CN1543804A - Method for extracting and separating insecticidal active substance from Tripterygium wilfordii - Google Patents
Method for extracting and separating insecticidal active substance from Tripterygium wilfordii Download PDFInfo
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- CN1543804A CN1543804A CNA2003101088223A CN200310108822A CN1543804A CN 1543804 A CN1543804 A CN 1543804A CN A2003101088223 A CNA2003101088223 A CN A2003101088223A CN 200310108822 A CN200310108822 A CN 200310108822A CN 1543804 A CN1543804 A CN 1543804A
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Abstract
The invention discloses a process for extracting and separating Tripterygium wilfordii insecticidal active compound, wherein pure alkaloid can be extracted and at least five different Tripterygium wilfordii alkaloid monomers can be further separated from Tripterygium wilfordii root, haulm and each tissues, the invention is carried out through preparation of Tripterygium wilfordii tissue portion, preparation of coarse extract, extraction of raw total alkaloid, extraction of pure total alkaloid, and isolation of alkaloid monomer.
Description
Technical field
The present invention relates to the preparation field of botanical pesticide, especially relate to the extraction and the separating method of thunder godvine insecticide active substance.
Background technology
Thunder godvine Tripterygium wilfordii Hook f. is the Celastraceae tripterygium plant, has another name called herba fibraureae recisae root, cabbageworm medicine, just is used for medical science and the various insects of control in China a long time ago, is one of famous medicinal and pesticide plant.Since the U.S. in 1936 extracts terpene pigment trypterygine (tripterine) from the thunder godvine root, so far extract more than 100 kind of composition, be mainly alkaloids, Diterpenes, triterpenes, sesquiterpenoids, dulcin, evonymine, cupreol, glucoside and sugar etc.Research about the thunder godvine active insecticidal components starts from the thirties in 20th century, priorities such as American Beroza are extracted from velamen of Tripterygium wilfordii and are separated to a series of macrolide alkaloid, and its insecticidal activity tested, find that these alkaloids have very strong cytotoxicity to the European corn borer newly hatched larvae.But real research aspect plant pesticide just obtains very big development in recent years, because of the thunder godvine complex chemical composition, the extraction of active ingredient or derivative and purifying difficulty are quite big, as number of patent application is that root skin powder and the crude extract thereof of thunder godvine plant are mainly still used in the patent application of CN01141888.5, CN00129820.8, CN03115750.5 etc., yet there are no the extraction of complete thunder godvine insecticide active substance and the report of separating method so far.
Summary of the invention
The extraction and the separating method that the purpose of this invention is to provide the thunder godvine insecticide active substance.
Purpose of the present invention is achieved by the following technical programs.
The extraction of thunder godvine insecticide active substance of the present invention and separating method comprise the steps:
(1) preparation of thunder godvine plant tissue part:, standby after crushed with radical center, root skin, stem skin, stem xylem or the leaf cleaning, drying of thunder godvine;
(2) preparation of thunder godvine crude extract: with (1) ready powder water or organic solvent, adopt normal temperature or add gentle routine or the extraction of modified Soxhlet method, extract is directly or after concentrated, add water or add sodium chloride, potassium chloride, ammonium chloride or ammonium sulfate solution, separate out solid, filter, dry;
(3) extraction of the thick total alkaloids of thunder godvine: with the crude extract of (2) gained directly or with behind the non-pure organic solvent dissolution, with the acid solution extraction once or once, combining water layer, water layer is adjusted the pH value to 6-12 with alkali or salt, separates out solid, filters the back drying;
(4) tripterygium total alkaloid extraction: with the thick total alkaloids of (3) gained with non-pure organic solvent dissolution after, with the acid solution extraction once or once, combining water layer, water layer is adjusted the pH value to 6-12 with alkali or salt, separates out solid, it is dry to filter the back;
(5) extraction of the pure total alkaloids of thunder godvine: adopt the mixed liquor of 0.1%-99.9% acetone and 99.9%-0.1% methyl alcohol to be recrystallized the total alkaloids of (4) gained and obtain;
(6) separation of thunder godvine alkaloid monomer: the pure total alkaloids of (5) gained is carried out column chromatography for separation, with in silica gel, aluminium oxide and the hydrochloric acid any for fixing mutually, be aided with flowing phase and carry out wash-out, substep is collected, detect through high performance liquid chromatogram, fling to flowing phase, merge the part that contains identical component, drying obtains 5 kinds of different thunder godvine alkaloids.
The described organic solvent of step of the present invention (2) is any among methyl alcohol, ethanol, ethyl acetate, chloroform, carrene, benzinum, acetone, ether, cyclohexanone, cyclohexane and the n-hexane;
Step of the present invention (3), (4) described non-pure organic solvent are one or more intermixtures in ethyl acetate, chloroform, carrene, benzinum, ether, cyclohexanone, cyclohexane and the n-hexane;
Step of the present invention (3), (4) described acid solution are the aqueous sulfuric acid of 0.1-40% or the aqueous hydrochloric acid solution of 0.1-20%;
Step of the present invention (3), (4) described alkali or salt are any in sodium hydroxide, potassium hydroxide, ammoniacal liquor, sodium carbonate, sodium bicarbonate, potash, saleratus, calcium carbonate or the calcium bicarbonate;
The described flowing phase of step of the present invention (6) is one or more mixed solvents in methyl alcohol, ethanol, ethyl acetate, chloroform, carrene, benzinum, acetone, ether, cyclohexanone, cyclohexane and the n-hexane; Described more than one mixed solvent flowing phases, the content of each one-component all can be 0.1%-99.9%, and the total amount of each component is 100%.
Beneficial effect of the present invention, the one, adopt complete thunder godvine alkaloid extraction and the separating method of a cover provided by the invention, can effectively extract and be separated at least 5 kinds of different thunder godvine alkaloid monomers, and purity all can reach more than 95%; The 2nd, tracking and measuring of the present invention each alkaloid monomer of above-mentioned thunder godvine to food refusal effect, stomach poison function and the effect of growing under selectivity such as lepidoptera pest such as cabbage caterpillar, diamond-back moth and striped rice borer, pink rice borer and the non-selective condition, thereby the difference between the clear and definite insecticidal activity of each alkaloid monomer, for each alkaloidal toxicologic study of thunder godvine and thunder godvine as the further exploitation of botanical pesticide with the technology that provides rationally is provided.
Embodiment
Below in conjunction with embodiment the inventive method is further described.
Embodiment 1: get 1 kilogram of velamen of Tripterygium wilfordii, after drying, the pulverizing, extract with the ethyl acetate Soxhlet.Extract is separated out solid after concentrating, and filters, dries, and obtains the thunder godvine crude extract.After this crude extract was dissolved with chloroform, with 8% hydrochloric acid solution extraction four times, combining water layer, water layer was adjusted pH value to 10 with sodium hydroxide, separates out solid, and filtration, drying obtain the thick total alkaloids of thunder godvine.With this thick total alkaloids of chloroform dissolving, 0.1% hydrochloric acid solution extracts four times again, combining water layer, and water layer is adjusted pH value to 8 with sodium hydroxide, separates out solid, and filtration, drying obtain the tripterygium total alkaloid.The tripterygium total alkaloid is recrystallized through 0.1% acetone and 99.9% methyl alcohol mixed solvent and obtains pure thunder godvine alkaloid.
Above-mentionedly obtain pure biology total alkali and carry out column chromatography for separation.Chromatographic column is fixing to be silica gel mutually, and flowing phase is methyl alcohol and ether (1: 1), and through the flowing phase wash-out, fraction collection detects through high performance liquid chromatogram, flings to flowing phase.Wherein the 19-22 pipe merges, carry out column chromatography for separation again, fixing is silica gel mutually, and flowing phase is ethyl acetate and chloroform (11: 1), through the flowing phase wash-out, fraction collection, detect through high performance liquid chromatogram, fling to flowing phase, merge the 11-13 pipe, drying obtains wilfordine (wilfordine) 480mg.After measured to the selective conditions AFC of diamond-back moth
50(concentration in the food refusal) is 82.7mg/L, non-selective condition AFC
50Be 170.1mg/L, lethal concentration of 50 LC during 96h
50Be 122mg/L, concentration is 161.4mg/L in the inhibition of growing; Under the selective conditions, to the AFC of Chilo spp larvae
50Be 46.1mg/L, under non-selective condition, to the AFC of Chilo spp larvae
50Be 188.3mg/L, LC
50Be 326.5mg/L, growing inhibiting rate is more than 88.79%.
Embodiment 2: get 1 kilogram of root skin, after drying, the pulverizing, extract with the ethanol Soxhlet.Extract adds water after concentrating, and separates out solid, filters, dries, and obtains the thunder godvine crude extract.After this crude extract was dissolved with chloroform, with 20% hydrochloric acid solution extraction four times, combining water layer, water layer was adjusted pH value to 10 with sodium bicarbonate solution, separates out solid, and filtration, drying obtain the thick total alkaloids of thunder godvine.With this thick total alkali of chloroform dissolving, 0.5% hydrochloric acid solution extracts four times again, combining water layer, and water layer is adjusted pH value to 8 with sodium hydroxide, separates out solid, and filtration, drying obtain the tripterygium total alkaloid.The tripterygium total alkaloid is recrystallized through 99.9% acetone and 0.1% methyl alcohol mixed solvent and obtains pure thunder godvine alkaloid.
Above-mentionedly obtain pure biology total alkali and carry out column chromatography for separation.Chromatographic column is fixing to be silica gel mutually, and flowing phase is methyl alcohol and ether (1: 1), and through the flowing phase wash-out, fraction collection detects through high performance liquid chromatogram, flings to flowing phase.Wherein the 25-28 pipe merges, carry out column chromatography for separation again, fixing is silica gel mutually, and flowing phase is ethyl acetate and chloroform (1: 19), through the flowing phase wash-out, fraction collection, detect through high performance liquid chromatogram, fling to flowing phase, merge the 15-16 pipe, drying obtains wilforine (wilforine) 220mg.After measured to the selective conditions AFC of diamond-back moth
50(concentration in the food refusal) is 483.6mg/L, non-selective condition AFC
50Be 670.5mg/L, lethal concentration of 50 LC during 96h
50Be 570.7 mg/L, concentration is 612.5mg/L in the inhibition of growing; Under the selective conditions, to the AFC of Chilo spp larvae
50Be 146.1mg/L, under non-selective condition, to the AFC of Chilo spp larvae
50Be 388.3mg/L, LC
50Be 826.5mg/L, growing inhibiting rate is more than 60.79%.
Embodiment 3: get 1 kilogram of root skin, after drying, the pulverizing, use extracted by ether.Extract is separated out solid after concentrating, and filters, dries, and obtains the thunder godvine crude extract.After this crude extract was dissolved with chloroform, with 10% sulfuric acid solution extraction four times, combining water layer, water layer was adjusted pH value to 10 with ammoniacal liquor, separates out solid, and filtration, drying obtain the thick total alkaloids of thunder godvine.With this thick total alkali of chloroform dissolving, 1% hydrochloric acid solution extracts four times again, combining water layer, and water layer is adjusted pH value to 8 with sodium hydroxide, separates out solid, and filtration, drying obtain the tripterygium total alkaloid.The tripterygium total alkaloid is recrystallized through 30% acetone and 70% methyl alcohol mixed solvent and obtains pure thunder godvine alkaloid.
Above-mentionedly obtain pure biology total alkali and carry out column chromatography for separation.Chromatographic column is fixing to be silica gel mutually, and flowing phase is methyl alcohol and ether (1: 1), and through the flowing phase wash-out, fraction collection detects through high performance liquid chromatogram, flings to flowing phase.Wherein the 35-37 pipe merges, carry out column chromatography for separation again, fixing is silica gel mutually, and flowing phase is ethyl acetate and chloroform (2: 15), through the flowing phase wash-out, fraction collection, detect through high performance liquid chromatogram, fling to flowing phase, merge the 22-23 pipe, drying obtains wilfornine (wilfornine) 280mg.After measured to the selective conditions AFC of diamond-back moth
50(concentration in the food refusal) is 117.6mg/L, non-selective condition AFC
50Be 234.2mg/L, lethal concentration of 50 LC during 96h
50Be 211.4mg/L, concentration is 285.0mg/L in the inhibition of growing; Under the selective conditions, to the AFC of Chilo spp larvae
50Be 95.7mg/L, under non-selective condition, to the AFC of Chilo spp larvae
50Be 266.5mg/L, LC
50Be 402.3mg/L, growing inhibiting rate is more than 74.5%.
Embodiment 4: get 1 kilogram of tripterygium leaf, after drying, the pulverizing, use methanol extraction.Extract concentrates the back step with example 1, wherein the 26-29 pipe merges, and carries out column chromatography for separation again, and fixing is aluminium oxide mutually, flowing phase is ethyl acetate and chloroform (5: 1), through the flowing phase wash-out, fraction collection detects through high performance liquid chromatogram, fling to flowing phase, merge the 28-30 pipe, drying obtains hot alkali (wilforzine) 46mg of thunder godvine.After measured to cabbage caterpillar have anesthesia and stomach poison function, get food on a small quantity after, polypide motionless and slowly begin the blackout; The selective conditions AFC of diamond-back moth
50(concentration in the food refusal) is 286.3mg/L, non-selective condition AFC
50Be 321.6mg/L, lethal concentration of 50 LC during 96h
50Be 482.7mg/L, growing inhibiting rate is more than 86%.
Embodiment 5: get 1 kilogram of root skin, after drying, the pulverizing, use alcohol extract.Extract is separated out solid after concentrating, and filters, dries, and obtains the thunder godvine crude extract.After this crude extract was dissolved with chloroform, with 40% sulfuric acid solution extraction four times, combining water layer, water layer was adjusted pH value to 10 with ammoniacal liquor, separates out solid, and filtration, drying obtain the thick total alkaloids of thunder godvine.With this thick total alkali of ether dissolution, 0.1% hydrochloric acid solution extracts four times again, combining water layer, and water layer is adjusted pH value to 8 with sodium hydroxide, separates out solid, and filtration, drying obtain the tripterygium total alkaloid.The tripterygium total alkaloid is recrystallized through 50% acetone and 50% methyl alcohol mixed solvent and obtains pure thunder godvine alkaloid.
Above-mentionedly obtain pure biology total alkali and carry out column chromatography for separation.Chromatographic column is fixing to be aluminium oxide mutually, and flowing phase is methyl alcohol and ether (1: 1), and through the flowing phase wash-out, fraction collection detects through high performance liquid chromatogram, flings to flowing phase.Wherein the 35-37 pipe merges, carry out column chromatography for separation again, fixing is silica gel mutually, and flowing phase is ether and chloroform (2: 15), through the flowing phase wash-out, fraction collection, detect through high performance liquid chromatogram, fling to flowing phase, merge the 27-29 pipe, drying obtains special alkali (wilfortrine) 165mg of thunder godvine.After measured to the selective conditions AFC of diamond-back moth
50(concentration in the food refusal) is 135.6mg/L, non-selective condition AFC
50Be 220.3mg/L, lethal concentration of 50 LC during 96h
50Be 282.6mg/L, concentration is 235.1mg/L in the inhibition of growing; Under the selective conditions, to the AFC of Chilo spp larvae
50Be 115.7mg/L, under non-selective condition, to the AFC of Chilo spp larvae
50Be 245.0mg/L, LC
50Be 416.2mg/L, growing inhibiting rate is more than 80%.
Claims (7)
1, the extraction of thunder godvine insecticide active substance and separating method is characterized in that comprising the steps and technology:
(1) preparation of thunder godvine plant tissue part:, standby after crushed with radical center, root skin, stem skin, stem xylem or the leaf cleaning, drying of thunder godvine;
(2) preparation of thunder godvine crude extract: with (1) ready powder water or organic solvent, adopt normal temperature or add gentle routine or the extraction of modified Soxhlet method, extract is directly or after concentrated, add water or add sodium chloride, potassium chloride, ammonium chloride or ammonium sulfate solution, separate out solid, filter, dry;
(3) extraction of the thick total alkaloids of thunder godvine: with the crude extract of (2) gained directly or with behind the non-pure organic solvent dissolution, with the acid solution extraction once or once, combining water layer, water layer is adjusted the pH value to 6-12 with alkali or salt, separates out solid, filters the back drying;
(4) tripterygium total alkaloid extraction: with the thick total alkaloids of (3) gained with non-pure organic solvent dissolution after, with the acid solution extraction once or once, combining water layer, water layer is adjusted the pH value to 6-12 with alkali or salt, separates out solid, it is dry to filter the back;
(5) extraction of the pure total alkaloids of thunder godvine: adopt the mixed liquor of 0.1%-99.9% acetone and 99.9%-0.1% methyl alcohol to be recrystallized the total alkaloids of (4) gained and obtain;
(6) separation of thunder godvine alkaloid monomer: the pure total alkaloids of (5) gained is carried out column chromatography for separation, with in silica gel, aluminium oxide and the hydrochloric acid any for fixing mutually, be aided with flowing phase and carry out wash-out, substep is collected, detect through high performance liquid chromatogram, fling to flowing phase, merge the part that contains identical component, drying obtains 5 kinds of different thunder godvine alkaloids.
2,, it is characterized in that the described organic solvent of step (2) is any in methyl alcohol, ethanol, ethyl acetate, chloroform, carrene, benzinum, acetone, ether, cyclohexanone, cyclohexane and the n-hexane according to the described method of claim 1;
3,, it is characterized in that step (3), (4) described non-pure organic solvent are one or more arbitrary proportion mixed liquors in ethyl acetate, chloroform, carrene, benzinum, ether, cyclohexanone, cyclohexane and the n-hexane according to the described method of claim 1;
4,, it is characterized in that step (3), (4) described acid solution are the sulfuric acid solution of 0.1%-40% or the hydrochloric acid solution of 0.1%-20% according to the described method of claim 1;
5,, it is characterized in that step (3), (4) described alkali or salt are any in sodium hydroxide, potassium hydroxide, ammoniacal liquor, sodium carbonate, sodium bicarbonate, potash, saleratus, calcium carbonate or the calcium bicarbonate according to the described method of claim 1;
6,, it is characterized in that the described flowing phase of step (6) is one or more mixed solvents compositions in methyl alcohol, ethanol, ethyl acetate, chloroform, carrene, benzinum, acetone, ether, cyclohexanone, cyclohexane and the n-hexane according to the described method of claim 1;
7, according to the described method of claim 6, it is characterized in that described more than one mixed solvent flowing phases, the content of each one-component all can be 0.1%-99.9%, and the total amount of each component is 100%.
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| CN 200310108822 CN1240292C (en) | 2003-11-25 | 2003-11-25 | Method for extracting and separating insecticidal active substance from Tripterygium wilfordii |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793737A (en) * | 2011-05-26 | 2012-11-28 | 澳门科技大学 | Method for preparing powder of effective part of thunder god vine |
| CN103814969A (en) * | 2012-11-19 | 2014-05-28 | 柳喆熙 | Method for preparing insect repellent |
| CN107873742A (en) * | 2017-12-06 | 2018-04-06 | 昭平县科学技术实验场 | Composite insecticide containing wilfordine and camptothecine and preparation method thereof |
| CN110447665A (en) * | 2018-05-07 | 2019-11-15 | 周帆 | A kind of crops pesticide and its manufacturing method |
| CN111484485A (en) * | 2019-05-16 | 2020-08-04 | 江西中医药大学 | Anti-inflammatory macrocyclic polyamine alkaloid celacarfurine |
-
2003
- 2003-11-25 CN CN 200310108822 patent/CN1240292C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793737A (en) * | 2011-05-26 | 2012-11-28 | 澳门科技大学 | Method for preparing powder of effective part of thunder god vine |
| CN103814969A (en) * | 2012-11-19 | 2014-05-28 | 柳喆熙 | Method for preparing insect repellent |
| CN107873742A (en) * | 2017-12-06 | 2018-04-06 | 昭平县科学技术实验场 | Composite insecticide containing wilfordine and camptothecine and preparation method thereof |
| CN110447665A (en) * | 2018-05-07 | 2019-11-15 | 周帆 | A kind of crops pesticide and its manufacturing method |
| CN111484485A (en) * | 2019-05-16 | 2020-08-04 | 江西中医药大学 | Anti-inflammatory macrocyclic polyamine alkaloid celacarfurine |
| CN111484485B (en) * | 2019-05-16 | 2022-04-05 | 江西中医药大学 | Anti-inflammatory macrocyclic polyamine alkaloid celacarfurine |
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| CN1240292C (en) | 2006-02-08 |
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