CN105418543A - Method for extracting baccatin III reference substance from taxus chinensis plants - Google Patents

Abstract

The invention discloses a method for extracting a baccatin III reference substance from taxus chinensis plants. The method comprises the following steps: preparing an extract from branches and leaves of taxus chinensis; then extracting the extract repeatedly by using chloroform/water, performing extraction for liquid separation to obtain an extracted substance of chloroform, and performing crude separation on the extracted substance by using a normal phase silica gel column to obtain a crude separation substance; performing fine separation on the crude separation substance by using the normal phase silica gel column to obtain a fine separation substance; and separating the fine separation substance by adopting high-efficiency liquid chromatography to prepare the baccatin III reference substance of which the purity is 99.1% and meets requirements on a traditional Chinese medicine chemical reference substance for content determination.

Description

The method of baccatin III reference substance is extracted from southerm yew plant

Technical field

The present invention relates to a kind of separating and purifying technology, especially a kind of method extracting baccatin III reference substance from southerm yew plant.

Background technology

Taxol is since U.S. FDA approval clinical application in 1992, just with the mechanism of action of its uniqueness, definite curative effect and the less feature of side effect gain a widespread reputation in international antitumor drug research field, become the No.1 medicine of the malignant tumours such as treatment advanced ovarian cancer, breast cancer, nonsmall-cell lung cancer and Kaposi ' S sarcoma.

But the content of natural Japanese yew alcohol in Ramulus et folium taxi cuspidatae is extremely low, is only 0.001-0.002%, the natural Japanese yew alcohol extracting 1 kilogram will consume bark of Ramulus et folium taxi cuspidatae 30 tons.And the Ba Kating-III very similar to taxol mother nucleus structure and 10-deacetylate baccatin III (10-DABIII) rich content in Ramulus et folium taxi cuspidatae; in Japanese yew class other plant, also find that it exists in a large number simultaneously; so just give the semi-synthetic condition of providing convenience of taxol; its method is the phenylisoserine side chain of synthetic taxol, itself and plant milk extract is clung to Ka Ting-III or 10-deacetylate baccatin III (10-DABIII) and carries out docking and obtain paclitaxel analog compound.

As can be seen from the distribution area of Afforestation of Taxus Plants in Guizhou Province, southerm yew having a very wide distribution compared with Ramulus et folium taxi cuspidatae.But at present bibliographical information is had no to the chemical constitution study of Guizhou product southerm yew.How from the product southerm yew plant of Guizhou, to obtain baccatin, be need to solve.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of method extracting baccatin III reference substance from southerm yew plant, and it can produce the baccatin III being separated southerm yew plant and obtaining very high purity from Guizhou.

The present invention is achieved in that the method extracting baccatin III reference substance from southerm yew plant, comprises the steps:

1) get southerm yew branches and leaves, after branches and leaves are dried, then pulverized, obtain dry particle; The particle of drying is adopted after sherwood oil carries out pre-treatment, then floods with methyl alcohol, carry out stirring while dipping, ultrasonic, steeping fluid is filtered, then by filtrate reduced in volume, obtains concentrated solution, after repeatedly being extracted by concentrated solution normal hexane, then evaporate to dryness makes medicinal extract;

2) by the extracting 6 times repeatedly of the medicinal extract chloroform/water that obtains in step 1), extraction separatory, obtain the extract of chloroform, extract and silica gel are pressed the mass ratio of 1:10, be that the sherwood oil-chloroform of 15:1 carries out gradient elution, pressurized column chromatography for eluent with volume ratio, collect containing the close mixture of baccatin III polarity, the scope of the Rf value of collecting is the scope in baccatin III standard substance Rf value ± 0.2, is separated and obtains roughing out thing;

3) roughing out thing is added silica gel, the raw material added first and segmentation from the mass ratio of silica gel be 1:10, be that the sherwood oil-chloroform of 12:1 carries out gradient elution for eluent with volume ratio, pressurized column chromatography, collect containing baccatin III polarity mixture closely, the scope of the Rf value of collecting is the scope in baccatin III standard substance Rf value ± 0.1, is separated and obtains thin isolate;

4) acetone is added by thin isolate, the volume ratio of thin isolate and acetone is 1:10, with first alcohol and water for moving phase wash-out, the volume of methyl alcohol and water is 80:20, and flow velocity is 8mL/min, simultaneously with UV spectrophotometer measuring, wash-out section effluent liquid goes out peak at wavelength 237nm, contrast baccatin III standard substance collection of illustrative plates, determines the absorption peak of baccatin III, collects and obtain the baccatin III reference substance that purity is 99.1%.

Baccatin III is white powder, fusing point 191.5-191.9 ^oc, ultraviolet absorption peak is 237nm, dissolves in methyl alcohol, ethanol, acetone, water insoluble, and its molecular formula is, molecular weight is, structural formula is:

Owing to have employed technique scheme, compared with prior art, the present invention makes medicinal extract with southern Ramulus et folium taxi cuspidatae; Then with chloroform/water extracting medicinal extract repeatedly, extraction separatory, obtain the extract of chloroform, the roughing out of extract normal phase silicagel column obtains roughing out thing; Roughing out thing segments from obtaining thin isolate with normal phase silicagel column again; Thin isolate adopts high performance liquid chromatography to be separated and prepares baccatin III reference substance, and purity is 99.1% requirement meeting assay traditional Chinese chemical contrast.

Accompanying drawing explanation

Accompanying drawing 1 and accompanying drawing 2 are the baccatin III nuclear magnetic spectrogram of embodiments of the invention 1;

Accompanying drawing 3 is the baccatin III high-efficient liquid phasor of embodiments of the invention 1.

Embodiment

Embodiments of the invention: the method extracting baccatin III reference substance from southerm yew plant, comprises the steps:

1) get 100g southerm yew branches and leaves, the branches and leaves of dry (drying) are pulverized, obtain particle; Adopted by the particle of drying 1L sherwood oil to carry out soak degreasing (to remove a large amount of this type of non polar impurities existed), then carry out cold soaking with 4L methyl alcohol, carry out stirring while flooding, ultrasonic 1 hour, naturally cooling half an hour; 3 times repeatedly (object of naturally cooling avoids because stirring and ultrasonicly making solution temperature too high and produce high-temperature extraction), steeping fluid is filtered, concentrate filtrate to 150mL, obtain concentrated solution, concentrated solution 100mL normal hexane is extracted after 3 times repeatedly, discard normal hexane layer, extraction liquid is spin-dried for solvent, makes medicinal extract;

2) by the extracting 6 times repeatedly of the medicinal extract chloroform/water that obtains in step 1), extraction separatory, obtain the extract of chloroform, 10g extract is mixed with 300-400 object silica gel 100g, be that the sherwood oil-chloroform of 15:1 carries out gradient elution, pressurized column chromatography for eluent with volume ratio, collect containing the close mixture of baccatin III polarity, the scope of the Rf value of collecting is the scope in baccatin III standard substance Rf value ± 0.2, and separation obtains roughing out thing and is about 1g;

3) 1g roughing out thing is mixed with 300-400 object silica gel 10g, be that the sherwood oil-chloroform of 12:1 carries out gradient elution for eluent with volume ratio, pressurized column chromatography, collect containing baccatin III polarity mixture closely, the scope of the Rf value of collecting is the scope in baccatin III standard substance Rf value ± 0.1, is separated and obtains thin isolate 0.2g;

4) add acetone by 0.2 thin isolate, the volume ratio of thin isolate and acetone is 1:10, and with first alcohol and water for moving phase wash-out, the volume of methyl alcohol and water is 80:20, and flow velocity is 8mL/min, and simultaneously with UV spectrophotometer measuring, pillar is HederacC ₁₈200mm, wash-out section effluent liquid shows the very high absorption peak of 1 intensity at wavelength 237nm, and contrast baccatin III standard substance collection of illustrative plates, determines the absorption peak clinging to card booth IV, collect and obtain the baccatin III reference substance 25mg that purity is 99.1%.

The results such as nucleus magnetic resonance and high resolution mass spectrum test are as follows: ¹hNMR (CDCl ₃, 400MHz) δ: 1.10 (s, 6H), 1.66 (s, 3H), 1.82-1.89 (m, 1H), 2.04 (s, 3H), 2.17 (s, 1H), 2.24 (s, 3H), 2.28 (s, 6H), 2.51-2.59 (m, 1H), 3.87 (d j=8.0Hz, 1H), 4.15 (d, j=8.0Hz, 1H), 4.30 (d, j=8.0Hz, 1H), 4.44-4.48 (m, 1H), 4.85-4.89 (m, 1H), 4.98 (d, j=8.0Hz, 1H), 5.61 (d, j=8.0Hz, 1H), 6.32 (s, 1H), 7.46-7.50 (m, 2H), 7.59-7.63 (m, 1H), 8.08-8.10 (m, 2H); ¹³cNMR (CDCl ₃, 100MHz) δ: 9.4,15.6,20.9,22.5,26.9,35.5,38.6,42.6,46.1,58.6,67.8,72.2,74.8,76.2,79.0,80.6,84.4,128.6,129.2,130.0,131.6,133.6,146.5,167.0,170.6,171.3,204.2; HRMS (ESI) Calcd.forC ₃₃h ₄₀naO ₁₂[M+Na] ⁺: 651.2417; Found:651.2419.

Claims (1)

1. from southerm yew plant, extract a method for baccatin III reference substance, it is characterized in that: comprise the steps:

1) get southerm yew branches and leaves, after branches and leaves are dried, then pulverized, obtain dry particle; The particle of drying is adopted after sherwood oil carries out pre-treatment, then floods with methyl alcohol, carry out stirring while dipping, ultrasonic, steeping fluid is filtered, then by filtrate reduced in volume, obtains concentrated solution, after repeatedly being extracted by concentrated solution normal hexane, then evaporate to dryness makes medicinal extract;

2) by the extracting 6 times repeatedly of the medicinal extract chloroform/water that obtains in step 1), extraction separatory, obtain the extract of chloroform, extract and silica gel are pressed the mass ratio of 1:10, be that the sherwood oil-chloroform of 15:1 carries out gradient elution, pressurized column chromatography for eluent with volume ratio, collect containing the close mixture of baccatin III polarity, the scope of the Rf value of collecting is the scope in baccatin III standard substance Rf value ± 0.2, is separated and obtains roughing out thing;

3) roughing out thing is added silica gel, the raw material added first and segmentation from the mass ratio of silica gel be 1:10, be that the sherwood oil-chloroform of 12:1 carries out gradient elution for eluent with volume ratio, pressurized column chromatography, collect containing baccatin III polarity mixture closely, the scope of the Rf value of collecting is the scope in baccatin III standard substance Rf value ± 0.1, is separated and obtains thin isolate;

4) acetone is added by thin isolate, the volume ratio of thin isolate and acetone is 1:10, with first alcohol and water for moving phase wash-out, the volume of methyl alcohol and water is 80:20, and flow velocity is 8mL/min, simultaneously with UV spectrophotometer measuring, wash-out section effluent liquid goes out peak at wavelength 237nm, contrast baccatin III standard substance collection of illustrative plates, determines the absorption peak of baccatin III, collects and obtain the baccatin III reference substance that purity is 99.1%.