CN102659734B - Triene antibiotic, preparation method thereof and application thereof - Google Patents

Triene antibiotic, preparation method thereof and application thereof Download PDF

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CN102659734B
CN102659734B CN201210129652.6A CN201210129652A CN102659734B CN 102659734 B CN102659734 B CN 102659734B CN 201210129652 A CN201210129652 A CN 201210129652A CN 102659734 B CN102659734 B CN 102659734B
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concentration
supernatant liquor
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macroporous resin
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CN102659734A (en
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李德舜
马井玉
陈琦
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Shandong University
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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

A kind of preparation method of triene antibiotic
Technical field
The present invention relates to a kind of microbiotic, particularly a kind of triene antibiotic and its preparation method and application, belongs to biological technical field.
Background technology
Plant diseases is all the formidable enemy of agriculture production all the time, the Plant diseases being caused by pathogenic fungi in three class Plant diseasess occupies first place (fungal disease, Micobial Disease and virus disease), in 200 multiple diseases of wheat, fungal disease has occupied 90% left and right, and the loss that therefore caused can account for the 10%-20% of its ultimate production every year.For a long time, people, for antagonism disease, have used a large amount of chemical pesticides, 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; Beneficial microorganism is killed, and has destroyed the balance of microbial population in animal and plant body, makes pathogenic fungi become dominant population, causes the outbreak of epidemic of disease.These toxic side effect based on chemical pesticide and the continuous enhancing of people's environmental consciousness and the raising gradually to green non-pollution food requirement, high-tech, harmless boilogical agricultural chemicals that research and development, production and use have independent intellectual property right have become the task of top priority.Agricultural antifungal antibiotic is as one of biological pesticide important branch, because it has efficiently, easily degraded, noresidue, the advantage such as pollution-free be subject to people's attention day by day, as jingganmycin, kasugamycin, Zhongshengmycin, Astromicin, new Polyoxin, polyoxin, pesticide corrosion 120, qingfengmeisu etc., be now widely used in preventing and treating in all kinds of plant pathogenic fungi diseases.
Agricultural antibiotic produces in microbial metabolism, it refers under lower concentration and just to suppress maybe to kill crop disease, worm, crop smothering or chemical substance that can regulating crop growth growth, the U.S., Britain, Japan and other countries early start application agricultural antibiotic.To the research of agricultural antibiotic, along with medical antibiotic development gets up, early application is as usually controlling plant diseases of medical antibiosis such as Streptomycin sulphate, grisovin, terramycin.Afterwards, the 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 in 1958 and kills rice blast fungus-S (Blasticidin-s), and large-scale application in 1961 in the control of rice blast, this has just substituted the use of organomercurial in rice blast control substantially, can say the discovery of blasticidin-S and the industrialization of production, be the milestone of agricultural antibiotic exploitation.After this, the fungicide new variety that have again a series of high-efficiency low-toxicities such as kasugamycin, polyoxin of in succession developing.China starts from early fifties in last century to the research of agricultural antibiotic, though starting development in evening is comparatively rapid.The agricultural antibiotic kinds such as jingganmycin, Gongzhuling mycin (Gongzhulingmycin), pesticide corrosion 120, Wuyiencin, kasugamycin are in succession developed out and are applied.Through the exploratory development of decades, China is enhanced in the antibiotic method of screening novel agricultural, develops again in recent years Tianzhu rhzomorph, Zhongshengmycin (Zhongshengmycin), new Polyoxin etc.
Polyenoid class antifungal antibiotic is the medicine that acts on fungi infestation, its mechanism of action is to carry out selective binding with the sterol part of fungal cell membrane, on cytolemma, form duct, thereby change the permeability of cytolemma, cause multiple small-molecule substance in cell to leak, finally cause necrocytosis and bring into play anti-microbial effect, but it to bacterium without effect, because bacterial cell membrane is containing sterol.Polyenoid class antifungal drug develops comparatively fast in recent years.In polyene antibiotics, having a class is polyene macrolide microbiotic, and their composition comprises a part of conjugated polyene structure on lactonic ring and the ring being made up of 25-37 carbon atom, has hydroxyl, and connect one or more aminosugars by glycosidic bond at its corresponding position.Can be divided into three, four, five, six, seven alkene by the difference of conjugated double bond number in structure, each has its special uv-absorbing peak value.This class antibiosis have much common character, as: broad-spectrum antifungal, chance light easily decompose, not soluble in water, intravenously administrable toxicity is high, oral toxicity is low etc.The macrolide with conjugation tetraene being produced by actinomycetes, ultra-violet absorption spectrum is at (291 ± 2) nm, (302 ± 2) nm, (320 ± 3) nm has charateristic avsorption band, and each absorption peak has anti-mycotic activity.Polyenoid class antifungal drug approximately has more than 50 to plant at present, as: amphotericin B (amphotericin B, AMB) new preparation AMB be at present unique clinical use can intravenous polyenoid class antifungal antibiotic, it can with fungal cell membrane in ergosterol (ergosterol) in conjunction with and make its oxidation, there is Fungicidally active strong, the features such as has a broad antifungal spectrum, weak point is that AMB has stronger renal toxicity, the AMB new preparation of clinical study at present and exploitation listing mainly contains 3 classes: liposome (liposome), lipid complex (lipid complex) and colloid dispersion agent (colloidal dispersion), they are all made taking lipid as medium.Nyotran Nyotran Nystatin (NYS) can be in conjunction with ergosterol, the stability that reduces cytolemma, has antibiosis activity to multiple fungi, but because it is difficult for being absorbed at gi tract, and the reasons such as toxicity is larger, are very restricted the clinical application of this medicine.The Johnson of Aronex company etc. is developed into the liposome of commodity Nyotran by name, has not only greatly improved curative effect, and has reduced its toxic side effect.These 2 compounds of 3874H1 and 3874H3 and AMB are similar, all belong to heptaenes microbiotic, wherein 3874H1 contains an aromatic nucleus, and 3874H3 is not containing aromatic nucleus, therefore 3874H3's is water-soluble very strong, both there is anti-microbial effect widely, various skin fungi, filamentous fungus and yeast are had to antibiosis activity, and active in AMB and NYS.
Because fungi is eukaryotic microorganisms, they are all similar to high vegeto-animal many biological natures, and therefore fungi microbiotic does not have that high-efficiency low-toxicity of bacteria antibiotic and the good characteristic of specificity, and generally their toxic side effect is large and curative effect is 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 serve as the considerably less of body innerlich anwenden.So screening sterilization (or antibacterial) efficiency antifungal antibiotic high, that side effect is little, specificity is good is significant.
Summary of the invention
The object of this invention is to provide a kind of triene antibiotic and its preparation method and application, this kind of microbiotic has good bacteriostatic activity, and extract time cycle obviously shorten, Financial cost is corresponding reduction also, purity is high.
The technical scheme that the present invention takes is:
A kind of triene antibiotic, its molecular formula is C 23h 34o 8s, structural formula is as follows:
The preparation method of described triene antibiotic comprises the steps:
(1) fermentation liquor pretreatment: scraping thalline is inoculated in No. 1 liquid nutrient medium of Gao Shi from the ripe inclined-plane of Shandong streptomycete (Streptomyces shandongensis), cultivate 70-75h for 25-30 DEG C, inoculate to 25-30 DEG C of agitation condition bottom fermentation 4-5 days in fermentor tank, regulate fermented liquid pH=3-6, remove thalline and stay supernatant liquor centrifugal fermented liquid;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: by supernatant liquor in macroporous resin X-5 post loading, first use deionized water rinsing (with the volume of 2 posts), then use 40% (v/v) alcohol flushing (volumes of 1.5 posts) to wash the impurity such as pigment off, use again 50% (v/v) ethanol to wash lower active ingredient, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into the 30-40% of supernatant liquor volume, obtain preliminary concentrated solution;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: by preliminary concentrated solution in macroporous resin HP-20 post loading, first use 45% (v/v) alcohol flushing (by 1.5 cylinder accumulated amounts) to wash impurity off, then use 50% (v/v) ethanol to wash lower active ingredient, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into 20 ± 5% of supernatant liquor volume, obtain secondary concentration liquid;
(4) the semi-preparative liquid phase separation of HPLC: by secondary concentration liquid liquid phase separation, HPLC semipreparative column is C18, 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), methyl alcohol initial concentration is 50% (v/v), stopping concentration is 90% (v/v), collect each peak in liquid phase, remove solvent methanol and ammonium acetate;
(5) concentrated collection liquid rotary evaporation corresponding to the peak at active ingredient place (i.e. 271 ± 1nm) postlyophilization is obtained to wanted sterling.
In above-mentioned preparation method:
The weight part ratio of No. 1 liquid nutrient medium of Gao Shi described in 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 41000 parts of 0.01 parts, water; PH 7.2～7.4.
The described stirring velocity of step (1) is 200-400r.min -1; Described centrifugal speed is 6000-12000r.min -1.
The described flash concentration temperature of step (2) is instantaneous 100 DEG C, and drip washing speed is according to practical situation manual regulation compression pump flow.
Instantaneous 100 DEG C of flash concentration temperature described in step (3), drip washing speed is according to practical situation manual regulation compression pump flow.
The elution speed 15ml/min that step (4) is described, sample size 2-3ml/min
The rotary evaporation temperature 50 C that step (5) is described, cryodesiccated time 24-32h.
Described triene antibiotic is in the application of preparing in fungistat.
The present invention is centrifugal under acidic conditions in the time removing in fermented liquid protein, avoids unstable under alkaline condition, easily decomposes; HPLC detected result shows, by X-5 and twice processing of HP-20 resin and only use the fermented liquid of X-5 resin primary treatment, can obtain impurity fewer, the thick sterling that purity is higher; Rotary evaporation is by volatile removal of solvents, but sample after rotary evaporation, through the HPLC meeting of detection, decomposed occurs at 45 DEG C, reduces sample purity, adopts 50 DEG C of rotary evaporations, and the degree of decomposition of sample reduces greatly, and decomposed substance distinctive " bimodal " disappears.Under same concentrations, microbiotic antibacterial circle diameter maximum of the present invention, fungistatic effect is the strongest, and its fungistatic effect is a little more than nystatin but far above amphotericin B.
Brief description of the 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;
Tri-structure fragment figure of A-C that Fig. 4 microbiotic of the present invention contains;
The fermented liquid HPLC that Fig. 5 processed through macroporous resin X-5 post and HP-20 column chromatography detects figure;
The fermented liquid HPLC that Fig. 6 processed through macroporous resin X-5 column chromatography detects figure;
The semi-preparative liquid phase separation liquid phase of Fig. 7 HPLC collecting zone;
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;
Tri-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 13c-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 illustrate below in conjunction with embodiment, but not limited by embodiment.In embodiment, macroporous resin X-5 post, macroporous resin HP-20 post used is that factory of Nankai University produces, 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 instrument originates from Japanese Shimadzu.
Embodiment 1
The preparation method of triene antibiotic:
(1) from the ripe inclined-plane of Shandong streptomycete, scraping thalline is inoculated in No. 1 liquid nutrient medium of Gao Shi, 28 DEG C of shaking flask concussion (90r.min -1) cultivate 72h, triangular flask liquid amount is 20%.Then seed liquor is forwarded in (5L) fermentor tank with 10% inoculum size, 28 DEG C of temperature, initial stirring velocity is 200r.min -1, after inoculation 5h, regulate stirring velocity to 300r.min -1, after inoculation 18h, regulate stirring velocity to 400r.min -1and keep; 5d secondary fermentation finishes, by the fermented liquid 6000r.min at 4 DEG C obtaining -1centrifugal 20min removes thalline, 12000r.min after gained supernatant liquor adjusting pH to pH=3 -1centrifugal 15min, stays supernatant liquor, and supernatant liquor pH regulator returns pH=6, is stored under 4 DEG C of conditions for subsequent use;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: by supernatant liquor in macroporous resin X-5 post loading, first use deionized water rinsing (with 2 column volumes), then use 40% (v/v) alcohol flushing (by the amount of 1.5 column volumes) to wash the impurity such as pigment off, use again 50% (v/v) ethanol to wash lower active ingredient, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into 30% of supernatant liquor volume, obtain preliminary concentrated solution;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: by preliminary concentrated solution in macroporous resin HP-20 post loading, first use 45% (v/v) alcohol flushing (by the amount of 1.5 column volumes) to wash the impurity such as pigment off, then use 50% (v/v) ethanol to wash lower active ingredient, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into 20% of supernatant liquor volume, obtain secondary concentration liquid;
(4) the semi-preparative liquid phase separation of HPLC: by secondary concentration liquid liquid phase separation, HPLC semipreparative column is C18, 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%, methyl alcohol initial concentration is 50%, stopping concentration is 90%, collects each peak in liquid phase, removes solvent methanol and ammonium acetate;
(5) concentrated 50 DEG C of rotary evaporations of collection liquid corresponding (271nm) peak at active ingredient place postlyophilization is obtained to wanted sterling.
Embodiment 2
The preparation method of triene antibiotic:
(1) from the ripe inclined-plane of Shandong streptomycete, scraping thalline is inoculated in No. 1 liquid nutrient medium of Gao Shi, 28 DEG C of shaking flask concussion (90r.min -1) cultivate 72h, triangular flask liquid amount is 20%.Then seed liquor is forwarded in (5L) fermentor tank with the inoculum size of 10% (about 400ml), 28 DEG C of temperature, initial stirring velocity is 200r.min -1, after inoculation 5h, regulate stirring velocity to 300r.min -1, after inoculation 18h, regulate stirring velocity to 400r.min -1and keep; 5d secondary fermentation finishes, by the fermented liquid 6000r.min at 4 DEG C obtaining -1centrifugal 20min removes thalline, 12000r.min after gained supernatant liquor adjusting pH to pH=3 -1centrifugal 15min, stays supernatant liquor, and supernatant liquor pH regulator returns pH=6, is stored under 4 DEG C of conditions for subsequent use;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: by supernatant liquor in macroporous resin X-5 post loading, first use deionized water rinsing (by 2 cylinder accumulated amounts), then use 40% (v/v) alcohol flushing (1.5 cylinder accumulated amounts) to wash the impurity such as pigment off, use again 50% (v/v) ethanol to wash lower active ingredient, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into 40% of supernatant liquor volume, obtain preliminary concentrated solution;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: by preliminary concentrated solution in macroporous resin HP-20 post loading, first use 45% (v/v) alcohol flushing (by 1.5 cylinder accumulated amounts) to wash the impurity such as pigment off, then use 50% (v/v) ethanol to wash lower active ingredient, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into 25% of supernatant liquor volume, obtain secondary concentration liquid;
(4) the semi-preparative liquid phase separation of HPLC: by secondary concentration liquid liquid phase separation, HPLC semipreparative column is C18, 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%, methyl alcohol initial concentration is 50%, stopping concentration is 90%, collects each peak in liquid phase, removes solvent methanol and ammonium acetate;
(5) concentrated collection liquid rotary evaporation corresponding (271nm) peak at active ingredient place postlyophilization is obtained to wanted sterling.
The structural analysis of the sterling of preparation:
Get embodiment 1 sterling and be dissolved in 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 peak value scope is 0～5.Uv scan figure is shown in Fig. 1, and as seen from Figure 1, this active substance is at 261nm, and there is characteristic absorbance at 271nm and 283nm place, and wherein there is maximum uv-absorbing at 271nm place.This characteristic absorbance is the peculiar uv-absorbing of conjugated triene compounds, therefore can judge and in this active substance, have conjugated triene structure.
Embodiment 1 sterling is before doing every detection analysis, carry out the analysis of HPLC purity detecting, method is: after the sterling pure water after preparing is on a small quantity dissolved, be HPLC and detect, 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; HPLC testing conditions: HPLC analytical column is Agela Venusil ASB C18 (4.6mm*250mm), sample size 10-45uL, detection wavelength is 271nm.The initial concentration of 20mM ammonium acetate pH3.0 is 35%, and stopping concentration is 5%, and methyl alcohol initial concentration is 65%, and stopping concentration is that 95%, LC time-program(me) arranges as table 1.As shown in Figure 2, Fig. 2 has reached more than 90% through the peak purity that calculates this sterling result, and normalization method peak area reaches more than 95%, sees Fig. 3, and this purity meets carries out the requirement that in structure elucidation, mass spectrum and nucleus magnetic resonance etc. detect.
Table 1
Get embodiment 1 sterling and be dissolved in after methyl alcohol, carry out ESI-MS positive ion mode and negative ion mode analysis, ESI-MS positive ion mode provides m/z 493[M+Na] +and 509[M+K] +, negative ion mode provides m/z 469[M-H] -quasi-molecular ion peak, prompting compound molecular weight be 470.Positive and negative ion mode annunciations molecular formula is C 30h 30o 5.ITMS ± ESI-MS positive ion mode and negative ion mode are analyzed collection of illustrative plates and are seen Figure 13-14.
After embodiment 1 sterling is dissolved with deuterated DMSO, pack in clean nuclear magnetic tube, 400MHz nuclear-magnetism carries out NMR test.The project of test comprises 1h-NMR, 13c-NMR, DEPT, gCOSY, HMBC and HMQC, Figure 15-20 are shown in by collection of illustrative plates, data are in table 2.
Table 2
Compound 1h NMR (DMSO-d 6, 400MHz) spectrum provided 10 alkene hydrogen proton signals between low place δ 5.40-7.03, provided at δ 1.38-2.68 place 7 even oxygen inferior/methene proton signals, provided 2 methyl proton signals at δ 0.86 and δ 0.96 place. 13c NMR (DMSO-d 6, 400MHz) and spectrum provides 23 carbon signals, comprises 15 methine carbon signals by known these carbon signals of DEPT spectrum, and wherein 10 is sp 2hydridization carbon, in conjunction with containing 5 two keys in 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.Be an esters of unsaturated fatty acids compound according to above carbon, hydrogen spectrum Notes of Key Data compound.
By HSQC, carbon and direct connected hydrogen signal thereof are carried out to accurate ownership, passed through 1h- 1the heteronuclear distant relation relevant and HMBC of the even proton of the neighbour of H COSY determines the structure of compound, shows to exist in compound structure tri-structure fragments of A～C (seeing Fig. 4).Compound 1h- 1in H COSY spectrum, demonstrate 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 (in figure shown in thick line), in conjunction with the existence of A segment in the chemical shift data prompting structure of its hydrocarbon signal.H-2 and C-3, the C-6 of this structural unit in being composed by HMBC, H-7 and C-23, C-6, C-18, H-23 and C-7, C-15, the distant relation peak of H-16 and C-15 and H-17 and C-11 is further confirmed.In addition, compound 1h- 1in H COSY spectrum, give H-24 and H-20, H-9, H-14 is relevant to H-9, H-19 and H-19 and H-21's, has unsaturated lactone structural unit B in the chemical shift prompting structure in conjunction with these carbon and proton.This structural unit also composed by HMBC in H-24 and C-9, C-25, C-14, the distant relation of H-20 and C-9, C-25 and H-19 and C-14 is confirmed.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 structure.
Show through ultimate analysis, in this compound, except containing C, H, O, also contain S element.Be 470 according to compound molecular weight, tool conjugated triene structure, the character such as soluble in water, infers that molecular formula is C 23h 34o 8s, structural formula is:
Purity check detects:
High 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.HPLC testing conditions: HPLC analytical column is Agela Venusil ASB C18 (4.6mm*250mm), sample size 10-45u L, detection wavelength is 271nm.According to previous work, moving phase is methyl alcohol: ammonium acetate (20mM pH3.0)=70: 30.In order to separate better each absorption peak of the rear sample of preparation, use program gradient elution method instead, the initial concentration of 20mM ammonium acetate (pH3.0) is 35%, stopping concentration is 5%, methyl alcohol initial concentration is 65%, and stopping concentration is that 95%, LC time-program(me) arranges as table 3:
Table 3
The fermented liquid HPLC that embodiment 1 processed through macroporous resin X-5 post and HP-20 column chromatography detects figure as Fig. 5, the fermented liquid HPLC that embodiment 2 processed through macroporous resin X-5 column chromatography detects figure as Fig. 6, can find out, the thick sterling purity of fermented liquid gained of processing through X-5 post and HP-20 post is higher, its purity 85% as calculated, its foreign matter content of fermented liquid of only processing with X-5 post is lower, and the resolution under this condition is also better, impurity peaks signal occurs early, can in preparation, distinguish with active substance absorption peak, therefore also can adopt only by the mode of X-5 post primary treatment.
Anti-mycotic activity test:
The semi-preparative liquid phase separation liquid phase of HPLC collecting zone is shown in Fig. 7, collects 1-4 peak liquid in Fig. 7 and does bacteriostatic test and UV scanning detection, and scanning result is shown in Fig. 8-11, known the 4th absorption peak that peak is Substance.
Respectively get solubility amphotericin B, nystatin, embodiment 1 sterling 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; With the phosphoric acid buffer of 0.1M pH=7.0, above-mentioned three kinds of antibiotic solutions being diluted is 80%, 60%, 40%, 20% of original content; Do bacteriostatic test (apple decay bacterium, geotrichum candidum) and measure inhibition zone by cup-plate method, see Figure 12, can find out, under same concentrations, the active substance antibacterial circle diameter maximum that the present invention produces, 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 with nystatin tire represent.
Adopting dilution method double, is original content by the embodiment of Shandong streptomycete fermentation liquid and 0.2mg/ml 1 sterling solution dilution 1/2,1/4,1/8,1/16,1/32,1/64,1/128; Test minimum inhibitory concentration by cup-plate method.
Adopt dilution method double, record MIC result and show, the minimum inhibitory concentration of thick sterling and fermented liquid is 1/128, and 0.78%.Can define this concentration as a potency unit.

Claims (4)

1. a preparation method for triene antibiotic, its molecular formula is C 23h 34o 8s, structural formula is as follows:
It is characterized in that, comprise the steps:
(1) fermentation liquor pretreatment: scraping thalline is inoculated in No. 1 liquid nutrient medium of Gao Shi from the ripe inclined-plane of Shandong streptomycete, cultivate 70-75h for 25-30 DEG C, inoculate to 25-30 DEG C of agitation condition bottom fermentation 4-5 days in fermentor tank, regulate fermented liquid pH=3-6, remove thalline and stay supernatant liquor centrifugal fermented liquid; The weight part ratio of described No. 1 liquid nutrient medium of Gao Shi consists of: 20 parts of Zulkovsky starches, KNO 31 part, NaCl0.5 part, K 2hPO 43H 2o0.655 part, MgSO 40.5 part, FeSO 41000 parts of 0.01 parts, water; PH7.2～7.4;
(2) with the pre-treatment of macroporous resin X-5 column chromatography: by supernatant liquor in macroporous resin X-5 post loading, first use deionized water rinsing, then wash the impurity such as pigment off with 40% alcohol flushing, wash lower active ingredient with 50% ethanol again, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into the 30-40% of supernatant liquor volume, obtain preliminary concentrated solution;
(3) with the pre-treatment of macroporous resin HP-20 column chromatography: by preliminary concentrated solution in macroporous resin HP-20 post loading, first wash impurity off with 45% alcohol flushing, then wash lower active ingredient with 50% ethanol, collect elutriant, by the elutriant flash concentration of collecting, be concentrated into 20 ± 5% of supernatant liquor volume, obtain secondary concentration liquid;
(4) the semi-preparative liquid phase separation of HPLC: by secondary concentration liquid liquid phase separation, HPLC semipreparative column is C18, 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%, methyl alcohol initial concentration is 50%, stopping concentration is 90%, collects each peak in liquid phase, removes solvent methanol and ammonium acetate;
(5) concentrated collection liquid the rotary evaporation corresponding peak at active ingredient place postlyophilization is obtained to wanted sterling.
2. the preparation method of triene antibiotic according to claim 1, is characterized in that, the described stirring velocity of step (1) is 200-400r.min -1; Described centrifugal speed is 6000-12000r.min -1.
3. the preparation method of triene antibiotic according to claim 1, is characterized in that, the rotary evaporation temperature 50 C that step (5) is described, cryodesiccated time 24-32h.
4. the preparation method of triene antibiotic according to claim 1, is characterized in that, the peak at step (5) active ingredient place is 271 ± 1nm.
CN201210129652.6A 2012-04-28 2012-04-28 Triene antibiotic, preparation method thereof and application thereof Expired - Fee Related CN102659734B (en)

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