CN107501199A - The extraction of fluorine thunder Rana monomeric compound and purification process - Google Patents
The extraction of fluorine thunder Rana monomeric compound and purification process Download PDFInfo
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- CN107501199A CN107501199A CN201710701591.9A CN201710701591A CN107501199A CN 107501199 A CN107501199 A CN 107501199A CN 201710701591 A CN201710701591 A CN 201710701591A CN 107501199 A CN107501199 A CN 107501199A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
- C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
- C07D261/04—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
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Abstract
The present invention discloses extraction and the purification process of a kind of fluorine thunder Rana monomeric compound, comprises the following steps：1) after Bravecto preparations are ground into powder, soxhlet extraction is carried out with petroleum ether, dichloromethane, methanol until phegma is clarified successively, low pressure rotary evaporation obtains crude extract respectively by dichloromethane extract or three kinds of extract solutions；2) silica gel with the quality such as crude extract is weighed, chromatographic column is poured into after petroleum ether dissolution, is designated as 1 height unit, adds the silica gel of 6~8 height units；3) crude extract for obtaining step 1) is added in chromatographic column；4) with different volumes than petroleum ether ethyl acetate mixture carry out chromatography, obtain target product.The method of the invention, which can succeed to extract from Bravecto, obtains the fluorine thunder Rana compound of high-purity, the problems such as meeting to examine the needs of problems of the compound or detection.
Description
Technical field
The present invention relates to pharmaceutical chemistry and biomedicine field, in particular to a kind of Xin Xing isoxazole compounds
The extraction of fluorine thunder Rana and purification process and its application in production, life.
Background technology
Iso-oxazoline (Isoxazoline) compound is in research phthalic acid formamide (phthalic
Diamide a kind of new type disinsection activity) and during anthranilic diamides (anthranilic diamide) obtained
Material.At present, the Research Team of world community, such as Japan, US and European area, have deployed to grind such compound
The research of hair and its mechanism of action.
Xin Xing isoxazole compounds fluorine thunders Rana, it is characterised in that：Research and development code name is A1443, chemical name 4,5-
[(3,5- dichlorophenyl) -4,5- dihydro -5- trifluoromethyl -3- isoxazolyls] -2- methyl-nitrogen-[2- oxos -2- [(2,2,2- tri-
Fluoro ethyl) amino] ethyl]-benzamide, there is enantiomter structure, its chemical structural formula is：
Fluorine thunder Rana research and develop course approximately as：2004, Japanese Nissan chemical industry M Co., Ltd. ita etc. was successfully closed
Into fluorine thunder Rana.2010, Ozoe etc. had studied in detail the part biological and electrophysiologic activity of fluorine thunder Rana.2013, Garc í
A-Reynaga etc. synthesized radioactive ligand [3H] fluorine thunder Rana, for study fluorine thunder Rana γ-aminobutyric acid (GABA) by
The action site and mechanism of body.2014, the comparative studies such as Zhao (the first inventor) fluorine thunder Rana and the like was in elder brother
Worm and the action site of mammal GABA acceptors；Gassel etc. and Rohdich etc. reports fluorine thunder Rana to lice and flea
Bioactivity；German animal health innovation research researcher dog has been cooked using fluorine thunder Rana serial " clinical test ", inspection
Bioactivity of the fluorine thunder Rana to dog parasite and toxicity to dog etc. are surveyed；The same year, fluorine thunder Rana was by U.S.'s food and medicine
Surveillance Authority's approval listing (trade name Bravecto) is sold in European ＆ American Market, and quarterly sales revenue is up to 100,000,000 dollars.
Fluorine thunder Rana is mainly played a role by disturbing GABA to gate chloride channel, with cyclopentadiene, Phenylpyrazole
The action target of the insecticides such as class and macrolides is similar.A kind of brand-new GABA doors have been started in the successful research and development of fluorine thunder Rana
Chloride channel agent interfering research direction is controlled, causes animal medicine and the concern and favor of pesticide science worker.Fluorine thunder is drawn
It is a kind of broad spectrum insecticide to receive, and good desinsection is respectively provided with to insects such as tick mesh, Siphonaptera, Anoplura, Semiptera and Dipteras and is lived
Property, its virulence are higher than or suitable with common insecticide.Activity of the fluorine thunder Rana to comb head flea (Ctenocephalides felis)
Better than conventional pesticides ethiprole and imidacloprid；It is higher than ethiprole, Di Shi for the activity of lucilia cuprina (Lucilia cuprina)
Agent, imidacloprid and decis；It is far above bromine cyanogen chrysanthemum for the activity of boophilus microplus (Rhipicephalus microplus)
Ester, resist small brown rice planthopper (Laodelphax striatellus) and the Tetranychus urticae (Tetranychus urticae) of ethiprole
Insecticidal activity it is also preferable.Fluorine thunder Rana is not only with existing insecticide without significant cross resistance, or even to partial resistance insect
Also there is preferable insecticidal activity.Du pont company Lahm etc. reports a series of isoxazoles similar with fluorine thunder Rana structure
Quinoline class compound, such compound is to Lepidoptera, such as Spodopterafrugiperda (Spodoptera frugiperda) and bollworm
(Helicoverpa zea)；Homoptera, such as the micro- leaf of silkworm (Empoasca fabae) and Thysanoptera, such as Frankliniella occidentalis
Insects such as (Frankliniella occidentalis) shows good insecticidal activity.In addition, Zhao (the first applications
People) etc. found by radioactive ligand competitive combination test, fluorine thunder Rana the people GABA of in-vitro recombination expression is gated chlorine from
Subchannel (subunits of β 3) is almost without effect.
At present, commercialization running has been completed in fluorine thunder Rana as veterinary drug, is applied to the preventing and treating of parasite.From 2013 extremely
The present, German animal health innovation research researcher series of experimental research has been carried out to fluorine thunder Rana, using oral
The methed comparision of feeding fluorine thunder Rana, ethiprole (Frontline) and decis (Scalibor) are to dog parasite
Comb head flea and the prevention effect of Dermacentor variabilis.As a result show, fluorine thunder Rana has to comb head flea and tick extraordinary kills off effect
Fruit.2014, Merck ＆ Co., Inc. was commercially produced using fluorine thunder Rana as veterinary drug Bravecto first, was posted for killing dog
Infested louse and flea etc..A series of research shows that the medicine can be absorbed rapidly by dog body, have quick, long half time,
Lasting period is long and the features such as metabolic rate is low.The medicine has obtained the Licensing Authority of U.S. FDA at present, and in the country such as Europe and the U.S.
Start selling and promote, it is contemplated that it also points the day and await for it in the popularization of Asia or even China.
Fluralaner is mainly sent out as a kind of new isoxazole class insecticide by disturbing GABA to gate chloride channel
The effect of waving, it is similar with the action target of the insecticide such as cyclopentadiene, phenyl pyrazoles and macrolides.But current grind
Study carefully discovery, traditional GABA acceptors target insecticide such as fluralaner and ethiprole, ethiprole, butene-fipronil, AVM
Mechanism of action is different, and the research and discovery for its mechanism of action have extremely important effect and meaning in insect resistance
Justice.Fluralaner monomeric compounds are different from Bravecto preparations, have its specific Research Significance in experimental study.Mesh
Before, fluralaner monomeric compounds are in great demand in experimental study, but there is no method buying on the market, and synthesize road
Line is complicated, and technical difficulty is high.Therefore, fluralaner acquisition turns into urgent problem to be solved.
Bravecto is a kind of new drug and composition is more complicated, and wherein fluralaner content only has 14%, passes through HPLC-
TOF-MS detections find the impurity Ji Weijiejined with fluralaner polarity wherein be present, therefrom extract high-purity
Fluralaner difficulty is larger, therefore, how to separate the impurity Ji Weijiejined with fluralaner polarity, obtains the change of high-purity
Compound fluorine thunder Rana, meet that scientific research and other needs for such as detecting inspection etc. are the urgent problems to be solved that presently, there are.
The content of the invention
The purpose of the present invention essentially consists in offer one kind and its effective active composition is simply, efficiently extracted from Bravecto
The method of fluorine thunder Rana monomeric compound.This method, which can succeed to extract from Bravecto, obtains the fluorine thunder Rana of high-purity
Compound, the problems such as meeting to examine the needs of problems of the compound or detection.
The extraction of fluorine thunder Rana monomeric compound provided by the invention and purification process, comprise the following steps：
1) after Bravecto preparations are ground into powder, successively with petroleum ether, dichloromethane, methanol carry out soxhlet extraction until
Phegma is clarified, and by dichloromethane extract or three kinds of extract solutions, low pressure rotary evaporation obtains crude extract respectively；
2) silica gel with the quality such as crude extract is weighed, chromatographic column is poured into after petroleum ether dissolution, is surveyed after silica gel completely precipitation
Height of the silica gel in chromatographic column is measured, is designated as 1 height unit, then the silica gel of 6~8 height units is added into chromatographic column；
3) crude extract for obtaining step 1) is added in chromatographic column；
4) with volume ratio it is successively (7.8~8.2):1st, (5.8~6.2):1st, (3.8~4.2):1st, (2.8~3.2):1、
(1.8~2.2):1st, (0.8~1.2):1、2:The petroleum ether-ethyl acetate mixed solution of (2.8~3.2) carries out chromatography,
Obtain target product.
Petroleum ether, dichloromethane, methanol must be selected to carry out soxhlet extraction in step 1) of the present invention successively, it is molten using other
Agent and different sequence of extraction, which carry out surname extraction, can not then reach more completely extraction effect, and be difficult to wherein property is similar
Impurity separates.Inventor detects through HPLC-TOF-MS, and in the present invention, petroleum ether carries out surname extraction can will be big in Bravecto
Partial impurity, including impurity similar in polarity and a small amount of fluralaner are extracted, and then carry out Soxhlet with dichloromethane
Extraction can not extract a small amount of in Bravecto including impurity similar in polarity and most fluralaner, finally
Surname extraction is carried out with methanol remaining fluralaner in Bravecto and a small amount of impurity can be extracted.
Preferably, during step 1) soxhlet extraction of the present invention, petroleum ether Extracting temperature is 85~90 DEG C；Dichloromethane carries
It is 59~64 DEG C to take temperature；Methanol Extracting temperature is 87~93 DEG C.It can not be carried out if less than extract solution if said temperature scope
Extraction, can cause extract solution to volatilize, cause to waste, or extract solution largely overflows from condenser pipe if above said temperature scope
Go out, cause certain dangerous and waste.It is preferred that petroleum ether Extracting temperature is 88 DEG C, dichloromethane Extracting temperature is 61 DEG C, and methanol carries
It is 90 DEG C to take temperature.
In step 2) of the present invention, from the silica gel of 7~9 height units, obtained fluralaner purity highests are chromatographed,
Impurity content is minimum.If changing the amount of silica gel, the fluralaner purity for chromatographing to obtain can reduce, and use petroleum ether-
The efficiency that ethyl acetate mixture rinses can reduce.Preferably, from the silica gel of 8 height units.
Preferably concrete operations mode is step 3) of the present invention：It is molten that the crude extract that step 1) is obtained adds dichloromethane
Solution, adds the silica gel with the quality such as crude extract, and drying and grinding is slowly added in chromatographic column into powdered.The present invention is by step
1) crude extract obtained adds dichloromethane dissolving, adds the silica gel with the quality such as crude extract, can make silica gel and crude extract
What is mixed is more uniform, while combines silica gel and crude extract, the material just meeting similar in polarity so subsequently when being chromatographed
Chromatographed with buffer solution, so as to which fluorine thunder Rana and other impurities be separated in the wash buffer of different proportion.
Preferably, the temperature dried in step 3) of the present invention is 50~55 DEG C.It can not pass through heating if temperature is too low
The mode of evaporation removes liquid；If temperature is too high, with the progress of heating, when the material in surface plate tends to be thick
When can largely spill, cause the loss of medicine, and possess certain danger, such as splash and scalded on skin.
Preferably, the filtrate containing target product that chromatography obtains in step 4) of the present invention, vacuum filtration, drying are ground
Mill can obtain target product.
Silica gel of the present invention is preferably 100~200 mesh.
To achieve the above object, the technical solution adopted in the present invention mainly includes the following steps that：
(1) by Bravecto with high-speed multifunctional pulverizer crush, with petroleum ether under 88 DEG C of water bath conditions surname extraction
Untill petroleum ether extraction phegma clarification；Medicinal powder is continuing with dichloromethane surname extraction under 61 DEG C of water bath conditions, directly
Untill dichloromethane extraction phegma clarification；Medicinal powder is continuing with methanol surname extraction under 90 DEG C of water bath conditions, until first
Untill alcohol extracting phegma is clarified；
(2) respectively by dichloromethane or dichloromethane, petroleum ether, methanol extract low pressure on a rotary evaporator respectively
Rotation is evaporated, and obtains the quality that rotation is evaporated rear material；
(3) silica gel of the mesh of the quality such as material 100~200 is weighed and be evaporated, with being poured into after petroleum ether dissolution in chromatographic column, is treated
Height of the silica gel in chromatographic column is measured with graduated scale after silica gel precipitation completely, is designated as 1 height unit, then add into chromatographic column
Enter the silica gel of 7 height units；
(4) material after being evaporated will be rotated and adds q. s. methylene chloride, it is completely dissolved in flask, pour into evaporating dish
It is interior, the silica gel of quality such as add, on 50~55 DEG C of water-bath heating be evaporated to being completely dried in mortar, then fully grind
Mill, makes it become powdered completely, is slowly added in chromatographic column；
(5) dose volume ratio is 8:1 petroleum ether-ethyl acetate mixed solution is separated, under being filtered in chromatographic column
Liquid in rotation is evaporated, and is then dissolved with the solution, is detected in uv analyzer after putting plate and is compared with standard items, chromatography is always
Proceed to after rotary evaporation that there is no solid matter precipitation；
(6) 6 are prepared respectively:1、4:1、3:1、2:1、1:1、2:3 petroleum ether-ethyl acetate mixed solutions, sequentially add layer
It is rinsed in analysis post；Liquid low rotation under being filtered in chromatographic column is evaporated, and is then dissolved with corresponding mixed solution, puts plate
Detect in uv analyzer and compared with standard items afterwards；Chromatography is performed until rotary evaporation there is no solid matter precipitation,
Then next gradient solution chromatography is changed；
(7) purpose product that chromatography obtains is poured into glass suction funnel to be filtered by vacuum, obtained product is poured into
In glass culture dish, grind into powder after 0.5-1.5h is dried.
The product of the present invention can be detected by HPLC-TOF-MS, so that it is determined that its purity.
The present invention extracts the fluorine thunder Rana compound of high-purity from Bravecto first, and method is simple and efficient, has
Stronger practical value and realistic meaning.Relative to prior art, there is following advantage：
1) based on the simple compound extracting methods such as soxhlet extraction, low pressure rotary evaporation, chromatography post separation, Ke Yijian
Just the effective active composition fluorine thunder Rana compound in Bravecto, is quickly extracted；Extraction process only needs soxhlet extraction, rotation
The operations such as evaporation, chromatography post separation, vacuum filtration, HPLC-TOF-MS detections, it is simple and efficient；
2) from petroleum ether-ethyl acetate mixed solution carry out chromatographic column filtering, high recycling rate few using solvent,
Efficient economizing；In addition, vacuum filtration is simple to operate, purification effect is good, and obtained compound purity is high；
3) present invention carries out surname extraction successively according to the order of petroleum ether, dichloromethane, methanol：Rope is carried out with petroleum ether
Family name's extraction can extract most impurity in Bravecto, including impurity similar in polarity and a small amount of fluralaner,
Then carrying out surname extraction with dichloromethane can be by a small amount of nonpolarity similar impurity in Bravecto and most
Fluralaner is extracted, and finally carrying out surname extraction with methanol can be by remaining fluralaner in Bravecto and a small amount of
Impurity extract.More completely extraction effect can not then be reached by carrying out shown extraction using other solvents and different sequence of extraction
Fruit, and HPLC-TOF-MS detections find that the similar impurity of property can not be separated；
4) the method for the invention fluorine thunder Rana that only the further extraction purification in dichloromethane surname extraction part obtains
Monomeric compound detects purity up to more than 90% under mass spectrum, and liquid chromatographic detection high purity 100%, recovery rate is 60%
~70%；If plus the part of petroleum ether and methanol surname extraction, recovery rate can reach 80% or so.
5) purpose thing that the present invention extracts has excellent touch to the sanitary insect pests such as Groton bug, striped rice borer and agricultural pests
Kill activity.
Brief description of the drawings
Fig. 1：Uv analyzer (254nm) comparison chart is used after chromatographic solution rotary evaporation in vacuo；
Fig. 2：HPLC-TOF-MS detection fluorine thunders Rana compound mass-spectrogram；
Fig. 3：Detect fluorine thunder Rana compound liquid chromatogram collection of illustrative plates；
Fig. 4：Detect fluorine thunder Rana compound nucleocytoplasmic ratio collection of illustrative plates；
Fig. 5：Detect fluorine thunder Rana compound ultraviolet absorption peak collection of illustrative plates；
Embodiment
Technical scheme and beneficial effect are understood for ease of those skilled in the art, spy is with reference to accompanying drawing pair
Embodiment is described below.It should be appreciated that detailed description below is all exemplary, it is intended to the present invention is provided into
The explanation of one step.Unless otherwise indicated, all scientific and technical terms for using of the present invention have with the present invention belonging to technology lead
The identical meanings that domain personnel are generally understood that.
Embodiment 1：The extraction and purifying of fluorine thunder Rana
1. main material and reagent
Point sample capillary (0.5 × 100) is purchased from Condar experiment equipment factory of Jiangyan City；Column chromatography silica gel (refined type) is purchased from green grass or young crops
Island subsidiary factory of marine chemical industry factory；Chromatographic column is purchased from Oriental Scientific Instruments inlet and outlet Group Co., Ltd.Bravecto is silent purchased from Germany
Gram company；Petroleum ether is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Dichloromethane, methanol and ethyl acetate are purchased from Guangdong brilliance
Science and Technology Co., Ltd..Electric heating constant-temperature blowing drying box is purchased from Shanghai Tian Heng Medical Devices Co., Ltd.s；High-speed multifunctional powder
Broken machine is purchased from Deqing Bai Jie Electrical Appliances Co., Ltd；Digital display thermostat water bath is purchased from the limited public affairs of Jintan City of Jiangsu Province high honour instrument manufacturing
Department；Cable type extractor according (150mL) and condenser pipe (150mL) are purchased from Oriental Scientific Instruments inlet and outlet Group Co., Ltd；ZF-20D
Dark box type uv analyzer and thermostatted water oil bath pan are purchased from Yuhua Instrument Co., Ltd., Gongyi City；Table balance is purchased from
BJ Medical Scales Factory；Rotary Evaporators are purchased from Shanghai Yu Ying Instrument Ltd.；Multiplex vavuum pump of circulating water type is purchased from Hangzhou
David's Science ＆ Teaching Instrument Co., Ltd；Sonicator is purchased from Kunshan Ultrasonic Instruments Co., Ltd..
2. experimental procedure
(1) 30g Bravecto tablets are taken, be ground into high-speed multifunctional pulverizer it is powdered, with petroleum ether 88
Surname extraction is untill petroleum ether extract is clarified under DEG C water bath condition；
(2) powdered medicinal powder is continuing with dichloromethane surname extraction under 61 DEG C of water bath conditions, until dichloromethane carries
Untill taking liquid clarification；
(3) powdered medicinal powder is continuing with methanol surname extraction under 90 DEG C of water bath conditions, until methanol extract liquid is clarified
Untill；
(4) by the crude extract of petroleum ether, dichloromethane, methanol, low pressure rotation is evaporated (rotation on a rotary evaporator respectively
Vavuum pump is kept to be constantly in opening during being evaporated, oil bath pan modulates 49 DEG C, and flask part is immersed in the water of oil bath pan,
And keep condenser pipe to be in water flowing state).With counter balance weigh be evaporated after material mass, respectively obtain 12.5g,
10.25g, 11.25g materials；
(5) glass chromatography column of appropriate length is selected, shutoff valve adds appropriate chloroform, detects whether leak behind the door,
Choose the closed chromatographic column of valve；
(6) 10.25g (dichloromethane fractions material) silica gel (100-200 mesh) is weighed, with pouring into chromatography after petroleum ether dissolution
In post, height of the silica gel in chromatographic column is measured with graduated scale after silica gel completely precipitation, 2.5cm, is designated as 1 height unit.
Add the silica gel of 7 height units into chromatographic column again with same method, i.e. 17.5cm, silica gel highly amounts in chromatographic column
20cm；
(7) the dichloromethane fractions material after being evaporated will be rotated and adds q. s. methylene chloride, make it completely molten in flask
Solution, is poured into mortar, the silica gel of quality such as adds, on 50-55 DEG C of water-bath heating be evaporated to being completely dried in mortar, so
After be fully ground, it is become powdered completely；
(8) powder mixture is poured slowly into chromatographic column, after its completely precipitation, cuts one and omited than chromatography column internal diameter
Small filter paper, is put into chromatographic column, makes its flat cover layer on the mixture, and an absorbent cotton is then filled in into chromatographic column.Open
Chromatographic column valve, the petroleum ether in chromatographic column is bled off into major part；
(9) petroleum ether-ethyl acetate (8 is prepared:1) mixed solution, add in chromatographic column, starting flushing (must in chromatographic column
Liquid must be remained).Liquid low rotation under being filtered in chromatographic column is evaporated, and is then dissolved with the solution, after putting plate,
Detect in uv analyzer and compared (Fig. 1) with standard items.Chromatography is performed until rotary evaporation, and there is no solid matter precipitation；
(10) 6 are prepared respectively:1、4:1、3:1、2:1、1:1、2:3 petroleum ether-ethyl acetate mixed solutions, sequentially add layer
Chromatographed in analysis post.Liquid in rotation under being filtered in chromatographic column is evaporated, is then dissolved with the solution, uses silica gel plate G254 points
After plate, detect at uv analyzer 254nm and compared (Fig. 1) with standard items.Chromatography be performed until rotary evaporation there is no
Solid matter separates out, and then changes next gradient solution and rinses；
(11) purpose product for obtaining chromatography is poured into glass suction funnel and is filtered by vacuum, the product that will be obtained
To pour into glass culture dish, (0.5-1.5h) obtains 2.62g solids, as fluorine thunder Rana compound after drying, i.e., of the invention
Purpose component.
The fluorine thunder Rana compound recovery rate for extracting to obtain by this method is 62.38%, and it is detected through HPLC-TOF-MS
Purity>99% (Fig. 2~Fig. 5).
Embodiment 2：The extraction and purifying of fluorine thunder Rana
1. test apparatus used in prepares essentially identical with example 1 with reagent；
2. experimental method：
(1) 30g Bravecto tablets are taken, be ground into high-speed multifunctional pulverizer it is powdered, with petroleum ether 88
Surname extraction is untill petroleum ether extract is clarified under DEG C water bath condition；
(2) powdered medicinal powder is continuing with dichloromethane surname extraction under 61 DEG C of water bath conditions, until dichloromethane carries
Untill taking liquid clarification；
(3) powdered medicinal powder is continuing with methanol surname extraction under 90 DEG C of water bath conditions, until methanol extract liquid is clarified
Untill；
(4) by the crude extract of petroleum ether, dichloromethane, methanol, low pressure rotation is evaporated (rotation on a rotary evaporator respectively
Vavuum pump is kept to be constantly in opening during being evaporated, oil bath pan modulates 49 DEG C, and flask part is immersed in the water of oil bath pan,
And keep condenser pipe to be in water flowing state).With counter balance weigh be evaporated after material mass, respectively obtain 12.5g,
10.25g, 11.25g materials；
(5) glass chromatography column of appropriate length is selected, shutoff valve adds appropriate chloroform, detects whether leak behind the door,
Choose the closed chromatographic column of valve；
(6) 10.25g (dichloromethane fractions material) silica gel (100-200 mesh) is weighed, with pouring into chromatography after petroleum ether dissolution
In post, height of the silica gel in chromatographic column is measured with graduated scale after silica gel completely precipitation, 2.5cm, is designated as 1 height unit.
Add the silica gel of 7 height units into chromatographic column again with same method, i.e. 17.5cm, silica gel highly amounts in chromatographic column
20cm；
(7) the dichloromethane fractions material after being evaporated will be rotated and adds q. s. methylene chloride, make it completely molten in flask
Solution, is poured into mortar, the silica gel of quality such as adds, on 50-55 DEG C of water-bath heating be evaporated to being completely dried in mortar, so
After be fully ground, it is become powdered completely；
(8) powder mixture is poured slowly into chromatographic column, after its completely precipitation, cuts one and omited than chromatography column internal diameter
Small filter paper, is put into chromatographic column, makes its flat cover layer on the mixture, and an absorbent cotton is then filled in into chromatographic column.Open
Chromatographic column valve, the petroleum ether in chromatographic column is bled off into major part；
(9) petroleum ether-ethyl acetate (8 is prepared:1) mixed solution, add in chromatographic column, starting flushing (must in chromatographic column
Liquid must be remained).Liquid low rotation under being filtered in chromatographic column is evaporated, and is then dissolved with the solution, after putting plate,
Detect in uv analyzer and compared (Fig. 1) with standard items.Chromatography is performed until rotary evaporation, and there is no solid matter precipitation；
(10) 6 are prepared respectively:1、4:1、3:1、2:1、1:1、2:3 petroleum ether-ethyl acetate mixed solutions, sequentially add layer
Chromatographed in analysis post.Liquid in rotation under being filtered in chromatographic column is evaporated, then dissolved with the solution, after putting plate, at ultraviolet point
Detect in analyzer and compared with standard items.Chromatography is performed until rotary evaporation there is no solid matter precipitations, then under replacing
One gradient solution rinses；
(11) the 12.5g crude extracts obtained after petroleum ether soxhlet extraction and first are chromatographed using (6)-(10) identical method
The 11.25g crude extracts obtained after alcohol soxhlet extraction, the purpose product that chromatography is obtained are poured into glass suction funnel and carried out very
Sky is filtered, and obtained product is poured into glass culture dish, and (0.5-1.5h) obtains 3.15g solids after drying, and as fluorine thunder is drawn
Receive compound, i.e. purpose of the present invention component.
The fluorine thunder Rana compound recovery rate for extracting to obtain by this method is 75.00%, purity>85%, wherein 2.20g
Purity>98%, 0.82g purity>91%, 0.13g purity>85%.
Activity experiment
First, active determination test of the fluorine thunder Rana to Groton bug
1. main material and reagent
Groton bug (Blattella germanica) indoor feeding strain, by this laboratory (Agricultural University Of Nanjing plant
Protect institute's insect Physiology and biochemistry and Molecular Biology Lab) provide, under conditions of not in contact with any insecticide manually
Feed is continuously raised.Ethiprole (92%), butene-fipronil (92%), DDVP (93%), provided by this laboratory；Efficiently
Cypermethrin (93%), arprocarb (>99%), piperonyl butoxide (95%), it is limited purchased from Shanghai Aladdin biochemical technology share
Company；Acetone, purchased from company of Logistics Group of Agricultural University Of Nanjing.Manual micro intravenous drip device, purchased from Burkard companies；Humidifier,
Disposal plastic cup, rubber band, cotton, vaseline, culture dish, square plastic material cylinder etc., being purchased from Nanjing longevity moral test equipment has
Limit company；Flour, milk powder, glucose etc., are purchased from supermarket.
Method for breeding：Raise indoors and mix raising in cylinder in a manner of regardless of age, the intraoral depths of wall 1/3 of raising cylinder applies
Vaseline oil band wide 3-5cm, to prevent escape.Raise and two culture dishes are placed in cylinder, a culture dish, which is used to hold to mix, raises
Expect (flour：Milk powder：Glucose ratio is 6:3:1 mixing), another culture dish is used to hold aqueous and cotton, is mended per 3-4 days
Fill feed and water.Separation cylinder bottom portion is put into multilayer folding hardboard, the habitat as Groton bug；Raise cylinder individual layer yarn
Cloth seals, to prevent escape.Raising cylinder periodically is swept, keeps clean feeding environment.The density of observation Groton bug in good time, 0.6-
1.0/cm2It is advisable, has both ensured the normal development of polypide, makes full use of raising cylinder to raise the maximum amount of test worm again, when density height
When the scope, cylinder should be divided to raise.Cultivating condition is temperature (26 ± 1) DEG C, humidity (60 ± 5) %, illumination condition (L:D is 16:
8h)。
2. medicine is prepared
The preparation of 2.1 fluorine thunder Rana solution
Using acetone as solvent prepare 10,000mg/L fluorine thunder Rana mother liquor, then with acetone be diluted to 1.25mg/L,
Six concentration gradients of 2.5mg/L, 5.0mg/L, 7.5mg/L, 10.0mg/L, 12.5mg/L.
The preparation of 2.2 common insecticide solution
10,000mg/L ethiprole, butene-fipronil, DDVP, High effect cypermethrin, residual is prepared by solvent of acetone
Prestige mother liquor is killed, then each medicament is diluted to 5.0mg/L respectively with acetone.
3. topical application method fluorine thunder Rana and common insecticide are to the activity of Groton bug
Groton bug male insect 30 is chosen, is randomly divided into 3 groups, every group 10.By test worm CO2After anesthesia, lie on the back
Be arranged in culture dish, with manual micro intravenous drip device successively by as little as high concentration (0.16mg/L, 0.80mg/L, 4.0mg/L,
20.0mg/L, 100.0mg/L) fluorine thunder Rana 1.0 μ L liquid medicine points are dropped on the 2nd, the 3rd pair of intercoxal web of foot of test worm,
Then it is transferred in the container of cleaning, is normally fed with mixed fodder and immersion cotton balls.If acetone does blank control, in 24h, 48h,
The death rate is checked after 72h, 96h, 120h.Faced upward with polypide and turn over, can not creep as death standard.
Medicament is diluted to the decoction of various concentrations gradient with standby with acetone.By test worm CO2Anesthesia, it is dense from low to high
Degree is successively dropped in 1.0 μ L liquid medicine points on the 2nd, the 3rd pair of intercoxal web of foot of test worm with manual micro intravenous drip device.Each concentration
Each drop 10 of level, is repeated 3 times.Blank control is used as using acetone.It is placed in after point medicine and includes feed and the disposable modeling containing white Hupeh anemone root and leaf
Normally raised in material cup, rim of a cup is sealed with mono layer gauze, to prevent escape.Checked after 24h, 48h, 72h, 96h, 120h dead
Rate.Faced upward with polypide and turn over, can not creep as death standard.Every kind of medicament is made more than 3 times and repeated.
4. data statistic analysis
Using softwares such as POLO Plus, SPSS 17.0, virulence regression equation y=a+bx, LC50, LD50 etc. are obtained.Respectively
Experimental data such as table -1 and table -2.
Toxicity test result of the test of the fluorine thunder Rana of table -1 to Groton bug
The sensitivity testing result of the test of the fluorine thunder Rana of table -2 and six kinds of conventional medicaments to Groton bug
Note：* significant difference is represented, * * represent to reach pole significant difference.
From data above, fluorine thunder Rana to Groton bug 24h, 48h, 72h, 96h, 120h LD50Value is respectively
0.0117th, 0.0076,0.0066,0.0046 and 0.0045 μ g/ heads.Toxicity action of the fluorine thunder Rana to Groton bug as can be seen here
Increase in 0-96h with the time and increase, and toxicity action tends towards stability after 96h.Using the softwares of SPSS 17.0 to several routines
Medicament and the virulence of fluorine thunder Rana carry out significance difference analysis, as a result show 5mg/L butene-fipronil, ethiprole, enemy enemy
Fear, fluorine thunder Rana, six kinds of High effect cypermethrin, arprocarb medicaments be respectively 80.00% to the fatal rate of Groton bug,
80.00%th, 58.89%, 51.11%, 42.22%, 21.11%.From table 2, ethiprole, butene-fipronil insecticidal activity phase
To preferable, higher than fluorine thunder Rana；DDVP, fluorine thunder Rana, High effect cypermethrin take second place, and the insecticidal activity of three is without significance difference
It is different；The insecticidal activity of arprocarb is relatively worst, and pole is substantially less than fluorine thunder Rana.
2nd, fluorine thunder Rana is tested the indoor vitality test of striped rice borer
1 main material and reagent
Striped rice borer (Chilo suppressalis) strain of indoor feeding, from the 4 instar larvae (9.00- of same generation
11.00mg/ heads), provided by plant protection institute of Agricultural University Of Nanjing insect Physiology and biochemistry and Molecular Biology Lab.Fluorine worm
Nitrile (92%), provided by plant protection institute of Agricultural University Of Nanjing insect Physiology and biochemistry and Molecular Biology Lab.
2 toxicity test methods
Toxicity test method reference《Rice-stem borer monitoring for resistance technical regulation capillary titration》(NY/T 2058-
2011), improved, suitable for the toxicity test of the instar larvae of striped rice borer 4.Active compound is weighed on electronic balance, it is molten using acetone
Solution, is configured to certain density mother liquor.Mother liquor after dissolving is sub-packed in 2mL centrifuge tubes, it is cold to be stored in -20 DEG C.Use liquid-transfering gun
A certain amount of mother liquor is drawn, standby into 6-7 concentration with acetone proportional diluted, using acetone as control, often pipe is no less than 2mL.
The picking 9.00-11.00mg instar larvae of striped rice borer 4, according to the sequential processes for examination liquor strength from low to high.With
Micro intravenous drip device draws decoction drop chest back side in test worm, every μ L of test worm drop 1, and each concentration handles 30.By point
Test worm after drop, which is placed in kind, to be had in the flat Boiling tube of rice seedlings, and each test tube places 10, if 3 repetitions, control group point
Drip equal-volume acetone.Test worm after processing is put back in former feeding environment, larvae alive situation is observed when 24h and 48h, with hair
Pen touches polypide, and it is death standard that polypide, which is unable to the coordinated movement of various economic factors, records death toll.
3 data statistic analysis
Virulence regression equation is calculated using POLO PLUS (LeOra Software, 2003) software.Calculating parameter includes b
Value, LC50And its 95% confidence limit.Each experimental data such as table -3.
Toxicity test result of the fluorine thunder Rana of table -3 to indoor striped rice borer strain
Striped rice borer product it can be seen from data above using topical application method fluorine thunder Rana and ethiprole to indoor feeding
The activity of system, the results showed that：Fluorine thunder Rana handles 24h and 48h LD50Value is respectively 0.022 and 0.003 μ g/ heads.At ethiprole
Manage 24h and 48h LD50Value is respectively 0.025 and 0.004 μ g/ heads.
It follows that after 48h is handled, fluorine thunder Rana and ethiprole are obvious to the toxic action of the instar larvae of striped rice borer 4
More than 24h.After handling 48h, the LD of fluorine thunder Rana and ethiprole to striped rice borer50Value respectively 0.003 and 0.004 μ g/ heads, explanation
Fluorine thunder Rana is more than ethiprole to the activity of the instar larvae of striped rice borer 4, has good insecticidal effect.
Claims (8)
1. extraction and the purification process of fluorine thunder Rana monomeric compound, it is characterised in that comprise the following steps：
1) after Bravecto preparations are ground into powder, soxhlet extraction is carried out with petroleum ether, dichloromethane, methanol successively until backflow
Liquid is clarified, and by dichloromethane extract or three kinds of extract solutions, low pressure rotary evaporation obtains crude extract respectively；
2) silica gel with the quality such as crude extract is weighed, chromatographic column is poured into after petroleum ether dissolution, silicon is measured after silica gel completely precipitation
Height of the glue in chromatographic column, 1 height unit is designated as, then the silica gel of 6~8 height units is added into chromatographic column；
3) crude extract for obtaining step 1) is added in chromatographic column；
4) with volume ratio it is successively (7.8~8.2):1st, (5.8~6.2):1st, (3.8~4.2):1st, (2.8~3.2):1、(1.8
~2.2):1st, (0.8~1.2):1、2:The petroleum ether-ethyl acetate mixed solution of (2.8~3.2) carries out chromatography, obtains
Target product.
2. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 1, it is characterised in that step 3)
Concrete operations mode be：The crude extract that step 1) is obtained adds dichloromethane dissolving, adds and the quality such as crude extract
Silica gel, drying and grinding are slowly added in chromatographic column into powdered.
3. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 1, it is characterised in that step 1)
During soxhlet extraction, petroleum ether Extracting temperature is 85~90 DEG C；Dichloromethane Extracting temperature is 59~64 DEG C；Methanol extraction temperature
Spend for 87~93 DEG C.
4. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 3, it is characterised in that step 1)
During soxhlet extraction, petroleum ether Extracting temperature is 88 DEG C, and dichloromethane Extracting temperature is 61 DEG C, and methanol Extracting temperature is 90
℃。
5. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 1, it is characterised in that step 2)
In, the silica gel with the quality such as crude extract is weighed, chromatographic column is poured into after petroleum ether dissolution, measurement silica gel exists after silica gel completely precipitation
Height in chromatographic column, 1 height unit is designated as, then the silica gel of 7 height units is added into chromatographic column.
6. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 1, it is characterised in that step 3)
The temperature of middle drying is 50~55 DEG C.
7. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 1, it is characterised in that step 4)
The filtrate containing target product that middle chromatography obtains, vacuum filtration, drying grinding can obtain target product.
8. extraction and the purification process of fluorine thunder Rana monomeric compound according to claim 1, it is characterised in that described
Silica gel is 100~200 mesh.
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