CN102659734A - Triene antibiotic, preparation method thereof and application thereof - Google Patents
Triene antibiotic, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention relates to a triene antibiotic. The formula of the triene antibiotic is C23H34O8S and the structural formula of the triene antibiotic is as follows. The preparation method comprises steps of using Shandong streptomycetes to cultivate a fermentation broth, performing centrifugation for the fermentation broth, removing thallus, reserving supernatant liquor, preprocessing the supernatant liquor by using a macroporous resin X-5 column chromatography, preprocessing the supernatant liquor by using a macroporous resin HP-20 column chromatography, performing High Performance Liquid Chromatography (HPLC) semi-preparation liquid phase separation, performing freezing and drying after rotating, evaporating and concentrating collection liquid corresponding to peaks of active constituents, and obtaining required purified products. Accordingly, products which have high purity and little impurity can be obtained. Under the same concentration, the triene antibiotic has a large-diameter inhibition zone and good inhibitory effects.
Description
Technical field
The present invention relates to a kind of microbiotic, particularly a kind of triene antibiotic belongs to biological technical field.
Background technology
Plant diseases all is the formidable enemy of agriculture prodn all the time; The Plant diseases that in three types of Plant diseasess, is caused by pathogenic fungi occupies first place (fungal disease, Micobial Disease and virus disease); In 200 multiple diseases of wheat; Fungal disease has occupied about 90%, and the loss that is therefore caused can account for the 10%-20% of its ultimate production every year.For a long time, people have used a large amount of chemical pesticides for the antagonism disease, have caused the generation of Three Difficult Issues: contaminate environment, accumulation residual hazard, final harm humans self health; The resistance of pathogenic fungi is explosive type and rises; Useful microbe is killed, and has destroyed the balance of microbial population in the animal and plant body, makes pathogenic fungi become dominant population, causes the outbreak of epidemic of disease.Based on the continuous enhancing of these toxic side effect of chemical pesticide and people's environmental consciousness with to the raising gradually of green non-pollution food requirement, research and development, produce and use high-tech, harmless boilogical agricultural chemicals to become the task of top priority with independent intellectual property right.Agricultural antifungal antibiotic is as one of biological pesticide important branch; Because of it has efficiently, is prone to degraded, noresidue, advantage such as pollution-free receives people's attention day by day; Like jingganmycin, kasugamycin, middle living rhzomorph, Astromicin, new Polyoxin, polyoxin, pesticide corrosion 120, qingfengmeisu etc., be widely used in preventing and treating in all kinds of plant pathogenic fungi diseases at present.
Agricultural antibiotic produces in microbial metabolism; It is meant the chemical substance with regard to suppressing maybe can kill crop disease, worm, crop smothering or can regulate crop growth under lower concentration, and the U.S., Britain, Japan and other countries early start are used agricultural antibiotic.Research to agricultural antibiotic grows up along with medical development of antibiotics, the early stage use like medical antibiosis such as Streptomycin sulphate, grisovin, terramycin controlling plant diseases usually.Afterwards, people such as Dekker had reported for example a collection of in succession: cycloheximide (Cycloheximide), antimycin A (ActimycinA) and some other polyenoid class agricultural antibiotic.Japan successfully developed rice blast fungus-S (Blasticidin-s) extremely in 1958; And large-scale application in 1961 in the control of rice blast; This has just substituted the use of organomercurial in the rice blast control basically; We can say the discovery of blasticidin-S and the industriallization of production, is the milestone of agricultural antibiotic exploitation.After this, the antimycotic pesticide new variety of developing in succession that a series of high-efficiency low-toxicities such as kasugamycin, polyoxin are arranged again.China starts from the early fifties in last century to the research of agricultural antibiotic, though starting development in evening is comparatively rapid.Agricultural antibiotic kinds such as jingganmycin, Gongzhuling mycin (Gongzhulingmycin), pesticide corrosion 120, Wuyiencin, kasugamycin are developed out in succession and are applied.Through the exploratory development of decades, China is enhanced on the antibiotic method of screening novel agricultural, develops Tianzhu rhzomorph, middle living rhzomorph (Zhongshengmycin), new Polyoxin etc. in recent years again.
Polyenoid class antifungal antibiotic is the medicine that acts on fungi infestation; Its mechanism of action is that the sterol with fungal cell membrane partly carries out selective binding, on cytolemma, forms the duct, thereby changes the permeability of cytolemma; Cause that multiple small-molecule substance leaks in the cell; Finally cause necrocytosis and bring into play anti-microbial effect, but it does not have effect to bacterium, because bacterial cell membrane does not contain sterol.Polyenoid class antifungal drug develops comparatively fast in recent years.Have one type to be the polyene macrolide microbiotic in the polyene antibiotics, their composition comprises has the lactonic ring of being made up of 25-37 carbon atom and a part of conjugated polyene structure of encircling hydroxyl at its corresponding position, and connects one or more aminosugars by glycosidic bond.Difference by conjugated double bond number in the structure can be divided into three, four, five, six, seven alkene, and each all has its special uv-absorbing peak value.This type antibiosis have many common character, as: broad-spectrum antifungal, meet light be prone to decompose, not soluble in water, intravenously administrable toxicity high, oral toxicity is low etc.By the macrolide with conjugation tetraene that actinomycetes produce, uv absorption spectrum is at (291 ± 2) nm, (302 ± 2) nm, and (320 ± 3) nm has charateristic avsorption band, and each absorption peak all has anti-mycotic activity.Kind surplus polyenoid class antifungal drug has 50 approximately at present; As: amphotericin B (amphotericin B; AMB) new preparation AMB be unique clinical use at present can intravenous polyenoid class antifungal antibiotic; It can combine with the ergosterol (ergosterol) in the fungal cell membrane and make its oxidation, has characteristics such as Fungicidally active is strong, has a broad antifungal spectrum, and weak point is that AMB has stronger renal toxicity; The AMB new preparation of clinical study at present and exploitation listing mainly contains 3 types: liposome (liposome), lipid complex (lipid complex) and colloid dispersion agent (colloidal dispersion), they all are that medium is processed with lipid.Nyotran Nyotran Nystatin (NYS) can combine ergosterol; Reduce cell membrane stability, have antibiosis active multiple fungi, but owing to it is difficult for being absorbed at gi tract; And reasons such as toxicity is bigger make the clinical application of this medicine receive very big restriction.The Johnson of Aronex company etc. is developed into the liposome of commodity Nyotran by name, has not only improved curative effect greatly, and has reduced its toxic side effect.These 2 compounds of 3874H1 and 3874H3 and AMB are similar; All belong to the heptaenes microbiotic, wherein 3874H1 contains an aromatic nucleus, and 3874H3 does not contain aromatic nucleus; Therefore 3874H3's is water-soluble very strong; The both has anti-microbial effect widely, all have antibiosis active to various skin fungi, filamentous fungus and yeast, and activity is higher than AMB and NYS.
Because fungi is eukaryotic microorganisms, they are all similar with high vegeto-animal many biological natures, so the fungi microbiotic do not have the sort of high-efficiency low-toxicity of bacteria antibiotic and the good characteristic of specificity, generally their toxic side effect greatly and curative effect lower.Therefore, although the medicine of anti-fungal infection has many kinds clinically, most chemical synthetic drugs can only be used for the treatment of surface infection, can be as the body innerlich anwenden considerably less.So screening sterilization (or antibacterial) efficient is high, spinoff antifungal antibiotic little, that specificity is good is significant.
Summary of the invention
The purpose of this invention is to provide a kind of triene antibiotic, this kind microbiotic has bacteriostatic activity preferably, and the time cycle of extracting obviously shorten, Financial cost also corresponding reduction, purity is high.
The technical scheme that the present invention takes is:
A kind of triene antibiotic, its molecular formula are C
23H
34O
8S, structural formula is following:
The preparation method of described triene antibiotic comprises the steps:
(1) fermentation liquor pretreatment: scrape from the ripe inclined-plane of Shandong streptomycete (Streptomyces shandongensis) and to get thalline and be inoculated in No. 1 liquid nutrient medium of Gao Shi; Cultivate 70-75h for 25-30 ℃; Inoculated to the fermentor tank 25-30 ℃ of agitation condition bottom fermentation 4-5 days; Regulate fermented liquid pH=3-6, the centrifugal thalline of removing of fermented liquid is stayed supernatant;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: with supernatant appearance on macroporous resin X-5 post; With deionized water rinsing (with the volume of 2 posts), use 40% (v/v) alcohol flushing (volumes of 1.5 posts) to wash impurity such as pigment off then earlier, use 50% (v/v) ethanol to wash active ingredient again; Collect elutriant; With the elutriant flash concentration of collecting, be concentrated into the 30-40% of supernatant volume, get preliminary liquid concentrator;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: with preliminary liquid concentrator appearance on macroporous resin HP-20 post; Earlier wash impurity off with 45% (v/v) alcohol flushing (with 1.5 column volume amounts); Use 50% (v/v) ethanol to wash active ingredient then, collect elutriant, with the elutriant flash concentration of collecting; Be concentrated into 20 ± 5% of supernatant volume, get secondary concentration liquid;
(4) HPLC half preparative liquid is separated: with secondary concentration liquid liquid phase separation, the HPLC semipreparative column is C18, and moving phase is methyl alcohol and 20mM ammonium acetate pH3.0; Employing program gradient elution (ammonium acetate solution volumetric concentration 50%-10%) method, the initial concentration of 20mM ammonium acetate pH3.0 is 50% (volume ratio), stopping concentration is 10% (v/v); The methyl alcohol initial concentration is 50% (v/v); Stopping concentration is 90% (v/v), collects each peak in the liquid phase, removes solvent methanol and ammonium acetate;
(5) (promptly 271 ± 1nm) corresponding collection liquid rotary evaporations concentrate postlyophilizations and promptly get the pure article of wanting at the peak that active ingredient is belonged to.
Among the above-mentioned preparation method:
The weight part ratio of No. 1 liquid nutrient medium of the described Gao Shi of step (1) consists of: 20 parts of Zulkovsky starches, KNO
31 part, 0.5 part of NaCl, K
2HPO
43H
20.655 part of O, MgSO
40.5 part, FeSO
40.01 1000 parts in part, water; PH 7.2～7.4.
The described stirring velocity of step (1) is 200-400r.min
-1Described centrifugal speed is 6000-12000r.min
-1
The described flash concentration temperature of step (2) is instantaneous 100 ℃, and drip washing speed is according to practical situation manual regulation compression pump flow.
Instantaneous 100 ℃ of the described flash concentration temperature of step (3), drip washing speed is according to practical situation manual regulation compression pump flow.
The described elution speed 15ml/min of step (4), sample size 2-3ml/min
50 ℃ of the described rotary evaporation temperature of step (5), cryodesiccated time 24-32h.
The application of described triene antibiotic in the preparation fungistat.
The present invention is centrifugal under acidic conditions during protein in removing fermented liquid, avoids unstable under alkaline condition, is prone to decomposition; The HPLC detected result shows, reaches the fermented liquid of only using the primary treatment of X-5 resin through X-5 and twice processing of HP-20 resin, and it is fewer to obtain impurity, and purity is than higher thick pure article; Rotary evaporation is with volatile removal of solvents, but sample through the HPLC meeting of detection the part decomposition takes place behind 45 ℃ of following rotary evaporations, reduce sample purity, adopts 50 ℃ of rotary evaporations, and the degree of decomposition of sample reduces greatly, decomposed substance distinctive " bimodal " disappearance.Under same concentrations, microbiotic antibacterial circle diameter of the present invention is maximum, and fungistatic effect is the strongest, and its fungistatic effect is a little more than nystatin but far above amphotericin B.
Description of drawings
Fig. 1 microbiotic uv scan of the present invention figure;
Fig. 2 microbiotic HPLC of the present invention purity detecting figure;
Fig. 3 microbiotic purity of the present invention normalization method view;
Three structure fragment figure of the A-C that Fig. 4 microbiotic of the present invention contains;
The fermented liquid HPLC that Fig. 5 handled through macroporous resin X-5 post and HP-20 column chromatography detects figure;
The fermented liquid HPLC that Fig. 6 handled through macroporous resin X-5 column chromatography detects figure;
Fig. 7 HPLC half preparative liquid liquid phase collecting zone that is separated;
Fig. 8 the 1st absorption peak UV scanning figure;
Fig. 9 the 2nd absorption peak UV scanning figure;
Figure 10 the 3rd absorption peak UV scanning figure;
Figure 11 the 4th absorption peak UV scanning figure;
Three kinds of microbiotic of Figure 12 antibacterial circle diameter graphic representation under different concns;
The ITMS+cESI-MS figure that Figure 13 is antibiotic;
The antibiotic ITMS-cESI-MS figure of Figure 14;
Figure 15 is antibiotic
1H NMR spectrogram;
Figure 16 is antibiotic
13The C-NMR spectrogram;
The antibiotic DEPT-NMR figure of Figure 17;
The antibiotic gCOSY figure of Figure 18;
The antibiotic gHSQC figure of Figure 19;
The antibiotic gHMBC figure of Figure 20.
Embodiment
Further specify below in conjunction with embodiment, but not limited by embodiment.Used macroporous resin X-5 post, macroporous resin HP-20 post is that factory of Nankai University produces among the embodiment; Solubility amphotericin B (Amresco); Nystatin (Sigma); LC-10ATvp type high performance liquid chromatograph originates from Japanese Shimadzu, and LC-10AD type half preparative high-performance liquid chromatographic appearance originates from Japanese Shimadzu.
The preparation method of triene antibiotic:
(1) scrape from the ripe inclined-plane of Shandong streptomycete and get thalline and be inoculated in No. 1 liquid nutrient medium of Gao Shi, 28 ℃ are shaken a bottle concussion (90r.min
-1) cultivate 72h, the triangular flask liquid amount is 20%.Then seed liquor is forwarded in (5L) fermentor tank with 10% inoculum size, 28 ℃ of temperature, initial stirring velocity is 200r.min
-1, regulate stirring velocity to 300r.min behind the inoculation 5h
-1, regulate stirring velocity to 400r.min behind the inoculation 18h
-1And keep; The 5d secondary fermentation finishes, with the fermented liquid that obtains at 4 ℃ of following 6000r.min
-1Centrifugal 20min removes thalline, 12000r.min behind the gained supernatant adjusting pH to pH=3
-1Centrifugal 15min stays supernatant, and the supernatant pH regulator returns pH=6, is stored under 4 ℃ of conditions subsequent use;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: with supernatant appearance on macroporous resin X-5 post; With deionized water rinsing (with 2 column volumes), use 40% (v/v) alcohol flushing (with the amount of 1.5 column volumes) to wash impurity such as pigment off then earlier, use 50% (v/v) ethanol to wash active ingredient again; Collect elutriant; Elutriant flash concentration with collecting is concentrated into 30% of supernatant volume, gets preliminary liquid concentrator;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: with preliminary liquid concentrator appearance on macroporous resin HP-20 post; Earlier wash impurity such as pigment off with 45% (v/v) alcohol flushing (with the amount of 1.5 column volumes); Use 50% (v/v) ethanol to wash active ingredient then, collect elutriant, with the elutriant flash concentration of collecting; Be concentrated into 20% of supernatant volume, get secondary concentration liquid;
(4) HPLC half preparative liquid is separated: with secondary concentration liquid liquid phase separation, the HPLC semipreparative column is C18, and moving phase is methyl alcohol and 20mM ammonium acetate pH3.0; Employing program gradient elution (ammonium acetate concentration 50%-10%) method, the initial concentration of 20mM ammonium acetate pH3.0 is 50%, stopping concentration is 10%; The methyl alcohol initial concentration is 50%; Stopping concentration is 90%, collects each peak in the liquid phase, removes solvent methanol and ammonium acetate;
(5) (271nm) peak at active ingredient place is the corresponding concentrated postlyophilization of 50 ℃ of rotary evaporations of collection liquid promptly gets the pure article of wanting.
The preparation method of triene antibiotic:
(1) scrape from the ripe inclined-plane of Shandong streptomycete and get thalline and be inoculated in No. 1 liquid nutrient medium of Gao Shi, 28 ℃ are shaken a bottle concussion (90r.min
-1) cultivate 72h, the triangular flask liquid amount is 20%.Then the inoculum size of seed liquor with 10% (about 400ml) is forwarded in (5L) fermentor tank, 28 ℃ of temperature, initial stirring velocity is 200r.min
-1, regulate stirring velocity to 300r.min behind the inoculation 5h
-1, regulate stirring velocity to 400r.min behind the inoculation 18h
-1And keep; The 5d secondary fermentation finishes, with the fermented liquid that obtains at 4 ℃ of following 6000r.min
-1Centrifugal 20min removes thalline, 12000r.min behind the gained supernatant adjusting pH to pH=3
-1Centrifugal 15min stays supernatant, and the supernatant pH regulator returns pH=6, is stored under 4 ℃ of conditions subsequent use;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: with supernatant appearance on macroporous resin X-5 post; With deionized water rinsing (with 2 column volume amounts), use 40% (v/v) alcohol flushing (1.5 column volume amounts) to wash impurity such as pigment off then earlier, use 50% (v/v) ethanol to wash active ingredient again; Collect elutriant; Elutriant flash concentration with collecting is concentrated into 40% of supernatant volume, gets preliminary liquid concentrator;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: with preliminary liquid concentrator appearance on macroporous resin HP-20 post; Earlier wash impurity such as pigment off with 45% (v/v) alcohol flushing (with 1.5 column volume amounts); Use 50% (v/v) ethanol to wash active ingredient then, collect elutriant, with the elutriant flash concentration of collecting; Be concentrated into 25% of supernatant volume, get secondary concentration liquid;
(4) HPLC half preparative liquid is separated: with secondary concentration liquid liquid phase separation, the HPLC semipreparative column is C18, and moving phase is methyl alcohol and 20mM ammonium acetate pH3.0; Employing program gradient elution (ammonium acetate concentration 50%-10%) method, the initial concentration of 20mM ammonium acetate pH3.0 is 50%, stopping concentration is 10%; The methyl alcohol initial concentration is 50%; Stopping concentration is 90%, collects each peak in the liquid phase, removes solvent methanol and ammonium acetate;
(5) (271nm) peak at active ingredient place is the corresponding concentrated postlyophilization of collection liquid rotary evaporation promptly gets the pure article of wanting.
The structural analysis of the pure article of preparation:
Get embodiment 1 pure article and be dissolved in the methyl alcohol, carry out the scanning of ultraviolet all wave band with UV-3100 type ultraviolet-visible spectrophotometer.It is 400～200nm that wavelength region is set, and the peak value scope is 0～5.Uv scan figure sees Fig. 1, can be found out by Fig. 1, and this active substance is at 261nm, and there is characteristic absorbance at 271nm and 283nm place, and wherein there is maximum uv-absorbing at the 271nm place.This characteristic absorbance is the peculiar uv-absorbing of conjugated triene compounds, therefore can judge that the conjugated triene structure is arranged in this active substance.
Table 1
After getting embodiment 1 pure article and being dissolved in methyl alcohol, carry out ESI-MS positive ion mode and negative ion mode analysis, the ESI-MS positive ion mode provides m/z 493 [M+Na]
+With 509 [M+K]
+, negative ion mode provides m/z 469 [M-H]
-Quasi-molecular ion peak, the prompting compound molecular weight be 470.Positive and negative ion mode annunciations molecular formula is C
30H
30O
5ITMS ± ESI-MS positive ion mode and negative ion mode are analyzed collection of illustrative plates and are seen Figure 13-14.
With embodiment 1 pure article with deuterium for after the DMSO dissolving, in the nuclear-magnetism pipe of packing into clean, the 400MHz nuclear-magnetism carries out the NMR test.The project of test comprises
1H-NMR,
13C-NMR, DEPT, gCOSY, HMBC and HMQC, collection of illustrative plates see Figure 15-20, and data are seen table 2.
Table 2
Compound
1H NMR (DMSO-d
6, 400MHz) spectrum has provided 10 alkene hydrogen proton signals between low place δ 5.40-7.03, has provided 7 even oxygen/methene proton signals at δ 1.38-2.68 place, has provided 2 methyl proton signals at δ 0.86 and δ 0.96 place.
13C NMR (DMSO-d
6, 400MHz) spectrum provides 23 carbon signals, can know that by the DEPT spectrum these carbon signals comprise 15 methine carbon signals, and wherein 10 is sp
2Hydridization carbon, in conjunction with containing 5 two keys in the hydrogen spectrum Notes of Key Data structure, other 5 is to connect oxygen sp
3Methine carbon; 5 mesomethylene carbon; 1 ester carbonyl group (δ 162.2) and 2 methyl carbon signals.According to above carbon, hydrogen spectrum Notes of Key Data compound is an esters of unsaturated fatty acids compound.
Through HSQC carbon and directly continuous hydrogen signal thereof have been carried out accurate ownership, through
1H-
1The relevant of the adjacent even proton of H COSY confirmed with the long-range relevant structure to compound of the heteronuclear of HMBC, shows to have three structure fragment (see figure 4)s of A～C in the compound structure.Compound
1H-
1Demonstrate H-2-H-3-H-6-H-4-H-7-H-23-H-18-H-15-H-16-H-17-H-22-H-11 relevant (among the figure shown in the thick line) in the H COSY spectrum, in conjunction with the pulsating existence of A in the chemical shift data prompting structure of its hydrocarbon signal.H-2 during this structural unit is composed by HMBC and C-3, C-6, H-7 and C-23, C-6, C-18, H-23 and C-7, C-15, the long-range relevant peaks of H-16 and C-15 and H-17 and C-11 is further proved conclusively.In addition, compound
1H-
1Give H-24 and H-20, H-9 in the H COSY spectrum, H-14 is relevant with H-9, H-19 and H-19 and H-21's, in conjunction with there being unsaturated lactone structural unit B in these carbon and the proton chemical shifts prompting structure.This structural unit is also by H-24 and C-9, C-25, C-14 in the HMBC spectrum, and H-20 proves conclusively with long-range relevant the obtaining of C-9, C-25 and H-19 and C-14.In addition,
1H-
1H COSY spectrum demonstrate H2-10 compose with H-13 and HMBC in the relevant peaks of H-13 and C-10, show the existence of unit C in the structure.
Show through ultimate analysis, except containing C, H, O, also contain the S element in this compound.According to compound molecular weight is 470, tool conjugated triene structure, and character such as soluble in water infers that molecular formula is C
23H
34O
8S, structural formula is:
Purity check detects:
Performance liquid chromatography (HPLC) testing product purity
High performance liquid chromatograph is configured to: LC-10ATvp high efficiency chromatography pump, SPD-M10Avp diode-array detector, RF-10Axl fluorimetric detector, Autosampler automatic sampler, temperature control column oven.The HPLC testing conditions: the HPLC analytical column is Agela Venusil ASB C18 (4.6mm*250mm), sample size 10-45u L, and the detection wavelength is 271nm.According to previous work, moving phase is methyl alcohol: ammonium acetate (20mM pH3.0)=70: 30.In order to separate each absorption peak of preparation back sample better, use the program gradient elution method instead, the initial concentration of 20mM ammonium acetate (pH3.0) is 35%; Stopping concentration is 5%; The methyl alcohol initial concentration is 65%, and stopping concentration is 95%, and the LC time-program(me) is provided with like table 3:
Table 3
The fermented liquid HPLC that embodiment 1 handled through macroporous resin X-5 post and HP-20 column chromatography detects figure like Fig. 5; The fermented liquid HPLC that embodiment 2 handled through macroporous resin X-5 column chromatography detects figure like Fig. 6, can find out, and is higher through the thick pure article purity of fermented liquid gained that X-5 post and HP-20 post were handled; Can reach 85% through calculating its purity; Its foreign matter content of fermented liquid of only handling with the X-5 post is lower, and the separating size under this condition is also better, and the impurity peaks signal occurs early; Can in preparation, distinguish, therefore also can adopt the mode of only passing through the primary treatment of X-5 post with the active substance absorption peak.
The anti-mycotic activity test:
The HPLC half preparative liquid liquid phase collecting zone that is separated is seen Fig. 7, collects among Fig. 7 that 1-4 peak liquid is done bacteriostatic test and UV scanning detects, and scanning result is seen Fig. 8-11, can know that the 4th peak is the absorption peak of bacteriostatic activity material.
Respectively get solubility amphotericin B, nystatin, embodiment 1 pure article 10mg, three kinds of microbiotic are dissolved in respectively in the phosphoric acid buffer of 50ml 0.1MpH=7.0, strength of solution is 0.2mg/ml; Use the phosphoric acid buffer of 0.1M pH=7.0 that above-mentioned three kinds of antibiotic solutions dilution is 80%, 60%, 40%, 20% of original content; Do bacteriostatic test (apple decay bacterium, geotrichum candidum) and measure inhibition zone with cup-plate method; See Figure 12, can find out, under same concentrations; The active substance antibacterial circle diameter that the present invention produces is maximum; Fungistatic effect is the strongest, and its fungistatic effect is a little more than nystatin but far above amphotericin B, its tire can be rough represent with tiring of nystatin.
Adopting dilution method double, is 1/2,1/4,1/8,1/16,1/32,1/64,1/128 of original content with the embodiment 1 pure article solution dilution of Shandong streptomycete fermentation liquid and 0.2mg/ml; Test minimum inhibitory concentration with cup-plate method.
Adopt dilution method double, record MIC result and show that the minimum inhibitory concentration of thick pure article and fermented liquid is 1/128, promptly 0.78%.Can define this concentration as a potency unit.
Claims (8)
1. triene antibiotic, its molecular formula is C
23H
34O
8S, structural formula is following:
2. the preparation method of the described triene antibiotic of claim 1 is characterized in that, comprises the steps:
(1) fermentation liquor pretreatment: scrape from the ripe inclined-plane of Shandong streptomycete and to get thalline and be inoculated in No. 1 liquid nutrient medium of Gao Shi; Cultivate 70-75h for 25-30 ℃; Inoculated to the fermentor tank 25-30 ℃ of agitation condition bottom fermentation 4-5 days; Regulate fermented liquid pH=3-6, the centrifugal thalline of removing of fermented liquid is stayed supernatant;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: with supernatant appearance on macroporous resin X-5 post; Earlier use deionized water rinsing, wash impurity such as pigment off with 40% alcohol flushing then, wash active ingredient with 50% ethanol again; Collect elutriant; With the elutriant flash concentration of collecting, be concentrated into the 30-40% of supernatant volume, get preliminary liquid concentrator;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: with preliminary liquid concentrator appearance on macroporous resin HP-20 post; Earlier wash impurity off with 45% alcohol flushing; Wash active ingredient with 50% ethanol then, collect elutriant, with the elutriant flash concentration of collecting; Be concentrated into 20 ± 5% of supernatant volume, get secondary concentration liquid;
(4) HPLC half preparative liquid is separated: with secondary concentration liquid liquid phase separation, the HPLC semipreparative column is C18, and moving phase is methyl alcohol and 20mM ammonium acetate pH3.0; Employing program gradient elution method, the initial concentration of 20mM ammonium acetate pH3.0 is 50%, stopping concentration is 10%; The methyl alcohol initial concentration is 50%; Stopping concentration is 90%, collects each peak in the liquid phase, removes solvent methanol and ammonium acetate;
(5) peak at active ingredient place is the corresponding concentrated postlyophilization of collection liquid rotary evaporation promptly gets the pure article of wanting.
3. the preparation method of triene antibiotic according to claim 2 is characterized in that, the weight part ratio of No. 1 liquid nutrient medium of the described Gao Shi of step (1) consists of: 20 parts of Zulkovsky starches, KNO
31 part, 0.5 part of NaCl, K
2HPO
43H
2O0.655 part, MgSO
40.5 part, FeSO
40.01 1000 parts in part, water; PH 7.2～7.4.
4. the preparation method of triene antibiotic according to claim 2 is characterized in that, the described stirring velocity of step (1) is 200-400r.min
-1Described centrifugal speed is 6000-12000r.min
-1
5. the preparation method of triene antibiotic according to claim 2 is characterized in that, 50 ℃ of the described rotary evaporation temperature of step (5), cryodesiccated time 24-32h.
6. the preparation method of triene antibiotic according to claim 2 is characterized in that, the peak at step (5) active ingredient place is 271 ± 1nm.
7. the application of the described triene antibiotic of claim 1 in the preparation fungistat.
8. the application of the described triene antibiotic of claim 1 in preparation apple decay bacterium, geotrichum candidum bacteria inhibitor.
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Cited By (2)
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| CN105010399A (en) * | 2014-04-21 | 2015-11-04 | 天津大学前沿技术研究院有限公司 | Separation and purification method of antifungal active substances in S.lydicus fermentation liquor |
| CN105601358A (en) * | 2015-10-22 | 2016-05-25 | 李德舜 | Microbial organic fertilizer for preventing and controlling strawberry red core disease, and production method thereof |
Citations (1)
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| CN102368998A (en) * | 2009-02-05 | 2012-03-07 | 靶向输送技术公司 | Methods of reducing the proliferation and viability of microbial agents |
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| CN102368998A (en) * | 2009-02-05 | 2012-03-07 | 靶向输送技术公司 | Methods of reducing the proliferation and viability of microbial agents |
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| CHRISTOPHER P. BURKE, ET AL.: "Total Synthesis and Evaluation of Phostriecin and Key Structural Analogues", 《THE JOURNAL OF ORGANIC CHEMISTRY》, vol. 75, 29 July 2010 (2010-07-29), pages 7505 - 7513 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105010399A (en) * | 2014-04-21 | 2015-11-04 | 天津大学前沿技术研究院有限公司 | Separation and purification method of antifungal active substances in S.lydicus fermentation liquor |
| CN105010399B (en) * | 2014-04-21 | 2018-07-10 | 天津大学前沿技术研究院有限公司 | The isolation and purification method of antifungus active substance in a kind of streptomyces lydicus zymotic fluid |
| CN105601358A (en) * | 2015-10-22 | 2016-05-25 | 李德舜 | Microbial organic fertilizer for preventing and controlling strawberry red core disease, and production method thereof |
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