CN103193735B

CN103193735B - Extraction method for taxus chinensis taxol activity extract

Info

Publication number: CN103193735B
Application number: CN201310099148.0A
Authority: CN
Inventors: 姜艳; 姜雪琪
Original assignee: ANHUI HEHUA BIOLOGICAL MEDICAL TECHNOLOGY Co Ltd
Current assignee: ANHUI HEHUA BIOLOGICAL MEDICAL TECHNOLOGY Co Ltd
Priority date: 2013-03-26
Filing date: 2013-03-26
Publication date: 2015-07-15
Anticipated expiration: 2033-03-26
Also published as: CN103193735A

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 Hydrolysis kinetics taxol from taxaceae (Taxaceae) Taxus (Tasus) plant.

Background technology

Ramulus et folium taxi cuspidatae is a kind of medicinal plant of preciousness, is world's rare and endangered tree species, and first-grade state protection plant, is described as " plant gold ".In the secondary metabolism meta-bolites of southerm yew branches and leaves, except being widely used in the various malignant tumour for the treatment of clinically containing the taxol composition of 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, is generally greater than 99.0%, and this just proposes strict requirement to the purification refine technique of taxol.

Summary of the invention

The object of this invention is to provide a kind of extracting method of taxus brevifolia alcohol activity extract, extraction conditions is gentle, and 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:

An extracting method for taxus brevifolia alcohol activity extract, is characterized in that, comprises following processing step:

(1) Ramulus et folium taxi cuspidatae is crushed to 20-50 order, adds the sherwood oil of 3-5 times amount and the mixture of normal hexane (2-3:1), at 0-5 DEG C, place 10-12 hour, filter and obtain filter residue;

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

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

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

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

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

(7) by content of taxol lower than the leacheate cut of 10%, put together repeating step (6);

(8) dissolved by the intermediates acetonitrile that step (6) obtains, be separated by high pressure liquid chromatography, chromatographic column is C18 silica filler, moving phase is acetonitrile and water, then evaporate containing the moving phase of taxol after being separated, dry, obtain high-purity taxol.

The present invention adopts technique; adopt leach at low temperature; the lixiviate of different concns gradient methanol; greatly protect taxol; it is made not transform in sepn process; taxol is as a kind of biomacromolecule material, and by the impact of the envrionment conditionss such as temperature, organic solvent, acid, alkali, easily degraded or isomery generate other taxane substances.Baccatin III can be degraded to as taxol or epi-position isomery occurs under strongly-acid or weakly alkaline environment condition and generate 7-Epitaxol; Also can there is DeR when temperature is higher, generate corresponding small-molecule substance.Present invention process substantially increases the yield of taxol.Product purity is high.

Embodiment

An extracting method for taxus brevifolia alcohol activity extract, comprises following processing step:

(1) Ramulus et folium taxi cuspidatae (cured leaf 3000g) is crushed to 20-50 order, adds the sherwood oil of 5 times amount and the mixture of normal hexane (2:1 volume ratio), at 0-5 DEG C, place 10-12 hour, degreasing, filter and obtain filter residue;

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

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

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

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

(8) the intermediates acetonitrile that step (6) obtains is dissolved, taxol is isolated by high pressure liquid chromatography, chromatographic column is C18 silica filler, moving phase is acetonitrile and water, then evaporate containing the moving phase of taxol after being separated, drying, obtains the taxol 7.57 grams of white high purity 99.4%.The checking of 400HZ nuclear magnetic resonance map is consistent with standard model, and liquid-mass chromatography stratographic analysis M/Z+ is 853.6.

Claims

1. an extracting method for taxus brevifolia alcohol activity extract, is characterized in that, comprises following processing step:

(1), by Ramulus et folium taxi cuspidatae be crushed to 20-50 order, add the sherwood oil of 3-5 times amount and the mixture of normal hexane, at 0-5 DEG C, place 10-12 hour, filter and obtain filter residue; Wherein, the volume ratio of sherwood oil and normal hexane is 2-3:1;

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

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

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

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

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

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

(8), by the intermediates acetonitrile that step (6) obtains dissolve, be separated by high pressure liquid chromatography, chromatographic column is C18 silica filler, and moving phase is acetonitrile and water, then evaporates containing the moving phase of taxol after being separated, dry, obtains high-purity taxol.