CN101492432A - Paclitaxel produced by separation purification of epiphyte with high-efficiency solid phase abstraction-HPLC method - Google Patents
Paclitaxel produced by separation purification of epiphyte with high-efficiency solid phase abstraction-HPLC method Download PDFInfo
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- CN101492432A CN101492432A CNA2008101536576A CN200810153657A CN101492432A CN 101492432 A CN101492432 A CN 101492432A CN A2008101536576 A CNA2008101536576 A CN A2008101536576A CN 200810153657 A CN200810153657 A CN 200810153657A CN 101492432 A CN101492432 A CN 101492432A
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Abstract
The invention relates to a highly efficient solid phase extraction-HPLC method for separating and purifying paclitaxel produced by fungi, comprising using a solid phase extraction principle to construct a new method of using the alkali alumina solid phase extraction combined with high performance liquid chromatography to separate and purify paclitaxel produced by fungi in a meliorative way. The recovery rate of 138 percent is obtained. The purity reaches 92 percent. RSD (relative standard deviation) is 1.4 percent. The method adopts a dry-column filling method for filling alkali alumina columella. Fillers and activated and eluted reagents are unnecessary to be further treated. Therefore, time and cost are saved. And the recovery rate of the paclitaxel is improved obviously.
Description
[technical field]:
The invention belongs to technical field of bioengineering, be specifically related to that a kind of Solid-Phase Extraction efficiently-HPLC method separation purification of epiphyte is paclitaxel produced.
[background technology]:
Taxol (paclitaxel, trade(brand)name Taxol) is a kind of natural cancer therapy drug of complexity, belongs to diterpene alkaloid.Its basic structure by Baccatine III (baccatin III) be connected its 13 carbon on a phenylalanine derivative constitute.
The early stage strategy of industrial production taxol, be to adopt the method for from the Chinese yew wood raw material, directly extracting, but because content of taxol is extremely low, water-soluble is poor, expansion along with this pharmaceutical applications scope, required Ramulus et folium taxi cuspidatae quantity will be very considerable, and this also brings serious threat to taxus resource, may cause the havoc of ecotope.
The complete synthesis taxol of chemical method has under lab been obtained success in 1994.But the taxol chiral radicals is more, complex synthetic route, and cost height, productive rate are low, can't be applied to industrial production so far.In recent years, Chinese scholars has been carried out a large amount of taxus callus cultivations and cell suspension culture research, and wherein major part has confirmed to produce taxol, but isolated culture production taxol realizes that the key issue of suitability for industrialized production is difficult to break through.
The current production technology that more generally adopts of international drugmaker then is biochemical semi-synthesis method.Though semi-synthesis method has improved taxol output, there is no difference in essence with the way of direct extraction taxol, still need to consume a large amount of Ramulus et folium taxi cuspidatae trees.
Because the market supply of taxol is limited by the restriction of raw material sources and technology of preparing two aspects, makes that the medicine high price is expensive.For this reason, new better raw material and method are being sought always by pharmaceutical industry and academia, replace existing production technology, with the output that improves taxol, satisfy clinical demand.The most significant progress that obtained in surplus past ten year is to have found to produce taxols with more symbiotic fungies of naked sub-xylophyta such as Ramulus et folium taxi cuspidatae, and its chemical structure and biological activity and Ramulus et folium taxi cuspidatae paclitaxel produced identical.According to statistics, the generation taxol fungal species of having reported surpasses 30 kinds both at home and abroad, and these fungi overwhelming majority are ascomycetes or imperfect fungi, and mostly are the endosymbiosis fungies.The same with the plant tissue extract, product is numerous in the fungal fermented filtrate, and the content of taxol is extremely low, has so just increased the cost of producing taxol to a great extent, and the viscosity of fermented liquid is very big simultaneously, as easy as rolling off a log obstruction chromatographic column.In order to address these problems, study efficient, economic extraction and separation method, with the purity and the rate of recovery of raising taxol, thus the industrial fermentation production of promotion taxol.
[summary of the invention]:
In order to overcome the deficiencies in the prior art and satisfy the demand in market, the object of the present invention is to provide that a kind of Solid-Phase Extraction efficiently-HPLC method separation purification of epiphyte is paclitaxel produced, to improve the yield and the purity of taxol in the tunning.
The loading method of a kind of solid phase extraction column provided by the invention, it comprises filler selection and loading method.
The loading method of described solid phase extraction column is dried column packing method, and filler is an alkali alumina.
The method of a kind of Solid-Phase Extraction provided by the invention, it comprises the activation of solid phase column, last sample and wash-out.
Described activation method in the solid phase pillar, add chloroform successively, methyl alcohol activates, last sample is that the fermented liquid of handling well is directly gone up sample, elutriant is an ethyl acetate.
The method of a kind of efficient liquid phase chromatographic analysis provided by the invention, it comprises the pre-treatment of sample, the selection of chromatographic column and the establishment of high-efficient liquid phase chromatogram condition.
The pre-treatment step of described sample is: the elutriant of collecting after the Solid-Phase Extraction, volatilize with nitrogen, and methyl alcohol redissolves, again through the membrane filtration of 0.45 μ m.
Described chromatographic column is C18 chromatographic column 5 μ m
Described high-efficient liquid phase chromatogram condition is that moving phase is methyl alcohol: water (v/v)=70: 30, and sample size 20 μ L, flow velocity 1.0mL/min, ultraviolet detection wavelength are 227nm, column temperature is 30 ℃.
Advantage of the present invention and positively effect:
The invention provides a kind of alkali alumina Solid-Phase Extraction of utilizing in conjunction with the paclitaxel produced method of high performance liquid chromatography separation purification of epiphyte, compare with the separation purification method of routine, the rate of recovery of taxol has obtained significant raising, reaches 138%, and taxol purity has reached 92%.This method adopts dried post to load method simultaneously, and filler and activation, elution reagent can not need further processing, have saved the time, have saved expense.
[description of drawings]:
Fig. 1 is the structure iron of solid phase extraction column.1, polypropylene column jecket 2, sieve plate 3, filler 4, joint
Fig. 2 is that the color atlas before and after the alkali alumina Solid-Phase Extraction compares.
Wherein the Taxol place is the taxol peak, and chromatogram elution curve 1 is the preceding collection of illustrative plates of extraction, and chromatogram elution curve 2 is the collection of illustrative plates after extracting.
The paclitaxel produced bacterial strain of energy provided by the invention is a strain saprophytic fungus plan dish stey, is preserved on October 8th, 2008 Chinese microorganism strain preservation administration committee common microorganism center, preservation address: Datun Road, Chaoyang District, Beijing City, the Chinese Academy of Sciences Institute of microbiology, culture presevation CGMCC No.2694, Classification And Nomenclature: Pestalotiopsis microspora.
[embodiment]:
Below will the invention will be further described by embodiment, these descriptions are not that content of the present invention is done further to limit.One skilled in the art will understand that invention technical characterictic being equal to of being done replaced or corresponding the improvement, still belong within protection scope of the present invention.
The filling of embodiment 1, solid phase extraction column
Adopt dried column packing method to carry out the filling of pillar.Pack into the earlier polypropylene screen (sieve plate) of lower end then adds load weighted filler 200mg alkali alumina in the pipe along funnel, the sieve plate of last covered layer, and the compacting of exerting oneself can be used.Solid phase extraction column (SPE post) structure iron is seen Fig. 1.
The Solid-Phase Extraction of embodiment 2, fermented liquid
Carry out the activation of solid phase extraction column earlier: in the solid phase pillar, add the 2mL chloroform successively and 2mL methyl alcohol activates, at last liquid level is maintained 1～2mm height on the sieve plate.Add the fermented liquid 0.1mL that handles well in the good post of activation, behind the upper prop, use the direct wash-out of 2mL ethyl acetate, flow rate control is 0.2mL/min, and wash-out is 5 times repeatedly, collects elutriant, volatilizes with nitrogen under the normal temperature and pressure, redissolves with 0.2mL methyl alcohol again.
The purifying of embodiment 3, high performance liquid chromatography
Sample redissolves through methyl alcohol, behind the membrane filtration of 0.45 μ m, carries out high performance liquid chromatography (HPLC) purifying.High-efficient liquid phase chromatogram condition: self-control C
18, 5 μ m,
Moving phase is methyl alcohol: water (v/v)=70: 30, sample size 20 μ L.
Flow velocity 1.0mL/min, ultraviolet detection wavelength are 227nm, and column temperature is 30 ℃.Sample is relatively seen Fig. 2 through the color atlas before and after the Solid-Phase Extraction.As seen from Figure 2, fermented liquid direct injection (chromatogram elution curve 1), unstability of base line, assorted peak is a lot, and a bit hangover of the peak of taxol correspondence; Through (chromatogram elution curve 2) after the Solid-Phase Extraction, not only baseline values reduces, and is more steady, and the ultraviolet absorption value in 5min obviously reduces, and other assorted peak everywhere also descends to some extent, and the peak shape of taxol correspondence improves.
The rate of recovery after embodiment 4, the Solid-Phase Extraction and purity testing
The mensuration of the rate of recovery after the Solid-Phase Extraction: to concentration known C
0Sample in add the taxol standard substance of isopyknic 0.025mg/mL, get 6 parts and carry out SPE-HPLC (Solid-Phase Extraction-HPLC), calculate ultimate density C according to typical curve
1Then the rate of recovery is: (C
1-C
0)/0.025 * 100%
Through determination of recovery rates, the result is as shown in table 1:
The rate of recovery after table 1 Solid-Phase Extraction
The detection of taxol purity is calculated purity according to the method for area percentage, and (Solid-Phase Extraction-HPLC), the taxol purity behind the purifying is 92% through SPE-HPLC.
Experimental result shows: utilize technological method of the present invention-alkali alumina Solid-Phase Extraction paclitaxel produced in conjunction with the high performance liquid chromatography separation purification of epiphyte, obtained the higher rate of recovery, the taxol purity of extraction has reached 92%.
Claims (10)
1, a kind of Solid-Phase Extraction-high performance liquid chromatography efficiently (HPLC) method separation purification of epiphyte is paclitaxel produced, it is characterized in that solid phase extraction techniques and HPLC combination, separating and purifying taxol from the fermented liquid of paclitaxel produced fungi, the yield and the purity of taxol are greatly improved.
2, method according to claim 1 is characterized in that solid phase extraction techniques comprises the filling of solid phase extraction column, the activation of solid phase column, last sample and wash-out
3, method according to claim 2, the loading method that it is characterized in that solid phase extraction column is dried column packing method, filler is an alkali alumina.
4, method according to claim 2, the activation method that it is characterized in that solid phase column is for adding chloroform successively and methyl alcohol activates in the solid phase pillar.
5, method according to claim 2, the last sample that it is characterized in that solid phase column are that the fermented liquid of handling well is directly gone up sample.
6, method according to claim 2, the elutriant that it is characterized in that solid phase column is an ethyl acetate.
7, method according to claim 1, the method that it is characterized in that high-efficient liquid phase chromatogram purification comprises pre-treatment, the selection of chromatographic column and the establishment of high-efficient liquid phase chromatogram condition of sample.
8, method according to claim 7 is characterized in that the pre-treatment step of sample is: collect elutriant after the Solid-Phase Extraction, volatilize with nitrogen, methyl alcohol redissolves, again through the membrane filtration of 0.45 μ m.
9, method according to claim 7 is characterized in that performance liquid chromatographic column is C18 chromatographic column 5 μ m
10, method according to claim 7 is characterized in that high-efficient liquid phase chromatogram condition is that moving phase is methyl alcohol: water (v/v)=70: 30, and sample size 20 μ L, flow velocity 1.0mL/min, ultraviolet detection wavelength are 227nm, column temperature is 30 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048400A (en) * | 2012-12-10 | 2013-04-17 | 吉林出入境检验检疫局检验检疫技术中心 | Solid-phase extraction column and preparation method thereof |
| CN111562322A (en) * | 2020-05-05 | 2020-08-21 | 大连润生康泰医学检验实验室有限公司 | Enrichment detection method and application of five anti-tumor drugs in blood sample |
| CN113917060A (en) * | 2021-10-08 | 2022-01-11 | 苏州圣苏新药开发有限公司 | Quantitative analysis method for paclitaxel in human plasma |
-
2008
- 2008-12-01 CN CNA2008101536576A patent/CN101492432A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048400A (en) * | 2012-12-10 | 2013-04-17 | 吉林出入境检验检疫局检验检疫技术中心 | Solid-phase extraction column and preparation method thereof |
| CN111562322A (en) * | 2020-05-05 | 2020-08-21 | 大连润生康泰医学检验实验室有限公司 | Enrichment detection method and application of five anti-tumor drugs in blood sample |
| CN111562322B (en) * | 2020-05-05 | 2021-03-26 | 大连润生康泰医学检验实验室有限公司 | Enrichment detection method and application of five anti-tumor drugs in blood sample |
| CN113917060A (en) * | 2021-10-08 | 2022-01-11 | 苏州圣苏新药开发有限公司 | Quantitative analysis method for paclitaxel in human plasma |
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