CN1895050A - Kosam seeds plant-resisting virusagent and preparation thereof - Google Patents
Kosam seeds plant-resisting virusagent and preparation thereof Download PDFInfo
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- CN1895050A CN1895050A CN 200510044114 CN200510044114A CN1895050A CN 1895050 A CN1895050 A CN 1895050A CN 200510044114 CN200510044114 CN 200510044114 CN 200510044114 A CN200510044114 A CN 200510044114A CN 1895050 A CN1895050 A CN 1895050A
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- java brucea
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Abstract
An antiviral chemical for plants is prepared from Bruceajavanica (L.) Merr by extracting Bruceine D and microemulsifying. It is used for suppressing the plant viruses TMV, CMV, PVY, etc without environmental pollution.
Description
Affiliated field the present invention relates to the application of a kind of anti-phytoviral activity plant, is specifically related to quassia Kosam seeds plant-resisting virusagent and preparation method thereof, and in the application that prevents and treats on the viroses of plant, belongs to technical field of pesticide.
Background technology Java brucea (Brucea javanica (L.) Merr) is a Simarubaceae brucea plant, has another name called crow courage, crow egg-shaped thing, duck's egg.Half evergreen shrubs or dungarunga, high 2～3m, complete stool is all by yellow pubescence.The odd number winglike compound leaf, alternate; Leaflet 5～11, ovum shape lanceolar, long 5～10cm, wide 2～4.5cm, there is bastard at the edge, and the two sides is by pubescence.Flower unisexuality, different strain or homophyletic, coniform cyme armpit is given birth to; Spend minimum, reddish yellow; Petal 4, stamen 4.Drupe is long avette or oval, black when ripe, tool reticulate pattern.3～August of florescence, really 4～September of phase.Be born in meadow, shrubbery and roadside area without shade.Java brucea contains multiple compositions such as alkaloid, glucoside, phenols, then mainly contain multiple quassinoids class material in its seed, not only have clearing heat and detoxicating, preventing malaria, end dysentery and erosion wart effect, and multiple cancers such as liver cancer, the carcinoma of the rectum, palace neck cancer, cutaneum carcinoma, the cancer of the esophagus are all had result of treatment.
At present, relevant Java brucea is used for the research report of prevention and elimination of disease and pests on agricultural less, and (Marvin's is gorgeous etc., Shandong forestry science and technology, 2000, (3): 20-22 to concentrate on the desinsection aspect substantially; Gao Lin etc., " environment friendly agricultural of a kind of efficient insecticide, safety, noresidue ", number of patent application: 00130230.2; Gao Lin etc., " plant pesticide of a kind of efficient insecticide, safety, noresidue ", number of patent application: 01144340.5; Surplus face south etc., " a kind of compound plant insecticide and compounding method thereof ", number of patent application: 03112811.4).Do not see with the Java brucea to be the report that primary raw material prepares the plant resource anti-plant virus agent so far as yet, do not see the report of Java brucea bitter principle D (Bruceine D) anti-phytoviral activity in the Java brucea yet.
Because the particularity of the viroses of plant, its control is a great problem in the agricultural production always, and existing antiviral agent belongs to chemical pesticide mostly, not high to the preventive effect of the viroses of plant on the one hand, in addition on the one hand, also there is certain environmental risk in long-term use, and therefore exploitation is that the antivirotic of active ingredient has broad prospects with the natural plants active substance.
Summary of the invention the object of the invention is to provide a kind of plant virus had both been had the inhibition dissemination, has Kosam seeds plant-resisting virusagent that suppresses proliferation function and preparation method thereof again.
Kosam seeds plant-resisting virusagent of the present invention comprises Fructus Bruceae extract and Java brucea Antiphytoviral microemulsion.The objective of the invention is to realize by the following method.
1, a kind of Kosam seeds plant-resisting virusagent is characterized in that directly utilizing the Fructus Bruceae extract of active ingredient for Java brucea bitter principle D (Bruceine D), and the preparation method comprises the following steps:
A, the Java brucea seed meal of drying is broken to 1～5mm;
B, with steps A gained dry powder with 5～8 times of amounts (w/v) organic solvent refluxing extraction, extracting temperature is 56～80 ℃, extraction time is 12～36h;
Described organic solvent is ethanol or ethyl acetate or acetone or methyl alcohol;
C, with behind the gained extract suction filtration among the step B, vacuum decompression is concentrated into 10%～40% of former dry powder weight under 30～50 ℃ of conditions, Fructus Bruceae extract.
2, a kind of Java brucea Antiphytoviral microemulsion is characterized in that described Fructus Bruceae extract and emulsifier, cosolvent are added water after mixing, and stirs to be prepared under 40 ℃～60 ℃; The percentage by weight of each component is:
Fructus Bruceae extract 2%～25%
Cosolvent 5%～20%
Emulsifier 10%～20%
Water 40%～75%
Described cosolvent is one or more in ethanol, methyl alcohol, dimethylbenzene, the dimethyl formamide; Described emulsifier is one or more in polyoxyethylene nonylphenol ether, calcium dodecyl benzene sulfonate, polyoxyethylenesorbitan sorbitan monooleate, phenethyl phenol polyethenoxy ether, the alkylaryl polyoxyethylene poly-oxygen propylene aether.
3, Java brucea bitter principle D (Bruceine D) extracting method:
Adopt the conventional plant chemical method, described Fructus Bruceae extract is divided into petroleum ether part, ethyl acetate extract and methyl alcohol position by polarity is ascending, and carries out biological activity determination respectively; Be tracked as guidance with biologically active, the ethyl acetate extract that the enantiopathy toxic action is the strongest carries out silica gel column chromatography, therefrom separate, be purified into Java brucea bitter principle D (Bruceine D), molecular weight is 410, molecular formula is C20H26O9, this material is the effective active composition of Java brucea Antiphytoviral, and its structural formula is as follows:
The existing chemical antiviral agent that uses of Kosam seeds plant-resisting virusagent of the present invention has very big advantage:
1, anti-phytoviral activity is remarkable, and inhibited to various plants virus;
2, the anti-phytoviral activity compound in the good Java brucea of environment compatibility is the structure that natural world itself just exists, easily degraded in atmosphere, soil, water body, crop, thereby after natural world is thrown in, environment is not had influence substantially;
3, to non-target organism safety;
4, the raw material source is abundant, and processing technology is simple, is suitable for applying;
The present invention is described in further detail by the following examples for embodiment, but be not limited to these embodiment.
Embodiment 1: a kind of preparation method of Fructus Bruceae extract
Take by weighing dry Java brucea seed 100Kg, be crushed to 1～5mm with the plant disintegrating machine, with 5 times of amounts (w/v) industrial alcohol 80 ℃ of following refluxing extraction three times, common 20h, the merging extract, under 40 ℃ of conditions vacuum decompression concentrated extract 20Kg.
Embodiment 2: a kind of preparation method of Fructus Bruceae extract
Take by weighing dry Java brucea seed 100Kg, be crushed to 1～5mm with the plant disintegrating machine, with 5 times of amounts (w/v) methyl alcohol 65 ℃ of following refluxing extraction three times, common 24h, the merging extract, under 38 ℃ of conditions vacuum decompression concentrated extract 30Kg.
Described Fructus Bruceae extract can be used as the raw material of preparation Java brucea Antiphytoviral microemulsion, also can be used as Kosam seeds plant-resisting virusagent, and working concentration is 0.25～0.05mg/mL, directly sprays.
Embodiment 3: Java brucea bitter principle D (Bruceine D) extracting method may further comprise the steps:
1. getting 100～200 order silica gel of described Fructus Bruceae extract of 100g and 300g mixes thoroughly, in 45 ℃ of oven dry, the rearmounted apparatus,Soxhlet's of pulverizing, it is closely colourless to be extracted into eluent with benzinum, ethyl acetate and three kinds of solvent refluxings of methyl alcohol successively under 60～90 ℃ condition; The extract concentrating under reduced pressure gets benzinum medicinal extract 10.8g, ethyl acetate medicinal extract 5.3g and methyl alcohol medicinal extract 6.5g respectively;
2. get Java brucea ethyl acetate medicinal extract 4.5g with after the small amount of methanol dissolving, be adsorbed in and go up silicagel column behind 100～200 order silica gel of 15g and carry out adsorpting column chromatography, the high 60cm of post, post footpath 6cm; Adopt the wet method upper prop, eluting solvent is selected petroleum ether-ethyl acetate-ethanol system gradient elution for use, and every 150mL collects 1 part, collects 150 stream parts altogether.Check through thin-layer chromatography (TLC), merge same composition, get an antiviral active components Fr3; Fr3 is carried out silica gel column chromatography again, made the chloroform-methanol wash-out by 9: 1, separate 15mg white needles compound crystal Y1, Y1 is Java brucea bitter principle D (Bruceine D).
Embodiment 4: a kind of preparation method of Java brucea Antiphytoviral microemulsion
Take by weighing described Fructus Bruceae extract 10Kg, with dimethyl formamide 20Kg, polyoxyethylenesorbitan sorbitan monooleate 30Kg fully mixed after, add distilled water 140Kg, under 45 ℃, stir and get final product.
Embodiment 5: a kind of preparation method of Java brucea Antiphytoviral microemulsion
Take by weighing described Fructus Bruceae extract 10Kg, with ethanol 15Kg, calcium dodecyl benzene sulfonate 6Kg, polyoxyethylene nonylphenol ether 14Kg fully mixed after, add distilled water 55Kg, under 50 ℃, stir and get final product.
The using method of Java brucea Antiphytoviral microemulsion: after diluting 500～1000 times, directly spray.
Experimental example 1: the mensuration of Java brucea ethanol extract anti-phytoviral activity
1. for trying the host, supplying the prelibation source
For trying the host: provide by plant virus research institute of University Of Agriculture and Forestry In Fujian.The host of TMV local lesion Nicotiana glutinosa (Nicotiana glutinosa), the common cigarette K of TMV systemic infection host
326(Nicotiana tabacum CV., K
326), CMV (Cucumber mosaic virus, CMV) and PVY
N(Potato virus Y
N, PVY
N) host of local lesion Chenopodium amaranticolor (Chenopodium amaranticolor), cultivate in the insect protected greenhouse.
Supply the prelibation source: the common strain of tobacco mosaic virus system, provide by plant virus research institute of University Of Agriculture and Forestry In Fujian, after Nicotiana glutinosa list spot separates, breed in common cigarette K
326On, the back of purifying is standby, and the virus inoculation final concentration is 10 μ g/mL; Cucumber mosaic virus, the downright bad strain of marmor upsilon system are provided by plant virus research institute of University Of Agriculture and Forestry In Fujian.Take by weighing CMV, PVY
NSick each 1g of leaf adds 10 times of amount 0.01mol/L phosphate buffers (pH=7.0) and fully grinds, and gets supernatant respectively as CMV and PVY after centrifugal
NInoculation liquid.
2. test method
Adopt local lesion's method.Select healthy, the eugonic host of local lesion, the mixed liquid of Zuo Banye inoculation Java brucea ethanol extract and viral equal-volume, the mixed liquid of right half leaf inoculation distilled water and viral equal-volume compares, the water flushing of inoculation back.4～5 blades of every processing inoculation repeat 3 times, and statistics withered spot number order calculates inhibiting rate behind the 3d.
Inhibiting rate (%)=[(contrast withered spot number-processing withered spot number)/contrast withered spot number] * 100.
3. result of the test
With local lesion's method to the anti-TMV of Java brucea ethanol extract, CMV and PVY
NActivity is measured, and the results are shown in Table 1.By table 1 as seen, the Java brucea ethanol extract is to TMV, CMV and PVY
NVery strong inhibitory action is arranged, for examination concentration when being 1mg/mL, to the inhibitory action of three plant species viruses all more than 90%.
Table 1 Java brucea ethanol extract is to TMV, CMV and PVY
NInhibitory action
| For examination concentration | Inhibiting rate (%) | ||
| TMV | CMV | PVY N | |
| 1mg/mL | 96.2 | 90.2 | 91.1 |
| 0.5mg/mL | 82.6 | 84.9 | 86.5 |
Experimental example 2: the bioactive mensuration of Java brucea bitter principle D (Bruceine D) anti-TMV
1. test method
1) suppresses the TMV dissemination and select healthy, eugonic 6～8 leaf phase Nicotiana glutinosas, the mixed liquid of Zuo Banye inoculation Java brucea bitter principle D and viral equal-volume (TMV inoculation final concentration is 10 μ g/mL), the mixed liquid of right half leaf inoculation distilled water and viral equal-volume compares, the water flushing of inoculation back.4～5 blades of every processing inoculation repeat 3 times, and statistics withered spot number order calculates inhibiting rate behind the 3d.Java brucea bitter principle D initial mass concentration is made as 0.4mg/mL, measures the inhibitory action that variable concentrations Java brucea bitter principle D infects TMV behind the doubling dilution.
2) suppress the TMV proliferation function behind virus inoculation certain hour on systemic infection host (common cigarette) blade of healthy growth, beat cut-off with card punch from mesophyll part (avoiding big vein) and directly be the disk of 12mm, (contain a small amount of methyl-sulfoxide to float on respectively for examination extract solution and distilled water, float in the distilled water as negative control as handling and positive control, being provided with simultaneously down together) with healthy leaf disk.Leaf disk is with the carbonate buffer solution bag quilt of 1: 20 (W/V) behind the 48h, and indirect elisa method is measured OD
405Value is obtained virus concentration according to the virus concentration calibration curve then, calculates inhibiting rate.Java brucea bitter principle D initial mass concentration is made as 0.4mg/mL, measures variable concentrations Java brucea bitter principle D behind the doubling dilution to the effect of TMV inhibition of proliferation.
2. the anti-TMV dissemination of result of the test measurement result shows (table 2), and Java brucea bitter principle D has significant inhibition dissemination to TMV, and along with the increase for examination, the inhibiting rate that TMV is infected raises gradually.When being 0.1mg/mL for examination concentration, Java brucea bitter principle D reaches 95.1% to the inhibiting rate that TMV infects.
The inhibitory action that table 2 Java brucea bitter principle D infects TMV
| Concentration (mg/mL) | 0.025 | 0.05 | 0.1 | 0.2 | 0.4 |
| Inhibiting rate (%) | 70.6 | 82.6 | 95.1 | 97.7 | 98.5 |
Anti-TMV proliferation function measurement result shows (table 3), and Java brucea bitter principle D has the obvious suppression effect to TMV propagation, 0.025mg/mL～0.4mg/mL for the examination concentration range in, to TMV inhibition of proliferation rate all more than 70%.
Table 3 Java brucea bitter principle D is to the effect of TMV inhibition of proliferation
| Concentration (mg/mL) | 0.025 | 0.05 | 0.1 | 0.2 | 0.4 |
| Inhibiting rate (%) | 71.5 | 72.2 | 75.5 | 78.9 | 80.2 |
Experimental example 3: Java brucea Antiphytoviral microemulsion is to the preventive and therapeutic effect of tobacco mosaic
1. the test method test is carried out in the insect protected greenhouse.Choose 5～6 leaf phase health, the consistent common cigarette K of growth
326, handle 10 strains for every group.Spray respectively at inoculation preceding 7d, 1d and inoculation back 2d, 9d, spray soup altogether 4 times.20d behind the last spray medicine, the investigation incidence calculates the incidence of disease, disease index and control efficiency.Test is established 20% virus and is got killed (moroxydine hydrochloride copper WP, Hua Feng chemical plant, Qiqihaer City, Heilongjiang Province product dilute 500 times) for contrasting medicament.State of an illness grade scale is: 0 grade-asymptomatic; 1 grade-blade has indivedual bottle green spots; 2 grades-indivedual blades are slight floral leaf; 3 grades-1～2 blades are heavier floral leaf; 4 grades-most blades present heavy floral leaf.
The incidence of disease (the %)=processing morbidity strain number/total strain number of processing * 100
Disease index=∑ (sick progression * this grade diseased plant number)/(the highest sick level * total strain number) * 100
Control efficiency (%)=[1-(handling disease index/contrast disease index)] * 100
2. result of the test pot experiment measurement result shows (table 4), and Java brucea Antiphytoviral microemulsion has the better prevention effect to tobacco mosaic virus disease, and control efficiency is higher than contrast medicament 20% virus and gets killed.
Table 4 Java brucea Antiphytoviral microemulsion is to the control efficiency of tobacco mosaic
| Handle | Extension rate | The incidence of disease (%) | Disease index | Control efficiency (%) |
| Java brucea Antiphytoviral microemulsion | 500 times | 100 | 40.0 | 51.5 |
| Java brucea Antiphytoviral microemulsion | 1000 times | 100 | 45.0 | 45.5 |
| 20% virus is got killed | 500 times | 100 | 55.0 | 33.3 |
| CK(+) | - | 100 | 82.5 | - |
| CK(-) | - | 0 | 0 | - |
CK (+): positive control; CK (-): negative control
Claims (4)
1, a kind of Kosam seeds plant-resisting virusagent is characterized in that directly utilizing the Fructus Bruceae extract of active ingredient for Java brucea bitter principle D (Bruceine D), and the preparation method comprises the following steps:
A, the Java brucea seed meal of drying is broken to 1～5mm;
B, with steps A gained dry powder with 5～8 times of amounts (w/v) organic solvent refluxing extraction, extracting temperature is 56～80 ℃, extraction time is 12～36h;
Described organic solvent is ethanol or ethyl acetate or acetone or methyl alcohol;
C, with behind the gained extract suction filtration among the step B, vacuum decompression is concentrated into 10%～40% of former dry powder weight under 30～50 ℃ of conditions, Fructus Bruceae extract.
2, a kind of Java brucea Antiphytoviral microemulsion is characterized in that the described Fructus Bruceae extract of claim 1 and cosolvent, emulsifier are added water after mixing, and stirs to be prepared under 40 ℃～60 ℃; The percentage by weight of each component is:
Fructus Bruceae extract 2%～25%
Cosolvent 5%～20%
Emulsifier 10%～20%
Water 40%～75%
Described cosolvent is one or more in ethanol, methyl alcohol, dimethylbenzene, the dimethyl formamide; Described emulsifier is one or more in polyoxyethylene nonylphenol ether, calcium dodecyl benzene sulfonate, polyoxyethylenesorbitan sorbitan monooleate, phenethyl phenol polyethenoxy ether, the alkylaryl polyoxyethylene poly-oxygen propylene aether.
3, a kind of Java brucea Antiphytoviral microemulsion according to claim 2, it is characterized in that the preparation method is as follows: get described Fructus Bruceae extract 10Kg, with ethanol 15Kg, calcium dodecyl benzene sulfonate 6Kg, polyoxyethylene nonylphenol ether 14Kg fully mixed after, add distilled water 55Kg, under 50 ℃, stir and get final product.
4, a kind of Java brucea Antiphytoviral microemulsion according to claim 2, it is characterized in that the preparation method is as follows: get described Fructus Bruceae extract 10Kg, with dimethyl formamide 20Kg, polyoxyethylenesorbitan sorbitan monooleate 30Kg fully mixed after, add distilled water 140Kg, under 45 ℃, stir and get final product.
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| CN 200510044114 CN1895050A (en) | 2005-07-13 | 2005-07-13 | Kosam seeds plant-resisting virusagent and preparation thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188459A (en) * | 2010-03-09 | 2011-09-21 | 胡清文 | Brucea javanica total terpenoid extractive, and preparation method and application thereof |
| CN102924467A (en) * | 2012-11-07 | 2013-02-13 | 福建农林大学 | Preparation method for extracting and purifying bruceine D form brucea javanica |
| CN103719161A (en) * | 2013-11-15 | 2014-04-16 | 安徽淮化股份有限公司 | Pesticide composition |
| CN109006841A (en) * | 2018-08-01 | 2018-12-18 | 华南农业大学 | It is a kind of can in plant transporting antifeedant for insect |
| CN112931496A (en) * | 2021-01-27 | 2021-06-11 | 仲恺农业工程学院 | Special pesticide containing brucea javanica oil for flying defense and preparation method thereof |
-
2005
- 2005-07-13 CN CN 200510044114 patent/CN1895050A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188459A (en) * | 2010-03-09 | 2011-09-21 | 胡清文 | Brucea javanica total terpenoid extractive, and preparation method and application thereof |
| CN102924467A (en) * | 2012-11-07 | 2013-02-13 | 福建农林大学 | Preparation method for extracting and purifying bruceine D form brucea javanica |
| CN102924467B (en) * | 2012-11-07 | 2015-04-01 | 福建农林大学 | Preparation method for extracting and purifying bruceine D form brucea javanica |
| CN103719161A (en) * | 2013-11-15 | 2014-04-16 | 安徽淮化股份有限公司 | Pesticide composition |
| CN109006841A (en) * | 2018-08-01 | 2018-12-18 | 华南农业大学 | It is a kind of can in plant transporting antifeedant for insect |
| CN109006841B (en) * | 2018-08-01 | 2021-05-04 | 华南农业大学 | Insect antifeedant capable of being transported in plant body |
| CN112931496A (en) * | 2021-01-27 | 2021-06-11 | 仲恺农业工程学院 | Special pesticide containing brucea javanica oil for flying defense and preparation method thereof |
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