CN110540506A - Preparation method of stellera chamaejasme total diterpene and application thereof in pharmacy - Google Patents
Preparation method of stellera chamaejasme total diterpene and application thereof in pharmacy Download PDFInfo
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Abstract
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.
Description
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
Example 1: method for enriching total diterpene of stellera chamaejasme L
pulverizing 1000g radix Euphorbiae Fischerianae, adding 7L 95% ethanol, reflux-extracting under heating for 3 times (1 hr each time), mixing extractive solutions, concentrating, and evaporating to obtain 174g extract (DFC-SC-E). Dispersing the extract in 3L warm water, extracting with cyclohexane for 4 times, 3L each time, to obtain 21.7g each cyclohexane extraction part (DFC-SC-C). The resulting extract was enriched by high speed counter current chromatography using n-hexane: ethyl acetate: methanol: taking a mixed solution of water (1: 1: 1: 1; v/v) as a solvent system, fully mixing, standing and layering, wherein an upper phase is a stationary phase, a 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 ℃, injecting samples, and respectively using normal hexane with the volume of 1-2 times of the column volume: ethyl acetate: methanol: eluting with water (1: 1: 1; 1: 1: 1.3: 0.7; 1: 1: 1.6: 0.4; v/v) as the lower phase, determining the chromatographic peak containing diterpene according to high performance liquid chromatography or mass spectrometry of the effluent, collecting and combining the corresponding effluents, and concentrating to obtain 0.65g (DFC-SC-C2) and 1.51g (DFC-SC-C3) of total diterpene crude extract. And (3) continuously carrying out high-speed countercurrent chromatography enrichment on the two parts respectively, wherein the two parts are respectively enriched by n-hexane: ethyl acetate: methanol: water (2: 1: 1.6: 1.4; v/v) and n-hexane: acetonitrile: further enriching the solvent system of water (2: 0.7: 1.3; v/v), determining the chromatographic peak containing diterpene according to the ultraviolet spectrum or mass spectrum of the effluent, collecting and combining the eluates containing diterpene components, and concentrating to obtain 34mg (DFC-SC-C21) and 466mg (DFC-SC-C31) of stellera chamaejasme total diterpene.
example 2: method for enriching total diterpene of stellera chamaejasme L
Taking 500g of stellera chamaejasme medicinal material, crushing by a small-sized crusher, adding 3L of methanol, ultrasonically extracting for 3 times, each time for 30 minutes, filtering, concentrating and evaporating to dryness to obtain 69g of extract. Dispersing the extract in 1L warm water, and extracting with n-hexane for 4 times, 1L each time, to obtain n-hexane extract parts each 8 g. The resulting extract was enriched by high speed counter current chromatography using n-hexane: ethyl acetate: methanol: taking a mixed solution of water (2: 1: 1.5: 1.5; v/v) as a solvent system, fully mixing, standing and layering, taking an upper phase as a stationary phase and a lower phase as 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 ℃, injecting samples, and respectively using n-hexane with the volume of 1-2 times of the column volume: ethyl acetate: methanol: eluting with water (2: 1: 1.5: 1.5; 2: 1: 2.1: 0.9; v/v) as lower phase, determining chromatographic peak containing diterpene according to high performance liquid chromatography or mass spectrometry of effluent, collecting and combining eluates containing diterpene components to obtain crude total diterpene 0.27g and crude total diterpene 0.72 g. And (3) continuously performing high-speed countercurrent chromatography enrichment on the two parts respectively, wherein the two parts are respectively prepared by the following steps of: ethyl acetate: methanol: water (1: 1: 1: 1; v/v) and petroleum ether: acetonitrile: further enriching the solvent system of water (2: 0.5: 1.5; v/v), determining the chromatographic peak containing diterpene according to the ultraviolet spectrum or mass spectrum of the effluent, collecting and combining the eluates containing diterpene components, and concentrating to obtain 12mg and 221mg of stellera chamaejasme total diterpene.
Example 3: method for enriching total diterpene of stellera chamaejasme L
Taking 500g of stellera chamaejasme medicinal material, crushing by a small crusher, adding 3L of acetonitrile, carrying out flash extraction for 5 times, 3 minutes each time, carrying out suction filtration, concentrating and evaporating to dryness to obtain 76g of extract. Dispersing the extract in 1L warm water, and extracting with ethyl acetate for 4 times, 1L each time, to obtain 16.2g each of ethyl acetate extract parts. The resulting extract was enriched by high speed counter current chromatography using petroleum ether: ethyl acetate: methanol: taking a mixed solution of water (3: 1: 2: 2; v/v) as a solvent system, fully mixing, standing and layering, wherein an upper phase is a stationary phase, a 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 ℃, injecting samples, and respectively using petroleum ether with a volume of 1-2 columns: ethyl acetate: methanol: eluting with water (3: 1: 2: 2; 3: 1: 2.5: 0.5; 3: 1: 3: 1; v/v) as lower phase, determining chromatographic peak containing diterpene according to high performance liquid chromatography or mass spectrum of effluent, collecting and combining eluates containing diterpene components to obtain crude total diterpene 0.32g and crude total diterpene 0.89 g. And (3) continuously carrying out high-speed countercurrent chromatography enrichment on the two parts respectively, wherein the two parts are respectively enriched by n-hexane: ethyl acetate: methanol: water (2: 1: 1.6: 1.4; v/v) and n-hexane: acetonitrile: further enriching a solvent system of water (2: 0.9: 1.1; v/v), determining a chromatographic peak containing diterpene according to an ultraviolet spectrum or a mass spectrum of an effluent, collecting and combining eluates containing diterpene components, and concentrating to obtain 13mg and 209mg of stellera chamaejasme total diterpene.
Example 4: method for enriching total diterpene of stellera chamaejasme L
Taking 500g of stellera chamaejasme medicinal material, crushing by a small-sized crusher, adding 3L of acetone, percolating and extracting for 3 times, each time for 4 hours, concentrating and evaporating filtrate to dryness to obtain 49g of extract. Dispersing the extract in 1L warm water, and extracting with petroleum ether for 4 times (1L each time) to obtain petroleum ether extraction parts each 6.1 g. The resulting extract was enriched by high speed counter current chromatography using petroleum ether: ethyl acetate: methanol: taking a mixed solution of water (1: 1: 1.2: 0.8; v/v) as a solvent system, fully mixing, standing and layering, taking an upper phase as a stationary phase and a lower phase as 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 ℃, injecting samples, and respectively using petroleum ether with a volume of 1-2 columns: ethyl acetate: methanol: eluting with water (1: 1: 1.2: 1.8; 1: 1: 1.5: 1.5; v/v) as lower phase, and determining the chromatographic peak containing diterpene according to high performance liquid chromatography or mass spectrometry of the effluent until no diterpene component flows out, to obtain crude total diterpene 0.23g and crude total diterpene 0.65 g. And (3) continuously performing high-speed countercurrent chromatography enrichment on the two parts respectively, wherein the two parts are respectively prepared by the following steps of: ethyl acetate: methanol: water (2: 1: 1.6: 1.4; v/v) and n-hexane: acetonitrile: further enriching with water (2: 0.7: 1.3; v/v) solvent system, determining the chromatographic peak containing diterpene according to the ultraviolet spectrum or mass spectrum of the effluent, collecting and combining the eluates containing diterpene component, and concentrating to obtain 15mg and 211mg of stellera chamaejasme total diterpene.
Example 5:
the content of the main diterpenes in each sample of example 1 was determined and the total daphnane diterpenes were semi-quantified by UPLC-MS using phorbol-12-benzoyloxy-13-decyl ester (1), nifedipine (2), simplex bacteriocin (3), saxitoxin a (4), and kularoxin (5) as controls. The content determination method adopts a standard curve method, and the liquid chromatogram conditions are as follows: 0-2 min, acetonitrile: 50% of water: 50 percent; 2-42 min, acetonitrile: 50% of water: 50% -90%: 10 percent; 42-45 min, acetonitrile: water 90%: 10% -100%: 0 percent; 45-67 min, 100% acetonitrile; the flow rate is 0.3 mL/min; the column temperature is 25 ℃; the detection wavelength is 230 nm; the contents of the respective samples are shown in table 1.
TABLE 1 Total diterpene enrichment method in stellera chamaejasme 1. the yield (%) of each sample and the diterpene component content (%)
remarking: NF means not detected; the yield represents the extract extraction rate or the yield of each step of sample compared with the previous step of sample.
Example 6: anti-HIV activity test of each sample in enrichment method 1 of total diterpenes in stellera chamaejasme
The key active fractions obtained in example 1 were subjected to an anti-HIV activity (EC50) test and a cytotoxic activity (CC50) test, and a therapeutic index (SI, CC50/EC50) was calculated. The anti-HIV activity test method comprises the steps of infecting MT4 cells (with infection times being 0.001) by using an HIV-1NL4-3 virus strain, adding medicines with different concentrations, adding a fresh matrix containing medicines with proper concentrations after infecting for 48 hours to maintain normal growth of the cells, analyzing the virus replication condition by using a P24ELISA kit after 4 days, and taking zidovudine (AZT) as a positive medicine in the experiment; the cell survival rate of the MT4 cells is determined by measuring the ATP content in the metabolized active cells by using a cell survival experiment. The results show (as shown in Table 2), when the total diterpene content in the components is obviously improved after one-step enrichment, the sample shows extremely strong anti-HIV activity, the EC50 value can reach 0.0051-0.0056 mu g/mL, the Selection Index (SI) is 2352-2857, after further enrichment, the EC50 value of the sample can reach 0.0025-0.0034 mu g/mL, and the Selection Index (SI) is more than 5882-8000.
TABLE 2 anti-HIV activity and cytotoxicity test results of various samples in the enrichment method 1 for total diterpenes of stellera chamaejasme
Claims (6)
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.
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Application publication date: 20191206 |