CN103497980B - The preparation method of polyetherin A - Google Patents

The preparation method of polyetherin A Download PDF

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CN103497980B
CN103497980B CN201310486479.XA CN201310486479A CN103497980B CN 103497980 B CN103497980 B CN 103497980B CN 201310486479 A CN201310486479 A CN 201310486479A CN 103497980 B CN103497980 B CN 103497980B
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polyetherin
wash
ethyl acetate
elutriant
preparation
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CN103497980A (en
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郑天凌
郑小伟
张帮周
黄丽萍
张金龙
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Xiamen University
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Abstract

The preparation method of polyetherin A, relates to and kills algae compound.The molecular formula of polyetherin A is C 40h 68o 11, relative molecular mass is 742.The inoculation of producing polyetherin A is separated in flat lining out, cultivates; Picking list colony inoculation is in Gause I liquid nutrient medium, and namely obtain fermented liquid after cultivation, fermented liquid is centrifugal, collects supernatant liquor; Described bacterial strain is Streptomyces malaysiensis O4-6, and deposit number is CCTCC NO:M2011051; Be extracted with ethyl acetate supernatant liquor, concentrating under reduced pressure, dry acetic acid ethyl ester extract, be dissolved in ethyl acetate again, join again in silicagel column and carry out wash-out, collect elutriant and carry out thin layer chromatography analysis, merge elutriant, evaporated in vacuo, product carries out the mensuration of algistatic activity, and obtaining can the active ingredient of algal control, then high performance liquid chromatography wash-out, collect elutriant, obtain polyetherin A.

Description

The preparation method of polyetherin A
Technical field
The present invention relates to and kill algae compound, particularly a kind of preparation method of polyetherin A.
Background technology
In recent years, along with widely using of molecular ecology method, marine actinomycete Study on Diversity, the especially huge progress that obtains of the actinomycetic Study on Diversity in deep-sea.Increasing marine actinomycete is found (1, Prof.Alan Claude, W., Diversity and biogeography of marine actinobacteria [J] .2006.9(3): 279-286.), comprise new 16SrDNA sequence cluster (2, Liesack, W.and E.Stackebrandt, Occurrence of novel groups of the domainBacteria as revealed by analysis of genetic material isolated from an Australian terrestrialenvironment.J.Bacteriol. [J], 1992.174 (15): 5072-5078.3, Rheims, H., et al., Molecularbiological evidence for the occurrence of uncultured members of the actinomycete line of descent indifferent environments and geographical locations.Microbiology [J], 1996.142 (10): 2863-2870.), actinomycetic kind and quantity constantly increase, 170 genus are increased by initial 3 genus.But, remain compared with the quantity that actinomycetic known species exists at nature with it inappreciable (4, Bull, A.T., et al., Marine actinobacteria:perspectives, challenges, future directions.Antonie Van Leeuwenhoek International Journal ofGeneral and Molecular Microbiology [J], 2005.87 (3): 259-262; 5, Jensen, P.R., et al., Marineactinomycete diversity and natural product discovery.Antonie van Leeuwenhoek [J], 2005.87 (1): 43-48).
Due to the singularity of ocean environment, marine microorganism has unique metabolic way, and the metabolic chemistry structure of generation has great complicacy and diversity.Over nearly 20 years, relevant marine microorganism produces the new report with bioactive secondary metabolite to be increased gradually, finds that the probability of new biological activity material is even more than Lu Sheng actinomycetes from marine actinomycete.The amount of activated material obtained from marine actinomycete has griseorhodin A, salt spore amine A, salt spore amine B, the blue element of smoked clothing cyanines, salt amine, tetraodotoxin, ocean mycin A, B, four and mycin D1, chromomycin A3, enterococcin, Quinomycin A, antimycin A, bar bifilomycin, Antibiotic U-5956, lipomycin, the lagosin, Deng (6, Xu Lihua etc., actinomycetes systematics---principle, method and put into practice [M]. Beijing: Science Press, 2007.).These results of study show, ocean environment finds and screens new actinomycetes species and the precious resources storehouse of meta-bolites, wherein contain a large amount of actinomycetes population and await development and utilize.
Since 20th century, along with coastland explosive population growth, developing rapidly of industrial or agricultural, the serious organic contamination of inner bay, river mouth and the stretch of coastal water and eutrophication.Harmful algal blooms (Harmful algae blooms, HABs) at the generation scale of China and frequency in sharply ascendant trend, to China coast cause serious ecology, Environmental and resource issue and great financial loss (7, Qin, S., et al., Two New Metabolites, Epoxydine A and B, from Phoma sp.Helvetica ChimicaActa [J], 2010.93 (1): 169-174).Research red-tide control method, formulates about Preventing Countermeasures has become the environmental practice of many countries and regions task of top priority.Use because the existence of physics and chemistry method improvement red tide is difficult to big area and easily causes the weak points such as secondary pollution, utilize biological Ecology Action each other administer red tide become current research focus (8, Skerratt, J.H., et al., Algicidal bacteria associated with blooms of a toxic dinoflagellate in a temperateAustralian estuary.Marine Ecology-Progress Series [J], 2002.244:1-15.).
Up to now, the algal control microorganism that great majority screen plays alga-inhibiting action by secreting the special material with algistatic activity.The algistatic activity material reported comprises: protein (containing extracellular enzyme), polypeptide, amino acid, microbiotic, nitrogenous compound etc. other not yet qualitatively algal control compound (9, Yoshikawa, K., et al., beta-cyanoalanine production bymarine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria.Applied and Environmental Microbiology [J], 2000.66 (2): 718-722).The new approaches of exploitation algae-inhibiting agent for red tide control are become by screening efficient, single-minded algistatic activity material.
Summary of the invention
The object of this invention is to provide the preparation method of polyetherin A.
The molecular formula of described polyetherin A (Nigericin) is C 40h 68o 11, relative molecular mass is 742, and structural formula is:
The present invention includes following steps:
1) inoculation of producing polyetherin A is separated in flat lining out, at 28 ~ 37 DEG C of temperature, cultivates 5 ~ 7d; Picking list colony inoculation is in Gause I liquid nutrient medium, and in 28 ~ 37 DEG C, namely 180 ~ 250rpm obtains fermented liquid after cultivating 5 ~ 7d; Gained fermented liquid is carried out centrifugal, collects supernatant liquor; The bacterial strain of described production polyetherin A is Streptomycesmalaysiensis O4-6, this bacterial strain is preserved in China typical culture collection center on February 28th, 2011, preservation center deposit number is: CCTCC NO:M2011051, address: China. Wuhan. and Wuhan University;
2) step 1) gained supernatant liquor is extracted with ethyl acetate, concentrating under reduced pressure, dry, obtain acetic acid ethyl ester extract;
3) acetic acid ethyl ester extract is dissolved in ethyl acetate, then joins in silicagel column and carry out wash-out, collect elutriant and carry out thin layer chromatography analysis, according to expansion collection of illustrative plates, merge the elutriant collected;
4) by step 3) gained elutriant evaporated in vacuo at 30 ~ 45 DEG C, product carries out the mensuration of algistatic activity, and obtaining can the active ingredient of algal control;
5) gained the active ingredient of algal control can be carried out high performance liquid chromatography wash-out, collect elutriant, obtain polyetherin A.
In step 1), described centrifugal condition can be: speed 10000 ~ 12000g, time 10 ~ 20min.
In step 3), the specification of described silicagel column can be 170mm × 30mm, 200 ~ 300 orders; The eluent of described wash-out can be the mixture etc. of normal hexane and ethyl acetate; The program of described wash-out is: the mixture wash-out 20min using normal hexane and ethyl acetate volume ratio 1: 2, then uses the mixture wash-out 30min of normal hexane and ethyl acetate volume ratio 1: 1, finally uses hexane 30min; The flow velocity of wash-out can be 1mL/min; The developping agent of described thin layer chromatography analysis can be ethyl acetate etc., and the developer of described thin layer chromatography analysis is the chloroformic solution etc. containing 0.5% iodine.
In step 5), the program of described wash-out can be:
0 ~ 7.5min 85% methanol/water (v/v);
7.5 ~ 10.5min 85% methanol/water ~ 100% methyl alcohol;
10.5 ~ 22.5min 100% methyl alcohol.
Collect 10.5 ~ 12.5min elutriant evaporated in vacuo at Rotary Evaporators 30 DEG C and be dissolved in DMSO checking algistatic activity.
Gained polyetherin A is a kind of potent algistatic activity compound, through algistatic activity component thin-layer chromatography (Thin LayerChromatography, TLC) detect, launch on silica gel thin-layer plate using ethyl acetate, methylene dichloride and chloroform as developping agent to be a spot, be defined as pure compound, and detect through 1H-NMR, determine the structural formula of potent algistatic activity compound, described potent algistatic activity compound can effectively kill phaeocystis globosa cell, and it has huge application potential in the preparation of algae-inhibiting agent.
The present invention uses ordinary method to screen and obtains a strain Streptomyces malaysiensis O4-6, pass through fermentation culture, obtain the fermented liquid containing strong algistatic activity compound, by centrifugal for described fermented liquid, collect supernatant liquor, then described supernatant liquor is carried out separation and purification, obtain the compound with strong algistatic activity.The compound of described strong algistatic activity efficiently, exclusively can kill frustule, has the potential being developed to algae-inhibiting agent, has a wide range of applications in biological degradation algae and improvement red tide etc.
Accompanying drawing explanation
Fig. 1 is the HPLC-UV detection of potent algistatic activity compound.In FIG, X-coordinate is time (min), and ordinate zou is absorbancy (mAU); Respectively compose peak from left to right and be respectively 2.948,10.491,10.910,12.042.
Embodiment
Following examples further illustrate of the present invention, but the invention is not restricted to following embodiment.
The separation screening of embodiment 1 marine streptomyces Streptomyces malaysiensis O4-6
One, the separation screening concrete steps of Streptomyces malaysiensis O4-6 are:
1) (particular location is Fujian sky Mangrove Wetlands: 117 ° of 24 '-117 ° of 30 ' E, 23 ° of 53 '-23 ° of 56 ' N) settling, dry 1 month is placed in room temperature, get the dry soil sample of 10g, be dissolved in autoclaved 90mL Gause I substratum, be placed in 150rpm shaking table concussion 20min, make soil sample dispersed;
2) by the dilution of 10 times, step 1) gained sample, Gause I (20g Zulkovsky starch, 1g NaNO is coated 3, 0.5gK 2hPO 4, 0.5g MgSO 47H 2o, 0.01g FeSO 47H 2o, 75 μ g K 2cr 2o 7, 10g agar, 1L seawater) and solid plate, cultivate 5d under being placed in 28 DEG C of temperature;
3) the dissimilar single bacterium colony of picking lines Gause I solid plate, cultivates 5d at being placed in 28 DEG C, verifies whether pure culture, repeats this step until obtain pure culture;
4) inoculate isolated pure growth list bacterium colony in 4mL Gause I liquid nutrient medium, be placed in 28 DEG C of shaking tables, 5d is cultivated in 180rpm concussion, gets culture centrifugal 10min under the centrifugal force of 10000g, removing bacterial sediment, and supernatant is saved to 4.5mL centrifuge tube;
5) joined by 1mL supernatant in 20mL exponential phase phaeocystis globosa nutrient solution, in 20 ± 1 DEG C, 12h illumination, 12h is dark, 50 μm of ol photons m -2s -1cultivate 2d under intensity of illumination condition, Gause I substratum is that contrast adds in algae liquid, arrange respectively 3 parallel; Observe the whether sedimentation of phaeocystis globosa cell, if sedimentation, represent containing algistatic activity material in the 7d culture supernatant of thalline, thus filter out algistatic activity bacterial strain.
Two, the separation screening concrete steps of Streptomyces malaysiensis O4-6 can be:
1) Fujian sky Mangrove Wetlands (117 ° of 24 '-117 ° of 30 ' E, 23 ° of 53 '-23 ° of 56 ' N) settling, dry 1 month is placed in room temperature, get the dry soil sample of 10g, be dissolved in autoclaved 90m L Gause I substratum, be placed in 200rpm shaking table concussion 30min, make soil sample dispersed;
2) by the dilution of 10 times, step 1) gained sample, Gause I (20g Zulkovsky starch, 1g NaNO is coated 3, 0.5gK 2hPO 4, 0.5g MgSO 47H 2o, 0.01g FeSO 47H 2o, 75 μ g K 2cr 2o 7, 10g agar, 1L seawater) and solid plate, cultivate 7d at being placed in 37 DEG C;
3) the dissimilar single bacterium colony of picking lines Gause I solid plate, cultivates 7d at being placed in 37 DEG C, verifies whether pure culture, repeats this step until obtain pure culture;
4) inoculate isolated pure growth list bacterium colony in 4mL Gause I liquid nutrient medium, be placed in 37 DEG C of shaking tables, 7d is cultivated in 250rpm concussion, gets culture centrifugal 20min under the centrifugal force of 12000g of 7d, removing bacterial sediment, and supernatant is saved to 4.5mL centrifuge tube;
5) joined by 1mL supernatant in 20mL exponential phase phaeocystis globosa nutrient solution, in 20 ± 1 DEG C, 12h illumination, 12h is dark, 50 μm of ol photons m -2s -1cultivate 2d under intensity of illumination condition, Gause I substratum is that contrast adds in algae liquid, arrange respectively 3 parallel; Observe the whether sedimentation of phaeocystis globosa cell, if sedimentation, represent containing algistatic activity material in the supernatant of thalline 7d culture, thus filter out algistatic activity bacterial strain.
The method of calculation of embodiment 2 algal control rate
1) phaeocystis globosa is at 20 ± 1 DEG C, 12h illumination, and 12h is dark, 50 μm of ol photons m -2s -1under intensity of illumination condition, in triangular flask, be cultured to exponential phase, be then dispensed in 24 porocyte culture plates, every hole packing 2mL frustule suspension, Adaptable growth 1d;
2) 100 μ L culture to be measured adds 24 orifice plates, cultivates 2d;
3) get phaeocystis globosa nutrient solution, 200 μ L samples, in 24 orifice plates, by the fluorescence intensity at 680nm place under microplate reader detection 460nm excitation, according to following formulae discovery algal control rate, observe frustule metamorphosis simultaneously:
Algal control rate=(F c-F t)/F c× 100%
In formula, F crepresent control group fluorescence intensity, F trepresent experimental group fluorescence intensity.
Embodiment 3 algistatic activity physical property is identified
1) marine streptomyces Streptomyces malaysiensis O4-6 is inoculated in Gause I substratum 28 ~ 37 DEG C of shaking tables, and 180 ~ 250rpm shakes cultivation 5 ~ 7d.Fermented liquid is centrifugal 10 ~ 20min under the centrifugal force of 10000 ~ 12000g, obtains streptomycete Streptomyces malaysiensis O4-6 nutrient solution supernatant; Be extracted with ethyl acetate gained supernatant liquor, concentrating under reduced pressure, dry, obtain acetic acid ethyl ester extract.
2) Temperature Treatment experimental design: 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C and 120 DEG C of high pressure 30min thermal treatment are carried out to the nutrient solution supernatant containing algistatic activity material, 3 parallel laboratory test groups are set; Control experiment group is: untreated nutrient solution supernatant; Getting 100 μ L nutrient solution supernatants adds in 2mL algae culturing liquid, after cultivating 2d, samples and calculates algal control efficiency by embodiment 2.Experimental result (see table 1) shows: after this nutrient solution supernatant carries out thermograde thermal treatment, and it does not vary widely the algal control efficiency of phaeocystis globosa, illustrates that algistatic activity material can not tolerate more than 100 DEG C pyroprocessing.
Table 1 Temperature Treatment is on the impact of algistatic activity material algal control efficiency
3) pH process experimental design: measure nutrient solution supernatant pH value, again its pH is recalled to process after nutrient solution supernatant pH being adjusted to respectively 1,3,5,7,9 and 11,2h, 3 parallel laboratory test groups are set; Getting 100 μ L bacteria-free filtrates adds in 2mL algae culturing liquid, after cultivating 24h, samples and calculates algal control efficiency by embodiment 2.Experimental result (see table 2) shows: after this nutrient solution supernatant carries out acidifying and alkalinisation treatment, and it does not vary widely the algal control efficiency of phaeocystis globosa, illustrates that algistatic activity material is all more stable under acidic conditions and alkaline condition.
Table 2pH process is on the impact of algistatic activity material algal control efficiency
4) dialysis treatment experimental design: utilize molecular retention amount to be about 1Kd dialysis tubing, carry out dialysis treatment to nutrient solution supernatant, after PBS damping fluid dialysis 3h, proceeds to fresh PBS damping fluid and continues dialysis 3h, arrange 3 parallel laboratory test groups; Control group is the nutrient solution supernatant of not dialysing; Getting 100 μ L nutrient solution supernatants adds in 2mL algae culturing liquid, after cultivating 24h, samples and calculates algal control efficiency by embodiment 2.Experimental result (see table 3) shows: this nutrient solution supernatant is after the dialysis tubing dialysis treatment that molecular retention amount is about 1Kd, it obviously reduces the algal control efficiency of phaeocystis globosa, its algal control efficiency is on average only 21.4%, illustrates that algistatic activity molecular weight of material is less than 1Kd.
Table 3 dialysis treatment is on the impact of algistatic activity material algal control efficiency
5) different organic solvents extraction experiments design: select propyl carbinol, methylene dichloride, ethyl acetate as extraction agent, respectively with 1: 1 volume ratio extraction 500mL nutrient solution supernatant, repeats 3 extractions.500mL nutrient solution supernatant is evaporated in vacuo at Rotary Evaporators 30 DEG C, selects 500mL acetonitrile to dissolve.Various extractive with organic solvent is evaporated in vacuo at Rotary Evaporators 30 DEG C, obtains extraction crude extract.5mL DMSO dissolves crude extract, gets 100 μ L DMSO solutes and adds in 2mL algae culturing liquid, after cultivating 24h, samples and calculates algal control efficiency by embodiment 2, arrange 3 parallel laboratory test groups, and adds DMSO to algae culturing liquid control group.Experimental result (table 4) shows: this nutrient solution supernatant is after opposed polarity organic solvent extraction, algal control efficiency display different, acetic acid ethyl ester extract algal control rate is higher, average out to 87.9%, illustrates that algistatic activity material polarity is more weak easily by extraction into ethyl acetate out.
Table 4 different organic solvents extract algal control efficiency
Embodiment 4 algistatic activity component silica gel column chromatography
Streptomycete Streptomyces malaysiensis O4-6 is inoculated in Gause I substratum 28 ~ 37 DEG C of shaking tables, and 180 ~ 250rpm shakes cultivation 5 ~ 7d.Fermented liquid is centrifugal 10 ~ 20min under the centrifugal force of 10000 ~ 12000g, obtains streptomycete Streptomyces malaysiensis O4-6 nutrient solution supernatant.Be extracted with ethyl acetate gained supernatant liquor, concentrating under reduced pressure, dry, obtain acetic acid ethyl ester extract.
100mg acetic acid ethyl ester extract is dissolved in 1mL ethyl acetate, is splined on silicagel column (170 × 30mm, 200-300 order), eluent: the mixture of normal hexane and ethyl acetate, elution program: volume ratio 1: 2,20min; 1: 1,30min; 1: 0,30min; Elution flow rate: 1mL/min; Elutriant 2mL/ pipe is collected with collection tube.
Embodiment 5 algistatic activity component thin-layer chromatography (Thin Layer Chromatography, TLC) is analyzed
1) as embodiment 4, elutriant utilizes TLC to analyze, and developping agent is ethyl acetate, developer: the chloroformic solution of 0.5% iodine, merges elutriant in collection tube according to expansion collection of illustrative plates.
2) merge elutriant evaporated in vacuo at Rotary Evaporators 30 DEG C, be dissolved in DMSO, by the method validation algistatic activity in embodiment 2, embodiment 3, obtain potent algistatic activity component.
Embodiment 6 algistatic activity component high performance liquid chromatography (High-performance liquid chromatography, HPLC) is analyzed
The potent algistatic activity component of embodiment 5 gained utilizes HPLC to analyze, elution program:
0 ~ 7.5min 85% methanol/water;
7.5min ~ 10.5min 85% methanol/water ~ 100% methyl alcohol;
10.5 ~ 22.5min 100% methyl alcohol.
As shown in Figure 1, elutriant evaporated in vacuo at Rotary Evaporators 30 ~ 45 DEG C of collecting 10.5 ~ 12.5min is dissolved in DMSO to experimental result, verifies algistatic activity by embodiment 2, embodiment 3.
The Structural Identification of the potent algistatic activity compound of embodiment 7:
Embodiment 6 algistatic activity detects, and detects, launch to be a spot with ethyl acetate, methylene dichloride and chloroform on silica gel thin-layer plate through TLC, and warp 1h-NMR detects, and be defined as pure compound, structural formula is as follows:
The ESI-MS spectrum of described pure compound is at m/z747.47 [M+Na] +the adduct ion peak of place's display molecule, in conjunction with 13C-NMR(DEPT) compose and 1H-NMR spectrum, releasing its molecular formula is C 40h 68o 11, relative molecular mass is 742.1H-NMR spectrum shows a large amount of methyl, methylene radical and 1 methoxyl group signal.What 13C-NMR composed shows 40 C, by the trace analysis with DEPT, can obviously obtain: 10 CH 3(δ C is respectively 59.53,29.00,22.76,16.98,16.41,16.12,14.38,13.39,13.07,11.48), 10 CH 2(δ C is respectively 66.88,41.65,37.08,35.77,32.30,31.99,29.47,26.27,25.86,23.51), 15 CH 1(δ C is respectively 85.17,81.39,79.40,76.75,76.43,73.20,68.37,60.38,45.79,39.58,36.69,36.40,35.07,31.80,27.65), 5 quaternary carbon (δ cbe respectively 183.83,107.49,97.15,84.78,82.31).From the two-dimensional map of HMBC, HSQC, H-H COSY and NOSEY, the associated home of each C and H can be determined.Through Scifinder database retrieval, then with corresponding document comparison, then its physico-chemical property comprehensive, determines consistent with polyetherin A.
In sum, the compound with algistatic activity that extraction purification of the present invention obtains is polyetherin A.

Claims (3)

1. the preparation method of polyetherin A, is characterized in that the molecular formula of described polyetherin A is C 40h 68o 11, relative molecular mass is 742, and structural formula is:
Described preparation method comprises the following steps:
1) inoculation of producing polyetherin A is separated in flat lining out, at 28 ~ 37 DEG C of temperature, cultivates 5 ~ 7d; Picking list colony inoculation is in Gause I liquid nutrient medium, and in 28 ~ 37 DEG C, namely 180 ~ 250rpm obtains fermented liquid after cultivating 5 ~ 7d; Gained fermented liquid is carried out centrifugal, collects supernatant liquor; The bacterial strain of described production polyetherin A is Streptomycesmalaysiensis O4-6, and this bacterial strain is preserved in China typical culture collection center on February 28th, 2011, and preservation center deposit number is CCTCC NO:M2011051;
2) step 1 is extracted with ethyl acetate) gained supernatant liquor, concentrating under reduced pressure, dry, obtain acetic acid ethyl ester extract;
3) acetic acid ethyl ester extract is dissolved in ethyl acetate, then joins in silicagel column and carry out wash-out, collect elutriant and carry out thin layer chromatography analysis, according to expansion collection of illustrative plates, merge the elutriant collected; The eluent of described wash-out is the mixture of normal hexane and ethyl acetate, the program of described wash-out is: the mixture wash-out 20min using normal hexane and ethyl acetate volume ratio 1: 2, use the mixture wash-out 30min of normal hexane and ethyl acetate volume ratio 1: 1 again, finally use hexane 30min, the flow velocity of wash-out is: 1mL/min; The developping agent of described thin-layer chromatography is ethyl acetate, and the developer of described thin-layer chromatography is the chloroformic solution containing 0.5% iodine;
4) by step 3) gained elutriant evaporated in vacuo at 30 ~ 45 DEG C, product carries out the mensuration of algistatic activity, and obtaining can the active ingredient of algal control;
5) gained the active ingredient of algal control can be carried out high performance liquid chromatography wash-out, collect elutriant, obtain polyetherin A;
The program of described wash-out:
0 ~ 7.5min 85% methanol/water;
7.5 ~ 10.5min 85% methanol/water ~ 100% methyl alcohol;
10.5 ~ 22.5min 100% methyl alcohol.
2. the preparation method of polyetherin A as claimed in claim 1, is characterized in that in step 1) in, described centrifugal condition is: speed 10000 ~ 12000g, time 10 ~ 20min.
3. the preparation method of polyetherin A as claimed in claim 1, is characterized in that in step 3) in, the specification of described silicagel column is 170mm × 30mm, 200 ~ 300 orders.
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