阅读说明：本技术 瑞香狼毒总二萜的制备方法及其在制药中的用途 (Preparation method of stellera chamaejasme total diterpene and application thereof in pharmacy ) 是由 陈道峰 赵化顶 卢燕 李国雄 于 2018-05-29 设计创作，主要内容包括：本发明属中药制药领域,涉及一种从瑞香狼毒中富集总二萜成分的方法及其在制备抗艾滋病毒药物中的用途。本发明采用高速逆流色谱的方法从瑞香科植物瑞香狼毒(Stellera chamaejasme L.)中富集总二萜类成分,并对所得的各组分进行了主要活性成分含量测定及抗HIV活性测试,结果显示,随着瑞香烷型总二萜成分含量的增加,抗HIV活性呈明显上升趋势。当富集组分中瑞香烷型总二萜含量大于29％时,即显示极强的抗HIV活性,EC<Sub>50</Sub>值可达到0.0034μg/mL,选择指数(SI)大于5800,高效低毒,与阳性对照药物齐多夫定相比具有明显的优势。本发明建立了从瑞香狼毒中快速富集总二萜的方法,方法简单,工艺合理,回收率高,弥补了现有技术中存在的固态支持物或载体的不可逆吸附、损耗等缺点,且富集的总二萜可以用于制备抗艾滋病毒药物。(The invention belongs to the field of traditional Chinese medicine pharmacy, and relates to a method for enriching total diterpene components from stellera chamaejasme and application of the diterpene components in preparation of anti-HIV drugs. The invention adopts a high-speed counter-current chromatography method to enrich total diterpenoid components from Stellera chamaejasme L (Thlerera chamaejasme L.) which is a Thymus plant, and the obtained components are subjected to content determination of main active components and anti-HIV activity test, and the result shows that the anti-HIV activity is in a trend of obviously increasing along with the increase of the content of the daphnane total diterpenoid components. When the content of daphnane type total diterpene in the enriched component is more than 29 percent, the enriched component shows extremely strong anti-HIV activity, the EC50 value can reach 0.0034 mu g/mL, the Selection Index (SI) is more than 5800, the enriched component has high efficiency and low toxicity, and the enriched component has obvious advantages compared with a positive control medicament zidovudine. The invention establishes a method for rapidly enriching the total diterpene from the stellera chamaejasme, has simple method, reasonable process and high recovery rate, makes up the defects of irreversible adsorption, loss and the like of a solid support or a carrier in the prior art, and the enriched total diterpene can be used for preparing the anti-HIV medicament.)

1. A preparation method of total diterpenoids in stellera chamaejasme is characterized by comprising the following steps:

(1) Mixing stellera chamaejasme medicinal material and organic solvent in a proper proportion, extracting for several times, combining extracting solutions, concentrating, recovering solvent and evaporating to dryness to obtain extract;

(2) Suspending the extract obtained in the step (1) with water, extracting for a plurality of times with an organic solvent, combining the extract liquor, and recovering the solvent to obtain an extract;

(3) Mixing petroleum ether or n-hexane: ethyl acetate: methanol: 1-3 parts of water: 1: 1-3: mixing at a ratio of 0.4-2 (v/v), taking an upper phase of the mixed solvent as a stationary phase and a lower phase as a mobile phase, separating the extract obtained in the step (2) by adopting high-speed counter-current chromatography, and collecting fractions containing diterpene components to obtain a total diterpene crude extract;

(4) And (3) continuously adopting high-speed countercurrent chromatography to separate the total diterpene crude extract obtained in the step (3) by using petroleum ether or n-hexane: ethyl acetate: methanol: 1-2 parts of water: 1: 1-1.6: 1-1.4 (v/v), or a mixture of petroleum ether or n-hexane: acetonitrile: the water content is 2: 0.5-1.3: and (3) separating by using a mixed solvent system of 0.7-1.5 (v/v), wherein the upper phase is a stationary phase, and the lower phase is a mobile phase, and collecting and combining fractions containing diterpene components to obtain the total diterpene.

2. The method of claim 1, wherein the organic solvent of step (1) is selected from methanol, ethanol, acetonitrile, and acetone.

3. The method of claim 1, wherein the extraction in step (1) is selected from the group consisting of thermal reflux extraction, flash extraction, ultrasonic extraction, and percolation extraction.

4. The method of claim 1, wherein the organic solvent in step (2) is selected from petroleum ether, cyclohexane, n-hexane, and ethyl acetate.

5. The process according to claim 1, wherein the total diterpenes obtained in step (4) are further purified by high-speed counter-current chromatography.

6. The method of claim 1, wherein the total diterpenes are used for preparing anti-HIV drugs.

Technical Field

The invention belongs to the field of traditional Chinese medicine pharmacy, relates to a preparation method of total diterpenoids in stellera chamaejasme and application thereof in pharmacy, and particularly relates to a method for enriching total diterpenoid components from stellera chamaejasme and application thereof in preparation of anti-HIV drugs.

Background

Stellera chamaejasme L is perennial herb of Stellera of Thymelaeaceae, commonly called tomahawood, steamed bread flower, radish, etc. its medicinal part is root, widely distributed in northern and southwest areas of China, and has the functions of expelling water, eliminating phlegm, breaking stagnated food and killing pests. Modern pharmacological studies show that the stellera chamaejasme has the effects of resisting tumors, AIDS, insects, bacteria and the like, and particularly the AIDS resistance is highly concerned by scholars at home and abroad. Researches show that diterpene components in stellera chamaejasme have extremely strong anti-AIDS activity but low content, and the chemical synthesis route is difficult to realize due to the complex structure of the diterpene components. Therefore, if a method for quickly enriching diterpenoid compounds from stellera chamaejasme can be established, the method has important practical significance for the research of high-efficiency and low-toxicity anti-HIV drugs. In the prior art (Chendaofeng, etc., a preparation method of stellera chamaejasme total diterpene and application thereof in pharmacy, application number: 201510489802.8, publication number: 106432263A) adopts solvent extraction and extraction, and the total diterpene is obtained by methods of reduced pressure column chromatography and gel column chromatography, and practice shows that the preparation method has the defects of large loss, low yield, complex operation, long time consumption and the like. The high-speed counter-current chromatography is a rapid enrichment chromatography technology without using a stationary phase as a carrier, has high recovery rate and high efficiency, is particularly suitable for components with lower content, can reduce the usage amount of an organic solvent, and is widely applied to enrichment separation of natural products at present.

based on the current situation of the prior art, the inventor of the application intends to provide a high-speed countercurrent chromatography method for rapidly enriching total diterpenoid components from stellera chamaejasme and application thereof in preparing anti-HIV drugs.

disclosure of Invention

The invention aims to provide a preparation method of total diterpenoids in stellera chamaejasme and application thereof in pharmacy based on the current situation of the prior art, and particularly relates to a method for enriching total diterpenoid components from stellera chamaejasme of Thymelaeaceae and application thereof in preparation of anti-HIV drugs.

The technical scheme adopted by the invention for realizing the purpose is as follows:

A method for preparing daphnane type total diterpene for resisting AIDS virus from stellera chamaejasme comprises the following steps:

(1) mixing stellera chamaejasme medicinal material and organic solvent in a proper proportion, extracting for several times, combining extracting solutions, concentrating, recovering solvent and evaporating to dryness to obtain extract;

The organic solvent is selected from methanol, ethanol, acetonitrile or acetone; the extraction method is selected from heating reflux extraction, flash extraction, ultrasonic extraction or percolation extraction;

(2) Suspending the extract obtained in the step (1) with water, extracting for a plurality of times with an organic solvent, combining the extract liquor, concentrating, recovering the solvent and evaporating to dryness to obtain an extract; the organic solvent is selected from petroleum ether, cyclohexane, n-hexane or ethyl acetate;

(3) and (3) enriching the extract obtained in the step (2) by high-speed counter-current chromatography, wherein the extraction is carried out by using n-hexane (petroleum ether): ethyl acetate: methanol: taking a mixed solution of water (1-3: 0.4-2; v/v) as a solvent system, fully mixing, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, filling the stationary phase into a chromatographic column of high-speed countercurrent chromatography, injecting the mobile phase at a flow rate of 1-3 mL/min under the conditions of a rotating speed of 600-1000 r/min and a water bath temperature of 15-30 ℃, introducing a sample, performing elution in a gradient elution mode, determining a chromatographic peak containing daphnane diterpene according to high performance liquid chromatography or mass spectrum of effluent liquid, collecting and combining corresponding effluent liquid, and concentrating to obtain crude total diterpene A and B;

(4) With petroleum ether (n-hexane): ethyl acetate: methanol: 1-2% of water: 1: 1-1.6: 1-1.4 (v/v) of a solvent system for crude total diterpene A obtained in the step (3) and petroleum ether (n-hexane): acetonitrile: the water content is 2: 0.5-1.3: 0.7-1.5 (v/v) of a solvent system, continuously separating the crude total diterpene B obtained in the step (3) by adopting high-speed countercurrent chromatography, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, filling the stationary phase into a chromatographic column of the high-speed countercurrent chromatography, injecting the mobile phase at a flow rate of 1-3 mL/min under the conditions of a rotating speed of 600-1000 r/min and a water bath temperature of 15-30 ℃, introducing a sample, performing elution by adopting a gradient elution mode, determining a chromatographic peak containing diterpene according to an ultraviolet spectrum or a mass spectrum of an effluent liquid, collecting and combining eluents containing diterpene components, and concentrating to obtain total diterpene C and D with higher contents;

(5) carrying out content measurement and anti-HIV (HIV) activity test on active ingredients such as phorbol-12-benzoyloxy-13-sunflower ester (1), nimodipine (2), simplex bacteriocin (3), saxitoxin A (4), hula toxin (5) and the like on each sample obtained in the steps (1) to (4); the result shows that the total diterpene content is greatly improved after the extract obtained in the step (2) is enriched; meanwhile, the anti-HIV activity is obviously enhanced, the EC50 value is reduced to 0.0056 mu g/mL from 0.53 mu g/mL of the initial extract, the activity is enhanced by nearly 100 times, and the selection index is also improved by more than 80 times. When the content of the daphnane type total diterpene in the enriched component is more than 29 percent, the enriched component shows extremely strong anti-HIV activity, the EC50 value can reach 0.0034 mu g/mL, the cytotoxicity is low (CC50 is more than 20), the Selection Index (SI) is more than 5800, and the enriched component can be used for preparing anti-HIV drugs.

the invention establishes a method for rapidly enriching the total diterpene from the stellera chamaejasme, the method is simple, the process is reasonable, the recovery rate is high, the defects of irreversible adsorption, loss and the like of a solid support or a carrier in the prior art are overcome, the obtained total diterpene has definite components, high content, strong anti-HIV activity and low toxicity, and is suitable for large-scale production. The method can be used for large-scale enrichment of total diterpenoid compounds in stellera chamaejasme, and the obtained total diterpenoid can be used for preparing anti-HIV drugs.

drawings

FIG. 1 is a flow diagram of the enrichment process of example 1.

FIG. 2 shows the structures of phorbol-12-benzoyloxy-13-sunflower ester (1), nimodimorph (2), simplex bacteriocin (3), saxitoxin A (4), and hula toxin (5).

FIG. 3 is a liquid chromatogram of each sample in the preparation process of the total diterpenes of stellera chamaejasme in example 1.

Detailed Description