CN1800188A - Method for separating and preparing triptolide diol from tripterygium wilfordii - Google Patents

Abstract

The invention discloses a method for separating and preparing triptolide diol from tripterygium wilfordii. It is made of Tripterygium wilfordii root bark extract containing triptolide diol, extracted several times with a mixed solvent, the obtained solid is dispersed in water and extracted several times with an organic solvent, the organic phase is collected, evaporated to dryness on the obtained solid Primary silica gel column or neutral alumina column, eluted with dichloromethane, chloroform or dichloroethane to obtain crude triptolide diol, crystallized with dichloromethane-ether to obtain coarse crystals of triptolide diol , recrystallized twice to obtain pure crystals. The raw materials used in the invention are cheap and easy to obtain, the environmental pollution is small, the process is simple and fast, and the obtained triptolide diol has low cost and high purity, and is suitable for industrial scale production.

Description

The method for separating and preparing triptolide diol from tripterygium wilfordii

technical field

The invention relates to a method for separating and preparing triptolide diol from tripterygium wilfordii.

Background technique

Tripterygium Wilfordii (Tripterygium Wilfordii Hook.f.), a plant of the genus Tripterygium Wilfordii, has 4 species in the world, 4 species produced in China, and 3 species used as medicine (Edited by Li Ruilin et al., Research and Clinical Application of Tripterygium wilfordii, first edition , Beijing, China Science and Technology Press, 1989: 11-12). Because Tripterygium wilfordii has significant physiological activities such as anti-inflammation, immunity, anti-tumor, anti-cancer, and anti-fertility, it has been widely used in the treatment of diseases such as rheumatism, skin disease, and kidney disease (Zhou Xue-ping et al., Journal of Traditional Chinese Medicine (Engl.), 2004, 24(2): 83-87; Hua Guo et al., World Journal of Gastroenterology (Engl.), 2003, 9(7): 1550-1553; Wang Li-Ling et al. , Chinese Journal of Integrative Medicine (Engl.), 2003, 9(3): 191-194; HuYong-Xiu et al., Chinese Medical Journal (Engl.), 2003, 116(2): 284-287; Qian, Shao Zhen et al., Contraception, 1995, 51(2): 121-129).

Triptolide diol (Tipdiolide), molecular formula is C ₂₀ H ₂₄ O ₇ , is a diterpenoid compound in Tripterygium wilfordii (Journal of the American Chemical Society, S.Morris Kupchan et al, 1972, 94(20): 7194 ). Triptolide diol (also known as triptolide) is one of the main active ingredients in Tripterygium wilfordii (Zhang Chongpu et al., Acta Pharmaceutica Sinica, 1993, 28 (2): 110-115; Zhang Jianwei et al., Journal of Practical Andrology, 1996, 2 (2): 81-84; Zhang Liang et al., Journal of China Pharmaceutical University, 1990, 21 (4): 251-256), which is the main effective compound in the traditional Chinese medicine Tripterygium wilfordii Polyglycoside Tablets (Zhang Chongpu et al., Chinese Journal of Pharmaceutical Sciences, 1994, 29(8):493-495).

Existing bibliographical reports separate and obtain the method for triptolide diol generally to adopt Tripterygium wilfordii extract through repeated column chromatography (Lin Sui et al., Acta Pharmaceutica Sinica, 2005, 40 (7): 632-635), and the process is cumbersome and time-consuming , there are also documents (Wen-Zhen Gu, et al. International Journal of Immunopharmacology, 1995, 17 (5): 351-356) reporting that the separation of triptolide diol is prepared by preparing high-performance liquid phase, which is a small number of samples for each injection operation. The cost is high, and it is also reported that triptolide diol was prepared by multidimensional countercurrent chromatography (Jun Wei et al., Journal of Liquid Chromatography & Related Technologies, 2005, 28: 1903-1911), but this method is not suitable for triptolide diol Large-scale separation preparation. The present invention uses common organic solvents to separate Tripterygium wilfordii extract, after several simple solvent extractions, and finally passing through a chromatographic column, eluting with a single organic solvent, crystallization can obtain triptolide diol coarse crystals, and recrystallization Pure crystals can be obtained twice.

Contents of the invention

The object of the present invention is to provide a method for separating and preparing triptolide diol from Tripterygium wilfordii.

It is made of Tripterygium wilfordii root bark extract containing triptolide diol, extracted several times with a mixed solvent, the obtained solid is dispersed in water and extracted several times with an organic solvent, the organic phase is collected, evaporated to dryness on the obtained solid Primary silica gel column or neutral alumina column, eluted with dichloromethane, chloroform or dichloroethane to obtain crude triptolide diol, crystallized with dichloromethane-ether to obtain coarse crystals of triptolide diol , recrystallized twice to obtain pure crystal

The advantages of the present invention are: Tripterygium wilfordii is widely distributed in southern China, so it is cheap and easy to obtain, and the process is simple, easy, and fast. It starts from the raw material and undergoes several simple extractions, and finally only passes through the chromatographic column once, and is eluted with a single organic solvent , crystallization can obtain triptolide diol coarse crystals, and recrystallization twice can obtain pure crystals. The used solvents are basically recyclable, have little environmental pollution, and the obtained triptolide diol has low cost and high purity, and is suitable for industrial scale production.

Detailed ways

The steps of the present invention are as follows:

1) Pulverize the root bark of Tripterygium wilfordii, crush the root bark of Tripterygium wilfordii (through a 60-mesh sieve), extract 3-5 times with 8 times the amount (80-95%) of ethanol, 10 hours each time, combine and concentrate to recover the ethanol to obtain the leaching The extract was extracted several times with ethyl acetate or chloroform, and the extract was subjected to silica gel or neutral alumina column chromatography, and gradient elution with methanol-chloroform (3:97, 5:95, 10:90) was performed step by step. Collect, concentrate under reduced pressure the part containing triptolide diol, evaporate to dryness and obtain solid as raw material

2) The raw material is extracted several times with a solvent system of n-butanol: ethanol: water = 30-35: 0.1-0.5: 30-35, detected by thin-layer chromatography, and the lower phase containing triptolide diol is collected and concentrated under reduced pressure The solvent was recovered to give a solid.

3) The solid obtained by extraction in step 2 is dispersed in water and extracted several times with ethyl acetate, chloroform or ether, the upper phase is collected, and the solvent is recovered under reduced pressure to obtain a solid.

4) Put the solid matter obtained in step 3 on a silica gel column or a neutral alumina column, elute with dichloromethane, chloroform or dichloroethane, collect the eluent, concentrate under reduced pressure to recover the organic solvent, and obtain triptolide di Crude alcohol.

5) The crude product of triptolide diol was crystallized with dichloromethane-diethyl ether to obtain coarse crystals of triptolide diol, which were recrystallized twice to obtain pure crystals.

Example 1:

The root bark of Tripterygium wilfordii is extracted by ethanol, ethyl acetate, silica gel column chromatography, gradient elution with methanol-chloroform (3:97, 5:95, 10:90), collected step by step, and concentrated under reduced pressure to contain triptolide The diol part was evaporated to dryness and the resulting solid was used as the raw material. The raw material was extracted with a solvent system of n-butanol:ethanol:water=30:0.1:30 (v/v). First, prepare 0.5l solvent according to the above volume ratio and place it in a separatory funnel, add 10g of raw materials and shake for 3 minutes, let it stand for a period of time, separate the upper and lower phases, and then follow the above steps after the upper phase is concentrated under reduced pressure. Extracted 3 times, combined the lower phases and concentrated to obtain a solid. The obtained solid is dispersed in water, 100ml of ethyl acetate is added, extracted twice according to the ratio of ethyl acetate: water=1:1 (v/v), the upper phase is collected and the solvent is recovered under reduced pressure, and the obtained triptolide diol The crude product was passed through a silica gel column (crude product/silica gel=1:30, w/w), eluted with dichloromethane, crystallized from dichloromethane-diethyl ether to obtain coarse crystals, recrystallized twice to obtain pure crystals of triptolide diol, and the yield 0.95% (based on raw materials).

Example 2:

The root bark of Tripterygium wilfordii is extracted by ethanol, ethyl acetate, silica gel column chromatography, gradient elution with methanol-chloroform (3:97, 5:95, 10:90), collected step by step, and concentrated under reduced pressure to contain triptolide The diol part was evaporated to dryness and the resulting solid was used as the raw material. The raw material was extracted with a solvent system of n-butanol:ethanol:water=30:0.2:30 (v/v). First, prepare 0.5l solvent according to the above volume ratio and place it in a separatory funnel, add 10g of raw materials and shake for 3 minutes, let it stand for a period of time, separate the upper and lower phases, and then follow the above steps after the upper phase is concentrated under reduced pressure. Extracted 3 times, combined the lower phases and concentrated to obtain a solid. The obtained solid was dispersed in water, 100ml of diethyl ether was added, extracted twice according to the ratio of diethyl ether: water=1:1 (v/v), the upper phase was collected and the solvent was recovered under reduced pressure, and the obtained triptolide diol crude product was passed through a silica gel column (crude product/silica gel=1: 30, w/w), eluting with chloroform, dichloromethane-diethyl ether crystallization obtains coarse crystal, recrystallization twice obtains triptolide diol pure crystal, yield rate is 0.91% ( based on raw materials).

Example 3:

The root bark of Tripterygium wilfordii is extracted by ethanol, ethyl acetate, silica gel column chromatography, gradient elution with methanol-chloroform (3:97, 5:95, 10:90), collected step by step, and concentrated under reduced pressure to contain triptolide The diol part was evaporated to dryness and the resulting solid was used as the raw material. The raw material was extracted with a solvent system of n-butanol:ethanol:water=35:0.1:35 (v/v). First, prepare 0.5l solvent according to the above volume ratio and place it in a separatory funnel, add 10g of raw materials and shake for 3 minutes, let it stand for a period of time, separate the upper and lower phases, and then follow the above steps after the upper phase is concentrated under reduced pressure. Extracted 3 times, combined the lower phases and concentrated to obtain a solid. The obtained solid is dispersed in water, 100ml of ethyl acetate is added, extracted twice according to the ratio of ethyl acetate: water=1:1 (v/v), the upper phase is collected and the solvent is recovered under reduced pressure, and the obtained triptolide diol The crude product was passed through a neutral alumina column (crude product/neutral alumina=1:30, w/w), eluted with dichloroethane, crystallized from dichloromethane-ether to obtain coarse crystals, and recrystallized twice to obtain triptolide The diol is pure crystal, and the yield is 0.92% (based on raw materials).

Example 4: Tripterygium wilfordii root bark was extracted with ethanol, ethyl acetate, silica gel column chromatography, gradient elution with methanol-chloroform (3:97, 5:95, 10:90), collected step by step, and concentrated under reduced pressure Containing triptolide diol part, evaporated to dryness to obtain solid as raw material. The raw material was extracted with a solvent system of n-butanol:ethanol:water=35:0.5:35 (v/v). First, prepare 0.5l solvent according to the above volume ratio and place it in a separatory funnel, add 10g of raw materials and shake for 3 minutes, let it stand for a period of time, separate the upper and lower phases, and then follow the above steps after concentrating the upper phase under reduced pressure Extract again 3 times, combine the lower phases and concentrate to obtain a solid. The obtained solid is dispersed in water, 100ml of ethyl acetate is added, extracted twice according to the ratio of ethyl acetate: water=1:1 (v/v), the upper phase is collected and the solvent is recovered under reduced pressure, and the obtained triptolide diol The crude product was passed through a silica gel column (crude product/silica gel=1:30, w/w), eluted with dichloromethane, crystallized from dichloromethane-diethyl ether to obtain coarse crystals, recrystallized twice to obtain pure crystals of triptolide diol, and the yield It is 0.85% (based on raw materials).

Claims (5)

1. one kind is separated the method for preparing Triptodiolide from trypterygine; it is characterized in that: it is that the tripterygium root peel extract that contains Triptodiolide is done raw material; with mixed extractant solvent for several times; the solids that obtains is dispersed in the water with the organic solvent extraction several; collect organic phase; last silicagel column of the solids that evaporate to dryness obtains or neutral alumina post; use methylene dichloride; trichloromethane or ethylene dichloride wash-out; obtain the Triptodiolide crude product; with methylene dichloride-ether crystallization; obtain the Triptodiolide coarse-grain, recrystallization twice, it is raw materials used cheap and easy to get promptly to get pure brilliant the present invention; environmental pollution is little; simple for process; fast, the Triptodiolide cost that is obtained is lower; the purity height is fit to industrial scale production.

2. a kind of method for preparing Triptodiolide of from trypterygine, separating according to claim 1, it is characterized in that: the described extracting method that contains the tripterygium root peel extract of Triptodiolide is: velamen of Tripterygium wilfordii is pulverized (crossing 60 mesh sieves), with (80-95%) ethanol lixiviate of 8 times of amounts 3-5 time, each 10 hours, merge the concentrated ethanol that reclaims and obtain medicinal extract, medicinal extract with ethyl acetate or chloroform extraction for several times, extract carries out silica gel or neutral alumina column chromatography, with methyl alcohol-chloroform (3: 97,5: 95,10: 90) gradient elution, substep is collected, concentrating under reduced pressure contains the Triptodiolide part, and evaporate to dryness gained solid is that raw material gets final product.

3. a kind of method for preparing Triptodiolide of separating from trypterygine according to claim 1, it is characterized in that: described mixed solvent is the mixture of propyl carbinol, ethanol, water.

4. mixed solvent according to claim 3 is characterized in that: the ratio of described mixed solvent is: propyl carbinol: ethanol: water=30-35: 0.1-0.5: 30-35

5. a kind of method for preparing Triptodiolide of separating from trypterygine according to claim 1, it is characterized in that: described organic solvent is ethyl acetate, trichloromethane or ether.