CN107522721A - A kind of technique of efficiently purification nimbin raw medicine - Google Patents
A kind of technique of efficiently purification nimbin raw medicine Download PDFInfo
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- CN107522721A CN107522721A CN201710752527.3A CN201710752527A CN107522721A CN 107522721 A CN107522721 A CN 107522721A CN 201710752527 A CN201710752527 A CN 201710752527A CN 107522721 A CN107522721 A CN 107522721A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
- A01N61/02—Mineral oils; Tar oils; Tar; Distillates, extracts or conversion products thereof
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Abstract
The invention discloses a kind of technique of efficiently purification nimbin raw medicine, belong to natural plant extracts purifying preparation field, comprise the following steps：1) it will be crushed with shell neem kernel, and remove kind of a shell and obtain benevolence powder；2) preparation of chinaberry medicinal extract is printed；3) nimbin coarse powder is prepared；4) Fast Field elution is carried out to print chinaberry coarse powder by " dodge post method ", merges the eluent containing nimbin and be concentrated under reduced pressure and obtain that high content is faint yellow, white nimbin raw medicine powder.Extraction purification nimbin raw medicine process schematic representation of the present invention is shown in accompanying drawing 2.The technique for the efficient purification nimbin raw medicine that the invention provides, technique is simple, and recovery rate is high, and content is high and can reduce cost, good processing scheme is provided for the industrial quick nimbin for obtaining high content, and prints chinaberry development for current biological agricultural chemicals and provides solid active compound basis.
Description
Technical field
The invention belongs to natural plant extracts to purify preparation field, and in particular to a kind of efficiently purification nimbin raw medicine
Technique.
Background technology
Nimbin be it is a kind of have wide spectrum, efficiently, low toxicity, noresidues, people and animals are had no toxic side effect, be safe to pest natural enemy
And the main component of the biological pesticide for the features such as being not likely to produce the resistance to the action of a drug.According to the literature, in neem seed nimbin content
It is very low, generally 0.2%~0.4%, it is high up to 0.6%, and fat content 30%~40%, generally use varsol or
It is that most nim oil such as CN1362020A, CN1184222C, CN1524858A are removed by the method for squeezing,
CN101440099A, CN1423939A, CN101029276, the neem cake for then removing nim oil are extracted with organic solvent,
The medicinal extract containing nimbin is obtained by liquid-liquid extraction again, but the technique extracted afterwards of first deoiling also have lost while deoiling
Part nimbin causes the content in last medicinal extract substantially relatively low and contains big polar molecule compound, such as protein, carbohydrate,
Stability is poor and is unfavorable for storing.In view of domestic nimbin extraction and purification process is relatively backward at present, content is generally below 15%,
Urgently research and develop a kind of method that efficiently Rapid Extraction purifying prepares high content nimbin raw medicine.
Compared to wasting time and energy, it is necessary to substantial amounts of stationary phase and eluent, operating efficiency for traditional classical column chromatography
Low, flash column chromatography technology " dodging post method " then makes eluant, eluent run through stationary phase so as to separate sample well using decompression after post
Product, have it is quick, simply, efficiently, the advantages that stationary phase dosage is few, more applied to organic preparation and natural products such as terpene, class
The separating-purifying of ester and a variety of biological samples.
The content of the invention
Instant invention overcomes it is above-mentioned the shortcomings that, it is an object of the invention to provide it is a kind of efficiently purification nimbin raw medicine work
Skill, specifically include following steps：
1) preparation of admixing medical solutions
Neem seed through drying process is crushed in high speed blade pulverizer, and crosses 20 mesh stainless steel mesh and removes print
Chinaberry kind shell, organic solvent adding, high-speed stirred extracts 2~4h at 0 DEG C~50 DEG C, obtains admixing medical solutions, wherein, through drying process
Neem seed and organic solvent feed liquid mass ratio be 1:3~1:10；
2) preparation of chinaberry medicinal extract is printed
The admixing medical solutions obtained in step 1 are filtered, obtain filtrate and filter residue, filter residue closes after being extracted again with organic solvent
And filtrate, filtrate is concentrated under reduced pressure below 50 DEG C, chinaberry medicinal extract must be printed, wherein, the volume ratio for printing chinaberry medicinal extract and filtrate is 1：5~
50；
3) preparation of nimbin coarse powder
The print chinaberry medicinal extract obtained in step 2) is dissolved with ethyl acetate, and with saturated common salt water washing 2~3 times, acetic acid second
Ester layer separates out flocculent deposit through being concentrated under reduced pressure and adding precipitating reagent, filters to obtain faint yellow nimbin coarse powder, wherein, precipitating reagent
Volume is 5~20 times of the ethyl acetate layer volume through being concentrated under reduced pressure；
4) print chinaberry coarse powder is isolated and purified using " dodging post method "
A. the installation of " sudden strain of a muscle post method " experimental provision
Reception sets grournd glass junction, grournd glass junction plan vertical installation tool grournd glass with container top
Filter plate tubular suction funnel, have and vacuum filtration mouth is horizontally disposed with grournd glass filter plate tubular suction funnel right sidewall, tool mill
Mouth fritillarine tubular suction funnel loads stationary phase on the inside of middle and upper part.
B. nimbin coarse powder is added in " sudden strain of a muscle post " purification devices equipped with stationary phase, is under reduced pressure stream with mixed solvent
It is dynamic mutually to carry out Fast Field elution, monitored with thin-layer chromatography, compareed with nimbin standard specimen, merge the elution containing nimbin
Liquid, dry, concentrate and produce high-purity nimbin raw medicine powder, wherein, the mass percentage content of nimbin is 20%~60%.
Preferably, organic solvent described in the step 1) is any one of methanol, ethanol or ethyl acetate.
Preferably, organic solvent described in the step 2) is any one of methanol, ethanol or ethyl acetate.
Preferably, the mass percentage content of nimbin is 5%~15% in step 3) the nimbin coarse powder.
Preferably, precipitating reagent is any one in petroleum ether or n-hexane in the step 3).
Preferably, the volume ratio of precipitating reagent and medicinal extract is 5~20 in the step 3)：1.
Preferably, the stationary phase in the step 4) b is any one in silica gel, macroreticular resin, aluminum oxide or polyamide
Kind, eluant, eluent is any one in petroleum ether or the mixture of n-hexane and ethyl acetate.
Preferably, stationary phase and the volume ratio of eluant, eluent are respectively 10 in the step 4) b:1,5:1,2:1,1:1,1:2,
1:5 and 1:10.
Preferably, high-purity nimbin raw medicine powder is any one in faint yellow or white in the step 4) b.
Second object of the present invention is to provide a kind of high-purity nimbin as claimed in claim 1 as biological pesticide
The purposes of active compound, particularly its application in print chinaberry biological insecticides are prepared.
Described print chinaberry biological insecticides include but is not limited to：Neem biotic insecticide, print chinaberry-AVM biological pesticide,
Chinaberry-emamectin benzoate insecticidal agent, print chinaberry-nicotine insecticide etc. are printed, such as prints the bitter nimbin of missible oil 1%, matrine, neem emulsifiable solution 0.5%
Nimbin, mite of rattling away print missible oil 10% pyridaben, nimbin, fluorine print missible oil 5% HEXAFLUMURON, nimbin, nitrile bacterium print missible oil 10.5%
Nitrile bacterium azoles, nimbin, the Acetamiprid of acetamiprid EC 3%, nimbin, 0.2% kill line pellet granule etc., wherein, above-mentioned percentage
Content is mass percentage content.
The beneficial effects of the invention are as follows：
The present invention is used first to crush and shelled afterwards, and degreasing after first extraction extraction, Direct precipitation separates out yellow-white nimbin powder
End, avoid squeezing and deoil and cause the loss of nimbin, then quick post of crossing purifies high to contain under reduced pressure using " dodging post method "
Nimbin is measured, technique is simple, and recovery rate is high, and content is high and can reduce cost, for the industrial quick nimbin for obtaining high content
Good processing scheme is provided, and prints chinaberry development for current biological agricultural chemicals and provides solid active compound basis.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is " sudden strain of a muscle post method " Experimental equipment in the present invention, and wherein A is stationary phase, and B is the tubular of tool grournd glass filter plate
Suction funnel, C are vacuum filtration mouth, and D grournd glasses junction, E is reception container.
Fig. 2 is the process chart of the present invention.
The high phase liquid chromatogram HPLC that Fig. 3 nimbin A standard items mass percentage content is 98% schemes.
The high phase liquid chromatogram HPLC figures of the high content nimbin raw medicine of Fig. 4 present invention process extraction.
Embodiment
Embodiment 1
Dry band shell nimbin benevolence is crushed with pulverizer, 20 eye mesh screens take 700g powder after removing most of kind shell, added
Enter 2L methanol, mechanical agitation 3 hours under normal temperature.Then 1 hour is stood to after precipitating completely, is separated by filtration, filter residue again with methanol
Extraction 1~2 time is repeated, extract is collected and is concentrated in vacuo below 50 DEG C, obtains printing chinaberry concentrate.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
About 1 hour is stood after mixing uniformly to be layered completely to ester-water.Ethyl acetate layer saturated common salt water washing, remove organic acid, water-soluble
Property protein, polysaccharide etc., water layer is extracted with ethyl acetate once to improve the extraction yield of nimbin, collects ethyl acetate layer and use again
Anhydrous sodium sulfate drying, then the Temperature Vacuum below 50 DEG C be concentrated to give the medicinal extract of original volume 1/20~1/50.Then with 15
The petroleum ether of~20 times of amounts is slowly added in medicinal extract and uniform stirring to container bottom forms flaxen flocculent deposit, filtering
And nimbin dry powder 14.25g, content 9.92% are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into ethyl acetate solution, silicagel column is then loaded to, adopts
Post is crossed with " dodging post method " decompression is quick, successively with petroleum ether than ethyl acetate 4:1,2:1,1:1 and each 1L of methanol carries out quick ladder
Degree elution, TLC detection, with standard control, merge the eluent containing nimbin, be concentrated under reduced pressure content be 43% high content
White nimbin powder 0.91g, yield 98.6%.
Embodiment 2
Dry band shell nimbin benevolence is crushed with pulverizer, 20 eye mesh screens take 700g powder after removing most of kind shell, added
Enter 2L methanol, mechanical agitation 3 hours under normal temperature.Then 1 hour is stood to after precipitating completely, is separated by filtration, filter residue again with methanol
Extraction 1~2 time is repeated, extract is collected and is concentrated in vacuo below 50 DEG C, obtains printing chinaberry concentrate.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
About 1 hour is stood after mixing uniformly to be layered completely to ester-water.Ethyl acetate layer saturated common salt water washing, remove organic acid, water-soluble
Property protein, polysaccharide etc., water layer is extracted with ethyl acetate once to improve the extraction yield of nimbin, collects ethyl acetate layer and use again
Anhydrous sodium sulfate drying, then the Temperature Vacuum below 50 DEG C be concentrated to give the medicinal extract of original volume 1/20~1/50.Then with 15
The petroleum ether of~20 times of amounts is slowly added in medicinal extract and uniform stirring to container bottom forms flaxen flocculent deposit, filtering
And nimbin dry powder 14.15g, content 9.87% are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into dichloromethane solution, silicagel column is then loaded to, adopts
Post is crossed with " dodging post method " decompression is quick, successively with dichloromethane than methanol 40:1,30:1,20:1、10:1 and each 1L of methanol is carried out
Fast Field elutes, and TLC detections, with standard control, merges the eluent containing nimbin, be concentrated under reduced pressure content is 38%
High content white nimbin powder 0.91g, yield 87.6%.
Embodiment 3
Dry band shell nimbin benevolence is crushed with pulverizer, 20 eye mesh screens take 700g powder after removing most of kind shell, added
Enter 2L methanol, mechanical agitation 3 hours under normal temperature.Then 1 hour is stood to after precipitating completely, is separated by filtration, filter residue again with methanol
Extraction 1~2 time is repeated, extract is collected and is concentrated in vacuo below 50 DEG C, obtains printing chinaberry concentrate.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
About 1 hour is stood after mixing uniformly to be layered completely to ester-water.Ethyl acetate layer saturated common salt water washing, remove organic acid, water-soluble
Property protein, polysaccharide etc., water layer is extracted with ethyl acetate once to improve the extraction yield of nimbin, collects ethyl acetate layer and use again
Anhydrous sodium sulfate drying, then the Temperature Vacuum below 50 DEG C be concentrated to give the medicinal extract of original volume 1/20~1/50.Then with 15
The petroleum ether of~20 times of amounts is slowly added in medicinal extract and uniform stirring to container bottom forms flaxen flocculent deposit, filtering
And nimbin dry powder 14.18g, content 9.89% are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into 30% methanol aqueous solution, is then loaded to macropore tree
Fat post, post is crossed using " dodging post method " decompression is quick, carries out Fast Field with 50%, 60%, 70% and 80% each 1L of methanol successively
Elution, TLC detection, with standard control, merge the eluent containing nimbin, be concentrated under reduced pressure content be 59% high content it is white
Color nimbin powder 0.41g, yield 61.2%.
Embodiment 4
700g powder is taken to be dissolved in solid-to-liquid ratio 1 after directly crushing dry band shell nimbin benevolence with pulverizer:3 ethanol is molten
In liquid, the lower 20 DEG C of uniform stirrings of normal temperature about 3 hours.Then about 1 hour is stood to after precipitating completely, filters to isolate precipitation
Thing, rear again with methanol repeat extraction 1~2 time, collect extract and are concentrated in vacuo below 50 DEG C.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
After mixing uniformly about 1 hour is stood to separate to ethyl acetate layer and water layer, ethyl acetate layer again with saturated aqueous common salt wash away organic acid,
Water soluble protein, polysaccharide etc., rear water layer of collecting are extracted with ethyl acetate once to improve the extraction yield of nimbin again, collect second
Ethyl acetate layer be concentrated in vacuo with anhydrous sodium sulfate drying and below 50 DEG C of temperature original volume 1/20~1/50 medicinal extract.So
It is slowly added to afterwards with the petroleum ether of 15~20 times of amounts in concentrate and uniform stirring to container bottom forms the cotton-shaped of yellow-white
Precipitation, it is 8.34% to filter and nimbin dry powder 12.18g contents are obtained after dry sediment.
The nimbin dry powder 4g that content is 5~7% is dissolved into ethyl acetate solution, is then loaded to silicagel column, is used
Quick post method of crossing is depressurized successively by petroleum ether than ethyl acetate 4:1,2:1,1:1 and each 1L of pure methanol carries out Fast Field elution,
It is concentrated and dried the 1 of its collection:1 eluent obtains the high content white nimbin powder 0.80g that content is 41%, yield 98.3%
Embodiment 5
700g powder is taken to be dissolved in solid-to-liquid ratio 1 after directly crushing dry band shell nimbin benevolence with pulverizer:3 ethanol is molten
In liquid, the lower 20 DEG C of uniform stirrings of normal temperature about 3 hours.Then about 1 hour is stood to after precipitating completely, filters to isolate precipitation
Thing, rear again with methanol repeat extraction 1~2 time, collect extract and are concentrated in vacuo below 50 DEG C.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
After mixing uniformly about 1 hour is stood to separate to ethyl acetate layer and water layer, ethyl acetate layer again with saturated aqueous common salt wash away organic acid,
Water soluble protein, polysaccharide etc., rear water layer of collecting are extracted with ethyl acetate once to improve the extraction yield of nimbin again, collect second
Ethyl acetate layer be concentrated in vacuo with anhydrous sodium sulfate drying and below 50 DEG C of temperature original volume 1/20~1/50 medicinal extract.So
It is slowly added to afterwards with the petroleum ether of 15~20 times of amounts in concentrate and uniform stirring to container bottom forms the cotton-shaped of yellow-white
Precipitation, it is 8.12% to filter and nimbin dry powder 12.09g contents are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into dichloromethane solution, silicagel column is then loaded to, adopts
Post is crossed with " dodging post method " decompression is quick, successively with dichloromethane than methanol 40:1,30:1,20:1、10:1 and each 1L of methanol is carried out
Fast Field elutes, and TLC detections, with standard control, merges the eluent containing nimbin, be concentrated under reduced pressure content is 37%
High content white nimbin powder 0.76g, yield 86.6%.
Embodiment 6
700g powder is taken to be dissolved in solid-to-liquid ratio 1 after directly crushing dry band shell nimbin benevolence with pulverizer:3 ethanol is molten
In liquid, the lower 20 DEG C of uniform stirrings of normal temperature about 3 hours.Then about 1 hour is stood to after precipitating completely, filters to isolate precipitation
Thing, rear again with methanol repeat extraction 1~2 time, collect extract and are concentrated in vacuo below 50 DEG C.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
After mixing uniformly about 1 hour is stood to separate to ethyl acetate layer and water layer, ethyl acetate layer again with saturated aqueous common salt wash away organic acid,
Water soluble protein, polysaccharide etc., rear water layer of collecting are extracted with ethyl acetate once to improve the extraction yield of nimbin again, collect second
Ethyl acetate layer be concentrated in vacuo with anhydrous sodium sulfate drying and below 50 DEG C of temperature original volume 1/20~1/50 medicinal extract.So
It is slowly added to afterwards with the petroleum ether of 15~20 times of amounts in concentrate and uniform stirring to container bottom forms the cotton-shaped of yellow-white
Precipitation, it is 8.27% to filter and nimbin dry powder 12.13g contents are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into 30% methanol aqueous solution, is then loaded to macropore tree
Fat post, post is crossed using " dodging post method " decompression is quick, carries out Fast Field with 50%, 60%, 70% and 80% each 1L of methanol successively
Elution, TLC detection, with standard control, merge the eluent containing nimbin, be concentrated under reduced pressure content be 54% high content it is white
Color nimbin powder 0.36g, yield 58.8%.
Embodiment 7
700g powder is taken to be dissolved in solid-to-liquid ratio 1 after directly crushing dry band shell nimbin benevolence with pulverizer:3 acetic acid second
In ester solution, the lower 20 DEG C of uniform stirrings of normal temperature about 3 hours.Then about 1 hour is stood to after precipitating completely, and it is heavy to filter to isolate
Starch, rear again with methanol repeat extraction 1~2 time, collect extract and are concentrated in vacuo below 50 DEG C.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
After mixing uniformly about 1 hour is stood to separate to ethyl acetate layer and water layer, ethyl acetate layer again with saturated aqueous common salt wash away organic acid,
Water soluble protein, polysaccharide etc., rear water layer of collecting are extracted with ethyl acetate once to improve the extraction yield of nimbin again, collect second
Ethyl acetate layer be concentrated in vacuo with anhydrous sodium sulfate drying and below 50 DEG C of temperature original volume 1/20~1/50 medicinal extract.So
It is slowly added to afterwards with the petroleum ether of 15~20 times of amounts in concentrate and uniform stirring to container bottom forms the cotton-shaped of yellow-white
Precipitation, it is 4.58% to filter and nimbin dry powder 12.48g contents are obtained after dry sediment.
The nimbin dry powder 4g that content is 5~7% is dissolved into ethyl acetate solution, is then loaded to silicagel column, is used
Quick post method of crossing is depressurized successively by petroleum ether than ethyl acetate 4:1,2:1,1:1 and each 1L of pure methanol carries out Fast Field elution,
It is concentrated and dried the 1 of its collection:1 eluent obtains the high content white nimbin powder 0.43g that content is 42%, yield
98.6%.
Embodiment 8
700g powder is taken to be dissolved in solid-to-liquid ratio 1 after directly crushing dry band shell nimbin benevolence with pulverizer:3 acetic acid second
In ester solution, the lower 20 DEG C of uniform stirrings of normal temperature about 3 hours.Then about 1 hour is stood to after precipitating completely, and it is heavy to filter to isolate
Starch, rear again with methanol repeat extraction 1~2 time, collect extract and are concentrated in vacuo below 50 DEG C.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
After mixing uniformly about 1 hour is stood to separate to ethyl acetate layer and water layer, ethyl acetate layer again with saturated aqueous common salt wash away organic acid,
Water soluble protein, polysaccharide etc., rear water layer of collecting are extracted with ethyl acetate once to improve the extraction yield of nimbin again, collect second
Ethyl acetate layer be concentrated in vacuo with anhydrous sodium sulfate drying and below 50 DEG C of temperature original volume 1/20~1/50 medicinal extract.So
It is slowly added to afterwards with the petroleum ether of 15~20 times of amounts in concentrate and uniform stirring to container bottom forms the cotton-shaped of yellow-white
Precipitation, it is 4.57% to filter and nimbin dry powder 12.36g contents are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into dichloromethane solution, silicagel column is then loaded to, adopts
Post is crossed with " dodging post method " decompression is quick, successively with dichloromethane than methanol 40:1,30:1,20:1、10:1 and each 1L of methanol is carried out
Fast Field elutes, and TLC detections, with standard control, merges the eluent containing nimbin, be concentrated under reduced pressure content is 39%
High content white nimbin powder 0.40g, yield 85.3%.
Embodiment 9
700g powder is taken to be dissolved in solid-to-liquid ratio 1 after directly crushing dry band shell nimbin benevolence with pulverizer:3 acetic acid second
In ester solution, the lower 20 DEG C of uniform stirrings of normal temperature about 3 hours.Then about 1 hour is stood to after precipitating completely, and it is heavy to filter to isolate
Starch, rear again with methanol repeat extraction 1~2 time, collect extract and are concentrated in vacuo below 50 DEG C.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 500mL of saturated aqueous common salt and stirred
After mixing uniformly about 1 hour is stood to separate to ethyl acetate layer and water layer, ethyl acetate layer again with saturated aqueous common salt wash away organic acid,
Water soluble protein, polysaccharide etc., rear water layer of collecting are extracted with ethyl acetate once to improve the extraction yield of nimbin again, collect second
Ethyl acetate layer be concentrated in vacuo with anhydrous sodium sulfate drying and below 50 DEG C of temperature original volume 1/20~1/50 medicinal extract.So
It is slowly added to afterwards with the petroleum ether of 15~20 times of amounts in concentrate and uniform stirring to container bottom forms the cotton-shaped of yellow-white
Precipitation, it is 4.55% to filter and nimbin dry powder 12.42g contents are obtained after dry sediment.
The nimbin dry powder 4g that content is 8~10% is dissolved into 30% methanol aqueous solution, is then loaded to macropore tree
Fat post, post is crossed using " dodging post method " decompression is quick, carries out Fast Field with 50%, 60%, 70% and 80% each 1L of methanol successively
Elution, TLC detection, with standard control, merge the eluent containing nimbin, be concentrated under reduced pressure content be 56% high content it is white
Color nimbin powder 0.19g, yield 58.5%.
Embodiment 10
Dry band shell nimbin benevolence is crushed with pulverizer, 20 eye mesh screens take 2kg powder after removing most of kind shell, added
6L methanol, mechanical agitation 3 hours under normal temperature.Then 1 hour is stood to after precipitating completely, is separated by filtration, filter residue again with methanol weight
Extract 1~2 time again, collect extract and be concentrated in vacuo below 50 DEG C, obtain printing chinaberry concentrate.
The ethyl acetate of equivalent is added to mix in the concentrate of previous step acquisition with each 1.5L of saturated aqueous common salt and stirred
About 1 hour is stood after mixing uniformly to be layered completely to ester-water.Ethyl acetate layer saturated common salt water washing, remove organic acid, water-soluble
Property protein, polysaccharide etc., water layer is extracted with ethyl acetate once to improve the extraction yield of nimbin, collects ethyl acetate layer and use again
Anhydrous sodium sulfate drying, then the Temperature Vacuum below 50 DEG C be concentrated to give the medicinal extract of original volume 1/20~1/50.Then with 15
The petroleum ether of~20 times of amounts is slowly added in medicinal extract and uniform stirring to container bottom forms flaxen flocculent deposit, filtering
And nimbin dry powder 42.45g, content 9.91% are obtained after dry sediment.
The nimbin dry powder 40g that content is 8~10% is dissolved into ethyl acetate solution, is then loaded to silicagel column,
Post is crossed using " dodging post method " decompression is quick, successively with petroleum ether than ethyl acetate 4:1,2:1,1:1 and each 1L of methanol carries out quick
Gradient elution, TLC detection, with standard control, merge the eluent containing nimbin, be concentrated under reduced pressure content be 42% height contain
Measure light yellow nimbin powder 8.94g, yield 94.7%.
The application of nimbin raw medicine：
1. the nimbin raw medicine prepared by this technique is configured into 0.3% neem emulsifiable solution, its insecticidal activity result of the test is such as
Under.
The nimbin emulsion of table 1 0.3% prevents and treats oriental tobacco budworm field control effectiveness test result
Field control effectiveness test is carried out to tobacco oriental tobacco budworm with plant insecticide nimbin, finds out from the result of the test of table 1, applies
Various processing in l days are not notable to the preventive effect of oriental tobacco budworm after medicine, all below 30%, illustrate that these medicaments do not show quick deinsectization
Effect.Each processing in 3 days substantially rises to oriental tobacco budworm preventive effect after medicine, all higher than more than 50%；Each processing in 7 days is all shown after medicine
Highest preventive effect, 0.3% nimbin emulsion dosage are 60mL/667m2Preventive effect and 80mL/667m2、100mL/667m2Have aobvious
The difference of work, concentration is higher, and its preventive effect is better, wherein 0.3% nimbin emulsion 100mL/667m2Preventive effect is best, reaches 93%,
80mL/667m2Processing preventive effect also reaches 90%；60mL/667m2The prevention effect of processing is then less than 70%.From preventive effect analysis result
It can be seen that, dosage is 80mL and 100mL/667m2The preventive effect of processing is considerably better than comparison medicament Su Yun bacterium bacillus.
2. the nimbin raw medicine prepared by this technique is configured to 1.1% AVM hereinafter nimbin suspending agent, its insecticidal activity
Result of the test is as follows.
Prevention effect and production-increasing function of the AVM hereinafter nimbin suspending agent of table 2 1.1% to yellow rice borer and rice leaf roller
1.1% AVM hereinafter print strain element suspending agent has good preventing and treating to yellow rice borer and rice leaf roller as can be seen from Table 2
Effect and production-increasing function.The time is retransmitted in rice leaf roller, preventive effect still up to more than 85%, is increased production up to more than 45%, better than print
Strain element and AVM are alone.
3.0.3% nimbin mineral oil mixture and 0.3% nimbin, mass percentage content are 35% 5a,6,9,9a-hexahydro-6,9-methano-2,4,40% pleasure
Fruit control insecticidal activity result of the test is as follows.
Field control effect of the 34 kinds of processing of table to smaller green leaf hopper
During experiment, 4 kinds of processing are tested respectively to the field insecticidal activity of smaller green leaf hopper, print chinaberry as known from Table 3
Element and mineral oil complex liquid and single dose nimbin, two kinds of chemical pesticide 5a,6,9,9a-hexahydro-6,9-methano-2,4s, Rogor different disposal are compared to there is preferable preventive effect
And the longer lasting period.I.e. the 2nd and 7 day after nimbin mineral oil mixture processing medicine, preventive effect respectively up to 78.54%, 89.12%,
Still reach 59.78% within the 14th day after medicine.The the 2nd and 7 day drug effect is up to 53.38% and 67.65% after single dose single dose nimbin medicine, drug effect
And quick-acting is all undesirable, the lasting period is shorter, and 1000 times of mass percentage contents are that 35% endosulfan ec preventive effect is little, the after medicine
2 days only up to 52%, Rogor was worse.
The analyzing detecting method of Azadirachtin Cotent：Analysis detection is carried out to Azadirachtin Cotent using high performance liquid chromatography.
From Agilent-C18 chromatographic columns 4.0mm × 250mm, 10 μm, with acetonitrile：Water V：V=37:63 be flowing
Phase, from UV-detector, Detection wavelength 218nm, flow velocity 1mL/min, under normal temperature condition, to nimbin A standard pair
According to product and extraction sample progress chromatography, most of peak in extraction sample is efficiently separated and favorable reproducibility, prints chinaberry
Plain A retention time is in 10min or so.Nimbin A standard reference material and the high content prepared with this technique extraction purification print
Chinaberry element high-efficient liquid phase chromatogram is shown in Fig. 3 and Fig. 4.
The general principle and principal character and advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (10)
1. a kind of technique of efficiently purification nimbin raw medicine, it is characterised in that specifically include following steps：
1) preparation of admixing medical solutions
Neem seed through drying process is crushed in high speed blade pulverizer, and crosses 20 mesh stainless steel mesh and removes print chinaberry kind
Shell, organic solvent adding, high-speed stirred extracts 2~4h at 0 DEG C~50 DEG C, obtains admixing medical solutions, wherein, the print through drying process
The mass ratio of the feed liquid of chinaberry seed and organic solvent is 1:3~1:10；
2) preparation of chinaberry medicinal extract is printed
The admixing medical solutions obtained in step 1) are filtered, obtain filtrate and filter residue, filter residue merges after being extracted again with organic solvent
Filtrate, filtrate is concentrated under reduced pressure below 50 DEG C, chinaberry medicinal extract must be printed, wherein, the volume ratio for printing chinaberry medicinal extract and filtrate is 1：5~
50；
3) preparation of nimbin coarse powder
The print chinaberry medicinal extract obtained in step 2) is dissolved with ethyl acetate, and with saturated common salt water washing 2~3 times, ethyl acetate layer
Flocculent deposit is separated out through being concentrated under reduced pressure and adding precipitating reagent, filters to obtain faint yellow nimbin coarse powder, wherein, the volume of precipitating reagent
For 5~20 times of the ethyl acetate layer volume through being concentrated under reduced pressure；
4) print chinaberry coarse powder is isolated and purified using " dodging post method "
A. the installation of " sudden strain of a muscle post method " experimental provision
Reception sets grournd glass junction, grournd glass junction plan vertical installation tool grournd glass filter plate with container top
Tubular suction funnel, have and vacuum filtration mouth is horizontally disposed with grournd glass filter plate tubular suction funnel right sidewall, have ground glass
Stationary phase is loaded on the inside of glass filter plate tubular suction funnel middle and upper part.
B. nimbin coarse powder is added in " sudden strain of a muscle post " purification devices equipped with stationary phase, is under reduced pressure mobile phase with mixed solvent
Fast Field elution is carried out, is monitored with thin-layer chromatography, is compareed with nimbin standard specimen, merge the eluent containing nimbin, is done
It is dry, concentrate and produce high-purity nimbin raw medicine powder, wherein, the mass percentage content of nimbin is 20%~60%.
2. the technique of efficiently purification nimbin raw medicine according to claim 1, it is characterised in that described in the step 1)
Organic solvent is any one of methanol, ethanol or ethyl acetate.
3. the technique of efficiently purification nimbin raw medicine according to claim 1, it is characterised in that described in the step 2)
Organic solvent is any one of methanol, ethanol or ethyl acetate.
4. the technique of efficiently purification nimbin raw medicine according to claim 1, it is characterised in that the step 3) nimbin
The mass percentage content of nimbin is 5%~15% in coarse powder.
5. the technique of efficiently purification nimbin raw medicine according to claim 1, it is characterised in that precipitated in the step 3)
Agent is any one in petroleum ether or n-hexane.
6. the technique of nimbin raw medicine is efficiently purified according to claim 1 or 5, it is characterised in that in the step 3)
The volume ratio of precipitating reagent and medicinal extract is 5~20：1.
7. the technique of efficiently purification nimbin raw medicine according to claim 1, it is characterised in that in the step 4) b
Stationary phase is any one in silica gel, macroreticular resin, aluminum oxide or polyamide, and eluant, eluent is petroleum ether or n-hexane and acetic acid
Any one in the mixture of ethyl ester.
8. the technique of the efficient purification nimbin raw medicine according to claim 1 or 7, it is characterised in that in the step 4) b
Stationary phase and the volume ratio of eluant, eluent are respectively 10:1,5:1,2:1,1:1,1:2,1:5 and 1:10.
9. the technique of efficiently purification nimbin raw medicine according to claim 1, it is characterised in that high in the step 4) b
Purity nimbin raw medicine powder is any one in faint yellow or white.
10. a kind of purposes of high-purity nimbin as biological pesticide active compound as claimed in claim 1, particularly it is in preparation print
Application in chinaberry biological insecticides.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874239A (en) * | 2022-04-29 | 2022-08-09 | 保山嘉宏生物科技有限公司 | Method for extracting high-purity azadirachtin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1907986A (en) * | 2006-08-08 | 2007-02-07 | 中国林业科学研究院林产化学工业研究所 | Method for extracting nimbin |
| CN101029276A (en) * | 2006-03-03 | 2007-09-05 | 中国林业科学研究院资源昆虫研究所 | Fast method for continuouslly extracting nim oil and nimbin raw medicine from nim seed |
| CN103450227A (en) * | 2013-08-30 | 2013-12-18 | 中国科学院昆明植物研究所 | Neem active compound and insecticide containing neem active compound as well as preparation method and application of neem active compound |
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2017
- 2017-08-28 CN CN201710752527.3A patent/CN107522721A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029276A (en) * | 2006-03-03 | 2007-09-05 | 中国林业科学研究院资源昆虫研究所 | Fast method for continuouslly extracting nim oil and nimbin raw medicine from nim seed |
| CN1907986A (en) * | 2006-08-08 | 2007-02-07 | 中国林业科学研究院林产化学工业研究所 | Method for extracting nimbin |
| CN103450227A (en) * | 2013-08-30 | 2013-12-18 | 中国科学院昆明植物研究所 | Neem active compound and insecticide containing neem active compound as well as preparation method and application of neem active compound |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114874239A (en) * | 2022-04-29 | 2022-08-09 | 保山嘉宏生物科技有限公司 | Method for extracting high-purity azadirachtin |
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