CN101053332A - Active constituent of curcuma longa for inhibiting and killing plant pathogenic fungi and its preparation and application - Google Patents
Active constituent of curcuma longa for inhibiting and killing plant pathogenic fungi and its preparation and application Download PDFInfo
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Abstract
The present invention discloses an inhibiting and killing plant pathogenic fungi active constituent of curcuma longa and its preparation and application. The active component is extract extracted by ethanol or methanol from curcuma longa or fine extract extracted further by n-hexane based on the extract extracted by ethanol or methanol. The inhibiting and killing plant pathogenic fungi active constituent of curcuma longa can be prepared inhibiting and killing plant pathogenic fungi and mushroom pathogenic fungi preparation, especially prepared preparation for inhibiting and killing phorna wasabiae Yokogi, sclerotinia sclerotiorum, tomato botrytis cinerea, verticillium, wheat phytoalexin, edible fungi mycogone perniciosa, olive green mycophenolate or P. pallidum. The preparation formulations can be water solution, soluble powder, wettable powder, thick suspending agent and granule. The active components disclosed by this present invention has extensive source of raw materials, no-toxic, easy degradation features, can be developed a non-pollution botanical inhibiting and killing fungi preparation.
Description
Technical field
The present invention relates to the plant pesticide technology, more particularly, relate to a kind of plant extracts active component and preparation method thereof and application of inhibiting and killing plant pathogenic fungi.
Background technology
Before chemical synthetic pesticide used, agricultural chemicals the earliest had much and extracts from plant, and the natural active matter of some plant has been developed to the commodity insecticide, as rotenone, nicotine etc.But since the forties in 20th century, the organic chemistry synthetic pesticide has replaced botanical pesticide one after another and has ranked market, causes the research of botanical pesticide once being absorbed in low ebb.The agricultural chemicals that uses in China at present is chemicals more than 80%, these chemical pesticides are to reducing plant pest, improving crop yield plays an important role, but also brought a series of problems such as environmental pollution, residual hazard harm, germ develop immunity to drugs, the person poultry safety is on the hazard simultaneously, brought hidden danger for people's health.Therefore develop the biologically active height, non-target organism safety, natural active matter that Environmental compatibility is good have been become the new trend of agricultural chemicals development, the exploitation of botanical pesticide becomes the focus of research again, is that the patent application of CN1218645C discloses a kind of Chinese herbal medicine insectofungicide of being made by extract and emulsifier, turpentine oil and the ethanol of the tuber of stemona, Wild Carrot Fruit, azedarach, the fruit of Rangoon creeper, kuh-seng, tarragon as notification number.Li Jingwu etc. are mixed with emulsion with fruit of cubeb litsea tree oil, and the main disease congo red of tea tree rust, tea verticillium wilt, tea moire blight and cotton wilt, verticillium wilt etc. are prevented and treated, and have obtained the better prevention effect.But Deng Ji botanical pesticide nearly all is an insecticide now, the formulation products of inhibiting and killing plant pathogenic fungi is but very few, and in agricultural production, plant pathogenic fungi is very harmful to plant, be the obstacle of agricultural produce, increasing peasant income, therefore, people press for the effectively inhibiting and killing plant pathogenic fungi that more kinds are provided, and environmental friendliness can ensure person poultry safety's pesticidal preparations product, to promote increasing peasant income.
Summary of the invention
At the very few present situation of existing plant resource inhibiting and killing plant pathogenic fungi preparation variety, the object of the invention aims to provide a kind of novel formulation that can be used for preparing inhibiting and killing plant pathogenic fungi, and preparation biologically active height, to non-target organism safety, active constituent of curcuma longa that Environmental compatibility is good, and the preparation method of this active component and application.
The active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi provided by the invention is for ethanol or methyl alcohol being the extract that extractant extraction root tuber of aromatic turmeric obtains.As inhibiting and killing plant pathogenic fungi extraction of active ingredients thing, can be to be the total extract that extractant extraction root tuber of aromatic turmeric obtains with ethanol or methyl alcohol, also can be to be extractant with the n-hexane to being the further essence extract that obtains of extraction of the total extract that obtains of extractant extraction root tuber of aromatic turmeric with ethanol or methyl alcohol.Total extract and essence extract all can be used for preparing the active component of inhibiting and killing plant pathogenic fungi preparation, preferentially select the active component of essence extract for use, the paste extract that the extract of particularly selecting for use extractant extraction root tuber of aromatic turmeric to obtain obtains through further concentration as preparation inhibiting and killing plant pathogenic fungi preparation.
In such scheme, extract the resulting extract of root tuber of aromatic turmeric with ethanol or methyl alcohol as extractant, pressing down of plant pathogenic fungi all there is good active extremely, that is to say, both can select for use ethanol as extractant, and also can select for use methyl alcohol, and select for use ethanol to make the extractant better effects if as extractant, preferentially select for use volume fraction to be not less than 75% ethanol, especially preferentially selecting volume fraction for use is 90%～98% ethanol.
The active constituent of curcuma longa of the inhibiting and killing plant pathogenic fungi that the present invention discloses can be prepared by the method that may further comprise the steps:
(1) with the root tuber of aromatic turmeric root-like stock of drying and crushing or piece root with ethanol or methyl alcohol lixiviate 12～24h, stirring in water bath is extracted 6～12h under 45～55 ℃ condition then, and the active component in the root tuber of aromatic turmeric is fully leached, and collects leaching liquor.
(2) leaching liquor of collecting is concentrated, obtain the root tuber of aromatic turmeric total extract, and reclaim ethanol or methyl alcohol as solvent.
The above-mentioned root tuber of aromatic turmeric total extract that obtains also can further be used n-hexane extraction, obtains the root tuber of aromatic turmeric essence extract.Extraction the root tuber of aromatic turmeric total extract and the essence extract that obtain, further simmer down to paste extract is as the active component of inhibiting and killing plant pathogenic fungi preparation.Paste extract can be with being directly used in the preparation for preparing inhibiting and killing plant pathogenic fungi behind the acetone solution.
The active constituent of curcuma longa of the inhibiting and killing plant pathogenic fungi that the present invention discloses, can be used for preparing inhibiting and killing plant pathogenic fungi and mushroom disease fungus preparation, especially for preparation press down to kill that the horseradish China ink goes into that bacterium, Sclerotinia sclerotiorum, botrytis cinerea, wheel branch pityrosporion ovale, gibberella saubinetii, edible mushroom wart spore are mould, preparations such as olive green mold or pale mould, as the active component of preparation.
The active constituent of curcuma longa of the inhibiting and killing plant pathogenic fungi that the present invention discloses, the plant pathogenic fungi that can be prepared into formulations such as aqua, soluble powder, wetting powder, strong suspending agent and granula presses down preparation extremely.The preparation method of various formulations is the conventional method of this area, and adjuvant used in each formulation preparation process is usual auxiliaries in the pesticide field.The solution that each formulation is prepared in use, wherein the concentration of Radix Curcumae extract should be in the scope of 0.1mg/ml～1000mg/ml.
Bacteriostatic active ingredients can also further adopt phytochemical method to separate and identify in the root tuber of aromatic turmeric, to find new compound with sterilization and bacteriostatic activity, and can be on this basis, with this compound structure is template, carry out molecular structure alteration, be developed to the new Fungicidal formulations kind that presses down.
Radix Curcumae extract provided by the invention to the horseradish China ink go into that bacterium, Sclerotinia sclerotiorum, botrytis cinerea, wheel branch pityrosporion ovale, gibberella saubinetii, edible mushroom wart spore are mould, olive green mold or pale mould etc. have tangible activity, and because the root tuber of aromatic turmeric raw material sources are extensive, nontoxic to human body, harmless to crop, press down the characteristics that Fungicidal formulations has easy degraded as plant resource, therefore be expected to be developed as a kind of non-harmful plant resource and press down Fungicidal formulations.
Embodiment
Describe the specific embodiment of the present invention by the following examples, content of the present invention is described in further detail again.But this should be interpreted as protection scope of the present invention only limits to example, on the contrary, all application based on the technology that content of the present invention realized all belongs to protection scope of the present invention.
What following examples were used is the root tuber of aromatic turmeric that produces available from Chongzhou City, Sichuan.Determination of activity is under the condition that exsomatizes, adopt growth rate method to measure the bacteriostatic activity of prepared sample to disease fungus, go into bacterium (Phomawasabiae Yokogi) for the examination bacterial classification for the horseradish China ink, Sclerotinia sclerotiorum (Sclerotinia sclerotiorum (Lib) de Bary), botrytis cinerea (Botrytis Cirerea), wheel branch pityrosporion ovale (Verticillium dahliae Kled), gibberella saubinetii (Fusariumgraminearum), edible mushroom wart spore mould (Mycogone peniciosa), olive green mold (Chaetomium olivaceumCooket Ell) or pale mould (penicillium pallidum Smith) are respectively by Sichuan Province Department of Agriculture Plant Quarantine Center, Chengdu Inst. of Biology, Chinese Academy of Sciences provides.
The preparation of embodiment 1 Radix Curcumae extract and bacteriostatic activity are measured
The step of preparation Radix Curcumae extract:
(1) dry root tuber of aromatic turmeric root-like stock (or piece root) is pulverized the back and cross 40 mesh sieves, obtain root tuber of aromatic turmeric dry powder.
(2) get 95% the ethanol that is equivalent to about 7 times of raw material quality (general control range be 5～10 times) volume room temperature (temperature is generally 18～24 ℃ of scopes) down the about 20h of lixiviate (generally control range is 12～24h).
(3) continuous stirring is extracted about 10h (generally control range is 6～12h) in about 50 ℃ (general control range be 45～60 ℃) waters bath with thermostatic control.Use the Buchner funnel negative pressure filtration, remove residue, get filtrate 1..
(4) residue is further stirred with 95% the ethanol that is equivalent to about 6 times of raw material quality (general control range be 5～10 times) volume again extract 8h (general control range is 6～12h), active component in the root tuber of aromatic turmeric is fully proposed, use the Buchner funnel negative pressure filtration, remove residue, get filtrate 2..
(5) merging filtrate 1. with filtrate 2., filtrate is concentrated down about 55 ℃ (general control range be 45～60 ℃) water-baths with Rotary Evaporators, obtains the lixiviate condensed cream, 4 ℃ of preservations.
(6) the root tuber of aromatic turmeric total extract that takes a morsel, it is 0.05gml that clear water is configured to concentration
-1Solution is as the standby sample of experiment.
The said extracted solvent also can be methyl alcohol.
Bioactive mensuration: under isolated condition, adopt the mycelial growth rate method to measure the bacteriostatic activity of Radix Curcumae extract.(mass concentration is 0.05gml to get the standby sample of experiment respectively
-1) the preparation medium, make 5 concentration gradients, i.e. 2.5mgml
-1, 2.0mgml
-1, 1.0mgml
-1, 0.5mgml
-1, 0.25mgml
-1Treat that flat board solidifies the back and goes into bacterium bacterium cake (d=6.0mm) at plate central authorities access horseradish China ink cultured, that growth is consistent.Every ware connects a bacterium cake, and to add sterile aqueous media inoculation back as blank (CK), 3 repetitions are established in each processing, cultivates record data when contrast culture ware bacterium colony is about to cover with culture dish in 26 ℃ of following constant incubators.Each bacterium colony measures twice according to the right-angled intersection method, represents the size of its bacterium colony with mean, and the computing formula of bacteriostasis rate is as follows:
Colony diameter (mm)=bacterium colony average diameter-bacterium cake diameter (6.0mm)
Bacteriostasis rate (%)=(contrast colony diameter-processing colony diameter) ÷ contrasts colony diameter * 100%
Experimental result is as shown in table 1.
Table 1 root tuber of aromatic turmeric is extracted medicinal extract is gone into bacterium to China ink inhibition effect
| Extract solvent Extraction solvent | Bacteriostasis rate (%) Inhibition rate | Virulence regression equation (Y=) Toxicity regression equatioin | Coefficient R 2 | EC 50 mg·ml -1 |
| Methanol | 65.29 67.08 | 1.3371x+5.2326 1.2923x+5.3148 | 0.9622 0.9781 | 0.6699 0.5707 |
Annotate: bacteriostasis rate is that liquor strength is at 1.0mgml
-1In time, measured
As can be seen from Table 1, root tuber of aromatic turmeric methyl alcohol, ethanol extract are gone into bacterium to the horseradish China ink and are shown stronger bacteriostatic activity, and concentration was EC during it suppressed
50Be respectively 0.6699mgml
-1, 0.5707mgml
-1
Embodiment 2 root tuber of aromatic turmeric ethanol total extracts, N-hexane extract are to the influence of pathogen spore germination
(1) for the examination bacterial classification: the horseradish China ink is gone into bacterium, olive green mold, pale mould, wheel branch pityrosporion ovale.
(2) adopt the concave slide method: (final concentration of soup is 10mgml to get the pastille spore suspension of preparation
-1) and each 100 μ l of spore suspension of blank, acetone contrast drip on concave slide, put into sterilized culture dish, place 26 ℃ of incubators to cultivate, soup is handled and control group is all established 4 repetitions.Check the spore germination situation after 12 hours.10 visuals field of microscopy, each is handled and approximately observes 200 spores, calculates spore germination rate.When spore germ tube length half (minimum diameter) above the spore diameter length, as the spore of having sprouted, computing formula is as follows:
Spore germination rate=sprouting spore count ÷ checks spore count * 100%
Spore germination inhibiting rate=(contrast germination rate-processing germination rate) ÷ contrasts germination rate * 100%
Experimental result is as shown in table 2.
Table 2 Radix Curcumae extract is to pathogen spore germination inhibitory action
| Pathogen Pathogeny | Ethanol extract is handled back germination rate % | Ethanol extract is handled back inhibiting rate % | N-hexane extract is handled back germination rate % | N-hexane extract is handled back inhibiting rate % | Acetone contrast germination rate % | Blank germination rate % |
| China ink is gone into the pale mould wheel of bacterium olive green mold branch pityrosporion ovale | 8.8 9.5 7.9 7.1 | 90.5 90.2 91.7 92.5 | 2.6 3.1 3.3 2.5 | 97.1 96.7 96.5 97.4 | 90.1 94.0 93.6 94.5 | 93.0 96.5 94.7 94.2 |
As can be seen from Table 2, when being 10mg/ml for the examination mass concentration, root tuber of aromatic turmeric ethanol crude extract is to supplying examination pathogen spore germination inhibiting rate all＞90%, and N-hexane extract to the spore germination inhibiting rate all＞96%.The thick cream of ethanol, n-hexane extraction medicinal extract that root tuber of aromatic turmeric is described all have stronger inhibition ability to the pathogen spore germination.
The preparation and the biological activity determination of embodiment 3 root tuber of aromatic turmeric N-hexane extracts.
(1) root tuber of aromatic turmeric ethanol total extract obtains n-hexane extract with 8 times of volume n-hexane extractions separation, and extract concentrates with Rotary Evaporators, and thickening temperature is about 40 ℃, obtains the paste extract.
(2) to be configured to concentration with acetone soln be 0.05gml to above-mentioned extraction paste
-1Soup is as the standby sample of experiment.With the horseradish China ink go into that bacterium, Sclerotinia sclerotiorum, botrytis cinerea, wheel branch pityrosporion ovale, gibberella saubinetii, edible mushroom wart spore are mould, olive green mold, pale mould serve as for the examination bacterial classification, measure the bacteriostatic activity under the different liquor strengths, the bacteriostatic activity assay method is with embodiment 1.
Experimental result is as shown in table 3.
Table 3 root tuber of aromatic turmeric N-hexane extract is to the inhibitory action of 8 kinds of pathogen mycelial growths
| Pathogen Pathogeny | Bacteriostasis rate (%) Inhibition rate | Virulence regression equation (Y=) Toxicity regression equatioinpic | Coefficient R 2 | EC 50 mg·ml -1 |
| The horseradish China ink enters the mould tomato gray mould bacterium wheel of the pale mould edible mushroom of bacterium gibberella saubinetii Sclerotinia sclerotiorum olive green mold wart spore branch pityrosporion ovale | 79.70 76.82 86.58 54.78 74.85 71.72 100 78.19 | 1.5427x+5.7430 1.1319x+5.6782 1.6137x+5.8899 1.3975x+5.0791 1.2270x+5.7781 2.1487x+5.3058 2.9576x+7.2392 1.6640x+5.6480 | 0.9468 0.9248 0.9475 0.9796 0.9561 0.9534 0.9172 0.9559 | 0.3299 0.2517 0.2809 0.8778 0.2322 0.7206 0.1749 0.4080 |
Annotate: bacteriostasis rate is that liquor strength is at 1.0mgml
-1In time, measured
As can be seen from Table 3, root tuber of aromatic turmeric ethanol total extract N-hexane extract all has the obvious suppression effect to 8 kinds of disease funguses, wherein the inhibiting rate to the tomato gray mould bacterium reaches 100%, the bacteriostasis rate of the horseradish China ink being gone into bacterium is compared with root tuber of aromatic turmeric ethanol total extract and is increased, and illustrates that the active ingredient of root tuber of aromatic turmeric is present in the N-hexane extract.
The mensuration of embodiment 4 minimum inhibitory concentration MIC and minimum bactericidal concentration MBC
Preparation potato liquid nutrient medium is sub-packed in some test tubes, and every pipe 4ml gets mother liquor (the concentration 0.05gml of root tuber of aromatic turmeric alcohol extract N-hexane extract configuration
-1), coubling dilution preparation medium makes and respectively manages content of dispersion and be followed successively by 25mgml
-1, 12.5mgml
-1, 6.25mgml
-1, 3.13mgml
-1, 1.56mgml
-1, 0.78mgml
-1, get respectively adjust concentration for examination bacteria suspension 40 μ l, add 6 and contain pencil and clear water, acetone are done the positive control pipe respectively, do not inoculate with clear water, acetone respectively in addition, make negative control.26 ℃ of shaking tables are cultivated 48h, are contrast with the negative tube, and are if muddiness appears in culture or the bottom floccule occurs for growing, promptly positive; Otherwise, clarify or do not have floccule then for not growing, promptly negative.Each pipe of doubling dilution is tried the minimum inhibitory concentration of bacterial classification with the high dilution of the root tuber of aromatic turmeric acetone solution liquid of asepsis growth as this, is the MIC value of this bacterium.Take out soup medium that fungi do not grow and be applied on the flat board that potato culture makes, cultivate weeks in 26 ℃, observed result again, the high dilution that fungi does not grow is the minimum bactericidal concentration (MBC) of this medicine.
Experimental result is as shown in table 4.
The MIC of table 4 root tuber of aromatic turmeric N-hexane extract and MBC value
| Pathogen Pathogeny | MIC(mg·ml -1) | MBC(mg·ml -1) | |||||||
| 0.78 | 1.56 | 3.13 | 6.25 | 12.5 | 1.56 | 3.13 | 6.25 | 12.5 | |
| The horseradish China ink enters the mould tomato gray mould bacterium wheel of the pale mould edible mushroom of bacterium gibberella saubinetii Sclerotinia sclerotiorum olive green mold wart spore branch pityrosporion ovale | + + + + + + + + | + + + + + + - + | + + - + - + - + | - - - + - - - - | - - - - - - - - | + + + + + + - + | + + - + - + - + | - - - - - - - - | - - - - - - - - |
Annotate :+: growth-: do not grow
The root tuber of aromatic turmeric N-hexane extract is for the inhibitory action that all has for the pathogen of trying in various degree as can be seen for table 4, and the high concentration medicament all has sterilizing ability to pathogen.
Claims (10)
1, a kind of active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi is characterized in that active component is is the extract that extractant extraction root tuber of aromatic turmeric obtains with ethanol or methyl alcohol.
2, the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 1, it is characterized in that described active component for being on the extract basis that obtains of extractant extraction root tuber of aromatic turmeric with ethanol or methyl alcohol, further is the extract that extractant extraction extract obtains with the n-hexane.
3, the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 1 and 2 is characterized in that described active component is the paste extract that extract obtains through concentration.
4, the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 1 and 2 is characterized in that described extractant is an ethanol.
5, the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 4 is characterized in that the ethanol as extractant is that volume fraction is not less than 75% ethanol.
6, the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 5 is characterized in that its volume fraction of ethanol as extractant is 90%～98% ethanol.
7, about the preparation method of the active constituent of curcuma longa of the described inhibiting and killing plant pathogenic fungi of each claim in the claim 1 to 6, it is characterized in that may further comprise the steps:
(1) with the turmeric rhizome of drying and crushing with ethanol or methyl alcohol lixiviate 12～24h, extract 6～12h 45～55 ℃ of stirring in water bath then, the active component in the root tuber of aromatic turmeric is fully leached, collect leaching liquor;
(2) leaching liquor of collecting is concentrated, obtain paste root tuber of aromatic turmeric total extract, and reclaim ethanol or methyl alcohol as solvent;
8, the preparation method of the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 7 is characterized in that concentrating the paste root tuber of aromatic turmeric total extract that obtains and obtains the root tuber of aromatic turmeric extract with the n-hexane extraction separation again.
9,, it is characterized in that the application in preparation inhibiting and killing plant pathogenic fungi and mushroom disease fungus preparation about the application of the active constituent of curcuma longa of the described inhibiting and killing plant pathogenic fungi of each claim in the claim 1 to 6.
10, the application of the active constituent of curcuma longa of inhibiting and killing plant pathogenic fungi according to claim 9 is characterized in that press down killing in preparation that the horseradish China ink goes into that bacterium, Sclerotinia sclerotiorum, botrytis cinerea, wheel branch pityrosporion ovale, gibberella saubinetii, edible mushroom wart spore are mould, the application in olive green mold or the pale mould preparation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206586A (en) * | 2011-04-22 | 2011-10-05 | 中国农业科学院植物保护研究所 | Culture method of entomogenous fungi sclerotium and application thereof |
| CN105341010A (en) * | 2015-12-09 | 2016-02-24 | 四川大学 | Curcuma aromatica active extract, sterilization preparation and application of curcuma aromatica extract |
| CN106614838A (en) * | 2016-11-28 | 2017-05-10 | 国营全椒县棉花原种总场 | Cotton seed seed-dressing agent |
| CN108703157A (en) * | 2018-07-06 | 2018-10-26 | 四川大学 | It the common fig leaf juice active constituent of inhibiting and killing plant pathogenic fungi and its produces and applies |
| CN113475534A (en) * | 2021-07-26 | 2021-10-08 | 海南师范大学 | Morinda citrifolia sterilizing preparation and preparation method thereof |
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2007
- 2007-05-08 CN CNB2007100490424A patent/CN100571518C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206586A (en) * | 2011-04-22 | 2011-10-05 | 中国农业科学院植物保护研究所 | Culture method of entomogenous fungi sclerotium and application thereof |
| CN102206586B (en) * | 2011-04-22 | 2013-09-18 | 中国农业科学院植物保护研究所 | Culture method of entomogenous fungi sclerotium and application thereof |
| CN105341010A (en) * | 2015-12-09 | 2016-02-24 | 四川大学 | Curcuma aromatica active extract, sterilization preparation and application of curcuma aromatica extract |
| CN105341010B (en) * | 2015-12-09 | 2018-11-13 | 四川大学 | A kind of Radix Curcumae activity extract, biocide preparation and its application |
| CN106614838A (en) * | 2016-11-28 | 2017-05-10 | 国营全椒县棉花原种总场 | Cotton seed seed-dressing agent |
| CN108703157A (en) * | 2018-07-06 | 2018-10-26 | 四川大学 | It the common fig leaf juice active constituent of inhibiting and killing plant pathogenic fungi and its produces and applies |
| CN113475534A (en) * | 2021-07-26 | 2021-10-08 | 海南师范大学 | Morinda citrifolia sterilizing preparation and preparation method thereof |
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| CN100571518C (en) | 2009-12-23 |
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