CN103193735B - Extraction method for taxus chinensis taxol activity extract - Google Patents

Abstract

A method for extracting the active extract of Taxus paclitaxel, first crushing the branches and leaves of Taxus chinensis to 20-50 mesh, adding 3-5 times the mixture of petroleum ether and n-hexane (2-3:1), at 0-5 Place it at ℃ for 10-12 hours, filter to obtain the filter residue; then carry out leaching, and finally carry out silica gel separation. The present invention adopts technology, adopts low-temperature leaching, methanol leaching with different concentration gradients, which greatly protects paclitaxel so that it does not undergo transformation during the separation process. As a biological macromolecular substance, paclitaxel is affected by temperature, organic solvents, acids, Under the influence of environmental conditions such as alkali, it is easy to degrade or isomerize to other taxanes. For example, paclitaxel will be degraded to baccatin III or undergo epitope isomerization to generate 7-epitaxel under strong acidic or weakly alkaline environmental conditions; degradation reactions will also occur at higher temperatures to generate corresponding small molecular substances. The process of the invention greatly improves the yield of paclitaxel. The product is of high purity.

Description

Extraction method of yew paclitaxel active extract

technical field

The invention relates to a method for extracting and refining paclitaxel from plants of the family Taxaceae (Taxaceae) of the genus Taxus (Tasus).

Background technique

Yew is a precious medicinal plant, a rare and endangered tree species in the world, and a national first-class protected plant, known as "plant gold". Among the secondary metabolites of the branches and leaves of Taxus chinensis, its medicinal value contains a very small amount of paclitaxel, which has been widely used clinically to treat various malignant tumors, and is considered to be one of the best anticancer drugs. Paclitaxel, as an anticancer drug, requires very high purity in clinical application, generally greater than 99.0%, which imposes strict requirements on the purification process of paclitaxel.

Contents of the invention

The object of the present invention is to provide a method for extracting the active extract of taxus paclitaxel, the extraction conditions are mild, the yield of paclitaxel extract reaches 0.2-0.3%, and the product purity reaches 99.3%.

Technical scheme of the present invention is as follows:

A method for extracting the active extract of Taxus paclitaxel is characterized in that it comprises the following processing steps:

(1) Crush the branches and leaves of yew to 20-50 mesh, add 3-5 times the mixture of petroleum ether and n-hexane (2-3:1), place it at 0-5°C for 10-12 hours, and filter to obtain filter residue;

(2) Put the filter residue in the extraction kettle, add 85-95% methanol of 6-10 times the volume of yew branches and leaves for extraction, the temperature of the extract is -10--5°C, the extraction time is 4-5 hours, and it is filtered to obtain The filtrate, the pH of the filtrate is adjusted to be neutral; the filter residue is extracted by adding 3-5 times the amount of 55-65% methanol, the temperature of the extract is -10-5°C, and the extraction time is 4-5 hours, and the filtrate is obtained by filtration. Adjust the pH to neutral; add 3-5 times the amount of 40-45% methanol to the filter residue for extraction, the temperature of the extract is -10-5°C, the extraction time is 10-15 hours, and the filtrate is obtained by filtration;

(3) Combine the filtrate of each filtration to obtain the crude extract;

(4) Concentrate the crude extract at low temperature and reduced pressure at 0-5°C to obtain a crude extract; dissolve the crude extract in hot water at 35-50°C, and the volume ratio of the crude extract to hot water is 1:2 -3;

(5) Add ethyl acetate to hot water for extraction, the volume ratio of the extract to ethyl acetate is 1:2-3, extract several times, combine the ethyl acetate extract; concentrate the ethyl acetate extract under reduced pressure Obtain the ethyl acetate extract containing paclitaxel;

(6) Mix the ethyl acetate extract containing paclitaxel with silica gel to prepare a dry sample, fill the dry sample onto the upper end of a silica gel 200-300 mesh column, rinse with ethyl acetate-methanol (2:1) mixture, rinse After the liquid flows out of the chromatographic column, the fractions are collected in sections, and the eluate fractions with a paclitaxel content between 10-30% are collected in sections, concentrated and dried to obtain an intermediate product;

(7) Collect the eluate fractions with paclitaxel content less than 10% and repeat step (6);

(8) Dissolve the intermediate product obtained in step (6) with acetonitrile and separate it with high-pressure liquid chromatography. The chromatographic column is filled with C18 silica gel, and the mobile phase is acetonitrile and water. After separation, the mobile phase containing paclitaxel is evaporated and dried. , to obtain high-purity paclitaxel.

The present invention adopts technology, adopts low-temperature leaching, methanol leaching with different concentration gradients, which greatly protects paclitaxel so that it does not undergo transformation during the separation process. As a biological macromolecular substance, paclitaxel is affected by temperature, organic solvents, acids, Under the influence of environmental conditions such as alkali, it is easy to degrade or isomerize to other taxanes. For example, paclitaxel will be degraded to baccatin III or undergo epitope isomerization to 7-epitaxel under strong acidic or weak alkaline environmental conditions; when the temperature is higher, the degradation reaction will also occur to generate corresponding small molecular substances. The process of the invention greatly improves the yield of paclitaxel. The product has high purity.

Detailed ways

A method for extracting the active extract of taxus paclitaxel, comprising the following process steps:

(1) Crush the branches and leaves of yew (3000g dry leaves) to 20-50 mesh, add 5 times the amount of petroleum ether and n-hexane (2:1 volume ratio) mixture, and place it at 0-5°C for 10-12 hours , defatted, and filtered to obtain the filter residue;

(2) The filter residue is placed in an extraction kettle, and 90% methanol of 6-7 times the volume of yew branches and leaves is added for extraction. The temperature of the extract is -7- -5°C, the extraction time is 4-5 hours, and the filtrate is obtained by filtration. Adjust the pH of the filtrate to be neutral; add 5 times the amount of 55% methanol to the filter residue for extraction, the temperature of the extract is -10-5°C, the extraction time is 4-5 hours, and the filtrate is obtained by filtration, and the pH of the filtrate is adjusted to neutral; Add 5 times the amount of 45% methanol to the filter residue for extraction, the temperature of the extract is -10-5°C, the extraction time is 15 hours, and the filtrate is obtained by filtration;

(3) Combine the filtrate of each filtration to obtain the crude extract;

(4) Concentrate the crude extract at low temperature and reduced pressure at 0-5°C to obtain a crude extract; dissolve the crude extract in hot water at 45-50°C, and the volume ratio of the crude extract to hot water is 1:3 ;

(5) Add ethyl acetate to hot water for extraction, the volume ratio of the extract and ethyl acetate is 1:3, extract several times, combine the ethyl acetate extract; concentrate the ethyl acetate extract under reduced pressure to obtain Ethyl acetate extract of paclitaxel;

(6) Mix the ethyl acetate extract containing paclitaxel with silica gel to prepare a dry sample, fill the dry sample onto the upper end of a silica gel 200-300 mesh column, rinse with ethyl acetate-methanol (2:1) mixture, rinse After the liquid flows out of the chromatographic column, the fractions are collected in sections, and the eluate fractions with a paclitaxel content between 10-30% are collected in sections, concentrated and dried to obtain an intermediate product;

(7) Collect the eluate fractions with paclitaxel content less than 10% and repeat step (6);

(8) Dissolve the intermediate product obtained in step (6) with acetonitrile, and separate paclitaxel by high-pressure liquid chromatography. The chromatographic column is filled with C18 silica gel, and the mobile phase is acetonitrile and water, and then the separated mobile phase containing paclitaxel is evaporated , dried to obtain 7.57 grams of white paclitaxel with a high purity of 99.4%. The 400HZ nuclear magnetic resonance spectrum verification is consistent with the standard sample, and the liquid-mass chromatography analysis M/Z+ is 853.6.

Claims (1)

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.