CN102260251B - A kind of macrolides compound and Synthesis and applications thereof - Google Patents
A kind of macrolides compound and Synthesis and applications thereof Download PDFInfo
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- CN102260251B CN102260251B CN201010185451.9A CN201010185451A CN102260251B CN 102260251 B CN102260251 B CN 102260251B CN 201010185451 A CN201010185451 A CN 201010185451A CN 102260251 B CN102260251 B CN 102260251B
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Abstract
The invention provides a kind of new 16-membered ring macrolides compound bafilomycin K with antagonistic plant pathogenic fungi activity that yellow three plain streptomycetes (Streptomyces flavotricini) the Y12-26 metabolism of marine microorganism produces.The invention still further relates to the application as agricultural chemicals of the preparation method of described compound and this compound.
Description
Technical field
The present invention relates to pesticide field.In particular, the present invention relates to the application of a kind of new macrolides compound, its preparation method, composition containing this compound and this compound.
Background technology
Ocean environment is unique, the feature of its high pressure, high salt, low nutrition, low temperature is considered to the extreme environment of vital movement, this environment creates the specificity of microbe species and pathways metabolism, can produce the novel biologically active substance being different from land microorganism completely.The active metabolite that marine microorganism produces has the feature of following 5 aspects: structure type is rich and varied; There is ecological significance (some can suppress the growth of invasive species, and some can provide provide protection to host); There is unique biological activity; Active substance ratio is high; Closely related with fermentation condition.Therefore relevant expert's prediction, marine microorganism will be the valuable source obtaining new medical, agricultural antibiotic from now on.R&D intensity is strengthened in this respect year by year in countries in the world, and obtain a series of microbiotic, the new antibiotic of the successful listing in such as first marine microorganism source: cynnematin, separation and Extraction from thalassiomycetes cephalosporium acremonium (Cephalosporium acermonium) and obtaining; Japanese researchers has been separated to again the microbiotic of wide spectrum, low toxicity from marine bacteria---she his mycin (istamycin), and manually modified and synthetic work is carried out to it.
Actinomycetes are unicellular prokaryotic that a class form difference has multinuclear greatly, in the outstanding Bacteria Identification handbook of uncle, belong to Firmicutes, actinobacteria guiding principle.Current mankind microbiotic major part used comes from terrestrial actinomycetes, due to current quite deep to terrestrial actinomycetic research, the probability of the new antibiotic found from terrestrial actinomycetes declines just gradually, therefore people from the end of the fifties in last century just develop marine actinomycete.At present, the active metabolite of the novel structure produced by marine actinomycete is maximum, and before coming genus bacillus, slime bacteria, Pseudomonas alba, and the active result more than 90% of marine actinomycete originates from streptomyces, originates from other Pseudomonas actinomycetic on a small quantity.
Lactone microbiotic is one of classification that in microbiotic, quantity is maximum, particularly Macrolide.At present all belong to Macrolide at pharmaceutically widely used many microbiotic, than erythromycin, Roxithromycin, clarithromycin if any 14 rings, the Azythromycin of 15 rings, the acetylspiramycin, mydecamycin, kitasamycin, josamycin, rokitamycin etc. of 16 rings.Above-mentioned microbiotic all originates from terrestrial microorganism, have also discovered the microbiotic of many Macrolidees at present from marine actinomycete, and wherein some compound has very strong biological activity.
Domestic and international many scientific research personnel are that activity is followed the tracks of with bacteriostatic activity, are separated the macrolide antibiotics obtaining some new textures.Such as, 2002, the people such as the Asolkar of Germany find that the tunning of marine actinomycete Streptomycete sp.B7064 has strong bacteriostatic activity, and extraction obtains the new macrolide antibiotics chalcomycinB with two glycosidic links, and known microbiotic chalcomycin, both inhibited to various bacteria, be 0.39 μ g/ml to the MIC of Staphylococcus aureus.2006, people's reports such as Fenical, be separated from the La Jolla coastal sediment thing of California and obtain a strain marine actinomycete CNQ140, through strain identification, this bacterial classification belongs to new genus, and called after Marinispora belongs to, the rear separation of fermentation obtains 4 new macrolide antibiotics marinomycins A-D, four compounds all have bacteriostatic activity and inhibiting tumor cell activity, 0.25 μ g/ml is all less than to the MIC of the streptococcus aureus of methicillin-resistant, 0.63 μ g/ml is all less than to the MIC of the faecium of anti-vancocin, marinomycins A is to the IC of colon cancer cell HCT-116
50be 0.18 μ g/ml.
In recent years, active in activity is followed the tracks of and be separated the report obtaining new compound and be on the increase with inhibiting tumor cell from marine actinomycete.2000, the people such as Canedo extract and obtain new macrolides compound IB-96212 from the mycelium after the fermentation of marine actinomycete Micromonospora sp., containing 26 rings and L-oleandrose in its structure, first spiro ketal Macrolide natural product found, IB-96212 has inhibiting tumor cell activity, has extremely strong cytotoxicity (IC to P388 cell
50=0.0001 μ g/ml), also there is obvious cytotoxicity (IC to A5-49, HT-29 and MEL-28 cell
50=1 μ g/ml).2002, the people such as Takeshi Yamada isolate a strain actinomycetes Streptomyces hygroscopicusOUPS-N92 from ocean fish, with to the cytotoxicity of P388 cell for activity is followed the tracks of, be separated from its tunning and obtain a kind of new Macrocyclic lactone compounds halichoblelide, cytotoxicity is had, to the ED of P388 cell to multiple cancer cells
50be 0.63 μ g/ml.2009, the people such as Hispanic Marta Perez extract and obtain new macrolides compound tartrolon D, to the GI of lung carcinoma cell, colon cancer cell, breast cancer cell from the fermented liquid of marine actinomycete Streptomyces sp.MDG-04-17-069
50be respectively 0.16,0.31,0.79 μ g/ml, and its analog tartrolone C has insecticidal activity, is also actinomycetes meta-bolitess.
Bafilomycins compounds has 16-membered macrocyclic lactone structure, and between 1983 to 1985 years, 8 kinds of bafilomycins compounds are separated from actinomycetes meta-bolites, are respectively bafilomycin A
1, A
2, B
1, B
2, C
1, C
2d, E, nineteen ninety-five has the derivative AH-758 of a kind of new bafilomycin B to be found, have again this new compounds to be found recently, the people such as Gavin Carr report in 2010, extracts and obtains 8 bafilomycins compounds from marine actinomycete Streptomyces sp.strains RJA71 and RJA635, wherein 5 is novel compound: bafilomycin F ~ J, and all the other 3 compounds are bafilomycin A
1, B
1, D.In above-mentioned bafilomycins compounds, bafilomycinA
1a kind of cavity H
+the specific inhibitor of-ATP enzyme (V-ATPase), its mechanism of action is furtherd investigate, can be used for treating osteoporosis, deafness, also can be used as cancer therapy drug; Bafilomycin A
1, B
1, C
1to multiple gram positive bacterium and fungi, there is antagonistic action; Bafilomycin D and E has insecticidal activity, and bafilomycin D also has weeding activity; Bafilomycins F, G, H and J have the activity of very strong T suppression cell autophagy.
Summary of the invention
Present inventor finds through research, 1 that is produced by yellow three plain streptomycete Y12-26 metabolism new ten hexa-atomic macrolides compound bafilomycin K have good inhibit activities to plant pathogenic fungis such as Pyricularia oryzaes (Pyricularia oryzae), the mycelium extract containing this compound of yellow three plain streptomycete Y12-26 has good prevention effect to the fungal diseases of plants such as powdery mildew of cucumber, cucumber anthracnose, therefore can prevent and treat these fungal diseases with used as pesticides.
One that the object of the present invention is to provide yellow three plain streptomycete Y12-26 to produce new ten hexa-atomic macrolides compounds with using value and its preparation method and application.
Specifically, first aspect present invention provides the ten hexa-atomic Macrocyclic lactone compounds bafilomycin K with new texture, and described compound structure is as shown below.
As used in this application, the implication of term " compound of the present invention " or similar terms not only includes the compound that structure above defines, and further comprises their salt.Described salt can be water-soluble, ester is molten or dispersible the form of product, its by with mineral acid or organic acid or alkali reaction form.The example of these acid salt comprises acetate, adipate, alginate, benzoate, benzene sulfonate, hydrosulfate, butyrates, Citrate trianion, dodecane sulfonate, hydrochloride, oxalate, propionic salt, succinate, tartrate.Basic salt comprises ammonium salt, an alkali metal salt, and as sodium salt and sylvite, alkaline earth salt, as calcium salt and magnesium salts, the salt of organic bases, as dicyclohexylamine salt etc.
Compound of the present invention also can its tautomeric form exist.Although explicitly point out these forms in compound not described here, they are also contained within scope of the present invention.Except as otherwise noted, the chemical name of compound described here comprises the mixture of the stereochemistry heterogeneous forms that all possible, described compound can comprise.Described mixture can contain all diastereomers and/or the enantiomer of described compound basic molecular structure.The all stereochemistry heterogeneous forms of the compounds of this invention can be purified form, or the form of mixing mutually, and this is included within the scope of the invention.
Second aspect present invention provides a kind of method preparing above-claimed cpd, and it is characterized in that, the method comprises the following steps:
(1) cultivate yellow three plain streptomycetes (Streptomyces flavotricini) (streptomycete Y12-26 as plain in the Huang three in the present invention), obtain fermented liquid;
(2) with one or more be selected from organic solvent extraction, the method for lixiviate or chromatography is separated and obtains described compound from described fermented liquid.
Bacterial strain involved in the present invention is yellow three plain streptomycete Y12-26, this bacterial strain is separated and obtains from the mangrove acanthus of tideland, Hainan, this bacterial strain is kept at China Microbiological bacterial strain preservation management committee's common micro-organisms center (CGMCC) (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica) on May 13rd, 2010, and preserving number is CGMCC No.3832.
Term used herein " fermentation " or " cultivation " have those skilled in the art and usually know and the implication of admitting.Described " mycelium ", " fermented liquid " or " nutrient solution ", by cultivating yellow three plain streptomycete Y12-26 of the present invention under the condition of applicable growth, make it grow to certain mycelium concentration and obtain.
Have no particular limits for the nutrition source cultivated in the substratum of bacterial strain of the present invention.Those skilled in the art can select suitable carbon source, nitrogenous source and other nutrition sources according to known technology.Such as, carbon source can be starch, dextrin, glucose, fructose, sucrose, glycerine, inositol, N.F,USP MANNITOL etc.Nitrogenous source can be peptone, soyflour, soybean cake powder, meat extract, protein powder, wheat skin, rice sugar, yeast powder, corn steep liquor, ammonium salt and other organism or inorganic nitrogen-containing compound.In addition, in substratum, also can suitably add some inorganic salts, as metal-salts such as sodium-chlor, phosphoric acid salt (as potassium primary phosphate and dipotassium hydrogen phosphate etc.), manganous sulfate, ammonium sulfate, magnesium sulfate, calcium carbonate.Usually various known conventional medium can be adopted, as LB nutrient agar, nutrient agar, glucose yeast cream nutrient agar and ox meat extract nutrient agar etc.
In a specific embodiment, the substratum for cultivating bacterial strain of the present invention has following composition (% represents mass/volume): glucose 0.1% ~ 3%, soybean cake powder 0.1% ~ 3%, W-Gum 0.1% ~ 2%, yeast powder 0.01% ~ 1%, extractum carnis 0.01% ~ 0.5%, NaCl 0.1% ~ 3%, K
2hPO
40.01% ~ 0.5%, pH 6.8 ~ 7.4.But, it will be appreciated by those skilled in the art that the present invention is not limited to these the concrete culture medium prescriptions enumerated herein.
The condition such as temperature, pH, vapour-liquid ratio, tank pressure, rotating speed of cultivating the bacterial strain in the present invention does not have restriction strict especially, as long as this condition is applicable to the growth of this bacterium.
The defoamer such as soya-bean oil, bubble enemy can be adopted froth breaking is carried out when cultivating.In some preferably embodiment, pH should control between 5.5 ~ 8.0, and culture temperature should between 20 ~ 35 DEG C.Incubation time is usually between 12h ~ 200h, and final bacteria concentration usually can up to 5 × 10
7cfu/ml ~ 1 × 10
11cfu/ml.
At one preferably in embodiment of the present invention, cultivate described yellow three plain streptomycetes by the following method to obtain fermented liquid and mycelium: containing carbon source, nitrogenous source, in the substratum of inorganic salt, controlling at vapour-liquid ratio at air flow is 0.2: 1 ~ 2: 1, rotating speed is at 100r/min or more, cultivate described yellow three plain streptomycete more than 24 hours, wherein said carbon source is selected from glucose, fructose, sucrose, 1 kind or several in starch and rice meal, described nitrogenous source is selected from extractum carnis, protein powder, yeast powder, 1 kind or several in groundnut meal and soybean cake powder, described inorganic salt comprise conventional inorganic salt, as NaCl, MgSO
4, (NH
4)
2sO
4, MgCl
2, KCl, KH
2pO
4, K
2hPO
4and CaCO
3.In a preferred scheme, the substratum for cultivating bacterial strain of the present invention has following composition (% represents mass/volume): glucose 0.1% ~ 3%, soybean cake powder 0.1% ~ 3%, W-Gum 0.1% ~ 2%, yeast powder 0.01% ~ 1%, extractum carnis 0.01% ~ 0.5%, NaCl0.1% ~ 3%, K
2hPO
40.01% ~ 0.5%, pH 6.8 ~ 7.4.
Above-mentioned these parameters enumerated just realize preferred version of the present invention.Therefore, those skilled in the art select suitable culture condition also can obtain fermented liquid of the present invention and mycelium beyond above-mentioned scope.
After obtaining above-mentioned fermented supernatant fluid or mycelium, with one or more (such as 2 kinds, 3 kinds, 4 kinds) be selected from organic solvent extraction, lixiviate, chromatography (as column chromatography, thin-layer chromatography, high performance liquid chromatography preparation) method be therefrom separated obtain as described in compound.Described fermented liquid comprises fermented supernatant fluid and mycelium, described compound is present in fermented supernatant fluid or mycelium, therefore, preferred version of the present invention obtains crude product or vat liquor from the fermented supernatant fluid after fermentation or mycelium, and then carry out further separation and purification.
As a preferred version of the present invention, first mycelium is obtained from described separation of fermentative broth, with acetone or methyl alcohol lixiviate, use chloroform, methylene dichloride or butanol solvent partition again, leach with anhydrous methanol, then use silica gel column chromatography with methylene chloride/methanol gradient elution, collect the wash-out position that methylene chloride/methanol is 80: 1 to 20: 1, prepare compound of the present invention with thin-layer chromatography preparation and HPLC successively.
Conventional separation means can be adopted as centrifuging etc. from separation of fermentative broth mycelium.Then, after obtaining mycelium, a kind of feasible mode carries out lixiviate one or many with organic solvent (as acetone, methyl alcohol etc.), extraction one or many is carried out again with low polar organic solvent (as propyl carbinol, methylene dichloride, chloroform etc.), leaching one or many is carried out again through too high polar organic solvent (as methyl alcohol, ethanol etc.), obtained mycelium extractive substance crude product, solvent for use lixiviate or extract 6 ~ 12 hours each time.
Subsequently, carry out the aforementioned crude product of methylene chloride/methanol gradient elution with column chromatography, collect the wash-out position (preferred methylene chloride/methanol is the wash-out position of 60: 1) that methylene chloride/methanol is 80: 1 to 20: 1.Column chromatography used can be such as silica gel column chromatography.Then, by thin layer chromatography board, aforementioned wash-out position is prepared (chromatographic solution proportioning is methylene chloride/methanol is 40: 1), collects R
fvalue is the band between 0.33 ~ 0.36, finally, prepares described compound with HPLC (analyze and prepare proportion of mobile phase used and be methyl alcohol/acetonitrile/water=40: 35: 25 (volumes)).
A kind of feasible mode is also had to be adopt the conventional meanses such as such as centrifugation to obtain fermented supernatant fluid.Then, adopt organic solvent extraction one or many, each extraction time is 6-12h.Described organic solvent be selected from chloroform, ethyl acetate, N-BUTYL ACETATE, methylene dichloride or propyl carbinol one or more.Then, as mentioned above, the extraction position obtained with methylene chloride/methanol gradient elution abovementioned steps with column chromatography, collects the wash-out position (preferred methylene chloride/methanol is 60: 1) that methylene chloride/methanol is 80: 1 to 20: 1.Column chromatography used can be such as silica gel column chromatography.Finally, compound of the present invention is obtained with thin-layer chromatography preparation and/or HPLC preparation (analyze conjunction preparation condition and be methyl alcohol/acetonitrile/water=40: 35: 25 (volumes)).
But, it will be appreciated by those skilled in the art that other separation means, solvent or eluent can be adopted to be separated according to routine techniques completely obtains compound of the present invention.Therefore, the scope of the inventive method is not limited to concrete grammar used in specification sheets embodiment.
And those skilled in the art also the reasonable structure design chemical synthesis route of compound disclosed according to the present invention can obtain this compound.
The present invention provides a kind of plain streptomycete bacterial strain of Huang three that can produce the separation of the compounds of this invention bafilomycin K on the other hand, this bacterial strain is kept at China Microbiological bacterial strain preservation management committee's common micro-organisms center (CGMCC) (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica) on May 13rd, 2010, and preserving number is CGMCCNo.3832.
As the embodiment of the present invention confirm, containing the active compound that can suppress the plant pathogenic fungi such as Pyricularia oryzae (Pyricularia oryzae) and melon anthrax bacteria (Collectotrichum lagenarium) in the fermented liquid (as above clear and/or mycelium) of yellow three plain streptomycetes (Streptomyces flavotricini), therefore, the invention still further relates to the application of above-claimed cpd used as pesticides.In preferred embodiments, described agricultural chemicals is used for control by botrytis cinerea pers (Botrytis cinerea, BC), Rhizoctonia solani Kuhn (Rhizoctonia solani, Rs), cucumber powdery mildew's pathogen (Sphaerotheca fuliginea, Sf), melon anthrax bacteria (Collectotrichum lagenarium, Cl), southern corn leaf blight (Helminthosporium maydis, Hm), fusarium graminearum (Gibberella zeae, Gz), Pyricularia oryzae (Pyricularia oryzae, the microbial disease of pathogenic such as Po), these diseases including, but not limited to, powdery mildew of cucumber, gray mold of cucumber, cucumber anthracnose, rice sheath blight disease etc.
In addition, yellow three plain streptomycete fermentation liquid of the present invention also can be used for preventing and treating the disease caused by above-mentioned plant pathogenic fungi.Therefore, the present invention additionally provides a kind of method of controlling plant diseases on the other hand, and the method comprises: cultivate yellow three plain streptomycetes (Streptomyces flavotricini), obtain fermented liquid; Described fermented liquid is applied on described plant.In a preferred embodiment, crude product or vat liquor should be obtained from the fermented supernatant fluid of fermented liquid or mycelium, then gained crude product or vat liquor are applied on plant.Such as, the separation means of available routine as centrifuging etc., from separation of fermentative broth mycelium.Then, lixiviate one or many is carried out with organic solvent (as acetone, methyl alcohol etc.), extraction one or many is carried out again with low polar organic solvent (as propyl carbinol, methylene dichloride, chloroform etc.), leaching one or many is carried out again through too high polar organic solvent (as methyl alcohol, ethanol etc.), obtained mycelium extractive substance crude product, solvent for use lixiviate or extract 6 ~ 12 hours each time.
The present invention provides a kind of pesticide composition on the other hand, and it is characterized in that, described composition comprises the compound of separation of the present invention as active ingredient, and acceptable carrier in Pesticide Science.Pesticide composition of the present invention can be used for control by botrytis cinerea pers (Botrytis cinerea, BC), Rhizoctonia solani Kuhn (Rhizoctonia solani, Rs), cucumber powdery mildew's pathogen (Sphaerotheca fuliginea, Sf), melon anthrax bacteria (Collectotrichum lagenarium, Cl), southern corn leaf blight (Helminthosporium maydis, Hm), fusarium graminearum (Gibberella zeae, Gz), Pyricularia oryzae (Pyricularia oryzae, the microbial disease of pathogenic such as Po), these diseases including, but not limited to, powdery mildew of cucumber, gray mold of cucumber, cucumber anthracnose, rice sheath blight disease etc.
Compound of the present invention or pesticide composition can directly be used with mycelium extract form, also its suitably dilution (such as diluting 10 times, 100 times, 1000 times or higher) can be used with diluent form.Compound of the present invention or its composition can be applied on the position such as root, stem, leaf of plant.Applying method is the routine techniques in this area, such as, can be soak seed at seeding time, is immersed in mycelium extract or its diluent by plant root before transplanting, or direct mycelium extract or its diluent being sprinkled is watered on seedbed.Filling root can be carried out during field planting, also can fill with root in growing process.Preserve if described pesticide composition is a carrier format, then can apply again after dilute with water before use.As for the suitableeest dispenser dosage of the present invention, those skilled in the art are without the need to determining through test of many times.Can include, but are not limited to the plant that method of the present invention, compound, composition or fermented liquid carry out preventing and treating, paddy rice, watermelon, cucumber, tomato, corn, wheat etc.
Embodiment
Embodiment 1 utilizes organic solvent extraction to be separated from yellow three plain streptomycete Y12-26 ferment rear mycelium extract and obtains new ten hexa-atomic Macrocyclic lactone compounds Bafilomycin K
The present embodiment obtains ten new hexa-atomic Macrocyclic lactone compounds Bafilomycin K by conventional separation means from mycelium after the fermentation of yellow three plain streptomycete Y12-26.
The present embodiment bacterial strain used is yellow three plain streptomycete Y12-26, and this bacterial strain is separated and obtains from the mangrove acanthus of tideland, Hainan.Concrete separating step is as follows:
Get pedotheque 1g, 5% (the v: in the triangular flask of v) sterile artificial's seawater filling 50ml is added under aseptic technique, after the abundant shaken well of turbula shaker, leave standstill after 200r/min shaking table vibration 10min and get supernatant liquor 5ml, add in the triangular flask that 45ml same salinity sterile artificial seawater is housed, the dilution of sesquialter method makes sample final concentration be 10
-2getting 0.1mL is spread evenly across on separating plate, each extent of dilution is coated with 3 flat boards, 26 DEG C of constant temperature culture 7-14 days, after 7 days, on the Gause I substratum being added with potassium bichromate, purifying cultivation is carried out to isolated actinomycetes, with the growth of anti-bacteria and fungi, until colonial morphology single stable, proceed to room temperature preservation in Gause I inclined-plane to after bacterium colony (numbering Y12-26).
Bacterial strain Y12-26 gelatine liquefication is fast, and milk first solidifies, and slowly peptonizes afterwards, and Starch Hydrolysis is fast, and Mierocrystalline cellulose does not grow, and nitrate reduction is strong, produces Melanoidins and H
2s.Utilize D-Glucose, D-Fructose; Do not utilize L-arabinose, D-wood sugar, sucrose, rhamnosyl, raffinose, inositol, PEARLITOL 25C.Verified, this bacterial strain is yellow three plain streptomycetes (Streptomycesflavotricini).
This bacterial strain is kept at China Microbiological bacterial strain preservation management committee's common micro-organisms center (CGMCC) (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica) on May 13rd, 2010, and preserving number is CGMCCNo.3832.
Concrete purification procedures is as follows:
(1) plain for Huang three streptomycete Y12-26 is cultivated under conventional actinomycete fermentation culture condition obtain 30L fermented liquid.Yellow three plain streptomycete Y12-26 to be inoculated in after seed culture medium shake-flask culture 12-24h as seed after slant medium activation, are inoculated in fermention medium by this seed.28 DEG C, cultivate under 220rpm and can terminate for about 7 days.
(2) 30L fermented liquid 4000rpm centrifugal after obtain fermented supernatant fluid and mycelium part.Fermented supernatant fluid part with 80% aqueous acetone solution with the consumption lixiviate 12 hours of volume ratio 1: 1,5000rpm frozen centrifugation after lixiviate, filter residue is removed after filtration, be evaporated to removing acetone and remain aqueous mixture, with equal-volume propyl carbinol by foregoing aqueous mixture extraction three times, each extraction time 12 hours, n-butanol extraction is merged mutually be evaporated to dry, again with anhydrous methanol leaching twice, each leaching time 12 hours, cross and filter insoluble methyl alcohol, be evaporated to dry, obtained mycelium crude extract 11.8g.
(3) by above-mentioned mycelium crude extract through silica gel column chromatography with methylene chloride/methanol 80: 1 to 0: 1 gradient elution (50ml/ flow point), biological activity test is carried out to the flow point of wash-out, indicator is Pyricularia oryzae (Pyricularia oryzae), result display Substance mainly concentrates on methylene chloride/methanol 60: 1 position, is merged by above-mentioned Active fractions after TLC detects.Active fractions after merging is prepared plate through thin-layer chromatography be prepared, chromatographic solution proportioning is methylene chloride/methanol 40: 1, will prepare R on plate
fvalue is that the band of the 256nm ultraviolet colour developing of 0.33 ~ 0.36 scrapes, 2 hours are soaked with anhydrous methanol, then filter four times, merging filtrate is evaporated to dry, be dissolved in 10ml anhydrous methanol and carry out HPLC analysis and prepare, analyze and prepare proportion of mobile phase used and be methyl alcohol/acetonitrile/water=40: 35: 25 (volume ratios), finally prepare compd B afilomycin K.
Embodiment 2 utilizes organic solvent extractionprocess to be separated from yellow three plain streptomycete Y12-26 ferment secondary fermentation supernatant liquor and obtains new ten hexa-atomic Macrocyclic lactone compounds Bafilomycin K
The present embodiment obtains ten new hexa-atomic Macrocyclic lactone compounds Bafilomycin K by conventional separation means from the fermentation secondary fermentation supernatant liquor of yellow three plain streptomycete Y12-26.Concrete purification procedures is as follows:
(1) plain for Huang three streptomycete Y12-26 is cultivated under suitable conditions obtain 30L fermented liquid.(concrete culture condition and step are with embodiment 1)
(2) 30L fermented liquid 4000rpm centrifugal after obtain fermented supernatant fluid and mycelium part.Fermented supernatant fluid equal-volume ethyl acetate extracts 3 times respectively, extracts 6-12h at every turn, is merged mutually by extraction into ethyl acetate and is evaporated to original fermented solution volume.With equal-volume water-saturated n-butanol, 3 times are extracted respectively to ethyl acetate extracting phase again, extract 6-12h at every turn.Carry out biological activity assay to above-mentioned ethyl acetate and n-butanol extraction position, indicator is Pyricularia oryzae (P.oryzae), determines that ethyl acetate extract is main antibacterial active site.
(3) by above-mentioned ethyl acetate reactive site through silica gel column chromatography with methylene chloride/methanol 80: 1 to 0: 1 gradient elution (50ml/ flow point), biological activity test is carried out to the flow point of wash-out, indicator is Pyricularia oryzae (P.oryzae), result display Substance mainly concentrates on methylene chloride/methanol 60: 1 position, is merged by above-mentioned Active fractions after TLC detects.Active fractions after merging is prepared plate through thin-layer chromatography be prepared, chromatographic solution proportioning is methylene chloride/methanol 40: 1, will prepare R on plate
fvalue is that the band of the 256nm ultraviolet colour developing of 0.33 ~ 0.36 scrapes, 2 hours are soaked with anhydrous methanol, then filter four times, merging filtrate is evaporated to dry, be dissolved in 10ml anhydrous methanol and carry out HPLC analysis and prepare, analyze and prepare proportion of mobile phase used and be methyl alcohol/acetonitrile/water=40: 35: 25 (volume ratios), finally prepare compd B afilomycin K.
The structural identification and analysis of the new ten hexa-atomic Macrocyclic lactone compounds Bafilomycin K of embodiment 3
The ten new hexa-atomic Macrocyclic lactone compounds Bafilomycin K that embodiment 1 and 2 obtains are carried out analysis and identification, and its result is as follows:
Bafilomycin K chemical structure is as shown below:
Bafilomycin K is colorless solid powder, has blackening under UV 254nm.Uv-absorbing λ
maxfor 247.2nm, 278.4nm; Mp > 250 DEG C; HR-ESI-MS m/z 629.3929 [M+Na]
+(calcd for C
35h
58o
8).
1h,
13c NMR,
1h-
1hCOSY, HMBC data are in table 1.
Structural Identification data sheet (the CDCl of table 1 compd B afilomycin K
3)
Embodiment 4Bafilomycin K is to the restraining effect of plant pathogenic fungi
The present embodiment has investigated the compd B afilomycin K of embodiment 1 acquisition to the restraining effect of plant pathogenic fungi.
Dull and stereotyped antagonism method is adopted to investigate the compd B afilomycin K of the purifying that embodiment 1 obtains to Pyricularia oryzae (Pyricularia oryzae), botrytis cinerea pers (Botrytis cinerea), botrytis cinerea (Botrytis cinerea), Rhizoctonia solani Kuhn (Rhizoctonia solani), melon anthrax bacteria (Collectotrichum lagenarium), southern corn leaf blight (Helminthosporium maydis), the restraining effect of the plant pathogenic fungis such as fusarium graminearum (Gibberella zeae).
The MIC (minimum inhibitory concentration) of Bafilomycin K to plant pathogenic fungi is as shown in table 2.
Table 2Bafilomycin K is to the restraining effect (μ g/ml) of plant pathogenic fungi
Bafilomycin K has stronger bacteriostatic activity to plant pathogenic fungis such as Pyricularia oryzae (Pyricularia oryzae) and melon anthrax bacterias (Collectotrichum lagenarium) as can be seen from Table 2, and therefore this compound can be used for preventing and treating the disease caused by above-mentioned plant pathogenic fungi.
Mycelium crude extract after the plain streptomycete Y12-26 of the Huang three of embodiment 5 containing Bafilomycin K ferments is to the prevention effect of fungal diseases of plants
Fermentation broth coarse extract after the present embodiment plain streptomycete Y12-26 of Huang three investigated containing compd B afilomycin K of the present invention ferments is to the preventive and therapeutic effect of fungal diseases of plants.
To above-mentioned fermentation broth coarse extract be investigated to the prevention effect of the fungal diseases of plants such as powdery mildew of cucumber, gray mold of cucumber, cucumber anthracnose, rice sheath blight disease with pot-culture method.Mycelium crude extract 500mg described in embodiment 1 adds in the 20ml water containing micro-emulsifying agent, be made into medicament to be measured, according to the southern pesticides discovery of country, SOP (Standard operation procedure SOP) carries out spray medicine in the heart, inoculation is brought out, investigated, and result is as shown in table 3.
Mycelium crude extract after the yellow three plain streptomycete Y12-26 of table 3 ferment is to the prevention effect of fungal diseases of plants
As shown in Table 3, the mycelium crude extract after the plain streptomycete Y12-26 of the Huang three containing compd B afilomycin K ferments can prevent and treat the fungal diseases of plants such as powdery mildew of cucumber and rice sheath blight disease effectively.
Although be described above object lesson of the present invention, having a bit is obvious to those skilled in the art, namely can make various changes the present invention and change under the premise without departing from the spirit and scope of the present invention.Therefore, claims cover all these variations within the scope of the present invention.
Claims (9)
1. macrolides compound, described compound has following structure:
2. prepare a method for compound according to claim 1, it is characterized in that, the method comprises the following steps:
(1) cultivate the plain streptomycete of Huang three (Streptomyces flavotricini) that preserving number is CGMCC No.3832, obtain fermented liquid; With
(2) (a) obtains supernatant liquor from described separation of fermentative broth, with the solvent extraction being selected from chloroform, ethyl acetate, N-BUTYL ACETATE, methylene dichloride or propyl carbinol, then use column chromatography with methylene chloride/methanol gradient elution, collect the wash-out position that methylene chloride/methanol is 80:1 to 20:1, the Active fractions of wash-out is prepared plate through thin-layer chromatography be prepared, chromatographic solution proportioning is methylene chloride/methanol 40:1, collects R
fvalue is the 256nm ultraviolet colour developing band of 0.33 ~ 0.36, soaks, carry out HPLC analysis and prepare, finally obtaining compound according to claim 1 with anhydrous methanol; Or
B () obtains mycelium from described separation of fermentative broth, with methyl alcohol or acetone extraction, use chloroform, methylene dichloride or butanol solvent partition again, leach with anhydrous methanol, gained extractive substance column chromatography, with methylene chloride/methanol gradient elution, is collected the wash-out position that methylene chloride/methanol is 80:1 to 20:1, is prepared by the Active fractions of wash-out through thin layer preparative chromatography, chromatographic solution proportioning is methylene chloride/methanol 40:1, collects R
fvalue is the band of the 256nm ultraviolet colour developing of 0.33 ~ 0.36, soaks, carry out HPLC analysis and prepare, finally obtaining compound according to claim 1 with anhydrous methanol.
3. method as claimed in claim 2, it is characterized in that, described column chromatography is silica gel column chromatography.
4. a method for controlling plant diseases, the method comprises: cultivate the plain streptomycete of Huang three (Streptomyces flavotricini) that preserving number is CGMCC No.3832, obtain the fermented liquid containing the compound described in claim 1; Described fermented liquid is applied on described plant.
5. a pesticide composition, is characterized in that, described composition includes the compound according to claim 1 of effective amount as active ingredient, and acceptable carrier in Pesticide Science.
6. pesticide composition as claimed in claim 5, it is characterized in that, described agricultural chemicals is used for preventing and treating the Plant diseases caused by Pyricularia oryzae (Pyricularia oryzae), botrytis cinerea pers (Botrytis cinerea), botrytis cinerea (Botrytis cinerea), Rhizoctonia solani Kuhn (Rhizoctonia solani), melon anthrax bacteria (Collectotrichum lagenarium), southern corn leaf blight (Helminthosporium maydis) or fusarium graminearum (Gibberella zeae).
7. compound according to claim 1 is as the application of agricultural chemicals.
8. apply as claimed in claim 7, it is characterized in that, described agricultural chemicals is for suppressing Pyricularia oryzae (Pyriculariaoryzae), botrytis cinerea pers (Botrytis cinerea), botrytis cinerea (Botrytis cinerea), Rhizoctonia solani Kuhn (Rhizoctonia solani), melon anthrax bacteria (Collectotrichum lagenarium), southern corn leaf blight (Helminthosporium maydis), fusarium graminearum (Gibberella zeae).
9. a method for controlling plant diseases, is characterized in that, the method comprises and being applied on described plant by the pesticide composition described in compound according to claim 1 or any one of claim 5 or 6.
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| CN102870820A (en) * | 2012-09-23 | 2013-01-16 | 江苏丘陵地区镇江农业科学研究所 | Application of metabolite of NF0919 strain of streptomyces corchorusii in wheat disease prevention and treatment |
| CN103205389B (en) * | 2013-05-13 | 2014-06-18 | 扬州大学 | Streptomyces flavotricini for antagonizing botryosphaeria dothidea |
| CN103882073B (en) * | 2014-03-13 | 2018-11-23 | 中国海洋大学 | A kind of preparation method of macrolides compound and the application as marine antifoulant |
| CN104817547B (en) * | 2015-03-17 | 2016-02-24 | 农业部环境保护科研监测所 | Novel bar bifilomycin, its extraction microorganism and extracting method |
| CN106370500B (en) * | 2016-08-26 | 2019-11-22 | 中国农业科学院农产品加工研究所 | The extraction separation method of lipid soluble metabolites in a kind of excrement |
| CN107058178B (en) * | 2017-03-13 | 2020-08-04 | 陕西博秦生物工程有限公司 | Streptomyces flavetris, microbial inoculum and application thereof |
| CN108977481B (en) * | 2017-11-08 | 2021-08-27 | 北大方正集团有限公司 | Method for producing meleumycin by fermentation |
| CN113372397A (en) * | 2021-06-22 | 2021-09-10 | 天方药业有限公司 | Preparation method of spiramycin adipate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4558139A (en) * | 1983-03-23 | 1985-12-10 | Bayer Aktiengesellschaft | Bafilomycin pesticides |
| WO1993018652A1 (en) * | 1992-03-20 | 1993-09-30 | Merck & Co., Inc. | Methods of achieving antitumor activity using 16-membered-macrolide-type compounds |
| CN101333206A (en) * | 2007-06-27 | 2008-12-31 | 国家海洋局第一海洋研究所 | Macrolide compounds, preparation and application thereof |
-
2010
- 2010-05-26 CN CN201010185451.9A patent/CN102260251B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4558139A (en) * | 1983-03-23 | 1985-12-10 | Bayer Aktiengesellschaft | Bafilomycin pesticides |
| WO1993018652A1 (en) * | 1992-03-20 | 1993-09-30 | Merck & Co., Inc. | Methods of achieving antitumor activity using 16-membered-macrolide-type compounds |
| CN101333206A (en) * | 2007-06-27 | 2008-12-31 | 国家海洋局第一海洋研究所 | Macrolide compounds, preparation and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Metabolic products of microorganisms.224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity;Werner G，等;《The Journal of Antibiotics》;19840229;第37卷(第2期);第110-117页 * |
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