CN110538243A - Preparation method of active total diterpene in Thymelaeaceae plant and application thereof in pharmacy - Google Patents
Preparation method of active total diterpene in Thymelaeaceae plant 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 preparation method of active total diterpene in a Thymelaeaceae plant and application thereof in pharmacy. The present invention relates to a method for enriching total diterpene components from Daphne genkwa (Daphne genkwa sieb. et Zucc), Wikstroemia canthus Wikstroemia canescens (Wall.) Meisn) and pseudo-langdu root (Stelleropsis tienschia Pobed) belonging to Thymelaeaceae (Thymelaeaceae), and the content of the main active component and anti-HIV activity of each component are measured. When the total diterpene content in the enriched component is more than 64 percent, the enriched component shows extremely strong anti-HIV activity, the EC50 value can reach 0.0047 mu g/mL, the Selection Index (SI) is more than 2000, the enriched component has high efficiency and low toxicity, and has obvious advantages compared with a positive control medicament zidovudine. The invention establishes a method for rapidly enriching the total diterpenoids from the Thymelaeaceae plants, the method is simple, the process is reasonable, the recovery rate is high, and the enriched total diterpenoids can be used for preparing anti-HIV drugs.
Description
Technical Field
the invention belongs to the field of traditional Chinese medicine pharmacy, relates to a preparation method of active total diterpenes in Thymelaeaceae plants and application thereof in pharmacy, and particularly relates to a method for enriching total diterpene components from Thymelaeaceae plants, Thymelaeaceae daphne genkwa, Wikstroelia wikstroma and Erythium chinense in Erythrosepalae and application thereof in preparing anti-AIDS virus drugs.
Background
daphnane diterpenoids, also called daphnotoxin compounds, mainly exist in plants of the Thymus family, and are found to have various biological activities such as anti-HIV and anti-tumor activities through modern pharmacological research, especially, a series of daphnane diterpenoids are found to have the characteristics of strong anti-HIV activity, small cytotoxicity, high therapeutic index and the like through recent research. The prior art discloses about 48 genus daphne of the family Thymelaeaceae, which is widely distributed in tropical and temperate regions of the northern and southern hemispheres, but the daphne diterpene component content is low, the traditional enrichment method such as column chromatography needs repeated separation and enrichment, the operation is complex, the time consumption is long, a large amount of organic solvent is consumed, the sample loss is large, the high-speed counter-current chromatography is a rapid enrichment chromatography technology which does not need a stationary phase as a carrier, the recovery rate is high, the efficiency is high, the method is particularly suitable for the components with low content, and the method is widely used for the enrichment and the separation of natural products at present.
Based on the current situation of the prior art, the inventor of the application intends to provide a method for rapidly enriching the diterpenoid components from the Thymelaeaceae plants, and further provides the application of the prepared daphnane diterpenoid compounds in preparing anti-HIV drugs. The method and the enriched total diterpenoid components have important practical significance for developing high-efficiency low-toxicity anti-HIV drugs.
Disclosure of Invention
the invention aims to provide a preparation method of total diterpenoids and application thereof in pharmacy based on the current situation of the prior art, in particular to a method for enriching total diterpenoid components from Thymelaeaceae plants and application thereof in preparing anti-HIV drugs.
The technical scheme adopted by the invention for realizing the purpose is as follows:
A method for preparing anti-HIV total diterpenes from Ruixianaceae plants comprises the following steps:
(1) Mixing Ruixianaceae plant materials with organic solvent at a proper ratio, extracting for several times, mixing extractive solutions, concentrating, recovering solvent, and evaporating 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 content of petroleum ether: ethyl acetate: methanol: water (1-3: 1: 1-3: 0.4-2; v/v) or n-hexane: 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 a 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 diterpene according to a high performance liquid chromatography or mass spectrum of an effluent liquid, collecting and combining the corresponding effluent liquid, and concentrating to obtain the total diterpene;
(4) If the enrichment is continued, separating the total diterpenes obtained in the step (3) by high-speed counter-current chromatography, and performing high-speed counter-current chromatography by using n-hexane: acetonitrile: water (2: 0.5-1.3: 0.7-1.5; v/v) or petroleum ether: acetonitrile: taking water (2: 0.5-1.3: 0.7-1.5; 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 a chromatographic column of high-speed countercurrent chromatography with the stationary phase, injecting the mobile phase at a flow rate of 1-3 mL/min under the conditions of a rotation 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 diterpene components according to an ultraviolet spectrum or a mass spectrum of effluent liquid, collecting and combining eluents containing the diterpene components, and concentrating to obtain total diterpene with higher content;
(5) And (3) carrying out content determination and anti-HIV (human immunodeficiency virus) activity test on active ingredients such as lilac daphne ester C (1), diterpene ortho ester-12-benzoyloxy daphne toxin (2), lilac daphne ester E (3), lilac daphne ester G (4), lilac daphne ester Heji (5), shanaquilaria resina (6), diterpene ortho ester-12-propionyloxy daphne toxin (7), lilac daphne flower ester Heji (8), diterpene ortho ester-12-butyryloxy daphne toxin (9), isolilac daphne ester A (10), lilac daphne ester A (11), sabotoxin A (12) and calatoxin (13) on each sample obtained in the steps (1) - (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 improved to 0.0036-0.047 mu g/mL from 0.31-2.12 mu g/mL of the initial extract, the Selection Index (SI) is more than 2000, and the anti-HIV drug can be prepared.
The invention has the following beneficial effects:
The enrichment method of the total diterpene provided by the invention is simple, convenient and rapid, the recovery rate is higher, the obtained total diterpene component 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 Thymelaeaceae plants, 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 is a flow chart of the enrichment process of example 2.
FIG. 3 is a flow chart of the enrichment process of example 3.
FIG. 4 shows the structures of Daphne genkwa ester C (1), diterpene ortho ester-12-benzoyloxy daphnetoxin (2), Daphne genkwa ester E (3), Daphne genkwa ester G (4), Daphne genkwa ester Hei (5), shanxiangqi essence (6), diterpene ortho ester-12-propionyloxy daphnetoxin (7), Daphne genkwa ester Hei (8), diterpene ortho ester-12-butyryloxy daphnetoxin (9), isodaphnetin A (10), Daphne genkwa ester A (11), Sambutoxin A (12) and kura toxin (13).
FIG. 5 is a liquid chromatogram of each sample in the preparation process of daphne genkwa total diterpenes in example 1.
FIG. 6 is a liquid chromatogram of each sample during the preparation of Wikstroelia wikstroelia total diterpene in example 2.
FIG. 7 is a liquid chromatogram of each sample in the preparation of the total diterpenes of Euphorbia fischeriana in example 3.
Detailed Description
Example 1: method for enriching total diterpene in lilac daphne
1000g of lilac daphne flower bud medicinal material is taken, crushed by a crusher, added with 7L of methanol, heated, refluxed and extracted for 3 times, each time lasts for 1 hour, the extracting solutions are combined, concentrated and evaporated to dryness, and 202g of extract is obtained. Dispersing the extract in 3L warm water, extracting with petroleum ether for 4 times (3L each time) to obtain petroleum ether extract part 23.2g (DFC-DG-P). The obtained extract is enriched by high-speed counter-current chromatography for multiple times, and the content of petroleum ether: 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 petroleum ether with 1-2 times of column volume: ethyl acetate: methanol: eluting with water (2: 1: 1.5: 1.5; 2: 1: 2.1: 0.9; v/v) to obtain eluate, and determining chromatographic peak containing diterpene component by high performance liquid chromatography or mass spectrometry. The corresponding eluates were pooled and concentrated to give 1.93g of total diterpenes (DFC-DG-P2).
example 2: method for enriching total diterpene in wikstroelia
Collecting 1000g of wikstroma, pulverizing with a small-sized pulverizer, adding methanol, ultrasonically extracting for 3 times, each time for 30 minutes, filtering, concentrating, and evaporating to dryness to obtain 248g of extract (DFC-WC). Dispersing the extract in 3L warm water, extracting with n-hexane for 4 times, 3L each time, to obtain n-hexane extract parts each 17.5g (DFC-WC-P). 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: 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 normal hexane with the volume of 1-2 times of the column volume: ethyl acetate: methanol: eluting with water (2: 1: 1: 2; 2: 1: 2.1: 0.9; v/v), and determining chromatographic peak containing diterpene component by high performance liquid chromatography or mass spectrometry of the eluate. Collecting and combining the corresponding effluent, and concentrating to obtain 1.01g of wikstrom flower total diterpene (DFC-WC-P2).
Example 3: method for enriching total diterpene in Tianshan pseudo-wolfsbane
Pulverizing 1000g of radix Euphorbiae Fischerianae, adding 7L of 95% ethanol, reflux-extracting under heating for 3 times (1 hr each time), mixing extractive solutions, concentrating, and evaporating to obtain 192g of extract (DFC-ST). Dispersing the extract in 3L warm water, extracting with cyclohexane for 4 times (3L each time) to obtain cyclohexane extract 18.2g (DFC-ST-P). The obtained extract is enriched by high-speed counter-current chromatography for multiple times, and the content of petroleum ether: 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 petroleum ether with 1-2 times of column volume: ethyl acetate: methanol: eluting with water (2: 1: 1.5: 1.5; 2: 1: 2.1: 0.9; v/v) to obtain eluate, and determining chromatographic peak containing diterpene component by high performance liquid chromatography or mass spectrometry. The corresponding eluates were pooled and concentrated to give 1.06g of total diterpenes (DFC-ST-P2). Continuously carrying out high-speed countercurrent chromatography enrichment on total diterpenoids (DFC-ST-P2) of the stellera chamaejasme, and carrying out high-speed countercurrent chromatography enrichment on the total diterpenoids by using n-hexane: acetonitrile: taking water (2: 0.7: 1.3; 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 ℃, injecting samples, and respectively using n-hexane with the volume of 1-2 times of the column volume: acetonitrile: separating lower phase of water (2: 0.7: 1.3; 2: 0.9: 1.1; 2: 1.1: 0.9; v/v) as mobile phase, determining component chromatographic peak containing diterpene according to ultraviolet spectrum or mass spectrum of effluent, collecting eluate containing diterpene component, mixing eluates, and concentrating to obtain total diterpene 550mg (DFC-ST-P22) of radix Euphorbiae Fischerianae.
Example 4: method for enriching total diterpene in lilac daphne
100g of lilac daphne flower bud medicinal material is taken, crushed by a small crusher, added with 500mL of acetonitrile, extracted by flash for 5 times, each time for 3 minutes, filtered, concentrated and evaporated to dryness, and 13.2g of extract is obtained. Dispersing the extract in 300mL warm water, and extracting with ethyl acetate for 4 times, each time 300mL, to obtain 4.6g of ethyl acetate extract. 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: 1.7: 1.3; 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: 1.7: 1.3; 2: 1: 2.5: 0.5; v/v) to obtain eluate, and determining chromatographic peak containing diterpene component by high performance liquid chromatography or mass spectrometry. Collecting and mixing the corresponding effluent, and concentrating to obtain 131mg of flos Genkwa total diterpene.
example 5: method for enriching total diterpene in wikstroelia
Taking 100g of wikstrom medicinal material, crushing by a small-sized crusher, adding 500mL of acetone, percolating and extracting for 3 times, each time for 4 hours, concentrating and evaporating filtrate to dryness to obtain 14.1g of extract. Dispersing the extract in 300mL warm water, and extracting with n-hexane for 4 times, each time 300mL, to obtain ethyl acetate extraction parts each 1.1 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: 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 ℃, until the two-phase solvent reaches an equilibrium state in the column, injecting samples, and using 1-2 times of the volume petroleum ether: ethyl acetate: methanol: eluting with water (1: 1: 1; 1: 1: 1.3: 0.7; 1: 1: 1.6: 0.4; v/v) as mobile phase, and determining chromatographic peak containing diterpene component by high performance liquid chromatography or mass spectrometry of effluent. Collecting and mixing the corresponding effluent, and concentrating to obtain 59mg of wikstrom flower total diterpene.
Example 6: method for enriching total diterpene in Tianshan pseudo-wolfsbane
Taking 100g of the radix euphorbiae lantu, crushing by a small-sized crusher, adding 500mL of methanol, heating, refluxing and extracting for 3 times, each time for 1 hour, combining extracting solutions, concentrating and evaporating to dryness to obtain 18.7g of extract. Dispersing the extract in 300mL warm water, and extracting with dichloromethane for 4 times, each time 300mL, to obtain 3.6g of extract. The resulting extract was enriched by high speed counter current chromatography using n-hexane: 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 1-2 column volumes of n-hexane: ethyl acetate: methanol: eluting with water (3: 1: 1: 2; 3: 1: 2: 2; 3: 1: 3: 1; v/v) as mobile phase, and determining chromatographic peak containing diterpene component by high performance liquid chromatography or mass spectrometry of effluent. Collecting and mixing the corresponding effluent, and concentrating to obtain 94mg of total diterpene. The total diterpene of the stellera chamaejasme is continuously enriched by high-speed countercurrent chromatography, and the total diterpene is obtained by the steps of: acetonitrile: taking water (2: 0.5: 1.5; 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 a 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 1-2 times of column volume: acetonitrile: separating the lower phase of water (2: 0.5: 1.5; 2: 1: 1; 2: 1.5: 0.5; v/v) as mobile phase, determining chromatographic peak containing diterpene component according to ultraviolet spectrum or mass spectrum of effluent, collecting and combining eluates containing diterpene component, and concentrating to obtain total diterpene 43mg of radix Euphorbiae Fischerianae.
Example 7:
The content of the main diterpene in each sample of example 1, example 2 and example 3 was measured and the total diterpene was semi-quantified by UPLC-MS using daphne ester c (1), diterpene ortho ester-12-benzoyloxy daphnetoxin (2), daphne ester E (3), daphne ester hepta (4), isodaphne ester already (5), daphne ester already (6), linarin (7), diterpene ortho ester-12-propionyloxy daphnetoxin (8), daphne ester hexyl (9), diterpene ortho ester-12-butyryloxy daphnetoxin (10), isodaphne ester methyl (11), daphne ester methyl (12), sabotoxin a (13) and calatoxin (14) as control. 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; 45-70 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 sample yield (%) and diterpene component content (%)
remarking: the yield represents the extract extraction rate or the yield of each step of sample compared with the previous step of sample.
Example 8: anti-HIV activity test of each sample in pilot enrichment method of total diterpenes in Thymelaeaceae plants
The key active fractions obtained in examples 1, 2 and 3 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 enriched component is more than 64 percent, the sample shows extremely strong anti-HIV activity, the EC50 value can reach 0.0047 mu g/mL, the cytotoxicity is small, and the Selection Index (SI) is more than 2000.
TABLE 2 anti-HIV Activity and cytotoxicity test results of samples in Total diterpene enrichment method
Claims (6)
1. A method for preparing active total diterpenes in a Thymus plant is characterized by comprising the following steps:
(1) Pulverizing Rutaceae plant materials, mixing with organic solvent (methanol, ethanol, acetonitrile or acetone) at a proper ratio, extracting (heating reflux extraction, or flash extraction, or ultrasonic extraction, or percolation extraction) for several times, mixing extractive solutions, concentrating, recovering solvent, and evaporating to obtain extract.
(2) suspending the extract obtained in the step (1) with water, extracting with an organic solvent (petroleum ether, cyclohexane, n-hexane, ethyl acetate or dichloromethane) for several times, combining the extract solutions, concentrating, recovering the solvent and evaporating to dryness to obtain the extract.
(3) With petroleum ether (or n-hexane): ethyl acetate: methanol: 1-3 parts of water: 1: 1-3: and (3) mixing at a ratio of 0.4-2 (v/v), wherein the upper phase is a stationary phase, the lower phase is a mobile phase, separating the extract obtained in the step (2) by adopting high-speed counter-current chromatography, and collecting and combining diterpene fractions 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 (3) are further purified by high-speed counter-current chromatography.
6. The method of claim 1, wherein the total diterpenes obtained are used in the preparation of anti-HIV drugs.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252285A (en) * | 1998-10-23 | 2000-05-10 | 沈阳药科大学 | Preparation of AIDS resisting medicine from indian stringbush root extract |
| CN101531644A (en) * | 2009-02-18 | 2009-09-16 | 沈阳药科大学 | New daphnane diterpene compounds in Daphne genkwa as well as preparation method and application of same |
| CN101787004A (en) * | 2010-03-05 | 2010-07-28 | 云南民族大学 | Lignanoid compound contained in Yunnan daphne herb, as well as preparation method and application thereof |
| CN102180852A (en) * | 2011-01-24 | 2011-09-14 | 南京泽朗医药科技有限公司 | Method for extracting genkwanin from Daphne genkwa Sieb.et Zucc. |
| CN102344455A (en) * | 2011-08-13 | 2012-02-08 | 中国科学院昆明植物研究所 | Daphnane diterpene orthoester compound, medicinal composition thereof and preparation method and application thereof |
| CN102875506A (en) * | 2012-10-11 | 2013-01-16 | 南京泽朗医药科技有限公司 | Method for preparing daphnoretin by using high-speed counter-current chromatography |
| CN105198899A (en) * | 2014-06-30 | 2015-12-30 | 复旦大学 | 1-alkylated daphnane diterpene and application thereof to preparation of anti-HIV drugs |
| CN105287497A (en) * | 2014-06-30 | 2016-02-03 | 复旦大学 | Application of daphnetoxin diterpene in preparing anti-HIV drug |
| CN106176716A (en) * | 2016-07-15 | 2016-12-07 | 山西大学 | The new application of daphane diterpene compound pimelotide C |
| CN106432263A (en) * | 2015-08-11 | 2017-02-22 | 复旦大学 | Preparation method of total diterpenoids of stellera chamaejasme L. and application of total diterpenoids in medicine preparation |
-
2018
- 2018-05-29 CN CN201810531203.1A patent/CN110538243A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252285A (en) * | 1998-10-23 | 2000-05-10 | 沈阳药科大学 | Preparation of AIDS resisting medicine from indian stringbush root extract |
| CN101531644A (en) * | 2009-02-18 | 2009-09-16 | 沈阳药科大学 | New daphnane diterpene compounds in Daphne genkwa as well as preparation method and application of same |
| CN101787004A (en) * | 2010-03-05 | 2010-07-28 | 云南民族大学 | Lignanoid compound contained in Yunnan daphne herb, as well as preparation method and application thereof |
| CN102180852A (en) * | 2011-01-24 | 2011-09-14 | 南京泽朗医药科技有限公司 | Method for extracting genkwanin from Daphne genkwa Sieb.et Zucc. |
| CN102344455A (en) * | 2011-08-13 | 2012-02-08 | 中国科学院昆明植物研究所 | Daphnane diterpene orthoester compound, medicinal composition thereof and preparation method and application thereof |
| CN102875506A (en) * | 2012-10-11 | 2013-01-16 | 南京泽朗医药科技有限公司 | Method for preparing daphnoretin by using high-speed counter-current chromatography |
| CN105198899A (en) * | 2014-06-30 | 2015-12-30 | 复旦大学 | 1-alkylated daphnane diterpene and application thereof to preparation of anti-HIV drugs |
| CN105287497A (en) * | 2014-06-30 | 2016-02-03 | 复旦大学 | Application of daphnetoxin diterpene in preparing anti-HIV drug |
| CN106432263A (en) * | 2015-08-11 | 2017-02-22 | 复旦大学 | Preparation method of total diterpenoids of stellera chamaejasme L. and application of total diterpenoids in medicine preparation |
| CN106176716A (en) * | 2016-07-15 | 2016-12-07 | 山西大学 | The new application of daphane diterpene compound pimelotide C |
Non-Patent Citations (3)
| Title |
|---|
| JIZHONG YAN，ET AL: ""Preparative Isolation and Purification of Two Coumarins from Edgeworthia chrysantha Lindl by High Speed Countercurrent Chromatography"", 《JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES》 * |
| 王祖林，等。: ""高速逆流色谱在天然产物有效成分分离中的应用"", 《山东科学》 * |
| 陈道峰，等。: ""瑞香狼毒的生物活性成分研究"", 《全国药用植物和植物药学学术研讨会》 * |
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