CN103548892A - Compound synergistic composition resistant to plant viruses and use thereof - Google Patents
Compound synergistic composition resistant to plant viruses and use thereof Download PDFInfo
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- CN103548892A CN103548892A CN201310563296.3A CN201310563296A CN103548892A CN 103548892 A CN103548892 A CN 103548892A CN 201310563296 A CN201310563296 A CN 201310563296A CN 103548892 A CN103548892 A CN 103548892A
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- ningnanmycin
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Abstract
The invention relates to a compound synergistic composition resistant to plant viruses and particularly relates to a compound synergistic composition of a cedrela sinensis bark extract and ningnanmycin. The effective components of the composition are the cedrela sinensis bark extract and the ningnanmycin, the mass percent ratio of the cedrela sinensis bark extract to the ningnanmycin is (0.5-8):2, and experiments indicates that the activity of the composition of the cedrela sinensis bark extract and the ningnanmycin against tobacco mosaic virus is better than the effect of a single dosage. The invention provides mutual synergy between antiviral compositions, so that the dosage of the ningnanmycin can be decreased to reduce the cost, and meanwhile, the generation of resistance to medicaments of the plant viruses can be alleviated.
Description
Technical field
The present invention relates to a kind of compound synergic composition of Antiphytoviral, be specifically related to the compound synergic composition of a kind of bark of Toona sinensis extract and Ningnanmycin, belong to biological pesticide technical field.
Background technology
Tobacco mosaic virus (
tobacco mosaic virustMV) be a kind of plant virus generally occurring, can infect 65Ge section 885 various plants, the feature such as have that host range is wide, strong stress resistance, disserve to produce are large, it is reported, the whole world is often only the economic loss being caused by the harm of tobacco mosaic virus disease (TMV) item and is approximately reached more than 100,000,000 dollar.Because plant virus belongs to absolute host, self noenergy metabolic system, has high dependency to host plant cell, and its control is not also had to the very effective method of preventing and treating at present.There are some researches show, from plant, screening antiviral substance is one of important channel of exploitation plant virus inhibitor.
Ningnanmycin is a kind of Cystosine nucleocide type antibiotics, there is the wide-spectrum bactericide that efficient, low toxicity, nothing " three cause " and nothing are accumulated, the powdery mildews such as bacterial blight of rice, wheat, vegetables and tobacco mosaic virus are had to obvious control and volume increase, value-added effect.
Bark of Toona sinensis derive from Meliaceae plant Chinese toon (
toona sinensis) bark, Chinese toon skin is pained, cool, has the effects such as heat extraction, eliminating dampness, hemostasis, desinsection.Modern pharmacology research shows, that Cedrela plant has is antibiotic, anticancer, reducing blood lipid, the effect such as hypoglycemic, anti-oxidant.2008, to Chinese toon, the application in agricultural is studied in this seminar, find that Chinese toon crude extract has good inhibitory action to tobacco mosaic virus, and crude extract and Ningnanmycin are carried out composite with mass ratio 1:1, find that its inhibition activity to TMV has humidification.The antiviral active substance producing in a lot of plants has that environment compatibility is good, safety and the advantage such as development cost is low, but single active components of plants drug effect generally can not reach ideal effect, itself and other agriculture chemical compounding can suitably be improved to drug effect, reduce the harm of chemical agent to the mankind and environment, also can slow down the drug-fast generation of plant virus.
Compound synergic composition is disclosed in prior art, the preparation that comprises Chinese toon crude extract and Ningnanmycin.Can be referring to application number 200810069089.1, the full text of this patent is incorporated herein by reference.
Summary of the invention
Object of the present invention is in original patent (a kind of medicine of Antiphytoviral and compound method, application number 200810069089.1) on the composite basis of Chinese toon crude extract and Ningnanmycin, further Chinese toon crude extract is carried out to separation and Extraction, discovery has the good component of antiviral activity, has developed the composition of new different proportionings.
The present invention carries out separation by active tracing to Chinese toon antiviral substance, by composite test, finds that complex composition can produce unexpected synergistic effect.
The compound synergic composition of a kind of Antiphytoviral of the present invention, comprise described in the number of applying for a patent 200810069089.1 by bark of Toona sinensis ethanol extract, add a small amount of water, in Ultrasound Instrument, disperse, use successively benzinum, chloroform, ethyl acetate and extracting n-butyl alcohol, the various extracts of gained, reduced pressure concentration obtains corresponding medicinal extract, it is characterized in that ethyl acetate extraction medicinal extract or extracting n-butyl alcohol medicinal extract and Ningnanmycin are composite, during mass ratio 0.5～8:2, anti-phytoviral activity extracts medicinal extract or extracting n-butyl alcohol medicinal extract higher than independent ethyl acetate under same concentrations, when mass ratio is 1:2, higher than Ningnanmycin, ethyl acetate extraction medicinal extract is through column chromatography for separation, the anti-phytoviral activity of separator Fr1 increases substantially than ethyl acetate extraction medicinal extract under same concentrations, the composite quality proportioning of Fr1 and Ningnanmycin 1:1～4 o'clock, anti-phytoviral activity is under same concentrations, higher than independent Fr1 or Ningnanmycin, extracting n-butyl alcohol medicinal extract is through column chromatography for separation, the anti-phytoviral activity of separator Fr11, Fr13 increases substantially than extracting n-butyl alcohol medicinal extract under same concentrations, the composite quality proportioning of Fr11 or Fr13 and Ningnanmycin 1:1～3 o'clock, anti-phytoviral activity is under same concentrations, higher than independent Fr11, Fr13 or Ningnanmycin.
Ethyl acetate extraction medicinal extract column chromatography for separation thing Fr1 of the present invention refers to ethyl acetate is extracted to medicinal extract acetic acid ethyl dissolution, 200～300 order silica gel mixed samples, with 200～300 order silica gel dress posts, carry out column chromatography for separation, with the eluent gradient elution of petrol ether/ethyl acetate=1:1～10 (V:V), with TLC point plate, follow the tracks of, collect the eluent of identical polar, obtain Fr1 to 8 components of Fr8, Fr1 is the product obtaining after first part of eluent reduced pressure concentration, described extracting n-butyl alcohol medicinal extract column chromatography for separation thing Fr11, Fr13 refers to n-butanol medicinal extract is dissolved with the aqueous solution containing 10% methyl alcohol, through D101 macroporous absorbent resin, adsorb, hold over night, then the eluant, eluent of preparing with methyl alcohol and water: methyl alcohol, 80% methanol aqueous solution, 60% methanol aqueous solution, 40% methanol aqueous solution, 6 kinds of eluents of 20% methanol aqueous solution and pure water carry out gradient elution, with the color of eluent approach colourless till, collect the eluent of identical polar, obtain 6 component Fr9 to Fr14, wherein Fr11 and Fr13 are the products obtaining after its 3rd component and BSA eluent reduced pressure concentration.
The composition of a kind of Antiphytoviral of the present invention, the raw material that it is characterized in that composition is the ethyl acetate extraction medicinal extract of bark of Toona sinensis ethanol extract, column chromatography for separation thing Fr1, the extracting n-butyl alcohol medicinal extract of Chinese toon ethanol extract, the column chromatography for separation thing Fr11 of extracting n-butyl alcohol medicinal extract and Fr13 and the Ningnanmycin of ethyl acetate extraction medicinal extract, and the best in quality proportioning of compound synergic composition is:
Ethyl acetate extraction medicinal extract: Ningnanmycin equals 1:2,
Extracting n-butyl alcohol medicinal extract: Ningnanmycin equals 1:2,
Fr1: Ningnanmycin equals 1:1,
Fr11: Ningnanmycin equals 1:2,
Fr13: Ningnanmycin equals 1:1.
The application of antiviral compound synergic composition described in the present invention should provide aspect control corps diseases.
Described crops comprise tobacco, vegetables and cereal crop.
Described corps diseases comprises tobacco mosaic virus, cucumber mosaic virus, maize stripe virus and rice dwarf virus disease viral disease.
Method of the present invention has following outstanding beneficial effect:
According to biologically active tracing, we have carried out effective extraction to bark of Toona sinensis anti-phytoviral activity composition, and have prepared the compound synergic composition of bark of Toona sinensis extract and Ningnanmycin.Bioactivity research shows, forms the relation of mutual synergy between compound synergic composition provided by the invention, compares with single dose, and drug effect is high, and the lasting period is long, slow down the drug-fast generation of plant virus, uses like this consumption that has reduced agricultural chemicals, reduced cost.
Accompanying drawing explanation
The therapeutic activity of the built agent of Fig. 1 ethyl acetate and n-butyl alcohol extract and Ningnanmycin to TMV
As can be seen from Figure 1, Chinese toon ethyl acetate extraction medicinal extract or extracting n-butyl alcohol medicinal extract and Ningnanmycin rear have obvious synergistic effect to tobacco mosaic virus by different quality mark than composite, and when composite ratio is 1:2, its synergistic effect is best, and inhibiting rate is higher than Ningnanmycin.
The therapeutic activity of the built agent of Fig. 2 component Fr1, Fr11 and Fr13 and Ningnanmycin to TMV
From Fig. 2, draw, built agent 0.5 mg/mL for examination concentration under, after component Fr1, component Fr11 or component Fr13 are composite with Ningnanmycin respectively, proportioning is between 1:1～1:4 time, the anti-TMV result for the treatment of of built agent obviously strengthens.Component Fr11 is when proportioning is 1:2 and 1:4, and its inhibition activity to TMV is better than single dose, and when proportioning is 1:2, treatment inhibiting rate reaches 68.20%, and synergistic effect is best; Component Fr1 or component Fr13 are between 1:1～1:2 time in proportioning, and anti-TMV activity is obviously better than single dose, and when proportioning is 1:1, its treatment inhibiting rate is respectively: 66.58%, 74.23%, and synergistic effect is best.
Embodiment
The present invention carries out separation by active tracing to Chinese toon antiviral substance, by composite test, finds that complex composition can produce unexpected synergistic effect.
This concrete embodiment is only explanation of the invention; but do not limit the present invention; the timely personnel in this area can make to this embodiment the modification that there is no creative contribution as required after reading this specification, but as long as the protection that is all subject to Patent Law within the scope of claim of the present invention.
Embodiment one
The preparation of Chinese toon crude extract:
Take bark of Toona sinensis 10 kg and pulverize and cross after 40 mesh sieves, do solvent soaking extract three times with 95% ethanol of 5 times of amounts, each 7 d, merge filtrate three times, and reduced pressure concentration is dry, obtains ethanol crude extract 1.9 kg; Ethanol crude extract adds a small amount of water, in Ultrasound Instrument, disperse, successively with benzinum, chloroform, ethyl acetate, each extraction of n-butanol, until extract is colourless, and respectively by extract reduced pressure concentration, obtain respectively the Chinese toon petroleum ether extraction medicinal extract of approximately 0.03 kg, the Chinese toon extracting n-butyl alcohol medicinal extract of the Chinese toon ethyl acetate of the Chinese toon chloroform extraction medicinal extract of approximately 0.06 kg, approximately 0.5 kg extraction medicinal extract, approximately 0.6 kg is standby.
Embodiment two
The preparation of Chinese toon antiviral activity composition:
Biologically active is followed the tracks of and is found, ethyl acetate extraction medicinal extract and extracting n-butyl alcohol medicinal extract are better active to the inhibition of tobacco mosaic virus, therefore adopt column chromatography further separated to it.Get ethyl acetate extraction medicinal extract 10 g, appropriate acetic acid ethyl dissolution, 200～300 order silica gel mixed samples, with 200～300 object silica gel 200 g dress posts, carry out column chromatography for separation, take petrol ether/ethyl acetate=1:1～10 (V:V) is eluant, eluent, with TLC point plate, follow the tracks of, the eluent of collecting identical polar, reduced pressure concentration, obtains 8 component Fr1 to Fr8; Get n-butanol layer extraction medicinal extract (10 g), with the appropriate aqueous solution containing 10% methyl alcohol, dissolve, through 20 g D101 macroporous resin adsorption, hold over night, the aqueous solution with methyl alcohol and water preparation: methyl alcohol, 80% methanol aqueous solution, 60% methanol aqueous solution, 40% methanol aqueous solution, 20% methanol aqueous solution, 6 kinds of eluents of pure water carry out gradient elution, with the color of eluent approach colourless till, collect the eluent of identical polar, obtain 6 component Fr9 to Fr14.
Embodiment three
The preparation of bark of Toona sinensis extract and Ningnanmycin compound synergic composition:
A kind of antiviral Recompounded pesticide, it is to be the composite aqua forming of active ingredient by bark of Toona sinensis extract and Ningnanmycin, wherein, total concentration for examination is 0.5 mg/mL, complex composition is with bark of Toona sinensis extract (the ethyl acetate extraction medicinal extract of bark of Toona sinensis ethanol extract, the column chromatography for separation thing Fr1 of ethyl acetate extraction medicinal extract, the extracting n-butyl alcohol medicinal extract of bark of Toona sinensis ethanol extract, column chromatography for separation thing Fr11 and the Fr13 of extracting n-butyl alcohol medicinal extract) press mass fraction than being 4:1 with Ningnanmycin, 2:1, 1:1, 1:2, 1:4 carries out composite, the aqueous solution of take containing 1% polysorbas20 is solvent.
Embodiment four
The antiviral indoor biological activity determination of bark of Toona sinensis extract and complex composition
Experimental technique:
1) live body therapeutic test
Adopt the withered spot method of half leaf to measure.Selecting the Nicotiana glutinosa that growing way is consistent, first with writing brush, dipping viral juice, in full leaf frictional inoculation virus, after 30 min, with clear water, rinsing out diamond dust, after blade is dry, at left half leaf daubing medicament, the solvent that right half leaf is smeared matched doses compares.In illumination box, moisturizing is cultivated subsequently, and illumination 10000 Lux control 23 ± 1 ℃ of temperature, and 4～5 leaves of inoculation are processed in every strain, and experiment in triplicate, observes and record the number that produces withered spot after 2～3 d, calculate inhibiting rate.
2) live body passivation test
Selecting the Nicotiana glutinosa that growing way is consistent, with phosphate buffer solution, TMV is being diluted to 6 * 10
-3mg/mL, by compound and isopyknic virus liquid mixing passivation 30 min, with the artificial frictional inoculation of writing brush, in left half leaf of the of the right age Nicotiana glutinosa sprinkled with diamond dust, corresponding solvent and virus liquid combined inoculation are in right half leaf of the of the right age Nicotiana glutinosa sprinkled with diamond dust.In illumination box, moisturizing is cultivated subsequently, and illumination 10000 Lux control 23 ± 1 ℃ of temperature, and 4～5 leaves of inoculation are processed in every strain, and experiment in triplicate, observes and record the number that produces withered spot after 2～3 d, calculate inhibiting rate.
3) control drug: 8% Ningnanmycin aqua (Heilungkiang Qiang Er biochemical technology Co., Ltd).
4) virus inoculation: tobacco mosaic virus (TMV), is purchased from Chinese Academy of Sciences's Wuhan virus research.
The anti-TMV of table 1 bark of Toona sinensis extraction medicinal extract is active
As can be seen from Table 1, in the extraction medicinal extract of bark of Toona sinensis ethanol extract, the anti-TMV of ethyl acetate extraction medicinal extract and extracting n-butyl alcohol medicinal extract is better active, its treatment inhibiting rate is respectively: 45.56%, 51.88%, passivation inhibiting rate is respectively: 85.99%, 86.83%, and close to commercialization medicament Ningnanmycin.
The therapeutic activity of the built agent of table 2 ethyl acetate and extracting n-butyl alcohol medicinal extract and Ningnanmycin to TMV
Note: when proportioning is 1:0, represent Chinese toon extraction medicinal extract; When proportioning is 0:1, representative has Ningnanmycin, is all contrast.
From accompanying drawing 1 and table 2, can find out, Chinese toon ethyl acetate extraction medicinal extract and extracting n-butyl alcohol medicinal extract and Ningnanmycin rear have obvious synergistic effect to tobacco mosaic virus by different quality than composite, and when composite ratio is 1:2, its synergistic effect is best, inhibiting rate is respectively: 60.04%, 60.07%, higher than Ningnanmycin, be worth further research.
Table 3 is active to the inhibition of TMV for the segmentation component of examination ethyl acetate and extracting n-butyl alcohol medicinal extract
As can be seen from Table 3, the inhibition to tobacco mosaic virus of component Fr1, component Fr11 and component Fr13 is better active, and its treatment inhibiting rate is respectively: 52.30%, 58.40%, 51.94%, and passivation inhibiting rate is respectively: 88.34%, 86.53%, 89.46%.
The composite rear therapeutic activity to TMV of table 4 component Fr1, Fr11 and Fr13 and Ningnanmycin
From accompanying drawing 2 and table 4, draw, built agent 0.5 mg/mL for examination concentration under, after component Fr1, component Fr11 and component Fr13 are composite with Ningnanmycin respectively, proportioning is between 1:1～1:4 time, the anti-TMV result for the treatment of of built agent obviously strengthens.Component Fr11 is when proportioning is 1:2 and 1:4, and its inhibition activity to TMV is better than single dose, and when proportioning is 1:2, treatment inhibiting rate reaches 68.20%, and synergistic effect is best; Component Fr1 and component Fr13 are between 1:1～1:2 time in proportioning, and anti-TMV activity is obviously better than single dose, and when proportioning is 1:1, its treatment inhibiting rate is respectively: 66.58%, 74.23%, and synergistic effect is best.
Claims (6)
1. the compound synergic composition of an Antiphytoviral, comprise by bark of Toona sinensis ethanol extract, add a small amount of water, in Ultrasound Instrument, disperse, use successively benzinum, chloroform, ethyl acetate and extracting n-butyl alcohol, the various extracts of gained, reduced pressure concentration obtains corresponding medicinal extract, it is characterized in that ethyl acetate extraction medicinal extract or extracting n-butyl alcohol medicinal extract and Ningnanmycin are composite, during mass ratio 0.5～8:2, anti-phytoviral activity extracts medicinal extract or extracting n-butyl alcohol medicinal extract higher than independent ethyl acetate under same concentrations, when mass ratio is 1:2, higher than Ningnanmycin; Ethyl acetate extraction medicinal extract is through column chromatography for separation, the anti-phytoviral activity of separator Fr1 increases substantially than ethyl acetate extraction medicinal extract under same concentrations, the composite quality proportioning of Fr1 and Ningnanmycin 1:1～4 o'clock, anti-phytoviral activity is under same concentrations, higher than independent Fr1 or Ningnanmycin; Extracting n-butyl alcohol medicinal extract is through column chromatography for separation, the anti-phytoviral activity of separator Fr11, Fr13 increases substantially than extracting n-butyl alcohol medicinal extract under same concentrations, the composite quality proportioning of Fr11 or Fr13 and Ningnanmycin 1:1～3 o'clock, anti-phytoviral activity is under same concentrations, higher than independent Fr11, Fr13 or Ningnanmycin.
2. the compound synergic composition of a kind of Antiphytoviral according to claim 1, ethyl acetate extraction medicinal extract column chromatography for separation thing Fr1 described in it is characterized in that refers to ethyl acetate is extracted to medicinal extract acetic acid ethyl dissolution, 200～300 order silica gel mixed samples, with 200～300 order silica gel dress posts, carry out column chromatography for separation, eluent gradient elution with petrol ether/ethyl acetate=1:1～10 (V:V), with TLC point plate, follow the tracks of, collect the eluent of identical polar, obtain Fr1 to 8 components of Fr8, Fr1 is the product obtaining after first part of eluent reduced pressure concentration, described extracting n-butyl alcohol medicinal extract column chromatography for separation thing Fr11, Fr13 refers to n-butanol medicinal extract is dissolved with the aqueous solution containing 10% methyl alcohol, through D101 macroporous absorbent resin, adsorb, hold over night, then the eluant, eluent of preparing with methyl alcohol and water: methyl alcohol, 80% methanol aqueous solution, 60% methanol aqueous solution, 40% methanol aqueous solution, 6 kinds of eluents of 20% methanol aqueous solution and pure water carry out gradient elution, with the color of eluent approach colourless till, collect the eluent of identical polar, obtain 6 component Fr9 to Fr14, wherein Fr11 and Fr13 are the products obtaining after its 3rd component and BSA eluent reduced pressure concentration.
3. the composition of a kind of Antiphytoviral according to claim 1, the raw material that it is characterized in that composition is the ethyl acetate extraction medicinal extract of bark of Toona sinensis ethanol extract, column chromatography for separation thing Fr1, the extracting n-butyl alcohol medicinal extract of bark of Toona sinensis ethanol extract, the column chromatography for separation thing Fr11 of extracting n-butyl alcohol medicinal extract and Fr13 and the Ningnanmycin of ethyl acetate extraction medicinal extract, and the best in quality proportioning of compound synergic composition is:
Ethyl acetate extraction medicinal extract: Ningnanmycin equals 1:2,
Extracting n-butyl alcohol medicinal extract: Ningnanmycin equals 1:2,
Fr1: Ningnanmycin equals 1:1,
Fr11: Ningnanmycin equals 1:2,
Fr13: Ningnanmycin equals 1:1.
4. the application in preventing corps diseases according to the antiviral synergistic composition described in claim 1 or 3.
5. application according to claim 4, is characterized in that crops comprise tobacco, vegetables and cereal crop.
6. application according to claim 5, is characterized in that: described corps diseases comprises tobacco mosaic virus, cucumber mosaic virus, maize stripe virus and rice dwarf virus disease viral disease.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106358960A (en) * | 2016-09-29 | 2017-02-01 | 张建委 | High-reproductive-rate seedling culture method of cedrela sinensis |
| CN108522548A (en) * | 2018-04-09 | 2018-09-14 | 贵州省果树科学研究所 | The antiviral compound synergic composition of one Plant Extracts |
| CN111109076A (en) * | 2020-01-22 | 2020-05-08 | 佛山科学技术学院 | Screening method of anti-TMV (Tetramethylbenzidine) medicament |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101438718A (en) * | 2008-12-30 | 2009-05-27 | 贵州大学 | Medicament for resisting plant virus and formulating method |
| CN103349007A (en) * | 2013-07-26 | 2013-10-16 | 海南正业中农高科股份有限公司 | Agricultural composition containing antibiotics and plant source materials and application thereof |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101438718A (en) * | 2008-12-30 | 2009-05-27 | 贵州大学 | Medicament for resisting plant virus and formulating method |
| CN103349007A (en) * | 2013-07-26 | 2013-10-16 | 海南正业中农高科股份有限公司 | Agricultural composition containing antibiotics and plant source materials and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106358960A (en) * | 2016-09-29 | 2017-02-01 | 张建委 | High-reproductive-rate seedling culture method of cedrela sinensis |
| CN108522548A (en) * | 2018-04-09 | 2018-09-14 | 贵州省果树科学研究所 | The antiviral compound synergic composition of one Plant Extracts |
| CN108522548B (en) * | 2018-04-09 | 2020-07-07 | 贵州省果树科学研究所 | Antiviral compound synergistic composition of plant extracts |
| CN111109076A (en) * | 2020-01-22 | 2020-05-08 | 佛山科学技术学院 | Screening method of anti-TMV (Tetramethylbenzidine) medicament |
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