CN103304640B - A kind of method extracting Echinocandin compound from fermented liquid - Google Patents

A kind of method extracting Echinocandin compound from fermented liquid Download PDF

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CN103304640B
CN103304640B CN201210066411.1A CN201210066411A CN103304640B CN 103304640 B CN103304640 B CN 103304640B CN 201210066411 A CN201210066411 A CN 201210066411A CN 103304640 B CN103304640 B CN 103304640B
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solvent
membrane
extraction
liquid
echinocandin compound
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CN103304640A (en
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张磊
杨洋
郭冬初
王小勇
孙新强
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Zhejiang Changhai Pharmaceutical Co., Ltd.
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Zhejiang Medicine Co Ltd Xinchang Pharmaceutical Factory
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Abstract

The present invention discloses a kind of method extracting Echinocandin compound from fermented liquid, said method comprising the steps of: a) fermented liquid microfiltration membrane equipment carries out cross flow filter, first with water cleaning in filtration procedure, then add extraction agent and extract, after filtration, obtain the filtrate containing target compound; B) filtrate containing target compound is adsorbed with macroporous resin, then uses the prewashing of the second solvent, parsing, obtains the desorbed solution containing target compound; C) the concentrated desorbed solution containing target compound, removing the second solvent; D), after adding the third solvent precipitation target compound, the solidliquid mixture containing target compound is obtained; E) solidliquid mixture of separating out target compound is filtered, after drying, obtain target compound dry powder.The inventive method, technical process is short, and the solvent system of use is simple, through simple nanofiltration enrichment step, extraction agent can turn back in microfiltration membrane and continue extraction, achieves the recycled of extraction agent, greatly reduce solvent usage quantity, be applicable to large-scale industrial production.

Description

A kind of method extracting Echinocandin compound from fermented liquid
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of industrialized preparing process extracting Echinocandin compound from fermented liquid.
Background technology
Fungi is conditioned pathogen for healthy human body, harmless under normal circumstances; But when Abwehrkraft des Koepers reduces, just likely cause local or Systemic fungal infections.In recent years owing to abusing cytotoxic drug, immunosuppressor, Broad spectrum antibiotics and hormone medicine clinically, not only the immunizing power of patient's body is suppressed, and the microecological balance in body is damaged, makes non-pathogenic fungi originally become dominant bacteria, thus bring out fungi infestation; Add carrying out of the Open Treatment means such as organ transplantation in modern medical service, interventional therapy, intubation technique, more increase the morbidity risk rate of fungi infestation.
According to the current standard of international medical community, fungi infestation can be divided into shallow fungi infestation, subcutaneous fungi infestation and systemic fungal infection.Wherein first two fungi infestation is the most common, but not fatal, systemic fungal infection most serious of all, i.e. deep fungal infection, if can not be controlled timely and effectively after catching an illness, and often threat to life.Since first antifungal drug amphotericin B comes out, the struggle of the mankind and fungal infection disease continue for over half a century, now, the mankind make a breakthrough in the fungi infestation of prevention and therapy shallow, but for subcutaneous fungi infestation and systemic fungal infection, due to the existence of the factors such as its difficulty for the treatment of, the resistance of fungi and toxic side effect of existing medicine, not yet obtain remarkable break-throughs.The medicine of modern clinic being treated fungi infestation use mainly contains amphotericin B and triazole antifungal agent thing (as fluconazole, itraconazole and Voriconazole etc.), but all there is certain problem in them in security, pharmacokinetics or antimicrobial spectrum etc., the therefore appearance of the urgently New-type wide-spectrum antifungal drug of efficient, low toxicity clinically.
Echinocandin (Echinocandins), also known as echinocandin, it is the novel antifungal drug of a class, belonging to acetyl six lopps, is glucan synthase inhibitors, can the β-1 of noncompetitive ground Antifungi cell walls, the synthesis of 3-D-dextran, thus destroy the integrity of cell walls and make its osmotic pressure change and play germicidal action, and the acellular wall of mammalian cell, therefore on mammalian cell without impact.Echinocandin class medicine by the glucan synthase in Antifungi body with killing fungus, to Cytochrome P450 without effect, untoward reaction is few, with amphotericin B and triazole antifungal agent thing without cross resistance, low to the toxicity of human body, mainly act on Candida (Candidas) and Eurotium (Aspergillus), also can act on parasite type pathogenic agent as Pneumocystis carinii (Pneumocystiscarinii) etc.
Echinocandin compound basic structure is similar, all be combined with different substituting groups in the structure of acetyl six ring, but the compound anti-mycotic activity of different substituents is different, through screening targetedly, researchist finds that some compound anti-mycotic activity is stronger, comprise lung and read rhzomorph B0 (PneumocandinB0), WF11899A and ECB (EchinocandinB), they are all obtained by the method for fermentation, carry out semi-synthetic using it as intermediate, antifungal drug Caspofungin can be obtained respectively, MFG and anidulafungin.Lung is read rhzomorph B0 (PneumocandinB0) and is carried out biosynthesizing generation by the fermentation of Zalerionarboricola thalline, Merck & Co., Inc. carries out semi-synthetic using it as intermediate, obtain antifungal drug caspofungin acetate, go on the market in the U.S. February calendar year 2001, be used for the treatment of the infection of Candida albicans and aspergillus fumigatus; WF11899A is fermented by ColeophomaempetriF-11899 thalline and carries out biosynthesizing generation, Teng Ze company carries out semi-synthetic using it as intermediate, obtain antifungal drug MFG, went on the market in 2002 months in Japan, be mainly used in Candida albicans and aspergillin infection, anti-candida albicans infection effect is better, especially stronger to the candidiasis activity of resistance to fluconazole; ECB (EchinocandinB) by Aspergilusnidulans or with Xinchang actual bacterium same names Aspergillusrugulovalvus thalline fermenting organism synthesize and produce, using it as intermediate, through presenting betrothal gifts to the bride's family before marriage, company and the exploitation of VicuronPharmaceuticals company joint research and development obtain antifungal drug anidulafungin, listing in 2006, its anti-mycotic activity is stronger to aspergillus fumigatus, relatively weak to Candida albicans.
The domestic technique for extracting Echinocandin compound from fermented liquid is not almost reported at present, external mainly some large drugmakers have carried out correlative study, but mainly concentrate on the small-scale extraction in laboratory, and extraction process route is longer, the yield of product is very low, the solvent species used is many, and the impact of some solvent on human body and environment is larger, is not suitable for large-scale industrial production.
WO2011/121599A1 extracts the technique of Echinocandin compound from fermented liquid, and main method is: first Echinocandin compound is extracted from fermented liquid with solvent; Carry out liquid-liquid extraction remove portion impurity again; After activated carbon decolorizing, utilize sorbent material to adsorb, eventually pass parsing, concentrated, precipitation obtains Echinocandin compound.The subject matter of this technique is, in extraction process, the usage quantity of solvent is larger; Fermented liquid is carried out to liquid-liquid extraction, carries out, in the process of liquid-liquid extraction, all producing mixed solvent layer to the extraction liquid containing target components, and can dissolve organic phase in various degree in aqueous phase, direct discharge can pollute, and reclaims and can increase cost again; The sorbent material price used, be not easy thorough regeneration, work-ing life is limited.
US5194377 and US5202309 extracts the technique of Echinocandin compound from fermented liquid, and main method is: first Echinocandin compound is extracted from mycelium with the first solvent; Carry out several times of liquid liquid extraction again, make Echinocandin compound in two-phase, carry out transfer remove portion impurity; Extraction liquid containing Echinocandin compound carries out chromatography through multiple different chromatographic column again, eventually passes parsing, concentrated, solvent deposition obtains Echinocandin compound.The subject matter of this technique is, in extraction process, the usage quantity of solvent is larger; Fermented liquid is carried out to liquid-liquid extraction, carries out, in the process of liquid-liquid extraction, all producing mixed solvent layer to the extraction liquid containing target components, and can dissolve organic phase in various degree in aqueous phase, direct discharge can pollute, and reclaims and can increase cost again; The adsorbent species simultaneously used is many, price, and be not easy thorough regeneration, work-ing life is limited; Operational path is long, and last total recovery is lower.
US661082282 and US2008/0108806A1 extracts the technique of Echinocandin compound from fermented liquid, and main method is: first Echinocandin compound is extracted from mycelium with isopropylcarbinol; Wash after concentrated containing the isopropylcarbinol of Echinocandin compound, add methyl alcohol-normal heptane again and carry out liquid-liquid extraction, form two-phase: organic phase (isopropylcarbinol-normal heptane) and aqueous phase (methanol-water), Echinocandin compound proceeds in aqueous phase; Add isopropylcarbinol again after aqueous phase concentrated removing methyl alcohol and carry out liquid-liquid extraction, Echinocandin compound proceeds in isopropylcarbinol; After concentrated removing isopropylcarbinol, add acetonitrile and carry out precipitation and obtain Echinocandin compound.The subject matter of this technique is, in extraction process, the usage quantity of solvent is larger; Isopropylcarbinol extraction liquid containing target components is washed, and and add methyl alcohol-normal heptane and carry out, in the process of liquid-liquid extraction, mixed solvent layer being produced, and organic phase can be dissolved in various degree in aqueous phase, direct discharge can pollute, and reclaims and can increase cost again; The organic phase that in leaching process, liquid-liquid extraction uses is mixed solvent, and recovery difficult is larger.
From fermented liquid, extract the technique of Echinocandin compound in US2010/0249371A1, main method is: first Echinocandin compound is extracted from mycelium with the first solvent; Then, after vacuum concentration remove portion solvent, add metallic salt sorbent material (such as calcium phosphate) and obtain Echinocandin compound-calcium phosphate complex; From this mixture, Echinocandin compound is dissolved out with the second solvent again, after concentrated removing the second solvent, adds the third solvent and carry out precipitation and obtain Echinocandin compound.The subject matter of this technique is, in extraction process, the usage quantity of solvent is larger; Add metal salt compound in leaching process, inevitably have residual in the product, need to be further purified to remove metal ion.
From fermented liquid, extract the technique of Echinocandin compound in CN101659693 and CN101659692A, main method is: first Echinocandin compound is extracted from mycelium with the first solvent; After concentrated the first solvent of removing, again extract with the second solvent; After concentrated removing the second solvent, again by the first dissolution with solvents Echinocandin compound, carry out chromatographic runs with acidic alumina column, adsorption resin column, reversed-phase resin post successively; After desorbed solution is concentrated, adds solvent deposition and obtain Echinocandin compound.The subject matter of this technique is, the usage quantity of solvent is larger; The route extracted is longer, through repeatedly chromatography, and total recovery can be lower; Adsorbent species is many, and regenerative process can use different kinds of liquid solvents, increases recovery difficult, improves extraction cost.
From fermented liquid, extract the technique of Echinocandin compound in WO9611210, main method is: first Echinocandin compound is extracted from mycelium with acetone; After concentrated removing acetone, after being extracted with ethyl acetate, use n-butanol extraction again; After concentrated remove portion propyl carbinol, successively through twice silica gel column chromatography, centrifugal chromatography chromatography, then twice silica gel column chromatography, last desorbed solution is concentrated into dry, and adjust pH to 7.0 again after adding a small amount of water dissolution, freeze-drying obtains Echinocandin compound.The subject matter of this technique is, in extraction process, the usage quantity of solvent is larger; With in ethyl acetate and butanol extraction liquid process, can produce mixed solvent layer, reclaim, recovery difficult is larger; Chromatographic step is many, and last yield is lower, and adding centre has a step centrifugal chromatography chromatography, is not suitable for suitability for industrialized production and uses; Chromatography media kind is many, and regenerative process can use different kinds of liquid solvents, increases recovery difficult, further increases extraction cost.
From fermented liquid, the technique of Echinocandin compound is extracted in US4024245, US4024246 and US4288549, main method is: first extract fermented liquid with methyl alcohol, with chloroform, liquid-liquid extraction is carried out to methanol extraction liquid again, after concentrated removing chloroform, add ether and make Echinocandin compound generate precipitation; After precipitation is dissolved again, through reversed-phase resin column chromatography, again concentrate removing chloroform after desorbed solution chloroform extraction, then add trimethyl carbinol dissolving, then freeze-drying obtains Echinocandin compound.The subject matter of this technique is that extraction step is many, and last yield is lower; Employ chloroform and so on hazardous chemical solvent in leaching process, be not suitable for use of large-scale production.
Summary of the invention
At present for the method for Echinocandin compound from fermented liquid, mainly several large drugmaker has carried out correlative study both at home and abroad, but mainly concentrate on the small-scale extraction in laboratory, and extraction process route is longer, the yield of product is very low, the solvent species used is many, and the impact of some solvent on human body and environment is larger, is not suitable for large-scale industrial production.
The present invention discloses a kind of method of extraction Echinocandin compound newly, by using membrane technique, extraction process route is short, the solvent species used is also relatively less, and through simple nanofiltration enrichment step in leaching process, extraction agent just can turn back in microfiltration membrane and continue extraction, achieves the recycled of solvent in leaching process, greatly reduce the usage quantity of solvent, reduce the cost of solvent recuperation; Meanwhile, use membrane concentration technology, achieve cryoconcentration, not only remove small molecular weight impurity, also reduce vacuum decompression heating concentrated time temperature drift and produce the problem of new impurity, improve quality product.Present invention process, high to the product purity obtained after target compound extraction purification, yield is high, use quantity of solvent few, and the cyclic utilization rate of solvent is high, is applicable to large-scale industrial production.
The invention provides a kind of method extracting Echinocandin compound from Echinocandin compound fermented liquid, the method comprises the following steps: a) adopt microfiltration equipment to carry out cross flow filter to Echinocandin compound fermented liquid, obtain the mycelium suspended liquid concentrated, then add the water being adjusted to pH=3.0 ~ 8.0 to clean mycelium suspended liquid, the mycelium suspended liquid after cleaning is obtained after micro-filtration, then at 5 DEG C ~ 50 DEG C temperature, add extraction agent extract the mycelium suspended liquid after cleaning, the filtrate containing Echinocandin compound is obtained after micro-filtration, wherein, described extraction agent is the mixed solvent of the first solvent and water, and the first solvent described is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone, b) by the Echinocandin compound in filtrate described in macroporous resin adsorption, then use the prewashing of the second solvent, parsing, obtain the desorbed solution containing Echinocandin compound, wherein, described macroporous adsorbent resin is nonpolar macroporous adsorption resin or low-pole macroporous adsorbent resin, and described the second solvent is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone, c) first concentrate described desorbed solution with nanofiltration, continue concentrating under reduced pressure to remove the second solvent, d), after adding the third solvent precipitation Echinocandin compound, the solidliquid mixture containing Echinocandin compound is obtained, wherein, the third solvent described is acetone, acetonitrile, ethyl acetate, isobutyl acetate, n-butyl acetate, hexane, ether or water, and e) described solidliquid mixture is filtered, obtain Echinocandin compound dry powder after wet-milling drying.
Preferably, described Echinocandin compound comprises lung and reads rhzomorph B0 (PneumocandinB0), WF11899A (FR901379) and ECB (EchinocandinB).
Preferably, step a) in, use respectively phosphoric acid, oxalic acid, hydrochloric acid or ammoniacal liquor regulate water to pH=3.0 ~ 8.0, described phosphoric acid, oxalic acid, hydrochloric acid or ammonia concn are 10wt.% ~ 30wt.%.
Preferably, step a) in, described micro-filtration adopts the multiple microfiltration membrane compounding in series in a kind of micro-filtrate membrane filtration in ceramic membrane, stainless steel membrane, ultra-filtration membrane or ceramic membrane, stainless steel membrane, ultra-filtration membrane to filter; Described microfiltration membrane to be aperture the be ceramic membrane of 0.05 ~ 0.5 μm or stainless steel membrane or molecular weight cut-off are the ultra-filtration membrane of 30KDalton ~ 3000KDalton.
Preferably, step a) in, the volume ratio of the first solvent described in described mixed solvent is 20% ~ 90%, preferably 40 ~ 60%, more preferably 20% ~ 50%.
Preferably, step a) in, add at 20 ~ 40 DEG C of temperature extraction agent to cleaning after mycelium suspended liquid extract.
Preferably, step a) in, before by the Echinocandin compound in filtrate described in macroporous resin adsorption, the nanofiltration membrane that molecular weight cut-off is 200Dalton ~ 800Dalton is used to concentrate, nanofiltration permeate turns back in solvent extraction, and nanofiltration concentrated solution then carries out step b) operation.
Preferably, in step b) in, described low-pole macroporous adsorbent resin is AmberliteXAD-7HP or DiaionSP-207; Described nonpolar macroporous adsorption resin is AmberliteXAD-1180N, AmberliteXAD-1600, AmberliteXAD-2, AmberliteXAD-4, DiaionHP-20 or DiaionSP-825.
Preferably, in step b) in, at extraction liquid after the Echinocandin compound in filtrate described in macroporous resin adsorption, after macroporous resin adsorption waste liquid first concentrates by nanofiltration membrane, nanofiltration permeate as extraction agent, and turn back to step a) again circulation extract.
Preferably, in step b) in, before macroporous resin carries out prewashing, parse operation, first wash resin column with water.
Preferably, in step b) in, when macroporous resin carries out pre-wash operation, pre-washing agent is the mixed solvent of organic solvent and water, and wherein organic solvent volume ratio control is 20% ~ 50%.
Preferably, in step b) in, when macroporous resin carries out parse operation, resolve the mixed solvent that agent is organic solvent and water, wherein organic solvent volume ratio control is 50% ~ 90%.
Preferably, in step c) in, described desorbed solution carry out concentrated before, or desorbed solution is through partial concentration, but when Echinocandin compound precipitation is not separated out, add discoloring agent to decolour at 0 DEG C ~ 30 DEG C temperature, described discoloring agent is gac, aluminum oxide or decolorizing resin, and the discoloring agent ratio added is 0.5 ~ 5 times that treats Echinocandin compound gross weight in decolouring feed liquid.
Preferably, in step c) in, at 20 DEG C ~ 60 DEG C temperature, desorbed solution carries out vacuum-concentrcted.
Preferably, in step e) in, described solidliquid mixture is directly filtered, obtains Echinocandin compound wet-milling.
Preferably, in step e) in, carry out drying to described Echinocandin compound wet-milling, described drying adopts vacuum decompression heat drying or vacuum decompression lyophilize.
Preferably, in step e) in, described vacuum decompression heat drying carries out drying at 20 DEG C ~ 60 DEG C temperature.
Preferably, in steps d) in, at described desorbed solution after concentrated removing the second solvent, then when to add the third solvent be water, after separating out Echinocandin compound precipitation, described solidliquid mixture is directly carried out vacuum decompression lyophilize.
Method of the present invention is by using membrane technique, extraction process route is short, the solvent species used is also relatively less, and through simple nanofiltration enrichment step in leaching process, extraction agent just can turn back in microfiltration membrane and continue extraction, the solvent cycle achieved in leaching process is applied mechanically, and greatly reduces the usage quantity of solvent, reduces the cost of solvent recuperation; Meanwhile, use membrane concentration technology, realize cryoconcentration, not only remove small molecular weight impurity, also reduce vacuum decompression heating concentrated time temperature drift and produce the problem of new impurity, improve quality product.Present invention process, high to the product purity obtained after target compound extraction purification, yield is high, use quantity of solvent few, and the cyclic utilization rate of solvent is high, is applicable to large-scale industrial production.
Embodiment
Further illustrate the present invention below by embodiment, embodiments of the invention are only used for the present invention being described and providing, instead of limitation of the present invention.
Embodiment 1 ~ embodiment 3 illustrates by fermentable, obtains the embodiment that lung reads rhzomorph B0 (PneumocandinB0), WF11899A (FR901379), ECB (EchinocandinB) three kinds of product fermented liquids.
Embodiment 1: lung reads the preparation of rhzomorph B0 (PneumocandinB0) fermented liquid
Bacterial strain: Zalerionarboricola (HCCB30111, CGMCCNo.5412) (see the application of No. 201210041247.9th, Chinese patent application);
Slant medium: PDA;
Slant culture condition: 28 DEG C, 5 days;
Seed culture medium (%): glucose 2.5, KH 2pO 40.9, yeast powder 0.5, medicine matchmaker 1.0,85% lactic acid 0.2ml, micro-0.1ml, pH6.0;
Seed culture condition: dig block inoculation, 25 DEG C, 220rpm, 3 days;
Fermention medium (%): sucrose 12.5, Sodium Glutamate 0.8, yeast powder 0.8, proline(Pro) 1.5, KH 2pO 40.15, bitter salt 0.04, micro-1ml, MES buffering salt 1.5, pH5.3;
Fermentation condition: transferred species amount 10%, 25 DEG C, 220rpm, fermentation culture 7 days;
Trace element composition (g/L): iron vitriol 1.0, manganese sulfate monohydrate 0.2, Calcium dichloride dihydrate 0.1, boric acid 0.056, copper chloride dihydrate 0.025, four water ammonium molybdate 0.019,12N hydrochloric acid 50ml.
Lung reads the product validation of rhzomorph B0 (PneumocandinB0): detect through HPLC, in fermented liquid, lung reads rhzomorph B0 (PneumocandinB0) content is 4.5g/L.
The preparation of embodiment 2:WF11899A (FR901379) fermented liquid
Bacterial strain: ColeophomaempetriWF11899A (FR901379) (see " improvementofFR901379productionbymutantselectionandmediu moptimizationbyMunekazuKandapublishedinVol.107No.5; 530-534; 2009, JournalofBioscienceandBioengineering ")
Slant medium: PDA;
Slant culture condition: 28 DEG C, 7 days;
Seed culture medium (%): sucrose 1.0, medicine matchmaker 2.0, yeast powder 1.0, peptone 1.0, KH 2pO 40.2, CaCO 30.2, Tween800.1, pH6.5;
Seed culture condition: dig block inoculation, 25 DEG C, 220rpm, fermentation culture 5 days;
Fermention medium (%): starch 3.0, glucose 1.0, wheatgerm 0.2, medicine matchmaker 2.0, Zein powder 0.5, KH 2pO 42.0, NaHPO 41.5, ZnSO 4.7H 2o0.001, pH6.7;
Fermentation condition: transferred species amount 10%, 25 DEG C, 220rpm, 7 days.
The product validation of WF11899A (FR901379): detect through HPLC, in fermented liquid, WF11899A (FR901379) content is 1.03g/l.
Embodiment 3: the preparation of ECB (EchinocandinB) fermented liquid
Bacterial strain: Aspergillusrugulovalvus (HCCB30110, CGMCCNo.5413) (see the application of No. 201210041876.1st, Chinese patent application);
Slant medium: PDA;
Slant culture condition: 28 DEG C, 5 days;
Seed culture medium (%): glucose 1.0, glycerine 1.0, cottonseed meal 2.5, pH6.3;
Seed culture condition: dig block inoculation, 25 DEG C, 220rpm, fermentation culture 8 days;
Fermention medium (%): maltose 6.0, dextrin 2.0, cottonseed meal 3.0, yeast powder 1.5, soya-bean oil 0.2, L-glutamic acid 0.3, KH 2pO 40.1, pH7.0;
Fermentation condition: transferred species amount 10%, 25 DEG C, 220rpm, 7 days.
The product validation of ECB (EchinocandinB): detect through HPLC, in fermented liquid, ECB (EchinocandinB) content is 0.87g/l.
Embodiment 4 ~ embodiment 27 illustrates that the fermented liquid obtained with three kinds of fermentable in embodiment 1 ~ embodiment 3 is respectively for raw material, extracts the embodiment that lung reads rhzomorph B0 (PneumocandinB0), WF11899A (FR901379), ECB (EchinocandinB) three kinds of products.
Embodiment 4: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 30L obtained by embodiment 1, obtain the mycelium suspended liquid concentrated, in 0.05 μm of ceramic membrane, utilize the water of 10% phosphorus acid for adjusting pH to 3.0 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in ceramic membrane with methyl alcohol (20%) and water mixed extractant, temperature controls, at 5 DEG C, to complete extraction-filter operation simultaneously.After the nanofiltration membrane of the extraction liquid 200Dalton containing lung Colistin B 0 is concentrated into original volume 1/5, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction.Nanofiltration concentrated solution directly adsorbs with 3LXAD-1180N macroporous resin, and again extract in the waste back-cycling after absorption to ceramic membrane, resin column first washes with water, then by concentration be 20% methyl alcohol prewashing after, the methyl alcohol of 60% is resolved.After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of lung Colistin B 0, then add the gac of active principle 0.5 times of weight, temperature remains on 0 DEG C and decolours; Feed liquid after decolouring 40 DEG C of vacuum concentration, add water after removing methyl alcohol and precipitate; Containing the direct vacuum lyophilization of solidliquid mixture that lung Colistin B 0 precipitates, obtain lung Colistin B 0 dry powder, product yield is 84.3%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=78.9% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 5: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 20L obtained by embodiment 1, obtain the mycelium suspended liquid concentrated, in 0.5 μm of stainless steel membrane, utilize the water of 20% phosphorus acid for adjusting pH to 6.3 to wash mycelium suspended liquid; After being filtered to 10L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 90%, temperature controls, at 50 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 400Dalton of lung Colistin B 0, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; It is after 45% that nanofiltration concentrated solution is diluted with water to alcohol concn, and adsorb with 2.5LXAD-1600 macroporous resin, resin column concentration is after the ethanol prewashing of 45%, and the ethanol of 90% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of sour jujube lung Colistin B 0, then add the aluminum oxide of active principle 2 times of weight, temperature remains on 30 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add ethyl acetate after removing ethanol and precipitate; The solidliquid mixture filtration precipitated containing lung Colistin B 0 obtains wet-milling, 60 DEG C of vacuum decompression dryings, and obtain lung Colistin B 0 dry powder, product yield is 82.7%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=79.0% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 6: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 21L obtained by embodiment 1, obtaining the mycelium suspended liquid concentrated, is utilize the water of 20% careless acid for adjusting pH to 4.6 to wash mycelium suspended liquid in the ultra-filtration membrane of 30KDalton at molecular weight cut-off; After being filtered to 10L, the n-propyl alcohol with 80%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature controls, at 20 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/4 containing the nanofiltration membrane of the extraction liquid 800Dalton of lung Colistin B 0, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; It is after 40% that nanofiltration concentrated solution is diluted with water to n-propyl alcohol concentration, and adsorb with 2LXAD-7HP macroporous resin, resin column first washes with water, then by concentration be 40% n-propyl alcohol prewashing after, the n-propyl alcohol of 80% is resolved; Desorbed solution containing lung Colistin B 0 adds the decolorizing resin of active principle 3 times of weight, and temperature remains on 20 DEG C and decolours; After the first nanofiltration of feed liquid after decolouring is concentrated into original volume 1/15, then 60 DEG C of vacuum concentration, add acetone after removing n-propyl alcohol and precipitate; The solidliquid mixture filtration precipitated containing lung Colistin B 0 obtains wet-milling, 20 DEG C of vacuum decompression dryings, and obtain lung Colistin B 0 dry powder, product yield is 85.1%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=80.1% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 7: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 25L obtained by embodiment 1, obtaining the mycelium suspended liquid concentrated, is utilize the water of 30% careless acid for adjusting pH to 5.8 to wash mycelium suspended liquid in the ultra-filtration membrane of 3000KDalton at molecular weight cut-off; After being filtered to 12L, directly extract in ultra-filtration membrane with the isopropanol-water mixed extractant of 80%, temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 600Dalton of lung Colistin B 0, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; It is after 40% that nanofiltration concentrated solution is diluted with water to isopropyl alcohol concentration, and adsorb with 2LHP-20 macroporous resin, resin column concentration is after the Virahol prewashing of 40%, and the Virahol of 80% is resolved; After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of lung Colistin B 0, then add the gac of active principle 5 times of weight, temperature remains on 10 DEG C and decolours; Feed liquid after decolouring 60 DEG C of vacuum concentration, add acetonitrile after removing Virahol and precipitate; The solidliquid mixture filtration precipitated containing lung Colistin B 0 obtains wet-milling, 30 DEG C of vacuum decompression dryings, and obtain lung Colistin B 0 dry powder, product yield is 85.9%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=79.5% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 8: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 23L obtained by embodiment 1, obtain the mycelium suspended liquid concentrated, in 0.5 μm of ceramic membrane, utilize 20% ammoniacal liquor to regulate the water of pH to 8.0 to wash mycelium suspended liquid; After being filtered to 10L, directly extract in ceramic membrane with the acetone-water mixed extractant of 30%, temperature controls, at 25 DEG C, to complete extraction-filter operation simultaneously; Extraction liquid containing lung Colistin B 0 directly adsorbs with 2.5LSP-207 macroporous resin, and resin column concentration is after the acetone prewashing of 30%, and the acetone of 50% is resolved; Desorbed solution containing lung Colistin B 0 adds the aluminum oxide of active principle 4 times of weight, and temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 30 DEG C of vacuum concentration, add isobutyl acetate after removing acetone and precipitate; The solidliquid mixture filtration precipitated containing lung Colistin B 0 obtains wet-milling, 30 DEG C of vacuum decompression dryings, and obtain lung Colistin B 0 dry powder, product yield is 84.9%
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=75.7% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 9: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 32L obtained by embodiment 1, obtain the mycelium suspended liquid concentrated, in 0.2 μm of ceramic membrane, utilize the water of 10% careless acid for adjusting pH to 3.6 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in ceramic membrane with the methanol-water mixed extractant of 30%, temperature controls, at 10 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/7 containing the nanofiltration membrane of the extraction liquid 400Dalton of lung Colistin B 0, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution directly adsorbs with 3LSP-825 macroporous resin, and absorption waste liquid nanofiltration membrane concentrates, and nanofiltration permeate turns back in stainless steel membrane and extracts, and resin column first washes with water, then with concentration be 30% methyl alcohol prewashing after, the methyl alcohol parsing of 60%; Desorbed solution containing lung Colistin B 0 adds the gac of active principle 1 times of weight, and temperature remains on 10 DEG C and decolours; After the first nanofiltration of feed liquid after decolouring is concentrated into original volume 1/10, then 50 DEG C of vacuum concentration, add n-butyl acetate after removing methyl alcohol and precipitate; Obtain wet-milling containing after the solidliquid mixture filtration that lung Colistin B 0 precipitates, 45 DEG C of vacuum decompression dryings obtain lung Colistin B 0 dry powder, and product yield is 86.6%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=75.5% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 10: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 26L obtained by embodiment 1, obtain the mycelium suspended liquid concentrated, in 0.1 μm of stainless steel membrane, utilize the water of 20% salt acid for adjusting pH to 5.0 to wash mycelium suspended liquid; After being filtered to 13L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 60%, temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/4 containing the nanofiltration membrane of the extraction liquid 600Dalton of lung Colistin B 0, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; It is after 30% that nanofiltration concentrated solution is diluted with water to alcohol concn, and adsorb with 2.5LXAD-2 macroporous resin, resin column concentration is after the ethanol prewashing of 30%, and the ethanol of 70% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of lung Colistin B 0, then add the aluminum oxide of active principle 2.5 times of weight, temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 40 DEG C of vacuum concentration, add ether after removing ethanol and precipitate; The solidliquid mixture filtration precipitated containing lung Colistin B 0 obtains wet-milling, 35 DEG C of vacuum decompression dryings, and obtain lung Colistin B 0 dry powder, product yield is 86.3%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=78.7% of lung Colistin B 0 (PneumocandinB0) in dry powder.
Embodiment 11: the extraction of lung Colistin B 0 (PneumocandinB0)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 27L obtained by embodiment 1, obtaining the mycelium suspended liquid concentrated, is utilize 10% ammoniacal liquor to regulate the water of pH to 7.5 to wash mycelium suspended liquid in the ultra-filtration membrane of 500KDalton at molecular weight cut-off; After being filtered to 15L, feed liquid puts into the ceramic membrane of 0.1um, directly extracts in the n-propyl alcohol with 50%-water mixed extractant, and temperature controls, at 30 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/4 containing the nanofiltration membrane of the extraction liquid 200Dalton of lung Colistin B 0, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution 3LXAD-4 macroporous resin adsorbs, and resin column concentration is after the n-propyl alcohol prewashing of 50%, and the n-propyl alcohol of 90% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of lung Colistin B 0, then add the gac of active principle 3 times of weight, temperature remains on 15 DEG C and decolours; Feed liquid after decolouring 60 DEG C of vacuum concentration, add hexane after removing n-propyl alcohol and precipitate; The solidliquid mixture filtration precipitated containing lung Colistin B 0 obtains wet-milling, 40 DEG C of vacuum decompression dryings, and obtain lung Colistin B 0 dry powder, product yield is 85.1%.
the HPLC testing conditions of lung Colistin B 0 (PneumocandinB0) is as follows:
Chromatographic column: LichrocartSilica5 μm 250 × 4.6mm
Determined wavelength: 278nm
Moving phase: ethyl acetate: methyl alcohol: water=86: 8: 6
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of PneumocandinB0 (lung reads rhzomorph B0): detect through HPLC, content=75.9% of lung Colistin B 0 (PneumocandinB0) in dry powder.
The extraction of embodiment 12:WF11899A (FR901379)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 20L obtained by embodiment 2, obtaining the mycelium suspended liquid concentrated, is utilize the water of 10% salt acid for adjusting pH to 4.8 to wash mycelium suspended liquid in the ultra-filtration membrane of 1000KDalton at molecular weight cut-off; After being filtered to I0L, feed liquid puts into the stainless steel membrane of 0.2um, directly extracts with the isopropanol-water mixed extractant of 90%, and temperature controls, at 30 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 600Dalton of WF11899A, nanofiltration permeate turns back in stainless steel membrane as extraction agent, proceeds extraction; It is after 45% that nanofiltration concentrated solution is diluted with water to isopropyl alcohol concentration, and adsorb with 2LHP-20 macroporous resin, resin column first washes with water, then by concentration be 45% Virahol prewashing after, the Virahol of 80% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of WF11899A, then add the gac of active principle 1.5 times of weight, temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add acetonitrile after removing Virahol and precipitate; Solidliquid mixture containing WF11899A precipitation filters and obtains wet-milling, and 30 DEG C of vacuum decompression dryings, obtain WF11899A dry powder, and product yield is 87.9%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=73.0% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 13:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 25L obtained by embodiment 2, obtain the mycelium suspended liquid concentrated, in 0.2 μm of ceramic membrane, utilize the water of 10% careless acid for adjusting pH to 4.0 to wash mycelium suspended liquid; After being filtered to 12L, directly extract in ceramic membrane with the methanol-water mixed extractant of 30%, temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 200Dalton of WF11899A, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution directly adsorbs with 2.5LXAD-1600 macroporous resin, and absorption waste liquid nanofiltration membrane concentrates, and nanofiltration permeate turns back in ceramic membrane and extracts, and resin column concentration is after the methyl alcohol prewashing of 30%, and the methyl alcohol of 60% is resolved; Desorbed solution containing WF11899A first adds the aluminum oxide of active principle 0.5 times of weight, and temperature remains on 0 DEG C and decolours; After feed liquid nanofiltration after decolouring is concentrated into original volume 1/10, then 40 DEG C of vacuum concentration, after removing methyl alcohol.Add n-butyl acetate to precipitate; Solidliquid mixture containing WF11899A precipitation filters and obtains wet-milling, and 50 DEG C of vacuum decompression dryings, obtain WF11899A dry powder, and product yield is 83.5%
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=74.6% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 14:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 23L obtained by embodiment 2, obtain the mycelium suspended liquid concentrated, in 0.2 μm of stainless steel membrane, utilize the water of 30% phosphorus acid for adjusting pH to 5.3 to wash mycelium suspended liquid; After being filtered to 10L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 80%, temperature controls, at 50 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 200Dalton of WF11899A, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; It is after 40% that nanofiltration concentrated solution is diluted with water to alcohol concn, and adsorb with 2LXAD-1180N macroporous resin, resin column first washes with water, then by concentration be 40% ethanol prewashing after, the ethanol of 80% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of WF11899A, then add the gac of active principle 2 times of weight, temperature remains on 25 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add water after removing ethanol and precipitate; Containing the direct vacuum lyophilization of solidliquid mixture of WF11899A precipitation, obtain WF11899A dry powder, product yield is 81.3%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=76.7% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 15:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 28L obtained by embodiment 2, obtain the mycelium suspended liquid concentrated, in 0.3 μm of stainless steel membrane, utilize the water of 10% salt acid for adjusting pH to 3.5 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 50%, temperature controls, at 30 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/4 containing the nanofiltration membrane of the extraction liquid 600Dalton of WF11899A, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution 3LXAD-2 macroporous resin adsorbs, and resin column concentration is after the ethanol prewashing of 50%, and the ethanol of 80% is resolved; After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of WF11899A, then add the decolorizing resin of active principle 2.5 times of weight, temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 40 DEG C of vacuum concentration, add hexane after removing ethanol and precipitate; Solidliquid mixture containing WF11899A precipitation filters and obtains wet-milling, and 30 DEG C of vacuum decompression dryings, obtain WF11899A dry powder, and product yield is 87.1%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=76.3% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 16:WF11899A (FR901379)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 20L obtained by embodiment 2, obtaining the mycelium suspended liquid concentrated, is utilize the water of 25% careless acid for adjusting pH to 5.1 to wash mycelium suspended liquid in the ultra-filtration membrane of 300KDalton at molecular weight cut-off; After being filtered to 10L, the n-propyl alcohol with 90%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/4 containing the nanofiltration membrane of the extraction liquid 600Dalton of WF11899A, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; It is after 45% that nanofiltration concentrated solution is diluted with water to n-propyl alcohol concentration, and adsorb with 2LXAD-7HP macroporous resin, resin column concentration is after the n-propyl alcohol prewashing of 45%, and the n-propyl alcohol of 80% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of WF11899A, then add the aluminum oxide of active principle 3 times of weight, temperature remains on 25 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add acetone after removing n-propyl alcohol and precipitate; Solidliquid mixture containing WF11899A precipitation filters and obtains wet-milling, and 25 DEG C of vacuum decompression dryings, obtain WF11899A dry powder, and product yield is 85.6%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=80.6% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 17:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 30L obtained by embodiment 2, obtain the mycelium suspended liquid concentrated, in 0.3 μm of ceramic membrane, utilize the water of 20% phosphorus acid for adjusting pH to 4.9 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in ceramic membrane with the methanol-water mixed extractant of 40%, temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/6 containing the nanofiltration membrane of the extraction liquid 800Dalton of WF11899A, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution directly adsorbs with 3LSP-825 macroporous resin, and resin column concentration is after the methyl alcohol prewashing of 40%, and the methyl alcohol of 60% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of WF11899A, then add the gac of active principle 2 times of weight, temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 45 DEG C of vacuum concentration, add isobutyl acetate after removing methyl alcohol and precipitate; Obtain wet-milling containing after the solidliquid mixture filtration of WF11899A precipitation, 45 DEG C of vacuum decompression dryings obtain WF11899A dry powder, and product yield is 83.6%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=79.5% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 18:WF11899A (FR901379)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 25L obtained by embodiment 2, obtain the mycelium suspended liquid concentrated, in 0.05 μm of stainless steel membrane, utilize the water of 10% careless acid for adjusting pH to 5.3 to wash mycelium suspended liquid; After being filtered to 10L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 80%, temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 400Dalton of WF11899A, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; It is after 40% that nanofiltration concentrated solution is diluted with water to alcohol concn, and adsorb with 2.5LXAD-2 macroporous resin, resin column concentration is after the ethanol prewashing of 40%, and the ethanol of 85% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of WF11899A, then add the gac of active principle 2.5 times of weight, temperature remains on 30 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add ethyl acetate after removing ethanol and precipitate; Solidliquid mixture containing WF11899A precipitation filters and obtains wet-milling, and 60 DEG C of vacuum decompression dryings, obtain WF11899A dry powder, and product yield is 83.7%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=77.3% of WF11899A (FR901379) in dry powder.
The extraction of embodiment 19:WF11899A (FR901379)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 24L obtained by embodiment 2, obtaining the mycelium suspended liquid concentrated, is utilize 20% ammoniacal liquor to regulate the water of pH to 7.4 to wash mycelium suspended liquid in the ultra-filtration membrane of 100KDalton at molecular weight cut-off; After being filtered to 10L, the n-propyl alcohol with 45%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 200Dalton of WF11899A, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution 2.5LXAD-4 macroporous resin adsorbs, and resin column concentration is after the n-propyl alcohol prewashing of 45%, and the n-propyl alcohol of 90% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of WF11899A, then add the gac of active principle 1.5 times of weight, temperature remains on 15 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add ether after removing n-propyl alcohol and precipitate; Solidliquid mixture containing WF11899A precipitation filters and obtains wet-milling, and 40 DEG C of vacuum decompression dryings, obtain WF11899A dry powder, and product yield is 86.1%.
the HPLC testing conditions of WF11899A is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of WF11899A: detect through HPLC, content=76.9% of WF11899A (FR901379) in dry powder.
Embodiment 20: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 27L obtained by embodiment 3, obtain the mycelium suspended liquid concentrated, in 0.1 μm of stainless steel membrane, utilize the water of 10% phosphorus acid for adjusting pH to 3.3 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in stainless steel membrane with the methanol-water mixed extractant of 20%, temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/6 containing the nanofiltration membrane of the extraction liquid 400Dalton of ECB, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution 2.5LXAD-1600 macroporous resin adsorbs, and extract in absorption waste back-cycling to stainless steel membrane, resin column first washes with water, then with concentration be 20% methyl alcohol prewashing after, the methyl alcohol parsing of 75%; After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of ECB, then add the decolorizing resin of active principle 1.5 times of weight, temperature remains on 30 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add n-butyl acetate after removing methyl alcohol and precipitate; Solidliquid mixture containing ECB precipitation filters and obtains wet-milling, and 50 DEG C of vacuum decompression dryings, obtain ECB dry powder, and product yield is 83.9%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=64.1% of ECB (EchinocandinB) in dry powder.
Embodiment 21: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 30L obtained by embodiment 3, obtaining the mycelium suspended liquid concentrated, is utilize the water of 25% salt acid for adjusting pH to 5.8 to wash fermented liquid in the ultra-filtration membrane of 2000KDalton at molecular weight cut-off; After being filtered to 15L, directly extract in ultra-filtration membrane with the isopropanol-water mixed extractant of 80%, temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 600Dalton of ECB, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; It is after 40% that nanofiltration concentrated solution is diluted with water to n-propyl alcohol concentration, and adsorb with 2.5LXAD-4 macroporous resin, resin column first washes with water, then by concentration be 40% n-propyl alcohol prewashing after, the n-propyl alcohol of 80% is resolved; Desorbed solution containing ECB first adds the gac of active principle 3 times of weight, and temperature remains on 15 DEG C and decolours; After feed liquid nanofiltration after decolouring is concentrated into original volume 1/15, then 50 DEG C of vacuum concentration, add acetone after removing Virahol and precipitate; Solidliquid mixture containing ECB precipitation filters and obtains wet-milling, and 35 DEG C of vacuum decompression dryings, obtain ECB dry powder, and product yield is 82.6%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=68.3% of ECB (EchinocandinB) in dry powder.
Embodiment 22: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 28L obtained by embodiment 3, obtain the mycelium suspended liquid concentrated, in 0.3 μm of stainless steel membrane, utilize the water of 20% careless acid for adjusting pH to 6.3 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 80%, temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 400Dalton of ECB, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; It is after 40% that nanofiltration concentrated solution is diluted with water to alcohol concn, and adsorb with 2LXAD-7HP macroporous resin, resin column first washes with water, then by concentration be 40% ethanol prewashing after, the ethanol of 80% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of ECB, then add the aluminum oxide of active principle 2.5 times of weight, temperature remains on 25 DEG C and decolours; Feed liquid after decolouring 40 DEG C of vacuum concentration, add acetonitrile after removing ethanol and precipitate; Solidliquid mixture containing ECB precipitation filters and obtains wet-milling, and 35 DEG C of vacuum decompression dryings, obtain ECB dry powder, and product yield is 83.9%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=71.6% of ECB (EchinocandinB) in dry powder.
Embodiment 23: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 31L obtained by embodiment 3, obtaining the mycelium suspended liquid concentrated, is utilize the water of 20% careless acid for adjusting pH to 5.2 to wash mycelium suspended liquid in the ultra-filtration membrane of 1500KDalton at molecular weight cut-off; After being filtered to 15L, directly extract in ultra-filtration membrane with the isopropanol-water mixed extractant of 90%, temperature controls, at 30 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 200Dalton of ECB, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; It is after 45% that nanofiltration concentrated solution is diluted with water to isopropyl alcohol concentration, and adsorb with 3LSP-207 macroporous resin, resin column concentration is after the Virahol prewashing of 45%, and the Virahol of 80% is resolved; After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of ECB, then add the gac of active principle 1.5 times of weight, temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 60 DEG C of vacuum concentration, add water after removing Virahol and precipitate; Containing the direct vacuum lyophilization of solidliquid mixture of ECB precipitation, obtain ECB dry powder, product yield is 84.9%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=70.1% of ECB (EchinocandinB) in dry powder.
Embodiment 24: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 25L obtained by embodiment 3, obtain the mycelium suspended liquid concentrated, in 0.2 μm of ceramic membrane, utilize the water of 10% careless acid for adjusting pH to 4.6 to wash mycelium suspended liquid; After being filtered to 10L, directly extract in ceramic membrane with the methanol-water mixed extractant of 35%, temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/6 containing the nanofiltration membrane of the extraction liquid 200Dalton of ECB, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution directly adsorbs with 2.5LXAD-1180N macroporous resin, and resin column concentration is after the methyl alcohol prewashing of 35%, and the methyl alcohol of 60% is resolved; After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of ECB, then add the gac of active principle 0.5 times of weight, temperature remains on 10 DEG C and decolours; Feed liquid after decolouring 40 DEG C of vacuum concentration, after removing methyl alcohol, add ether and precipitate; Solidliquid mixture containing ECB precipitation filters and obtains wet-milling, and 50 DEG C of vacuum decompression dryings, obtain ECB dry powder, and product yield is 83.5%
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=72.0% of ECB (EchinocandinB) in dry powder.
Embodiment 25: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (device for ultrafiltration membrane: SanDa film Science company limited, model SMRXM-100) cross flow filter is carried out to the Echinocandin compound fermented liquid 28L obtained by embodiment 3, obtaining the mycelium suspended liquid concentrated, is utilize 15% ammoniacal liquor to regulate the water of pH to 7.2 to wash mycelium suspended liquid in the ultra-filtration membrane of 200KDalton at molecular weight cut-off; After being filtered to 15L, the n-propyl alcohol with 50%-water mixed extractant directly extracts in ultra-filtration membrane, and temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 400Dalton of ECB, nanofiltration permeate turns back in ultra-filtration membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution 3LHP-20 macroporous resin adsorbs, and resin column concentration is after the n-propyl alcohol prewashing of 50%, and the n-propyl alcohol of 85% is resolved; After being concentrated into original volume 1/15 containing the first nanofiltration of desorbed solution of ECB, then add the gac of active principle 1 times of weight, temperature remains on 15 DEG C and decolours; Feed liquid after decolouring 50 DEG C of vacuum concentration, add isobutyl acetate after removing n-propyl alcohol and precipitate; Solidliquid mixture containing ECB precipitation filters and obtains wet-milling, and 40 DEG C of vacuum decompression dryings, obtain ECB dry powder, and product yield is 87.3%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=71.4% of ECB (EchinocandinB) in dry powder.
Embodiment 26: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the stainless steel membrane pipe of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 20L obtained by embodiment 3, obtain the mycelium suspended liquid concentrated, in 0.3 μm of stainless steel membrane, utilize the water of 15% careless acid for adjusting pH to 3.9 to wash mycelium suspended liquid; After being filtered to 10L, directly extract in stainless steel membrane with the alcohol-water mixed extractant of 45%, temperature controls, at 35 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/4 containing the nanofiltration membrane of the extraction liquid 400Dalton of ECB, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution 2LXAD-2 macroporous resin adsorbs, and resin column first washes with water, then by concentration be 45% ethanol prewashing after, the ethanol of 70% is resolved; Desorbed solution containing ECB first adds the gac of active principle 1.5 times of weight, and temperature remains on 20 DEG C and decolours; After feed liquid nanofiltration after decolouring is concentrated into original volume 1/10, then 40 DEG C of vacuum concentration, add hexane after removing ethanol and precipitate; Solidliquid mixture containing ECB precipitation filters and obtains wet-milling, and 30 DEG C of vacuum decompression dryings, obtain ECB dry powder, and product yield is 85.4%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=66.4% of ECB (EchinocandinB) in dry powder.
Embodiment 27: the extraction of ECB (EchinocandinB)
Adopt microfiltration membrane equipment (ceramic membrane equipment: SanDa film Science company limited customizes, the ceramic-film tube of two φ 3cm × 100cm is housed) cross flow filter is carried out to the Echinocandin compound fermented liquid 23L obtained by embodiment 3, obtain the mycelium suspended liquid concentrated, in 0.1 μm of ceramic membrane, utilize the water of 20% salt acid for adjusting pH to 4.3 to wash mycelium suspended liquid; After being filtered to 15L, directly extract in ceramic membrane with the methanol-water mixed extractant of 40%, temperature controls, at 40 DEG C, to complete extraction-filter operation simultaneously; After being concentrated into original volume 1/5 containing the nanofiltration membrane of the extraction liquid 800Dalton of ECB, nanofiltration permeate turns back in ceramic membrane as extraction agent, proceeds extraction; Nanofiltration concentrated solution directly adsorbs with 2LSP-825 macroporous resin, and resin column concentration is after the methyl alcohol prewashing of 40%, and the methyl alcohol of 70% is resolved; After being concentrated into original volume 1/10 containing the first nanofiltration of desorbed solution of ECB, then add the aluminum oxide of active principle 2.5 times of weight, temperature remains on 20 DEG C and decolours; Feed liquid after decolouring 45 DEG C of vacuum concentration, add ethyl acetate after removing methyl alcohol and precipitate; Obtain wet-milling containing after the solidliquid mixture filtration of ECB precipitation, 45 DEG C of vacuum decompression dryings obtain ECB dry powder, and product yield is 84.6%.
the HPLC testing conditions of ECB (EchinocandinB) is as follows:
Chromatographic column: PhenomenexLuna5 μm C18 (2) 250 × 4.6mm
Determined wavelength: 275nm
Moving phase: acetonitrile: water=45: 55 (adding the formic acid-ammonium formiate buffering salt of 0.3%)
Flow velocity: 1mL/min
Column temperature: 30 DEG C
The product validation of ECB (EchinocandinB): detect through HPLC, content=68.2% of ECB (EchinocandinB) in dry powder.
It is to be understood that foregoing invention content and embodiment are intended to the practical application proving technical scheme provided by the present invention, should not be construed as limiting the scope of the present invention.Those skilled in the art in spirit of the present invention and principle, when doing various amendment, equivalent replace or improve.

Claims (18)

1. from Echinocandin compound fermented liquid, extract a method for Echinocandin compound, the method comprises the following steps:
A) microfiltration equipment is adopted to carry out cross flow filter to Echinocandin compound fermented liquid, obtain the mycelium suspended liquid concentrated, then add the water being adjusted to pH=3.0 ~ 8.0 to clean mycelium suspended liquid, the mycelium suspended liquid after cleaning is obtained after micro-filtration, then at 5 DEG C ~ 50 DEG C temperature, add extraction agent extract the mycelium suspended liquid after cleaning, after micro-filtration, obtain the filtrate containing Echinocandin compound; Wherein, described extraction agent is the mixed solvent of the first solvent and water, and the first solvent described is methyl alcohol, ethanol, n-propyl alcohol, Virahol or acetone; Described micro-filtration adopts the multiple microfiltration membrane compounding in series in a kind of micro-filtrate membrane filtration in ceramic membrane, stainless steel membrane, ultra-filtration membrane or ceramic membrane, stainless steel membrane, ultra-filtration membrane to filter; Described microfiltration membrane to be aperture the be ceramic membrane of 0.05 ~ 0.5 μm or stainless steel membrane or molecular weight cut-off are the ultra-filtration membrane of 30KDalton ~ 3000KDalton;
B) by the Echinocandin compound in filtrate described in macroporous resin adsorption, then use the prewashing of the second solvent, parsing, obtain the desorbed solution containing Echinocandin compound; Wherein, described macroporous adsorbent resin is nonpolar macroporous adsorption resin or low-pole macroporous adsorbent resin, and described low-pole macroporous adsorbent resin is AmberliteXAD-7HP; Described nonpolar macroporous adsorption resin is AmberliteXAD-1180N, AmberliteXAD-2, AmberliteXAD-4 or DiaionSP-825; Described the second solvent is methyl alcohol, ethanol, n-propyl alcohol or Virahol;
C) first concentrate described desorbed solution with nanofiltration, continue concentrating under reduced pressure to remove the second solvent;
D), after adding the third solvent precipitation Echinocandin compound, the solidliquid mixture containing Echinocandin compound is obtained; Wherein, the third solvent described is acetone, ethyl acetate, isobutyl acetate, n-butyl acetate, hexane, ether or water; And
E) described solidliquid mixture is filtered, after wet-milling drying, obtain Echinocandin compound dry powder.
2. method according to claim 1, it is characterized in that, described Echinocandin compound comprises lung and reads rhzomorph BO (PneumocandinBO), WF11899A (FR901379) and ECB (EchinocandinB).
3. method according to claim 1, is characterized in that, step a) in, use respectively phosphoric acid, oxalic acid, hydrochloric acid or ammoniacal liquor regulate water to pH=3.0 ~ 8.0, described phosphoric acid, oxalic acid, hydrochloric acid or ammonia concn are 10wt.% ~ 30wt.%.
4. method according to claim 1, is characterized in that, step a) in, the volume ratio of the first solvent described in described mixed solvent is 20% ~ 90%.
5. method according to claim 4, is characterized in that, step a) in, the volume ratio of the first solvent described in described mixed solvent is 40 ~ 60%.
6. method according to claim 4, is characterized in that, step a) in, the volume ratio of the first solvent described in described mixed solvent is 20% ~ 50%.
7. method according to claim 1, is characterized in that, step a) in, add at 20 ~ 40 DEG C of temperature extraction agent to cleaning after mycelium suspended liquid extract.
8. method according to claim 1, it is characterized in that, step a) in, before by the Echinocandin compound in filtrate described in macroporous resin adsorption, the nanofiltration membrane that molecular weight cut-off is 200Dalton ~ 800Dalton is used to concentrate, nanofiltration permeate turns back in solvent extraction, and nanofiltration concentrated solution then carries out step b) operation.
9. method according to claim 1, it is characterized in that, in step b) in, at extraction liquid after the Echinocandin compound in filtrate described in macroporous resin adsorption, after macroporous resin adsorption waste liquid first concentrates by nanofiltration membrane, nanofiltration permeate as extraction agent, and turn back to step a) again circulation extract.
10. method according to claim 1, is characterized in that, in step b) in, before macroporous resin carries out prewashing, parse operation, first wash resin column with water.
11. methods according to claim 10, is characterized in that, in step b) in, when macroporous resin carries out pre-wash operation, pre-washing agent is the mixed solvent of organic solvent and water, and wherein organic solvent volume ratio control is 20% ~ 50%.
12. methods according to claim 10, is characterized in that, in step b) in, when macroporous resin carries out parse operation, resolve the mixed solvent that agent is organic solvent and water, wherein organic solvent volume ratio control is 50% ~ 90%.
13. methods according to claim 1, it is characterized in that, in step c) in, described desorbed solution carry out concentrated before, or desorbed solution is through partial concentration, but when Echinocandin compound precipitation is not separated out, adds discoloring agent and decolour at 0 DEG C ~ 30 DEG C temperature, described discoloring agent is gac, aluminum oxide or decolorizing resin, and the discoloring agent ratio added is 0.5 ~ 5 times that treats Echinocandin compound gross weight in decolouring feed liquid.
14. methods according to claim 13, is characterized in that, in step c) in, at 20 DEG C ~ 60 DEG C temperature, desorbed solution carries out vacuum-concentrcted.
15. methods according to claim 1, is characterized in that, in step e) in, described solidliquid mixture is directly filtered, obtains Echinocandin compound wet-milling.
16. methods according to claim 15, is characterized in that, in step e) in, carry out drying to described Echinocandin compound wet-milling, described drying adopts vacuum decompression heat drying or vacuum decompression lyophilize.
17. methods according to claim 16, is characterized in that, in step e) in, described vacuum decompression heat drying carries out drying at 20 DEG C ~ 60 DEG C temperature.
18. methods according to claim 1, it is characterized in that, in steps d) in, at described desorbed solution after concentrated removing the second solvent, when to add the third solvent be again water, after separating out Echinocandin compound precipitation, described solidliquid mixture is directly carried out vacuum decompression lyophilize.
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