CN1240292C - 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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- CN1240292C CN1240292C CN 200310108822 CN200310108822A CN1240292C CN 1240292 C CN1240292 C CN 1240292C CN 200310108822 CN200310108822 CN 200310108822 CN 200310108822 A CN200310108822 A CN 200310108822A CN 1240292 C CN1240292 C CN 1240292C
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Abstract
The present invention discloses a method for extracting and separating an insecticidal active substance from tripterygium wilfordii. The insecticidal active substance, specifically pure alkaloid, in the tripterygium wilfordii is extracted from the tissues of roots, stems, leaves and the like of the tripterygium wilfordii with the method. Besides, at least five different tripterygium wilfordii alkaloid monomers are further separated, and the purity can reach more than 95%. The method is implemented by the steps of preparation of tissue parts of the tripterygium wilfordii, the preparation of crude extract, the extraction of the coarse total alkaloid, the extraction of the pure total alkaloid, the separation of alkaloid monomers, etc. Besides, the method further tracks and detects the insecticidal activity of each alkaloid monomer and provides the technology for the toxicity research, the further development and the reasonable utilization of the tripterygium wilfordii.
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, are 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. as number of patent application; (separation of tripterygium total alkaloid and the insecticidal activity research Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology journal (natural science edition) that retrieve recently, 29 (2), the 61-64 page or leaf) disclose that a kind of tripterygium total alkaloid separates and the method for insecticidal activity research, but the document disclosed only be the concrete grammar that obtains pure product of thunder godvine total alkali and insecticidal activity research thereof from the velamen of Tripterygium wilfordii extraction; The process of extracting belongs to the method for traditional lixiviate or refluxing extraction, so its solvent load is big, extraction time is longer; Direct elutriation behind the thunder godvine alcohol extract very easily forms flocculent deposit or deposit seed is superfine, poor filtration effect, and yield is lower; This method is carried in the process in the alkaloidal acid of thunder godvine, directly adopts this meal of HCl solution diacolation, and its coarse biometric alkaline extraction yield is lower; This method only is to be target to extract the tripterygium total alkaloid simultaneously, can not be the difference between the further clear and definite thunder godvine different structure alkaloid monomer insecticidal activity, toxicologic study and further exploitation and rationally utilizing provide support.
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 step: the preparation of (1) thunder godvine plant tissue part: with thunder godvine radical center, root skin, stem skin, stem xylem or leaf cleaning, drying, standby after crushed; (2) preparation of thunder godvine crude extract: with (1) ready powder water or organic solvent, adopt normal temperature or add gentle conventional Soxhlet method and extract, extract adds water directly or after concentrating, and separates out solid, filters, dries; (3) extraction of thick total alkaloids of thunder godvine and total alkaloids: with the crude extract of (2) gained directly 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, is drying to obtain thick total alkaloids after the filtration; (4) extraction of the pure total alkaloids of thunder godvine: adopt the mixed liquor of percent by volume 0.1%-99.9% acetone and 99.9%-0.1% methyl alcohol to be recrystallized (3) gained total alkaloids and obtain; Wherein, described step (2) adopts modified Soxhlet method to extract (1) powder, and extract adds sodium chloride, potassium chloride, ammonium chloride or ammonium sulfate solution directly or after concentrating, and separates out solid, filters, dries; Described step (3) with (2) crude extract with non-pure organic solvent dissolution after, with the aqueous hydrochloric acid solution extraction of the aqueous sulfuric acid of percentage by weight 0.1%-40% or 0.1%-20% once or once, combining water layer, adjust its pH value to 6-12 with alkali or salt, separate out solid, filtration drying gets the thick total alkaloids of thunder godvine, and thick total alkaloids repeats once promptly to get the tripterygium total alkaloid by the same method; After getting the pure total alkaloids of thunder godvine, described step (4) carries out the separation of step (5) thunder godvine alkaloid monomer, with hydrochloric acid is fixing phase, 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 wilfordine (wilfordine), wilforine (wilforine), wilfornine (wilfomine), 5 kinds of different thunder godvine alkaloids of hot alkali of thunder godvine (wilforzine) and the special alkali (wilfortrine) of thunder godvine.
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;
The described non-pure organic solvent of step of the present invention (3) is one or more intermixtures in ethyl acetate, chloroform, carrene, benzinum, ether, cyclohexanone, cyclohexane and the n-hexane;
The described acid solution of step of the present invention (3) the be weight percentage aqueous sulfuric acid of 0.1-40% or the aqueous hydrochloric acid solution of 0.1-20%;
Described alkali of step of the present invention (3) 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 (5) 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 percent by volume 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: after getting 1 kilogram of root skin, drying, 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 570.7mg/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 (wilfomine) 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 (1)
1, the extraction of thunder godvine insecticide active substance and separating method comprise step: the preparation of (1) thunder godvine plant tissue part: with radical center, root skin, stem skin, stem xylem or the leaf cleaning, drying of thunder godvine, standby after crushed; (2) preparation of thunder godvine crude extract; (3) extraction of thick total alkaloids of thunder godvine and total alkaloids; (4) extraction of the pure total alkaloids of thunder godvine: adopt the mixed liquor of percent by volume 0.1%-99.9% acetone and 99.9%-0.1% methyl alcohol to be recrystallized (3) gained total alkaloids and obtain; It is characterized in that described step (2) adopts modified Soxhlet method to extract (1) powder, extract adds sodium chloride, potassium chloride, ammonium chloride or ammonium sulfate solution directly or after concentrating, and separates out solid, filters, dries; Described step (3) with (2) crude extract with non-pure organic solvent dissolution after, with the aqueous hydrochloric acid solution extraction of the sulfuric acid solution of percentage by weight 0.1%-40% or 0.1%-20% once or once, combining water layer, adjust its pH value to 6-12 with alkali or salt, separate out circle, filtration drying gets the thick total alkaloids of thunder godvine, and thick total alkaloids repeats once promptly to get the tripterygium total alkaloid by the same method; After getting the pure total alkaloids of thunder godvine, described step (4) carries out the separation of step (5) thunder godvine alkaloid monomer, with hydrochloric acid is fixing phase, 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 wilfordine (wilfordine), wilforine (wilforine), wilfornine (wilfornine), 5 kinds of different thunder godvine alkaloids of hot alkali of thunder godvine (wilforzine) and the special alkali (wilfortrine) of thunder godvine.
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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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| CN102793737B (en) * | 2011-05-26 | 2014-01-08 | 澳门科技大学 | Method for preparing powder of effective part of thunder god vine |
| KR101276241B1 (en) * | 2012-11-19 | 2013-06-26 | 류원희 | Manufacturing method of an 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 |
| 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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