is long, and
Overlaped with the chromatographic peak of another known sesquiterpene lactone, though the novel sesquiterpene lactone has been obtained to a certain extent,
But yield reduces about 1 times than embodiment 1, the novel sesquiterpene lactone obtained by embodiment 3 carries out nuclear magnetic resonance
Collection of illustrative plates finds that the nuclear magnetic resonance figures spectral purity obtained by test is poor when characterizing.It can be seen that to the novel sesquiterpene lactone
Crude product carry out preparative efficient liquid phase column chromatography when, with acetonitrile water (such as 30% acetonitrile water) be mobile phase, with target into
Get rate high, the obvious advantage such as purity height.
Embodiment 4
Novel sesquiterpene lactone compound anticancer experiment in vitro：
Take the logarithm the people source tumor cell line in growth period, after digestion is counted, by 3 × 103The μ L/ holes of individual cell/100 are inoculated in
In 96 porocyte culture plates, culture 24h is after after cell attachment, with the novel sequiterpene of the preparation of embodiment 1 of various concentrations
Lactone processing, three multiple holes are set per hole.Medicine is incubated after 24h with tumour cell, plus MTT, and 37 DEG C are continued to be incubated after 4h, terminate training
Support, nutrient solution is gently sopped up with liquid-transfering gun, add DMSO (150 μ L/ holes), after shaking is uniform, determined with ELIASA at 570nm
The optical density OD values in each hole, the OD values of each parallel hole are taken the mean, and the OD values of each instrument connection are subtracted into background OD values.Calculate medicine
Inhibiting rate of the thing to tumour cell.
Inhibiting rate=(1- dosing holes average optical density value/control group average optical density value) × 100%.According to inhibiting rate meter
Calculate half-inhibition concentration (IC of the medicine to tumour cell50), IC50Calculated with origin softwares.Experiment is repeated 3 times, IC50Take
mean±SD。
Two plants of people source tumor cell lines of human breast carcinoma MDA-MB-231, MDA-MB-468 are tested respectively, it is above-mentioned thin
Born of the same parents' strain is purchased from ATCC, and concrete outcome is as shown in table 2.The result of table 2 shows that the novel sesquiterpene lactone is external to two plants of people sources
Breast cancer cell is respectively provided with the effect of significantly inhibiting.
Table 2：Half-inhibition concentration IC of the novel sesquiterpene lactone to people source breast carcinoma cell strain50It is worth (mean ± SD, n=
3)
Novel sesquiterpene lactone compound can be with commercially available or conventional load in the feverwort that the present invention is prepared
Body combine, for prepare prevention, treatment, synergistic treatment cancer medicine.The medicine can be fat emulsion, injection, powder
The forms such as injection, tablet, capsule.
The present invention prepares sesquiterpene lactone compound in gained feverwort, when being administered in the treatment (administration)
When, it is possible to provide different effects.Generally, the sesquiterpene lactone compound that can be prepared the present invention is formulated in nontoxic, inert
In pharmaceutically acceptable aqueous carrier medium.The medicine prepared can be administered by conventional route, including (but do not limit
In)：Vein, muscle, intraperitoneal, subcutaneous, intracutaneous or local administration.Used carrier medium includes (but being not limited to)：Physiology salt
Water, buffer solution, glucose, water, glycerine, ethanol and combinations thereof.Sesquiterpene lactone compound of the present invention can be made into Fat Emulsion
Form, such as with the aqueous solution of injection soybean oil, lecithin, glycerine and other assistant agents, prepared by conventional method.
The medicine of such as tablet and capsule etc, can also be prepared by conventional method.Medicine is as injected agent solution preferably sterile
Under the conditions of manufacture.The dosage of active constituents of medicine is therapeutically effective amount, such as daily 1 μ g/kg body weight~2000mg/kg bodies
Weight.In addition, sesquiterpene lactone compound prepared by the present invention, can also cooperate with other antineoplastics and use.
, can be by times for the treatment of effective dose when sesquiterpene lactone compound is used as medicine in feverwort of the present invention
Sesquiterpene lactone compound is applied to mammal, wherein the treatment effective dose typically at least 10 μ g/kg body weight, is preferably administered
Dosage is about 10 μ g/kg body weight~30mg/kg body weight.Specific dosage is also contemplated that the factors such as administering mode, patient health situation,
Within these category skilled practitioners skills.
Specific embodiment described herein is only to spirit explanation for example of the invention.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode
Generation, but without departing from the spiritual of the present invention or surmount scope defined in appended claims.
Although more having used term herein, the possibility using other terms is not precluded from.Use these terms
Just for the sake of more easily describing and explaining the essence of the present invention；Be construed as any additional limitation be all with
What spirit of the present invention was disagreed.
Claims (5)
1. a kind of Sesquiterpene lactones compound, it is characterised in that shown in structure such as formula (I)：
2. a kind of preparation method of Sesquiterpene lactones compound as claimed in claim 1, it is characterised in that including following steps
Suddenly：
(A) extract：Medicinal material is extracted using alcoholic solvent, extract solution is obtained；
Described medicinal material is the aerial part and/or herb of eupatorium lindleynun var. trifoliolatum；
(B) extract：The extract solution that step (A) is obtained is concentrated, obtained concentrate adds water suspension, it is then molten using extracting
Agent carries out that extract is obtained by extraction, and extract obtains crude extract by concentration；
In step (B), extraction is carried out respectively using petroleum ether and ethyl acetate, and acetic acid ethyl acetate extract is carried out to be concentrated to give slightly
Extract；
(C) column chromatography：Normal phase column is eluted with eluant, eluent on the crude extract that step (B) is obtained, and is collected eluent, is recovered under reduced pressure
Solvent, obtains the extract rich in the sesquiterpene lactone；
In step (C), the filler used in described normal phase column is the column chromatography silica gel of 100~200 mesh；
In step (C), type of elution is gradient elution, and eluant, eluent is 20~0 by volume ratio:1 petroleum ether and ethyl acetate group
Into the eluent containing target product carries out inspection knowledge by TLC or LC-MS；
(D) medium pressure column chromatography：The extract that step (C) is obtained carries out medium pressure column chromatography separation, obtains the compound as shown in formula (1)
Crude product；
(E) preparative efficient liquid phase column chromatography：The crude product that step (D) is obtained, carries out preparative efficient liquid phase column chromatography, obtains
Compound as shown in formula (1)；
In step (D) and (E), filler used in medium pressure column chromatography and efficient liquid phase column chromatography is Octadecylsilane bonded phase silica gel；
The mobile phase of medium pressure column chromatography is 40~70% methanol aqueous solution, and gradient is carried out successively by polarity is descending during elution
Elution；
The mobile phase of preparative efficient liquid phase column chromatography is 20~40% acetonitrile solution.
3. the preparation method of Sesquiterpene lactones compound according to claim 2, it is characterised in that in step (A), institute
The medicinal material stated is extracted again after beating powder, crossing 40 mesh sieves.
4. the preparation method of Sesquiterpene lactones compound according to claim 2, it is characterised in that in step (A), institute
The extracting method stated is percolation, and Extraction solvent is 80~95% ethanol waters, and the amount ratio of Extraction solvent and medicinal material is 15
~20L ﹕ 1kg.
5. a kind of application of Sesquiterpene lactones compound as claimed in claim 1 in treatment anticancer medicine is prepared.
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| CN109265420B (en) * | 2018-11-30 | 2022-09-20 | 湖南中医药大学 | Process for the preparation of guaiane-type sesquiterpene compounds |
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