CN106749329A - A kind of method that ascosin is isolated and purified in the liquid from streptomycete fermentation - Google Patents
A kind of method that ascosin is isolated and purified in the liquid from streptomycete fermentation Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000007788 liquid Substances 0.000 title claims abstract description 27
- 238000000855 fermentation Methods 0.000 title claims abstract description 25
- 230000004151 fermentation Effects 0.000 title claims abstract description 25
- 241001655322 Streptomycetales Species 0.000 title claims abstract description 22
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 239000003960 organic solvent Substances 0.000 claims abstract description 21
- 239000011347 resin Substances 0.000 claims abstract description 14
- 229920005989 resin Polymers 0.000 claims abstract description 14
- 238000002386 leaching Methods 0.000 claims abstract description 11
- 239000012043 crude product Substances 0.000 claims abstract description 10
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 10
- 238000002425 crystallisation Methods 0.000 claims abstract description 9
- 230000008025 crystallization Effects 0.000 claims abstract description 4
- 239000012141 concentrate Substances 0.000 claims description 46
- 239000003480 eluent Substances 0.000 claims description 36
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 19
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 238000010828 elution Methods 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 239000013078 crystal Substances 0.000 claims description 8
- 239000003643 water by type Substances 0.000 claims description 8
- 239000003208 petroleum Substances 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 239000012467 final product Substances 0.000 claims description 5
- 239000012263 liquid product Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000000638 solvent extraction Methods 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 239000011435 rock Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 abstract 2
- 239000012046 mixed solvent Substances 0.000 abstract 2
- 230000006835 compression Effects 0.000 abstract 1
- 238000007906 compression Methods 0.000 abstract 1
- 230000006872 improvement Effects 0.000 description 10
- 239000012071 phase Substances 0.000 description 7
- ZDQSOHOQTUFQEM-NURRSENYSA-N ascomycin Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C/C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 ZDQSOHOQTUFQEM-NURRSENYSA-N 0.000 description 5
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 229960001967 tacrolimus Drugs 0.000 description 4
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000001953 recrystallisation Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 2
- 239000013051 drainage agent Substances 0.000 description 2
- 229940020485 elidel Drugs 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 239000010451 perlite Substances 0.000 description 2
- 235000019362 perlite Nutrition 0.000 description 2
- KASDHRXLYQOAKZ-XDSKOBMDSA-N pimecrolimus Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C/C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@H](Cl)[C@H](OC)C1 KASDHRXLYQOAKZ-XDSKOBMDSA-N 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- -1 add Eluant Substances 0.000 description 1
- 208000002029 allergic contact dermatitis Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- ZDQSOHOQTUFQEM-XCXYXIJFSA-N ascomycin Natural products CC[C@H]1C=C(C)C[C@@H](C)C[C@@H](OC)[C@H]2O[C@@](O)([C@@H](C)C[C@H]2OC)C(=O)C(=O)N3CCCC[C@@H]3C(=O)O[C@H]([C@H](C)[C@@H](O)CC1=O)C(=C[C@@H]4CC[C@@H](O)[C@H](C4)OC)C ZDQSOHOQTUFQEM-XCXYXIJFSA-N 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 208000037896 autoimmune cutaneous disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/18—Bridged systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A kind of method the invention provides ascosin is isolated and purified in liquid from streptomycete fermentation, streptomycete fermentation liquid plate compression is obtained thalline by the method, thalline is extracted with the multiple room temperature of organic solvent, merge leaching liquor, macroreticular resin and silica gel column chromatography are transferred to after leaching liquor is concentrated, the ascosin of gained carries out condensing crystallizing with mixed solvent, and the crude product after crystallization is transferred to dynamic axial high-pressure column chromatographic column and is separated, and the ascosin for obtaining carries out condensing crystallizing again with mixed solvent.In the present invention, up to more than 98%, yield is up to 80~90% for the purity of the ascosin prepared；Extraction time can be greatly shortened by the present invention, efficiency is improve, with easy to operate, process is simple and many advantages, such as low cost, it is easy to industrialized production, for the production of ascosin provides feasible separating technology.
Description
Technical field
The present invention relates to biological pharmacy technical field, more particularly to one kind to isolate and purify ascus from streptomycete fermentation liquid mould
The method of element.
Background technology
Ascosin (Ascomycin, Immunomycin, FK-520) is the macrolides compound of 23 carbon, is
Immunodepressant FK-506 (tacrolimus, tacrolimus) ethyl analog.Since being immunized for the similar tacrolimus of its structure
After inhibitory activity is found, the immunosuppressive activity research of ascosin and derivative has been promoted rapidly.Research finds John
Ascosin has very strong immunosuppressive action, and current ascosin has been used as producing semi-synthetic API (such as Elidel)
Intermediate.Elidel is mainly used in atopic dermatitis, allergic contact dermatitis, psoriasis, lupus erythematosus, flat tongue
The treatment of the autoimmune diseases such as moss, leucoderma, graft versus host disease(GVH disease).
The domestic research to ascosin is still in starting stage, the Chinese invention patent of Publication No. CN101036649A
Disclose ascosin and suppress rejection in preparation treatment autoimmune diabetes and skin disease as immunodepressant
Purposes in medicine.Kunming University of Science and Technology Mu Yunlong is by the Primary Study to ascosin strain improvement and fermentation condition, hair
Ferment culture 96h, fermentation titer reaches as high as 131.7mg/L.But for how separation and Extraction product is not illustrated.
The method of Teng Ze drugmakers of Japanese Japan report extraction ascosin in 1988.It is many due to that can exist in zymotic fluid
Ascomycin analog and because ascosin has an isomerism in water phase, and many aqueous phases in extraction separation method,
During this allows for zymotic fluid there is larger technical difficulty in purification ascosin major technique research.
Ascosin fermentation, ascomycin derivative is concentrated on for most of research work of ascosin at present to open
The aspects such as hair, antibacterial activity and clinical practice.The subject matter of ascosin separation and Extraction is the yield of ascosin at this stage
It is not high, the problems such as separation costs are high.Further, since there is isomerism, current extraction separation method in water phase in ascosin
In contain water phase, ascosin analogue is contained in product, influence product purity and product form.
The content of the invention
A kind of method it is an object of the invention to isolate and purify ascosin in disclosing liquid from streptomycete fermentation, to reality
Now efficient from streptomycete fermentation liquid, quick and inexpensive extraction simultaneously purifies ascosin, improves yield.
For achieving the above object, the invention provides isolating and purifying ascosin in a kind of liquid from streptomycete fermentation
Method, comprises the following steps：
Step 1), to added in streptomycete fermentation liquid equivalent to streptomycete fermentation liquid product 1~4% filter aid, stirring
30~45 minutes, separation of solid and liquid collected mycelium；
Step 2), with organic solvent soak extraction mycelium 2~5 times at room temperature, each 2~6h of soak extraction is obtained
Leaching liquor, merges leaching liquor, and be concentrated under reduced pressure to give concentrate；
Step 3), by step 2) obtained by concentrate adsorbed using the bed of filling non-polar macroporous resin, add
Eluant, eluent carries out gradient elution, collects eluent and concentrates；
Step 4), to step 3) obtained by concentrate in add organic solvent extraction, phase extract in extractions, to upper phase extraction
Take liquid and be concentrated under reduced pressure to give oily concentrate；
Step 5), by step 4) obtained by oily concentrate silica gel column chromatography, add eluant, eluent to carry out gradient elution, receive
Collection eluent is simultaneously concentrated, and obtains concentrate；
Step 6), by step 5) obtained by concentrate add organic solvent to be recrystallized, obtain ascosin crude product；
Step 7), by step 6) obtained by ascosin crude product separated using dynamic axial high-pressure column chromatographic column, plus
Entering eluant, eluent carries out gradient elution, collects eluent, and concentrate eluant；
Step 8), by step 7) in after eluent after concentration adds organic solvent extraction, recrystallized, and dry,
Obtain final product the crystal containing ascosin.
As a further improvement on the present invention, the step 1) in filter aid be selected from diatomite, perlite in one kind
Or two kinds of mixtures of arbitrary proportion.
As a further improvement on the present invention, the step 2) in organic solvent be selected from ethyl acetate, ethanol or third
The mixture of one or more arbitrary proportions in ketone；The concentrate that is concentrated under reduced pressure to give is specially：0.06~
Under the subnormal ambient of 0.1MPa, concentration and recovery organic solvent, to obtain concentrate.
As a further improvement on the present invention, the step 3) in non-polar macroporous resin be selected from X-5 macroreticular resins,
D1300 macroreticular resins or HZ818 macroreticular resins；The step 3) in eluant, eluent for 20~55%vol aqueous acetone solutions or
Person's 25~70%vol ethanol waters.
As a further improvement on the present invention, the step 4) in organic solvent be selected from ethyl acetate or chloroform.
As a further improvement on the present invention, the step 5) in silica gel column chromatography be 200~300 mesh silica gel column chromatography
Post；The step 5) in eluant, eluent for volume ratio be 15:85~70:30 ethanol and the mixed solution of n-hexane, 30~
50%vol acetonitrile solutions or 40~50%vol ethanol waters.
As a further improvement on the present invention, the step 6) in organic solvent be selected from petroleum ether, ether in one kind
Or two kinds of mixed solutions of arbitrary proportion.
As a further improvement on the present invention, the step 7) in dynamic axial high-pressure column chromatographic column in sieve plate particle diameter
It is 10um, pillar height 100cm, diameter 10cm；The step 7) in eluant, eluent for volume ratio be 15:85~70:30 acetone and
The mixed solution of n-hexane, 30~50%vol acetonitrile solutions or 40~50%vol ethanol waters；The step 7) in
Organic solvent be selected from petroleum ether, ether in one or two kinds of arbitrary proportion mixed solution.
As a further improvement on the present invention, the step 8) in organic solvent be selected from n-hexane, hexamethylene in one
Plant or two kinds of mixed solutions of arbitrary proportion.
As a further improvement on the present invention, the step 8) in crystallization mode be crystallisation by cooling, crystallization temperature be 0~
4℃。
Compared with prior art, the beneficial effects of the invention are as follows：The present invention can be greatly shortened relative to prior art
Extraction time, efficiency is improve, with easy to operate, process is simple and many advantages, such as low cost, it is easy to industrialized production, be
The production of ascosin provides feasible separating technology.
Specific embodiment
With reference to each implementation method, the present invention is described in detail, but it should explanation, these implementation methods are simultaneously
Non- limitation of the present invention, those of ordinary skill in the art are according in these implementation method institutes works energy, method or structure
Equivalent transformation or replacement, belong within protection scope of the present invention.
Unless there is specified otherwise in specification, component, the raw material in each embodiment in the present invention are pure using analysis
Rank.In addition, " g " in each embodiment is unit of weight " gram "；" h " is chronomere's " hour "；" ml " is volume unit " milli
Rise "；" room temperature " is 23 DEG C.
Embodiment one：
A kind of method that ascosin is isolated and purified in liquid from streptomycete fermentation, comprises the following steps.
Step 1), to added in streptomycete fermentation liquid equivalent to streptomycete fermentation liquid product 1% perlite as drainage
Agent, stirs 30 minutes, separation of solid and liquid, filtering, and collects mycelium.
Step 2), with ethyl acetate soak extraction mycelium 2 times at room temperature, each soak extraction 2h obtains leaching liquor,
Merge leaching liquor, and under the subnormal ambient of 0.06MPa, be concentrated under reduced pressure to give concentrate.
Step 3), by step 2) concentrate that obtains adsorbed with the bed for being filled with X-5 macroporous resin adsorptions, added
20%vol aqueous acetone solutions carry out gradient elution as eluant, eluent, collect eluent, concentration.
Step 4), to step 3) obtained by concentrate add ethyl acetate extracted, phase extract in extraction, extract
Suction filtration, 0.08MPa is concentrated under reduced pressure to give oily concentrate.
Step 5), the oily concentrate silica gel column chromatography of 200 mesh, be 15 with volume ratio:85 acetone and n-hexane it is mixed
Close solution carries out gradient elution as eluant, eluent, collects eluent, and concentration obtains concentrate.
Step 6), by step 5) concentrate add petroleum ether dissolution, recrystallization to obtain ascosin crude product.
Step 7), ascosin crude product separated using dynamic axial high-pressure column chromatographic column, with volume ratio be 15:85 third
The mixed solution of ketone and n-hexane carries out gradient elution as eluant, eluent, collects eluent, and concentrate eluant.
Step 8), by step 7) in eluent after concentration add petroleum ether, the crystallisation by cooling at 0~4 DEG C, obtain final product containing
The crystal of ascosin.In the present embodiment, the purity of ascosin is 99% in crystal, and yield is 85%.
Embodiment two：
A kind of method that ascosin is isolated and purified in liquid from streptomycete fermentation, comprises the following steps.
Step 1), to added in streptomycete fermentation liquid equivalent to streptomycete fermentation liquid product 4% diatomite as drainage
Agent, is stirred 40 minutes, separation of solid and liquid, filtering, and collects mycelium.
Step 2), with ethanol soak extraction mycelium 5 times at room temperature, each soak extraction 6h obtains leaching liquor, merges
Leaching liquor, and under the subnormal ambient of 0.1MPa, it is concentrated under reduced pressure to give concentrate.
Step 3), by step 2) obtained by concentrate adsorbed with the bed for being filled with D1300 macroporous resin adsorptions, use
The aqueous solution containing 70%vol ethanol carries out gradient elution as eluant, eluent, collects eluent, concentration.
Step 4), by step 3) obtained by concentrate add chloroform to be extracted, phase extract in extraction, extract is taken out
Filter, 0.08MPa is concentrated under reduced pressure to give oily concentrate.
Step 5), by step 3) obtained by oily concentrate with 300 mesh silica gel column chromatographies, use 40%vol acetonitrile solutions
Gradient elution is carried out as eluant, eluent, eluent is collected, concentration obtains concentrate.
Step 6), by step 5) obtained by concentrate by ether dissolution, recrystallization obtains ascosin crude product.
Step 7), by step 6) obtained by ascosin crude product separated using dynamic axial high-pressure column chromatographic column；With
The aqueous solution of 45%vol ethanol carries out gradient elution as eluant, eluent, collects eluent, and concentrate eluant.
Step 8), by step 7) obtained by concentrate eluant add ether, the crystallisation by cooling at 0~4 DEG C to obtain final product containing sub
The crystal of capsule mycin.In the present embodiment, the purity of ascosin is 98% in crystal, and yield is 90%.
Embodiment three：
A kind of method that ascosin is isolated and purified in liquid from streptomycete fermentation, comprises the following steps.
Step 1), to added in streptomycete fermentation liquid equivalent to streptomycete fermentation liquid product 3% by diatomite and pearl
The mixture that rock is constituted is stirred 45 minutes, separation of solid and liquid as filter aid, filtering, and collects mycelium.
Step 2), with acetone soak extraction mycelium 3 times at room temperature, each soak extraction 3h obtains leaching liquor, merges
Leaching liquor, and under the subnormal ambient of 0.07MPa, it is concentrated under reduced pressure to give concentrate.
Step 3), by step 2) concentrate that obtains adsorbed with the bed for being filled with X-5 macroporous resin adsorptions, contained
55%vol aqueous acetone solutions are eluted as eluent gradient, collect eluent, concentration.
Step 4), by step 3) concentrate add ethyl acetate to be extracted, extract suction filtration, 0.07MPa decompressions
It is concentrated into oily concentrate.
Step 5), by step 4) oily concentrate with 250 mesh silica gel column chromatographies, use 50%vol ethanol waters
Gradient elution is carried out as eluant, eluent, eluent, concentration is collected.
Step 6), by step 5), through ether dissolution, recrystallization obtains ascosin crude product for the concentrate that obtains.
Step 7), by step 6) ascosin that obtains separated using dynamic axial high-pressure column chromatographic column；Contain
45%vol ethanol waters are eluted as eluent gradient, collection liquid, and concentrate eluant.
Step 8), by step 7) in eluent after concentration add by mixing that the petroleum ether and ether of arbitrary proportion are constituted
Solution is closed, the crystallisation by cooling at 0~4 DEG C obtains final product the crystal containing ascosin.In the present embodiment, ascus in crystal
The purity of mycin is 99%, and yield is 90%.
Those listed above is a series of to be described in detail only for feasibility implementation method of the invention specifically
Bright, they simultaneously are not used to limit the scope of the invention, all equivalent implementations made without departing from skill spirit of the present invention
Or change should be included within the scope of the present invention.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each implementation method is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Specification an as entirety, the technical scheme in each embodiment can also be formed into those skilled in the art through appropriately combined
May be appreciated other embodiment.
Claims (10)
1. a kind of method that ascosin is isolated and purified in liquid from streptomycete fermentation, it is characterised in that comprise the following steps：
Step 1), to added in streptomycete fermentation liquid equivalent to streptomycete fermentation liquid product 1~4% filter aid, stirring 30~
45 minutes, separation of solid and liquid collected mycelium；
Step 2), with organic solvent soak extraction mycelium 2~5 times at room temperature, each 2~6h of soak extraction is extracted
Liquid, merges leaching liquor, and be concentrated under reduced pressure to give concentrate；
Step 3), by step 2) obtained by concentrate adsorbed using the bed of filling non-polar macroporous resin, add wash-out
Agent carries out gradient elution, collects eluent and concentrates；
Step 4), to step 3) obtained by concentrate in add organic solvent extraction, phase extract in extraction, to upper phase extract
It is concentrated under reduced pressure to give oily concentrate；
Step 5), by step 4) obtained by oily concentrate silica gel column chromatography, add eluant, eluent to carry out gradient elution, collection is washed
De- liquid is simultaneously concentrated, and obtains concentrate；
Step 6), by step 5) obtained by concentrate add organic solvent to be recrystallized, obtain ascosin crude product；
Step 7), by step 6) obtained by ascosin crude product separated using dynamic axial high-pressure column chromatographic column, addition is washed
De- agent carries out gradient elution, collects eluent, and concentrate eluant；
Step 8), by step 7) in after eluent after concentration adds organic solvent extraction, recrystallized, and dry, obtain final product
Crystal containing ascosin.
2. method according to claim 1, it is characterised in that the step 1) in filter aid be selected from diatomite, pearl
The mixture of the one or two kinds of arbitrary proportion in rock.
3. method according to claim 1, it is characterised in that the step 2) in organic solvent be selected from ethyl acetate,
The mixture of one or more arbitrary proportions in ethanol or acetone；The concentrate that is concentrated under reduced pressure to give is specially：
Under the subnormal ambient of 0.06~0.1MPa, concentration and recovery organic solvent, to obtain concentrate.
4. method according to claim 1, it is characterised in that the step 3) in non-polar macroporous resin be selected from X-5
Macroreticular resin, D1300 macroreticular resins or HZ818 macroreticular resins；The step 3) in eluant, eluent be 20~55%vol acetone
The aqueous solution or 25~70%vol ethanol waters.
5. method according to claim 1, it is characterised in that the step 4) in organic solvent be selected from ethyl acetate or
Person's chloroform.
6. method according to claim 1, it is characterised in that the step 5) in silica gel column chromatography be 200~300 mesh
Silica gel column chromatography；The step 5) in eluant, eluent for volume ratio be 15:85~70:The mixing of 30 ethanol and n-hexane is molten
Liquid, 30~50%vol acetonitrile solutions or 40~50%vol ethanol waters.
7. method according to claim 1, it is characterised in that the step 6) in organic solvent be selected from petroleum ether, second
The mixed solution of the one or two kinds of arbitrary proportion in ether.
8. method according to claim 1, it is characterised in that the step 7) in dynamic axial high-pressure column chromatographic column in
Sieve plate particle diameter be 10um, pillar height 100cm, diameter 10cm；The step 7) in eluant, eluent for volume ratio be 15:85~70:
30 acetone and the mixed solution of n-hexane, 30~50%vol acetonitrile solutions or 40~50%vol ethanol waters；Institute
State step 7) in organic solvent be selected from petroleum ether, ether in one or two kinds of arbitrary proportion mixed solution.
9. method according to claim 1, it is characterised in that the step 8) in organic solvent be selected from n-hexane, ring
The mixed solution of the one or two kinds of arbitrary proportion in hexane.
10. method according to claim 1, it is characterised in that the step 8) in crystallization mode be crystallisation by cooling, knot
Brilliant temperature is 0~4 DEG C.
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| CN108409751A (en) * | 2018-02-24 | 2018-08-17 | 海正药业(杭州)有限公司 | The purification process of one ascomycin |
| CN120383611A (en) * | 2025-04-24 | 2025-07-29 | 华东理工大学 | A method for extracting, separating and purifying ascomycin from mycelium |
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| CN107573362A (en) * | 2017-10-31 | 2018-01-12 | 无锡福祈制药有限公司 | A kind of method of the separating-purifying sirolimus from zymotic fluid |
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| CN120383611A (en) * | 2025-04-24 | 2025-07-29 | 华东理工大学 | A method for extracting, separating and purifying ascomycin from mycelium |
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