CN103193735A - Extraction method for taxus chinensis taxol activity extract - Google Patents

Abstract

The invention relates to an extraction method for a taxus chinensis taxol activity extract. The extraction method comprises the steps of: firstly, crushing taxus chinensis branches and leaves into 20-50 meshes, and adding 3-5 times of a petroleum ether and normal hexane mixture, wherein the ratio of the petroleum ether to normal hexane is (2-3):1; standing at 0-5 DEG C for 10-12 hours, and filtering to obtain filter residue; and then digesting and finally performing silica-gel separation. According to the technique, by adopting low-temperature digestion, as well as digestion with different concentration gradients of methanol, the taxol can be well protected, so as not to be transformed in the separation process, and as a biological macromolecule substance, the taxol is easily degraded or isomerized to produce other taxanes substances under the influences of conditions such as temperature, organic solvent, acid, and alkaline. For example, the taxol can be degraded into bacctain III or have epitope isomerization to produce 7-epi-taxol under the strong acid or weak alkaline environment condition; and the taxol can also have degradation reaction at a higher temperature to produce corresponding micromolecular substances. With the technique, the yield of the taxol can be greatly improved, and the purity of the product is high.

Description

The extracting method of taxus brevifolia alcohol activity extract

Technical field

The present invention relates to a kind of method of from taxaceae (Taxaceae) Taxus (Tasus) plant, extracting refining taxol.

Background technology

Ramulus et folium taxi cuspidatae is a kind of medicinal plant of preciousness, is world's rare and endangered tree species, and the first-grade state protection plant is described as " plant gold ".In the secondary metabolism meta-bolites of southerm yew branches and leaves, be widely used in the various malignant tumours for the treatment of clinically except the taxol composition that contains minute quantity in its pharmaceutical use, be considered to one of best anticarcinogen.Taxol is as a kind of anticancer drugs, and the requirement of its purity of clinical application is very high, generally is greater than 99.0%, and this has just proposed strict requirement to the purification refine technology of taxol.

Summary of the invention

The extracting method that the purpose of this invention is to provide a kind of taxus brevifolia alcohol activity extract, the extraction conditions gentleness, the yield of paclitaxel extract reaches 0.2-0.3%, and product purity reaches 99.3%.

Technical scheme of the present invention is as follows:

A kind of extracting method of taxus brevifolia alcohol activity extract is characterized in that, comprises following processing step:

(1) Ramulus et folium taxi cuspidatae is crushed to the 20-50 order, the sherwood oil that adding 3-5 doubly measures and the mixture of normal hexane (2-3:1) were placed 10-12 hour down in 0-5 ℃, filtered and obtained filter residue;

(2) filter residue places and extracts still, and the methyl alcohol that adds the 85-95% of 6-10 times of Ramulus et folium taxi cuspidatae volume extracts, and the extracting solution temperature be-10--5 ℃, extraction time is 4-5 hour, and filtration obtains filtrate, and filtrate PH is adjusted to neutrality; The methyl alcohol that filter residue adds the 55-65% that 3-5 doubly measures extracts, and the extracting solution temperature be-10--5 ℃, extraction time is 4-5 hour, and filtration obtains filtrate, and filtrate PH is adjusted to neutrality; The methyl alcohol that filter residue adds the 40-45% that 3-5 doubly measures again extracts, and the extracting solution temperature is-10--5 ℃, extraction time is 10-15 hour, filters to obtain filtrate;

(3) merge each filtrate of filtering and obtain crude extract;

(4) crude extract is carried out 0-5 ℃ of following concentrate under reduced pressure at low temperature and obtain crude extract; Crude extract is dissolved in 35-50 ℃ the hot water, the volume ratio of crude extract and hot water is 1: 2-3;

(5) add ethyl acetate in the hot water and extract, the volume ratio of medicinal extract and ethyl acetate is 1: 2-3 extracts repeatedly the combined ethyl acetate extraction liquid; The acetic acid ethyl acetate extract concentrating under reduced pressure is obtained containing the ethyl acetate medicinal extract of taxol;

(6) the ethyl acetate medicinal extract that will contain taxol is mixed with dry sample with silica gel, fill dry sample to silica gel 200-300 purpose chromatographic column upper end, with the drip washing of ethyl acetate-methyl alcohol (2:1) mixed solution, after leacheate flows out chromatographic column, the Fractional Collections cut, be that leacheate cut between the 10-30% puts together by section with content of taxol, through concentrate, dry, obtain intermediates;

(7) content of taxol is lower than 10% leacheate cut, puts together repeating step (6);

(8) intermediates that step (6) is obtained dissolve with acetonitrile, separate with high pressure liquid chromatography, and chromatographic column is the C18 silica filler, and moving phase is acetonitrile and water, and the moving phase that will contain taxol after then will separating is evaporated, and drying obtains high-purity taxol.

Adopting process of the present invention; adopt leach at low temperature; the lixiviate of different concns gradient methyl alcohol; greatly protected taxol; it is not transformed in sepn process; taxol is subjected to the influence of envrionment conditionss such as temperature, organic solvent, acid, alkali as a kind of biomacromolecule material, and easily degraded or isomery generate other taxanes material.Under strongly-acid or weakly alkaline environment condition, can be degraded to baccatin III or epi-position isomery generation 7-Epitaxol takes place as taxol; Also DeR can take place when temperature is higher, generate corresponding small-molecule substance.Technology of the present invention has improved the yield of taxol greatly.The product purity height.

Embodiment

A kind of extracting method of taxus brevifolia alcohol activity extract comprises following processing step:

(1) Ramulus et folium taxi cuspidatae (cured leaf 3000g) is crushed to the 20-50 order, adds the sherwood oil of 5 times of amounts and the mixture of normal hexane (2:1 volume ratio), placed 10-12 hour down in 0-5 ℃, degreasing is filtered and is obtained filter residue;

(2) filter residue places and extracts still, and 90% the methyl alcohol that adds 6-7 times of Ramulus et folium taxi cuspidatae volume extracts, and the extracting solution temperature be-7--5 ℃, extraction time is 4-5 hour, and filtration obtains filtrate, and filtrate PH is adjusted to neutrality; 55% the methyl alcohol that filter residue adds 5 times of amounts extracts, and the extracting solution temperature be-10--5 ℃, extraction time is 4-5 hour, and filtration obtains filtrate, and filtrate PH is adjusted to neutrality; 45% the methyl alcohol that filter residue adds 5 times of amounts again extracts, and the extracting solution temperature is-10--5 ℃, extraction time is 15 hours, filters to obtain filtrate;

(3) merge each filtrate of filtering and obtain crude extract;

(4) crude extract is carried out 0-5 ℃ of following concentrate under reduced pressure at low temperature and obtain crude extract; Crude extract is dissolved in 45-50 ℃ the hot water, the volume ratio of crude extract and hot water is 1: 3;

(5) add ethyl acetate in the hot water and extract, the volume ratio of medicinal extract and ethyl acetate is 1: 3, extracts repeatedly the combined ethyl acetate extraction liquid; The acetic acid ethyl acetate extract concentrating under reduced pressure is obtained containing the ethyl acetate medicinal extract of taxol;

(6) the ethyl acetate medicinal extract that will contain taxol is mixed with dry sample with silica gel, fill dry sample to silica gel 200-300 purpose chromatographic column upper end, with the drip washing of ethyl acetate-methyl alcohol (2:1) mixed solution, after leacheate flows out chromatographic column, the Fractional Collections cut, be that leacheate cut between the 10-30% puts together by section with content of taxol, through concentrate, dry, obtain intermediates;

(7) content of taxol is lower than 10% leacheate cut, puts together repeating step (6);

(8) intermediates that step (6) is obtained dissolve with acetonitrile, isolate taxol with high pressure liquid chromatography, chromatographic column is the C18 silica filler, moving phase is acetonitrile and water, the moving phase that will contain taxol after then will separating is evaporated, drying, the taxol 7.57 that obtains white high purity 99.4% restrains.The checking of 400HZ nuclear magnetic resonance map is consistent with standard model, and liquid-matter coupling stratographic analysis M/Z+ is 853.6.

Claims (1)

1. the extracting method of a taxus brevifolia alcohol activity extract is characterized in that, comprises following processing step:

(1) Ramulus et folium taxi cuspidatae is crushed to the 20-50 order, the sherwood oil that adding 3-5 doubly measures and the mixture of normal hexane (2-3:1) were placed 10-12 hour down in 0-5 ℃, filtered and obtained filter residue;

(2) filter residue places and extracts still, and the methyl alcohol that adds the 85-95% of 6-10 times of Ramulus et folium taxi cuspidatae volume extracts, and the extracting solution temperature be-10--5 ℃, extraction time is 4-5 hour, and filtration obtains filtrate, and filtrate PH is adjusted to neutrality; The methyl alcohol that filter residue adds the 55-65% that 3-5 doubly measures extracts, and the extracting solution temperature be-10--5 ℃, extraction time is 4-5 hour, and filtration obtains filtrate, and filtrate PH is adjusted to neutrality; The methyl alcohol that filter residue adds the 40-45% that 3-5 doubly measures again extracts, and the extracting solution temperature is-10--5 ℃, extraction time is 10-15 hour, filters to obtain filtrate;

(3) merge each filtrate of filtering and obtain crude extract;

(4) crude extract is carried out 0-5 ℃ of following concentrate under reduced pressure at low temperature and obtain crude extract; Crude extract is dissolved in 35-50 ℃ the hot water, the volume ratio of crude extract and hot water is 1: 2-3;

(5) add ethyl acetate in the hot water and extract, the volume ratio of medicinal extract and ethyl acetate is 1: 2-3 extracts repeatedly the combined ethyl acetate extraction liquid; The acetic acid ethyl acetate extract concentrating under reduced pressure is obtained containing the ethyl acetate medicinal extract of taxol;

(6) the ethyl acetate medicinal extract that will contain taxol is mixed with dry sample with silica gel, fill dry sample to silica gel 200-300 purpose chromatographic column upper end, with the drip washing of ethyl acetate-methyl alcohol (2:1) mixed solution, after leacheate flows out chromatographic column, the Fractional Collections cut, be that leacheate cut between the 10-30% puts together by section with content of taxol, through concentrate, dry, obtain intermediates;

(7) content of taxol is lower than 10% leacheate cut, puts together repeating step (6);

(8) intermediates that step (6) is obtained dissolve with acetonitrile, separate with high pressure liquid chromatography, and chromatographic column is the C18 silica filler, and moving phase is acetonitrile and water, and the moving phase that will contain taxol after then will separating is evaporated, and drying obtains high-purity taxol.