CN101759675B - Method for preparing active sesquiterpenes with functions of cancer resistance and bacterium inhibition from ligularia fischeri - Google Patents
Method for preparing active sesquiterpenes with functions of cancer resistance and bacterium inhibition from ligularia fischeri Download PDFInfo
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- CN101759675B CN101759675B CN2008101844806A CN200810184480A CN101759675B CN 101759675 B CN101759675 B CN 101759675B CN 2008101844806 A CN2008101844806 A CN 2008101844806A CN 200810184480 A CN200810184480 A CN 200810184480A CN 101759675 B CN101759675 B CN 101759675B
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Abstract
The invention relates to a method for preparing active sesquiterpenes with the functions of cancer resistance and bacterium inhibition from ligularia fischeri, which is used for preparing sesquiterpenoids with the structural formula as follows by using the feverfew ligularia fischeri as raw materials. The sesquiterpenes display inhibition activity to the growth of cancer cells of oral epitheliumsand obviously inhibit the growth of staphylococcus aureus and beta hemolytic streptococcus. The method can be used for quickly preparing a great amount of sesquiterpenes by adopting the combination of solvent extraction and the purification of a column chromatography through one-step chemical reaction. The preparation method has the advantages of high extracting transformation rate, low cost, convenient operation and high purity of products and is suitable for the development of standard substances and medicaments.
Description
Technical field
The present invention relates to a kind of is a kind of method with sesquiterpene of anticancer and antibiotic activity of feedstock production with composite family ligularia fischeri plant.
Background technology
Ligularia fischeri [Ligularia fischeri (Ledeb.) Turcz.] claims the renal lobe Farfugium kaemferi again, is that the composite family Farfugium kaemferi belongs to per nnial herb.Be distributed widely in Sichuan, Hubei, Guizhou, Hunan, Henan, Anhui, Zhejiang, Gansu, Shaanxi, North China and the Northeast.Its root and rhizome are used as medicine as RADIX ET RHIZOMA LIGULARIAE, have effects such as the warm lung therapeutic method to keep the adverse qi flowing downward, kobadrin, vital energy regualting and blood circulation-promoting, pain relieving, diuresis, hemostasis, have taken in the local drug standard in Jilin Province.
There are some researches show, sesquiterpenoids I, the chemical name 1 α-chloro-6 β-not fragrant sesquiterpene of isobutyl acyloxy-9-oxygen-10 beta-hydroxies-furans Airy, growth shows that inhibition is active to oral squamous carcinoma cell, can expect as the antitumor drug purposes.This sesquiterpene also obviously suppresses the growth of streptococcus aureus and beta hemolytic streptococcus, can expect and be used to prevent and treat the furuncle that this two infection causes, fester, pneumonia, osteomyelitis, acute myocarditis, endocarditis, meningitis, mazoitis, wing peptide inflammation, pelvic inflammatory disease, urinary tract inflammation, prostatitis, microbemia, toxic shock syndrome, poradenolymphitis, sacroiliitis, pharyngitis, trachelitis, and muscle, skin, urodeum, the abscess of central nervous system, prospect [Lee school Kun that medicinal exploitation is arranged, Deng. the chloride atom eremophilane in the black purple Farfugium kaemferi and antibiotic and cytotoxic activity (P) .2007 August 29 thereof, publication number CN101024636A].
Summary of the invention
The purpose of this invention is to provide the method that a kind of preparation has anticancer and antibiotic activity sesquiterpenoid I.
We are carrying out in the systematic study ligularia fischeri, find to be rich in this plant material a large amount of eremophilane type sesquiterpenoidss, and wherein the content of Compound I I can reach 2.94%.We study the preparation technology and the structure of modification of this compound, find the chemical combination II by the ligularia fischeri preparation, through obtaining the method for Compound I once the step simple chemical reaction.This method has solved the problem that Compound I content in plant is low, be difficult to obtain, realized quick, easy, prepare this compound in a large number, for further pharmaceutical research and drug development are laid a good foundation.
Because the content of this compound in plant is low, be difficult to obtain or the preparation trace, we have prepared its precursor compound II targetedly from feverfew ligularia fischeri raw material, and adopt simple one to go on foot chemical reaction, be translated into target compound I, and investigated processing condition, whole technology is suitable for a large amount of preparations.
A kind of is raw material with the ligularia fischeri plant, and preparation has the method for sesquiterpene of anticancer and antibiotic activity, it is characterized in that adopting that any one prepares in two kinds of methods of following A, B:
A, the ligularia fischeri plant is got total medicinal extract with solvent extraction, this medicinal extract (or with ethyl acetate extraction gained position medicinal extract) is through the method for a step silica gel column chromatography, mixed solvent gradient elution, separate purify Compound I I; With Compound I I and dilute hydrochloric acid reacting by heating, prepare Compound I again.
B, the total medicinal extract of ligularia fischeri plant (or ethyl acetate extraction position medicinal extract) directly and the dilute hydrochloric acid reacting by heating, reaction product is separated through silica gel column chromatography, the flow point of directly collecting Compound I is concentrated, promptly gets the Compound I crude product.
In above-mentioned each method, when total medicinal extract extracts the material ratio of solvent and raw material in 5: 1 to 10: 1 scopes, during extraction as lixiviate under the employing room temperature, then each 7 days, lixiviate 3～4 times; Also can adopt heating and refluxing extraction, each 2～3 hours, extract 3 times; Also or adopt excusing from death to extract and wait other prior art to extract; Extract solvent and can select 80-95% (V/V) ethanol or methyl alcohol, industrial ethyl acetate or acetone etc.
In above-mentioned each method, the solvent system of selecting for use during silica gel column chromatography is sherwood oil and ethyl acetate (20: 1 to 3: 1) or sherwood oil and acetone (25: 1 to 5: 1) gradient elution.Detect by thin-layer chromatography, collect under sherwood oil and ethyl acetate (5: 1) the expansion system, under the 254nm UV-light, have fluorescence, sulfuric acid-ethanol to show blue component, concentrating under reduced pressure under＜50 ℃ of conditions.
In above-mentioned each method, concrete operations step when reacting with dilute hydrochloric acid is: with reactants dissolved in small amount of ethanol or methyl alcohol, add 5%～10% aqueous hydrochloric acid (consumption of aqueous hydrochloric acid is about 80～100 times of Compound I I content in the reactant), reflux 1～2h, cold slightly putting a moment after reaction finishes, boil off a spot of ethanol or methyl alcohol in the solution, use ethyl acetate extraction, extraction liquid is concentrated into to do and gets final product.
The Compound I crude product for preparing through above-mentioned each side method can carry out purification refine by following steps: by a step purification by silica gel column chromatography, select sherwood oil and ethyl acetate (10: 1) or sherwood oil and acetone (15: 1) solvent elution for use, product purity can reach more than 90%; Also further recrystallization adopts sherwood oil and acetone or sherwood oil and ethyl acetate solvent system, the product crystal formation rule that obtains (no color lump crystalline substance), melting range narrow (84～85 ℃), and purity can reach more than 95%.
It is 72.5% that target compound of the present invention extracts transformation efficiency, has solved the problem that this compound is low at nature content, be difficult to obtain.And this method has advantages such as easy and simple to handle, that cost is low, and the product purity height of production is suitable to standard substance and pharmaceutical developments use.
Embodiment
Further specify the present invention by the following examples, but the present invention is not limited thereto embodiment.
Embodiment 1: the extraction purifying experiment of Compound I I
1. ligularia fischeri underground part dried plant 110g pulverizes, and extracts 2 hours with 1000mL 95% alcohol heating reflux, leaches extracting solution; Repeat altogether to extract three times, united extraction liquid, concentrating under reduced pressure get the about 13g of total medicinal extract.
2. take out 9g medicinal extract and add the 9.5g column chromatography silica gel and mix sample, volatilize solvent after, with 100g silica gel dry column-packing, last sample is with sherwood oil and ethyl acetate system (15: 1-5: 1) gradient elution.
3. detect by thin-layer chromatography, collect Rf value about 0.25 under petroleum ether-ethyl acetate (5: 1) the expansion system, and under the 254nm UV-light, having fluorescence, sulfuric acid-ethanol to show blue component, evaporated under reduced pressure solvent under＜50 ℃ of conditions promptly gets the crude product 0.23g of Compound I I;
Determine that it is through NMR (Nuclear Magnetic Resonance) spectrum and high resolution mass spectrum structure elucidation: 1 β, the 10 beta epoxides-6 β-isobutyl acyloxy-not fragrant sesquiterpene of 9-oxygen-furans Airy.
Embodiment 2: the amplification test of Compound I I extraction separation
1. the ligularia fischeri plant sample picks up from the Nanchuan, Chongqing, pulverizes behind natural air drying, and the ligularia fischeri rhizome 3.0Kg of pulverizing extracts with 95% ethanol 30L cold soaking, and the vat liquor decompression recycling ethanol extracts 4 times altogether, each 7 days, the back altogether total about 200g of medicinal extract;
2. total medicinal extract 1000mL water-dispersion, use ethyl acetate extraction again, the concentrating under reduced pressure acetic acid ethyl acetate extract obtains 125g medicinal extract, this medicinal extract is mixed sample with the 140g column chromatography silica gel, with 1000g silica gel wet method dress post, (20: 1-5: 1) gradient elution, thin-layer chromatography detect and merge same stream part for sherwood oil and ethyl acetate;
3. collect thin-layer chromatography and detect R under sherwood oil and ethyl acetate (5: 1) the expansion system
fBe worth approximately 0.25, and have fluorescence, sulfuric acid-ethanol to show blue component under the 254nm UV-light, evaporated under reduced pressure solvent under＜50 ℃ of conditions promptly gets the about 4g of crude product of Compound I I.
Embodiment 3: the assay experiment of Compound I I in the total medicinal extract of plant
1. laboratory apparatus and reagent:
Waters high performance liquid chromatograph: comprise Waters Delta 600 quaternary gradient pumps, 2996 diode-array detectors, manual injector, Millennium
32The data processing software system.
Acetonitrile (analytical pure, Tianjin chemical reagent company limited), water is the secondary redistilled water.Before all reagent use all through 0.45 μ m membrane filtration.
2. chromatographic condition
Chromatographic column: Kromasil C18 post (5 μ m, 4.6mm * 250mm, Dalian Inst of Chemicophysics, Chinese Academy of Sciences); Moving phase: acetonitrile and 0.1% phosphoric acid (50: 50, V/V); Flow velocity: 0.8mL/min; Detect wavelength: 280nm; Column temperature: room temperature; Sample size: 10 μ L.The online degassing of helium.
3. the configuration of solution
Accurately dispose the methanol solution of 0.1mg/mL Compound I I; Other gets total medicinal extract 30mg and is dissolved in methanol constant volume to the 10mL volumetric flask, gets 3.0mg/mL solution.All need filter before the sample solution test.
4. experimental result
Adopt external standard method, qualitative according to retention time, utilize peak area quantification, recording the content that Compound I I always extracts in the medicinal extract plant is 2.94%, illustrates that Compound I I has very high content in ligularia fischeri, truly have the prospect of development and use.1 result extracts the method extraction efficiency for preparing Compound I I and can reach 87.1% in conjunction with the embodiments, illustrates that Compound I I has realized the extraction of efficient greatly in the extraction separation step.
Embodiment 4: the preparation of target compound I
1. the pure product 50mg of Compound I is dissolved in the small amount of ethanol, adds the aqueous hydrochloric acid of 10mL 5%, reflux 1h; Cold slightly putting a moment boils off in the solution after the small amount of ethanol, uses ethyl acetate extraction, and extraction liquid is concentrated into dried, promptly gets the crude product of Compound I.
2. adopt purification by silica gel column chromatography, sherwood oil and acetone (20: 1) wash-out.Obtain the pure product 46.2mg of Compound I at last, purity is more than 95%.Theoretical yield is 55.5mg, and transformation efficiency can reach 83.2%.
3. select sherwood oil and acetone solvent system recrystallization for use, can obtain colourless bulk crystals, measuring its fusing point with X-4 digital micro-analysis melting point detector is 84～85 ℃.
Determine that it is through NMR (Nuclear Magnetic Resonance) spectrum and high resolution mass spectrum structure elucidation: 1 α-chloro-6 β-isobutyl acyloxy-9-oxygen-10 beta-hydroxies-furans eremophilane type sesquiterpene.
Embodiment 5: prepare Compound I by the total medicinal extract direct reaction of plant
1. the about 10g of the total medicinal extract of ligularia fischeri extraction using alcohol is dissolved in the small amount of ethanol, adds the aqueous hydrochloric acid of 40mL 10%, reflux 2h; Cold slightly putting a moment boils off small amount of ethanol in the solution, uses ethyl acetate extraction, admixes column chromatography silica gel behind the extraction liquid concentrating under reduced pressure, volatilizes solvent.
2. the sample that will mix silica gel is by column chromatography for separation, sherwood oil-acetone (20: 1-10: 1) gradient elution.The collection thin-layer chromatography detects, the Rf value is about 0.5 under sherwood oil and ethyl acetate (5: 1) the expansion system, and has fluorescence, sulfuric acid-ethanol to show blue component, evaporated under reduced pressure solvent under＜50 ℃ of conditions under the 254nm UV-light, get Compound I I 0.264g, productive rate is 81%.
Claims (5)
1. one kind is raw material with the feverfew ligularia fischeri, and preparation has the method for sesquiterpenoid I of anticancer and antibiotic activity, it is characterized in that adopting that any one prepares in two kinds of methods of following A, B:
A, the ligularia fischeri plant material is got total medicinal extract with solvent extraction, this medicinal extract or with the method for ethyl acetate extraction gained position medicinal extract through a step silica gel column chromatography, mixed solvent gradient elution, separation and purification obtains sesquiterpenoids II; With Compound I I and dilute hydrochloric acid reacting by heating, prepare sesquiterpenoids I again;
B, total medicinal extract that the ligularia fischeri plant is got with solvent extraction directly and the dilute hydrochloric acid reacting by heating, reaction product can directly be collected the flow point of Compound I through silica gel column chromatography separating purification, concentrates promptly to obtain the Compound I crude product.
2. the method for claim 1 is characterized in that: the extraction of total medicinal extract can be selected the method for solvent compartment's temperature cold soaking, thermal backflow or supersound extraction for use, and the extraction solvent can be selected ethanol or methyl alcohol, industrial ethyl acetate or the acetone of 80-95% (V/V).
3. the method for claim 1 is characterized in that: the solvent system of selecting for use during silica gel column chromatography is a sherwood oil than ethyl acetate is that 20: 1 to 3: 1 or sherwood oil are 25: 1 to 5: 1 gradient elutions than acetone.
4. the method for claim 1, it is characterized in that: the concrete operations step when reacting with dilute hydrochloric acid is: with reactants dissolved in small amount of ethanol or methyl alcohol, add 5%～10% aqueous hydrochloric acid, the consumption of aqueous hydrochloric acid is 80～100 times of Compound I I content in the reactant, reflux 1～2h, cold slightly putting a moment boiled off a spot of ethanol or methyl alcohol in the solution after reaction finished, use ethyl acetate extraction, extraction liquid is concentrated into to do and gets final product.
5. the method for claim 1, it is characterized in that: make the Compound I crude product, can carry out purification refine by following steps: by a step purification by silica gel column chromatography, is that 10: 1 or sherwood oil are 15: 1 solvent elution than acetone with sherwood oil than ethyl acetate, again through recrystallization, with sherwood oil and acetone or sherwood oil and ethyl acetate mixed solvent system, Compound I purity can reach more than 90%.
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| CN105695335A (en) * | 2014-11-25 | 2016-06-22 | 甘孜藏族自治州畜牧业科学研究所 | New aspergillus flavus and use thereof |
| CN109777622A (en) * | 2019-03-14 | 2019-05-21 | 武汉理工大学 | A kind of technique of high efficiency extraction net vein Farfugium kaemferi volatile oil |
| CN110194754B (en) * | 2019-04-29 | 2020-11-03 | 首都医科大学 | Ligularia fischeri fat-soluble extract and preparation method and application thereof |
| CN112876439B (en) * | 2021-01-15 | 2022-06-21 | 宁波大学 | Nano-docetaxel type sesquiterpenoids, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024636A (en) * | 2007-03-13 | 2007-08-29 | 温州医学院 | Eremophilane containing chlorine atom in Heizituowu and its anti-biotic and cell-toxin activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101024636A (en) * | 2007-03-13 | 2007-08-29 | 温州医学院 | Eremophilane containing chlorine atom in Heizituowu and its anti-biotic and cell-toxin activity |
Non-Patent Citations (3)
| Title |
|---|
| Jun Zhao et al..One chloro-furoeremophilanoid and two new natural dimers from Ligularia atroviolacea.《Chinese Chemical Letters》.2008,第19卷(第11期),1319-1322. * |
| Shu-Yun Shi et al..Furanoeremophilanes from Ligularia atroviolacea.《Fitoterapia》.2008,第79卷(第6期),476-478. * |
| 施树云 等.黑紫橐吾化学成分的分离与鉴定.《高等学校化学学报》.2008,第29卷(第5期),941-943. * |
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