CN107987118A - A kind of isolation and purification method of Celastrol - Google Patents

Abstract

The invention discloses a separation and purification method of tripterine, which is characterized in that it comprises the following steps: using an organic solvent ultrasonic extraction process to obtain an extract extract, and then using a degreasing and alcohol extraction-water precipitation process to treat the extract , to obtain the crude extract of tripterycin, and to separate the crude extract of tripterycin with a macroporous resin to obtain the crude product of tripterycin, and finally to recrystallize and purify with two solvents to obtain high-purity tripteryne. The crude extract separation process using macroporous resin has the advantages of good selectivity, high separation efficiency and easy large-scale preparation; the dual-solvent recrystallization purification process has the characteristics of simple process, product appearance and high purification.

Description

A kind of separation and purification method of tripterine

technical field

The invention relates to a separation and purification method of tripterine, which belongs to the technical field of plant active component separation technology.

Background technique

Tripterygium wilfordii is derived from the root bark of tripterygium wilfordii or southern snake vine, which is a natural product with various biological activities. Tripterygium wilfordii is one of the active ingredients in preparations such as tripterygium wilfordii tablets and tripterygium glycosides tablets for the treatment of rheumatoid diseases. Modern research shows that Tripterygium wilfordii has strong anti-oxidation and anti-cancer angiogenesis effects, as well as anti-rheumatoid and insecticidal effects, and also has outstanding effects on brain neuroprotection [Chinese herbal medicine, 2010, 41 (7): 1215-1218]. As a new type of natural proteasome inhibitor, tripterygium has great development potential in the prevention and treatment of cancer, and it is expected to become a new generation of highly effective anti-tumor plant drugs after paclitaxel [China Pharmaceutical Information, 2006, 22 (9) :15]. Our country has abundant tripterycin plant resources, therefore, it has important application prospect and practical significance to carry out the research on the extraction and separation and purification of tripterygium, obtain high-purity tripteryne products, and make full use of plant resources as much as possible. It can also provide a new way for the development of new drugs and the modernization of traditional Chinese medicine.

Triptolide belongs to pentacyclic triterpenoids, and its appearance is red or dark red crystals with certain toxicity. Its molecular formula and molecular weight are C ₂₉ H ₃₈ O ₄ and 450.61, respectively, and its melting point is 185-205°C. It is not easily soluble in water, but soluble in organic solvents such as methanol, ethanol, chloroform, ethyl acetate, etc. The physical and chemical properties are relatively stable, and the maximum absorption wavelength is 425nm.

The content of tripterycin in the plants that have been reported is generally low. Therefore, how to improve the extraction and separation efficiency of tripterygen in plants and make full use of limited resources is particularly important. There are many reports on the extraction process of tripterine, such as traditional organic solvent heating reflux extraction, supercritical fluid extraction and ultrasonic extraction. The separation techniques of tripteryglide mainly include droplet countercurrent chromatography [J Chromatogr A, 2004, 1028: 171-174] and silica gel column chromatography [Wang Ningqiang, Master Thesis of Shenyang Pharmaceutical University. 2008; Journal of Liaoning University of Traditional Chinese Medicine, 2013, 15(6): 48-50]. However, it is difficult to separate high-purity tripterine products by traditional column chromatography, and there are disadvantages of high solvent and energy consumption, complicated process and high cost.

Patent CN 10163842A uses low-level chlorinated alkanes as a solvent to heat and reflux extraction, and the extract is subjected to a degreasing-alcohol precipitation process to obtain a thick paste-like crude extract, which is then separated and purified with a normal-phase silica gel or reverse-phase silica gel column as the stationary phase, and collected and eluted Liquid, desolvated and dried to obtain powdery fine extract, and finally recrystallized with ethanol-water mixed solvent to obtain tripterine with higher purity. Patent CN 101311186A improves the deficiency that the traditional column chromatography method cannot separate high-purity tripterine, and uses chloroform ultrasonic extraction, dual-solvent elution silica gel column chromatography and mixed solvent recrystallization and other processes. In general, these separation and purification processes still have the disadvantages of complex process, low separation efficiency and high cost. Therefore, it is very necessary to explore a simple and effective separation and purification process of tripterycin, improve the separation efficiency of tripterygen, obtain high-purity tripteryne products, and make full use of tripteryne-related plant resources.

Contents of the invention

The technical problem to be solved by the present invention is to improve the separation and purification efficiency of tripterygol, simplify the separation and purification process, reduce the preparation cost of tripteryne, and obtain high-purity tripteryne products.

The technical scheme of the present invention is: a separation and purification method of tripterine, comprising the following steps: using an organic solvent ultrasonic extraction process to obtain the extract extract, and then using the degreasing and alcohol extraction-water precipitation process to process the extract, The crude tripterine extract is obtained, and the crude tripterine extract is separated with a macroporous resin to obtain the crude tripterine product, which is finally purified by double-solvent recrystallization to obtain high-purity tripterine.

The macroporous resin is AB-8 macroporous adsorption resin.

The mass ratio of the crude extract:macroporous resin is 1:(10-50).

The ethanol/water with a volume fraction of 60-90 is used as the eluent, and the pH value of the eluent is 5.5-6.5.

The dual solvent system is cyclohexane/dioxane or cyclohexane/THF, and the volume ratio of the mixed solvent is (85~98):(2~15).

Beneficial effects of the present invention: adopting the crude extract separation process of macroporous resin has the advantages of good selectivity, high separation efficiency and easy large-scale preparation; adopting dual-solvent recrystallization purification process has the advantages of simple process, product appearance and purification high feature.

Detailed ways

Example 1

Pulverize the root bark of tripterygium wilfordii through sufficient drying, and more than 80% of the powder is passed through a 30-40 mesh sieve. Take 200.0g of tripterygium twig root powder, add 800 ml of chloroform, soak for 2 h, then ultrasonically extract for 40 min, repeat the operation twice, combine the extracts, distill and recover the solvent chloroform, and dry at 50-60 ºC to obtain the extract 3.56 g.

Take 4.0 g of the above extract and add 60 ml of petroleum ether, heat to reflux for 30 min, filter while hot and pour out the petroleum ether, repeat twice to obtain a residue, volatilize the petroleum ether to obtain 1.22 g of the extract of depolarized components. Then add 50 ml of 95% ethanol, heat to about 70 ºC to dissolve, then slowly add the same volume of water, then evaporate most of the ethanol, place it in an ice-water bath to cool, and produce dark red deposits as the crude extract of tripterine. The dry weight was 0.32 g.

Take 0.5 g of the above-mentioned tripterygium crude extract, dissolve it with a small amount of ethanol solvent, pack it into a column with 20 g of pretreated AB-8 macroporous adsorption resin and perform column separation, and elute with 70% ethanol whose pH is adjusted to 6.5 , collected components with an Rf value of about 0.70, concentrated and dried, and obtained 0.12 g of crude tripterine.

Take 0.20 g of the crude tripterine obtained above, and recrystallize it with cyclohexane/dioxane at a volume ratio of 90:10 to obtain 0.182 g of tripterine as red needle crystals, melting point: 195~203ºC. The product was analyzed by HPLC, and the content was 98.5%.

Example 2

Pulverize the root bark of tripterygium wilfordii through sufficient drying, and more than 80% of the powder is passed through a 30-40 mesh sieve. Take 200.0g of tripterygium twig root powder, add 800 ml of chloroform, soak for 2 h, then ultrasonically extract for 40 min, repeat the operation twice, combine the extracts, distill and recover the solvent chloroform, and dry at 50-60 ºC to obtain the extract 3.56 g.

Take 4.0 g of the above extract and add 60 ml of petroleum ether, heat to reflux for 30 min, filter while hot and pour out the petroleum ether, repeat twice to obtain a residue, volatilize the petroleum ether to obtain 1.22 g of the extract of depolarized components. Then add 50 ml of 95% ethanol, heat to about 70 ºC to dissolve, then slowly add the same volume of water, then evaporate most of the ethanol, place it in an ice-water bath to cool, and produce dark red deposits as the crude extract of tripterine. The dry matter weighed 0.32 g.

Take 0.5 g of the above-mentioned tripterygium crude extract, dissolve it with a small amount of ethanol solvent, pack it into a column with 20 g of pretreated AB-8 macroporous adsorption resin and perform column separation, and elute with 70% ethanol whose pH is adjusted to 6.5 , collected components with an Rf value of about 0.70, concentrated and dried, and obtained 0.12 g of crude tripterine.

Take 0.20 g of the crude tripterine obtained above, and use cyclohexane/THF volume ratio 95: 5 for recrystallization, and obtain 0.175 g of tripterine as red needle crystals, melting point: 195~203 ºC. The product was analyzed by HPLC, and the content was 98.2%.

The invention provides a method for separating and purifying tripteryglide, which has the advantages of simple process, high separation and purification efficiency, can obtain tripteryglide product with a purity of 98.5%, and can meet the needs of medicine and various other uses.

Comparative Example 1

Pulverize the root bark of tripterygium wilfordii through sufficient drying, and more than 80% of the powder is passed through a 30-40 mesh sieve. Take 200.0g of tripterygium twig root powder, add 800ml of chloroform, soak for 2h, then ultrasonically extract for 40min, repeat the operation twice, combine the extracts, distill and recover the solvent chloroform, and dry at 50-60 ºC to obtain 3.56g of extract.

Take 4.0 g of the above extract and add 60 ml of petroleum ether, heat to reflux for 30 min, filter while hot and pour out the petroleum ether, repeat twice to obtain a residue, volatilize the petroleum ether to obtain 1.22 g of the extract of depolarized components. Then add 50 ml of 95% ethanol, heat to about 70 ºC to dissolve, then slowly add the same volume of water, then evaporate most of the ethanol, place it in an ice-water bath to cool, and produce dark red deposits as the crude extract of tripterine. The dry matter weighed 0.32 g.

Take 0.5 g of the above-mentioned tripterygium crude extract, dissolve it with a small amount of ethanol solvent, pack it into a column with 20 g of pretreated AB-8 macroporous adsorption resin and perform column separation, and elute with 70% ethanol whose pH is adjusted to 6.5 , collected components with an Rf value of about 0.70, concentrated and dried, and obtained 0.12 g of crude tripterine.

Take 0.20 g of the crude tripterine obtained above, and use cyclohexane/acetone with a volume ratio of 90:10 for recrystallization to obtain 0.164 g of tripterine as red needle-like crystals. The product was analyzed by HPLC, and the content was 93.6%.

Comparative Example 2

Pulverize the root bark of tripterygium wilfordii through sufficient drying, and more than 80% of the powder is passed through a 30-40 mesh sieve. Take 200.0g of tripterygium twig root powder, add 800 ml of chloroform, soak for 2 h, then ultrasonically extract for 40 min, repeat the operation twice, combine the extracts, distill and recover the solvent chloroform, and dry at 50-60 ºC to obtain the extract 3.56 g.

Take 4.0 g of the above extract and add 60 ml of petroleum ether, heat to reflux for 30 minutes, filter while hot and pour out the petroleum ether, repeat twice to obtain a residue, volatilize the petroleum ether to obtain 1.22 g of the extract of depolarized components. Then add 50ml of 95% ethanol, heat to about 70°C to dissolve, then slowly add the same volume of water, then evaporate most of the ethanol, place it in an ice-water bath to cool, and produce dark red deposits as the crude extract of tripterine. The dry weight was 0.32 g.

Take 0.5 g of the above-mentioned tripteryglide crude extract, dissolve it with a small amount of ethanol solvent, pack it into a column with 20 g of pretreated HPD100 macroporous adsorption resin, and carry out column separation, elute with 70% ethanol whose pH is adjusted to 6.5, and collect The component with an Rf value of about 0.70 was concentrated and dried to obtain 0.134 g of crude tripterine.

Take 0.20 g of the crude tripterine obtained above, and recrystallize it with cyclohexane/ethanol at a volume ratio of 90:10 to obtain 0.156 g of tripterine as red needle-like crystals. The product was analyzed by HPLC, and the content was 92.3%.

Claims (5)

1. a separation and purification method of tripterine, characterized in that: comprise the following steps: obtain extract extract with organic solvent ultrasonic extraction process, then use degreasing and alcohol extraction-water precipitation process to process extract, obtain The crude tripteryne extract is separated by a macroporous resin to obtain the crude tripteryne, which is finally purified by double-solvent recrystallization to obtain high-purity tripteryne. 2. the separation and purification method of a kind of tripterine according to claim 1, is characterized in that: described macroporous resin is AB-8 macroporous adsorption resin. 3. the separation and purification method of a kind of tripterine according to claim 2, is characterized in that: described crude extract: the mass ratio of macroporous resin is 1:(10～50). 4. the separation and purification method of a kind of tripterine according to claim 1, is characterized in that: the ethanol/water that is 60～90 with volume fraction is used as eluent, and the pH value of eluent is 5.5～6.5 . 5. the separation and purification method of a kind of tripterine according to claim 1, is characterized in that: described two-solvent system is hexanaphthene/dioxane or hexanaphthene/THF, the volume of mixed solvent The ratio is (85~98):(2~15).