CN106946907A - Method and the application of tacrolimus are isolated and purified from mycelium - Google Patents

Abstract

The invention discloses a method for separating and purifying tacrolimus from mycelium and its application. The invention dissolves the streptomyces tsukuba mycelium with hydrophilic ionic liquid, and then extracts and removes components such as fat and fatty acid with an organic solvent. Then, a hydrophobic ionic liquid-hydrophilic ionic liquid two-phase extraction method is used to transfer components such as tacrolimus into the hydrophobic ionic liquid phase. Finally, separation and purification were carried out by means of supercritical CO ₂ extraction to obtain tacrolimus crystals. The method provided by the invention can effectively recover tacrolimus, the recovery rate reaches 70-85%, and the obtained tacrolimus has a purity of more than 90%. The method is suitable for industrial separation and purification of tacrolimus.

Description

Method and application of separating and purifying tacrolimus from mycelium

technical field

The invention belongs to the field of compound separation and purification, in particular to a method and application for separating and purifying tacrolimus from mycelia.

Background technique

Tacrolimus (English: Tacrolimus) is a 23-membered macrolide antibiotic isolated from Streptomyces tsukubaensis. Chemical formula: C ₄₄ H ₆₉ NO ₁₂ H ₂ O, 3S-[3R[E(1S, 3S, 4S)], 4S, 5R, 8S, 9E, 12R, 14R, 15S, 16R, 18S, 19S, 26aR] ]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2- (4-Hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propene base)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazepine-1,7,20,21(4H,23H)- Tetraketone-monohydrate. Pure tacrolimus is a colorless prismatic crystal with a melting point of 127-129°C; it is easily soluble in methanol, ethanol, acetone, ethyl acetate, chloroform or ether, hardly soluble in hexane or petroleum ether, and insoluble in water.

Drugs with tacrolimus as the main raw material have a high degree of immunosuppressive activity, mainly by inhibiting the release of interleukin-2 (L-2), to fully inhibit the activation of T lymphocytes and the proliferation of T helper cells dependent on B cells; It will inhibit the production of lymphokines such as interleukin-2, interleukin-3 and gamma-interferon and the expression of interleukin-2 receptor. In recent years, as a first-line drug for liver and kidney transplantation, it has been marketed in 14 countries including Japan and the United States. Clinical experiments have shown that it has good curative effect in heart, lung, intestine, bone marrow and other transplants. At the same time, FK506 also plays an active role in the treatment of autoimmune diseases such as atopic dermatitis (AD), systemic lupus erythematosus (SLE), and autoimmune eye diseases.

Industrially, tacrolimus is mainly obtained by aerobic fermentation using Streptomyces tsukubaensis (S. tsukubaensis) at 30°C. The tacrolimus fermentation broth contains impurities such as residual soluble starch, corn steeping liquid, yeast powder and calcium carbonate. In addition, when tacrolimus is produced by fermentation, there will be by-products ascomycin and dihydrotacrolimus that are very similar to its structure. These two analogues are difficult to remove by chromatography and crystallization. Tacrolimus is easily converted to its isomer I/II in an aqueous environment. These factors lead to the difficulty of separation and purification of tacrolimus and the problems of low yield. At present, industrial separation and purification of tacrolimus from fermentation broth generally adopts the technical route of solvent extraction-macroporous resin adsorption-solvent extraction-silica gel column chromatography-crystallization (or resin specific adsorption). An example to illustrate the steps is as follows:

a. The fermentation broth is filtered through diatomaceous earth to obtain mycelium filter cake and filtrate.

b. The filter cake is extracted with acetone to obtain an extract. The extract is combined with the filtrate from the first step.

c. Pass through a column equipped with non-ionic absorbent resin. Gradient elution was performed with water and acetone in water.

d. After the eluate is concentrated under reduced pressure, it is extracted with ethyl acetate.

e. The ethyl acetate solution was concentrated under reduced pressure to obtain an oily product.

f. Adsorb the oily product in acidic silica gel, elute with a mixture of n-hexane and ethyl acetate and ethyl acetate, collect the eluate, and distill under reduced pressure.

g, repeat the above 3 times to obtain a white powdery product.

h. The product is dissolved and recrystallized using acetonitrile. Or use coordinative macroporous resins loaded with Ag ⁺ for adsorption and purification.

Exploring a fast, efficient and industrially applicable separation and purification method is of great significance for improving the quality of tacrolimus raw materials, production efficiency and reducing production costs.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a method for separating and purifying tacrolimus from mycelia.

Another object of the present invention is to provide the application of the method for separating and purifying tacrolimus from mycelia.

The object of the present invention is achieved through the following technical solutions: a method for separating and purifying tacrolimus from mycelia, comprising the following steps:

(1) Mix the mycelia of Streptomyces tsukubaensis (S.tsukubaensis) with a hydrophilic ionic liquid; heat and stir in an inert gas atmosphere or a vacuum environment under normal pressure, and the heating condition is to react at 40-90°C for 0.5-5 hours ;

(2) The mycelia-hydrophilic ionic liquid solution after the reaction in step (1) is mixed with the organic solvent A for extraction;

(3) Use a hydrophobic ionic liquid to mix with the mycelium-hydrophilic ionic liquid solution extracted in step (2), heat and stir in an inert gas atmosphere at normal pressure or in a vacuum environment, and the heating condition is to react at 40-90°C 0.5 to 5 hours, then let stand to separate layers, take the hydrophobic phase, and recover the hydrophilic phase containing the hydrophilic ionic liquid;

(4) washing the hydrophobic phase of step (3) with ultrapure water;

(5) The tacrolimus component in the hydrophobic phase after washing in step (4) is subjected to supercritical _CO2 extraction, the extract is collected, and the solvent containing the hydrophobic ionic liquid is reclaimed; the extract is dissolved with an organic solvent B after drying, Obtain an extract solution;

(6) Liquid carbon dioxide and the extract solution of step (5) are injected into the top of the supercritical _CO reactor simultaneously, and are sprayed into the reaction chamber by atomization; The sieve plate precipitator collects the tacrolimus crystals accumulated in the precipitator to obtain tacrolimus.

The method for separating and purifying tacrolimus from mycelium also includes the following steps: combining the hydrophilic phase recovered in step (3) containing the hydrophilic ionic liquid and the phase recovered in step (5) containing the hydrophobic ion After the liquid solvent is vacuum-dried and activated carbon is adsorbed, a hydrophilic ionic liquid and a hydrophobic ionic liquid that can be reused are respectively obtained.

The water content of the mycelia in step (1) is not higher than 50% (w/w); preferably 10-50% (w/w).

The amount of the hydrophilic ionic liquid described in step (1) is according to the dry weight of the mycelium: the weight of the hydrophilic ionic liquid=1:2～1:20 ratio; preferably according to the dry weight of the mycelium: Hydrophilic ionic liquid mass = 1:2 to 1:6 ratio.

The normal pressure described in steps (1) and (3) is 1 atmosphere.

The inert gas described in steps (1) and (3) is preferably nitrogen.

The heating condition described in step (1) is preferably to react at 50-90° C. for 0.5-2 hours.

The cation in the hydrophilic ionic liquid described in step (2) is preferably 1-methyl-3-methylimidazole, 1-methoxyl-3-methylimidazole, 1-carboxymethyl-3-methylimidazole Imidazole, 1-ethyl-3-methylimidazole, 1-vinyl-3-methylimidazole, 1-hydroxyethyl-3-methylimidazole, 1-ethoxy-3-methylimidazole, 1 -Carboxyethyl-3-methylimidazole, 1-acetoxyethyl-3-methylimidazole, 1-aminoethyl-3-methylimidazole, 1-propyl-3-methylimidazole, 1-amino Propyl-3-methylimidazole, 1-cyanopropyl-3-methylimidazole, 1-allyl-3-methylimidazole, 1-butyl-3-methylimidazole, 1-pentyl-3 -Methylimidazole, 1-hexyl-3-methylimidazole, 1-octyl-3-methylimidazole, 1-methyl-2,3-dimethylimidazole, 1-methoxy-2,3- Dimethylimidazole, 1-carboxymethyl-2, 3-dimethylimidazole, 1-ethyl-2, 3-dimethylimidazole, 1-vinyl-2, 3-dimethylimidazole, 1- Hydroxyethyl-2,3-dimethylimidazole, 1-ethoxy-2,3-dimethylimidazole, 1-carboxyethyl-2,3-dimethylimidazole, 1-acetate ethyl- 2, 3-dimethylimidazole, 1-aminoethyl-2, 3-dimethylimidazole, 1-propyl-2, 3-dimethylimidazole, 1-aminopropyl-2, 3-dimethyl Imidazole, 1-cyanopropyl-2, 3-dimethylimidazole, 1-allyl-2, 3-dimethylimidazole, 1-butyl-2, 3-dimethylimidazole, 1-pentane One or at least two of base-2,3-dimethylimidazole, 1-hexyl-2,3-dimethylimidazole and 1-octyl-2,3-dimethylimidazole; more preferably 1 - at least one of propyl-3-methylimidazole, 1-allyl-3-methylimidazole, 1-butyl-3-methylimidazole and 1-pentyl-3-methylimidazole.

The anion in the hydrophilic ionic liquid described in step (2) is preferably chlorine, bromine, iodine, tetrafluoroboric acid, acetic acid, nitric acid, perchloric acid, hydrogen sulfate, dihydrogen phosphate, trifluoromethanesulfonic acid, trifluoromethanesulfonic acid, One or at least two of fluoroacetic acid and p-toluenesulfonic acid; preferably one or both of chlorine and acetic acid.

The organic solvent A described in the step (2) is one or both of hexane and petroleum ether.

The hexane is preferably n-hexane.

The amount of the organic solvent A described in step (2) is preferably the ratio of the mycelia-hydrophilic ionic liquid solution to the organic solvent A in a volume ratio of 2:1 to 1:10;

The extraction described in step (2) is extraction at room temperature.

The room temperature is 10-30°C; preferably 20-30°C.

The cation in the hydrophobic ionic liquid described in step (3) is preferably 1-decyl-3-methylimidazole, 1-dodecyl-3-methylimidazole, 1-tetradecyl-3- Methylimidazole, 1-hexadecyl-3-methylimidazole, 1-octadecyl-3-methylimidazole, 1-ethyl-2,3-dimethylimidazole, 1-hexyl-2, One or at least two of 3-dimethylimidazole and 1-hexadecyl-2,3-dimethylimidazole; preferably 1-decyl-3-methylimidazole, 1-tetradecyl - one or at least two of 3-methylimidazole and 1-hexadecyl-2,3-dimethylimidazole.

The anion in the hydrophobic ionic liquid described in step (3) is preferably one or at least two of bromine, sulfonic acid, hexafluorophosphoric acid and bistrifluoromethanesulfonimide.

The amount of the hydrophobic ionic liquid described in step (3) is preferably 10:1 to 1:10 by mass ratio between the hydrophobic ionic liquid and the extracted mycelia-hydrophilic ionic liquid solution Proportion; preferably 5:1 to 1:2 by mass ratio.

The heating condition described in step (3) is preferably to react at 50-90° C. for 0.5-3 hours.

The standing stratification described in step (3) is preferably cooling to 30° C. and then standing stratification.

The above-mentioned ionic liquids are artificially synthesized compounds, and cations and anions can be combined freely according to needs. These ionic liquids can be synthesized by commercial means, such as Lanzhou Zhongke Kite Technology Industry and Trade Co., Ltd.

The conditions of the water washing in step (4) are preferably water washing at 20-60°C; more preferably water washing at 40-60°C.

The amount of ultrapure water used in step (4) is preferably 1-10 times the volume of the hydrophobic phase; preferably 2-5 times the volume of the hydrophobic phase.

The number of times of water washing described in step (4) is preferably 2 to 3 times.

The extraction conditions of the supercritical _CO2 extraction described in step (5) are preferably: the _CO2 amount and the hydrophobic phase are in a mass ratio of 3 to 20:1; the pressure of the extraction system is 9 to 30 MPa, and the extraction temperature is 35 ~60°C, add entraining solvent in an amount equivalent to 0~5% (w/w) of carbon dioxide mass, outlet temperature 35~55°C, flow rate 0.1~1L/min; more preferably: _CO2 dosage and hydrophobic phase The mass ratio is 3-15:1; the pressure of the extraction system is 10-30MPa, the extraction temperature is 40-55°C, and the entrained solvent is added in an amount equivalent to 0.5-1% (w/w) of carbon dioxide mass, and the outlet The temperature is 40-55°C, and the flow rate is 0.3-1L/min.

The entrained solvent used in the supercritical _CO2 extraction described in step (5) is preferably one or at least two of methanol, ethanol, acetone, ethyl acetate, chloroform and ether.

The extract collection solvent used in the supercritical _CO extraction described in step (5) is preferably one or at least one of methanol, ethanol, acetone, ethyl acetate, chloroform, ether, dimethylsulfoxide and acetonitrile two kinds.

The organic solvent B described in step (5) is preferably dimethyl sulfoxide or acetonitrile.

The concentration of the extract solution in step (5) is 0.5%-30% (w/w); preferably 10-15% (w/w).

The liquid carbon dioxide described in step (6) is high-pressure liquid carbon dioxide.

The liquid carbon dioxide and the extract solution described in step (6) are mixed in a ratio of 1 to 20:1 (m/v, g:mL); preferably mixed in a ratio of 8 to 10:1 by mass to volume .

The reaction conditions described in the step (6) are preferably as follows: the pressure of the reaction system is 10 to 50 MPa, the temperature is 40 to 65 ° C, and the flow rate is 0.5 to 2 L/min; more preferably As follows: the pressure of the reaction system is 10-45MPa, the temperature is 40-60°C, and the flow rate is 0.8-1.5L/min.

The method for separating and purifying tacrolimus from mycelium is suitable for industrial separation and purification to obtain tacrolimus.

Principle of the present invention: ionic liquid is a salt substance in a liquid state at or near room temperature, and is usually composed of organic cations and inorganic anions or organic anions. Ionic liquids have many characteristics: a wide range of liquid temperature, which can reach hundreds of degrees; high stability; low vapor pressure, almost non-volatile; similar properties to organic solvents, and good solubility for many organic and inorganic substances. Ionic liquids are very suitable as separation and purification media, especially in liquid-liquid extraction and separation: ionic liquids can dissolve certain organic compounds, inorganic compounds and organometallic compounds, but are immiscible with organic or inorganic solvents. The unique properties of ionic liquids are usually determined by their specific structures and interactions between ions. According to the different types of cation and anion groups in ionic liquids, ionic liquids can be divided into two types: hydrophilic and hydrophobic. Among them, the anions are Cl ^- and OAc ^- and other imidazole hydrophilic ionic liquids have the ability to dissolve cellulose and filamentous fungal cell walls efficiently. In this type of ionic liquid, the dissolution of the cell wall of S. tsukubaensis can effectively release the metabolites in the hyphae. Hydrophobic ionic liquids mainly include imidazoles whose cations are substituents with a chain length of more than 8 carbon atoms, and ionic liquids whose anions are hexafluorophosphate and trifluoromethylsulfonamide. Hydrophobic ionic liquids are not easily miscible with water, hydrophilic ionic liquids and other polar solvents. Hydrophobic ionic liquids with the same imidazolium cation and different anions can be blended to change their hydrophobicity and affinity to tacrolimus components, so as to achieve the optimal extraction effect for this system. Compared with conventional mechanical crushing methods and organic solvent dissolution methods, the use of ionic liquids for extraction and extraction has the characteristics of high efficiency and low toxicity.

The mycelium and its contents dissolved in the hydrophilic ionic liquid are first extracted with hexane or petroleum ether to remove components such as fat and fatty acid. Then, the hydrophobic ionic liquid-hydrophilic ionic liquid two-phase extraction method is used to transfer the components such as tacrolimus into the hydrophobic ionic liquid phase, so as to realize the separation from the extreme impurities in the fermentation broth and mycelium. The collected hydrophobic phase is washed with water, and the residual water-soluble substances (sugar, protein, amino acid, vitamin, etc.) are removed. Ionic liquids have extremely low saturated vapor pressure and are almost non-volatile. Non-volatile components (such as tacrolimus) dissolved in hydrophobic ionic liquids can be separated by supercritical CO ₂ extraction. Supercritical _CO2 has super strong penetration and solvency, and can be completely dissolved in ionic liquids, while ionic liquids are not dissolved in _CO2 . The separation and enrichment of tacrolimus raw material products can be achieved by controlling the pressure, temperature and entrained solvent of the reaction system. Compared with conventional methods such as distillation, evaporative concentration and chromatography, this method is simpler and more efficient. The tacrolimus product can be further purified using the gas antisolvent effect of supercritical crystallization technology. The gas anti-solvent effect refers to the process in which the dissolved _CO2 expands the organic solvent under high pressure conditions, the cohesive energy is significantly reduced, the solubility is reduced, and the dissolved substance forms a crystal or an amorphous precipitate. According to the characteristics that the gas anti-solvent effect is sensitive to temperature and reaction system pressure, supercritical fluid crystallization technology can prepare and purify ultrafine crystals while maintaining the natural structure and activity of the substance.

Compared with the prior art, the present invention has the following advantages and effects:

The invention has a high recovery rate of 70-85% for tacrolimus; the purity is also high, which is more than 90%, as high as 99%.

detailed description

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

1. The mycelium of Streptomyces Strptomyces GW-2-1920-1916-02 (Guangdong Provincial Microbial Germplasm Resource Bank) is obtained by liquid submerged fermentation, and the specific steps are as follows: the liquid fermentation medium (weight ratio) contains 1.5% Glucose, 1.5% soluble starch, 0.1% L-lysine, 0.1% (NH4) ₂ SO ₄ , 0.05% K ₂ HPO ₄ and MgSO ₄ , 0.005% VB ₁ , pH=6.5. Fermented at 30°C for 72 hours and filtered Collect mycelium. The collected Streptomyces tsukuba mycelium (water content is 10% (w/w)), and the hydrophilic ionic liquid 1-propyl-3-methylimidazolium chloride salt is in a ratio of 1:3 (dry weight w/ w) mixing. Heat and stir under normal pressure _N2 atmosphere, the heating temperature is 50°C, and the reaction time is 2 hours.

2. The mycelium-ionic liquid solution after the reaction in the first step is mixed with n-hexane at a ratio of 1:5 (v/v), and extracted at room temperature.

3. Use the hydrophobic ionic liquid 1-decyl-3-methylimidazole bromide to mix with the mycelium-ionic liquid solution extracted in the second step according to the ratio of 2:1 (w/w), and use the normal pressure N ₂ atmosphere Heat (50°C) and mechanically stir for 2 hours, then cool to 30°C and let stand to separate layers.

4. Use 3 times the volume of ultrapure water to wash the hydrophobic phase in step 3 at 40°C for 3 times.

5. Carry out supercritical CO ₂ extraction to the hydrophobic phase after water washing. The amount of _CO2 used is 3:1 (w/w) compared to the hydrophobicity, the pressure of the extraction system is 10MPa, the extraction temperature is 40°C, the entrained solvent is ethanol, the addition amount is 0.5% (w/w), and the outlet temperature is 40°C. The flow rate is 0.3L/min, and the extract collection solvent is ethanol.

6. The extract was dried in vacuum and dissolved in acetonitrile at a concentration of 10% (w/w).

7. Inject the high-pressure liquid carbon dioxide and the extract solution in step 6 into the atomizing nozzle at the top of the supercritical carbon dioxide reactor at a mass volume ratio of 10:1. The pressure of the reaction system is 10MPa, the temperature is 40°C, and the flow rate is 1L/min . Tacrolimus crystals were collected on a sieve module at the bottom of the reactor.

The calculated and analyzed components of tacrolimus had a recovery rate of 75% and a purity of 95%.

Recovery = (mass of tacrolimus component obtained by the separation method/total content of tacrolimus in mycelia) × 100%.

Purity = (mass of tacrolimus component in crystals obtained by this separation method/crystal mass) × 100%

Example 2

1. According to step 1 of the embodiment, the mycelium of Streptomyces tsukuba Strptomyces GW-2-1920-1916-02 with a water content of 20% was obtained by liquid submerged fermentation, and the obtained mycelium was mixed with the hydrophilic ionic liquid 1 - Allyl-3-methylimidazole acetate was mixed in a ratio of 1:6 (dry weight w/w). Heat and stir under _N2 atmosphere under normal pressure, the heating temperature is 70°C, and the reaction time is 1 hour.

2. The mycelium-hydrophilic ionic liquid solution after the reaction in the first step is mixed with petroleum ether in a ratio of 1:1 (v/v), and extracted at room temperature.

3. Use the hydrophobic ionic liquid 1-hexyl-2,3-dimethylimidazolium hexafluorophosphate and the mycelium-hydrophilic ionic liquid solution extracted in the second step according to the ratio of 5:1 (w/w) Mixed, heated (70°C) under normal pressure _N2 atmosphere and mechanically stirred for 3 hours, then cooled to 30°C and allowed to stand for stratification.

4. Use 5 times the volume of ultrapure water to wash the hydrophobic phase in step 3 at 50°C for 3 times.

5. Carry out supercritical CO ₂ extraction to the hydrophobic phase after water washing, and collect the extract. The amount of _CO2 used is 10:1 (w/w) compared to the hydrophobicity, the pressure of the extraction system is 25MPa, the extraction temperature is 50°C, the outlet temperature is 45°C, the flow rate is 0.8L/min, the entrained solvent is ethyl acetate, and the addition amount is 0.8% (w/w). The extract collection solvent was ethyl acetate.

6. The extract was dried in vacuum and dissolved in acetonitrile at a concentration of 15% (w/w).

7. Inject the high-pressure liquid carbon dioxide and the extract solution in step 6 into the atomizing nozzle at the top of the supercritical carbon dioxide reactor according to the mass volume ratio of 9:1. The pressure of the reaction system is 30MPa, the temperature is 55°C, and the flow rate is 0.8L/ min. Tacrolimus crystals were collected on a sieve module at the bottom of the reactor.

The calculated and analyzed components of tacrolimus had a recovery of 85% and a purity of 92%.

Example 3

1. According to step 1 of the embodiment, the mycelium of Streptomyces tsukuba Strptomyces GW-2-1920-1916-02 with a water content of 50% was obtained by submerged fermentation, and the obtained mycelium was mixed with the hydrophilic ionic liquid 1 - A mixture of butyl-3-methylimidazolium chloride and 1-pentyl-3-methylimidazolium chloride (1:1, v/v) was mixed in a ratio of 1:2 (dry weight w/w). Heating and stirring under normal pressure and vacuum, the heating temperature is 90° C., and the reaction time is 0.5 hours.

2. The mycelium-hydrophilic ionic liquid solution after the reaction in the first step is mixed with n-hexane at a ratio of 2:1 (v/v), and extracted at room temperature.

3. Use the hydrophobic ionic liquid as 1-dodecyl-3-methylimidazolium hexafluorophosphate and the mycelium after the extraction in the second step-hydrophilic ionic liquid solution according to 1:2 (w/w) Proportional mixing, vacuum heating (90°C) and mechanical stirring for 0.5 hours, cooling to 30°C and standing to separate layers.

4. Use 5 times the volume of ultrapure water to wash the hydrophobic phase in step 3 twice at 60°C.

5. Carry out supercritical CO ₂ extraction to the hydrophobic phase after water washing, and collect the extract. The amount of _CO used is 15:1 (w/w) compared to the hydrophobicity, the pressure of the extraction system is 30MPa, the extraction temperature is 55°C, the outlet temperature is 55°C, the flow rate is 1L/min, the entrained solvent is acetone, and the addition amount is 1% ( w/w), the extract collection solvent is acetone.

6. The extract was dried in vacuum and dissolved in dimethyl sulfoxide at a concentration of 5% (w/w).

7. The high-pressure liquid carbon dioxide and the extract solution in step 6 are jointly injected into the atomizing nozzle at the top of the supercritical carbon dioxide reactor in a ratio of 8:1 by mass to volume. The pressure of the reaction system is 45MPa, the temperature is 60°C, and the flow rate is 1.5L/min. Tacrolimus crystals were collected on a sieve module at the bottom of the reactor.

The calculated and analyzed components of tacrolimus had a recovery rate of 70% and a purity of 99%.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. A method for isolating and purifying tacrolimus from mycelium, characterized in that it comprises the following steps: (1) mixing Streptomyces tsukuba mycelium with a hydrophilic ionic liquid; heating and stirring in an inert gas atmosphere or a vacuum environment under normal pressure, and the heating condition is to react at 40-90° C. for 0.5-5 hours; (2) The mycelia-hydrophilic ionic liquid solution after the reaction in step (1) is mixed with the organic solvent A for extraction; (3) Use a hydrophobic ionic liquid to mix with the mycelium-hydrophilic ionic liquid solution extracted in step (2), heat and stir in an inert gas atmosphere at normal pressure or in a vacuum environment, and the heating condition is to react at 40-90°C 0.5 to 5 hours, then let stand to separate layers, take the hydrophobic phase, and recover the hydrophilic phase containing the hydrophilic ionic liquid; (4) washing the hydrophobic phase of step (3) with ultrapure water; (5) The tacrolimus component in the hydrophobic phase after washing in step (4) is subjected to supercritical _CO2 extraction, the extract is collected, and the solvent containing the hydrophobic ionic liquid is reclaimed; the extract is dissolved with an organic solvent B after drying, Obtain an extract solution; (6) Liquid carbon dioxide and the extract solution of step (5) are injected into the top of the supercritical _CO reactor simultaneously, and are sprayed into the reaction chamber by atomization; A sieve plate precipitator, collecting tacrolimus crystals gathered in the precipitator to obtain tacrolimus; The organic solvent A described in step (2) is one or both in hexane and sherwood oil; The organic solvent B described in step (5) is dimethyl sulfoxide or acetonitrile. 2. the method for separating and purifying tacrolimus from mycelium according to claim 1, is characterized in that also comprising the following steps: the hydrophilic phase containing the hydrophilic ionic liquid that step (3) reclaims and step After the solvent containing the hydrophobic ionic liquid recovered in (5) was vacuum-dried and activated carbon adsorption, the reusable hydrophilic ionic liquid and hydrophobic ionic liquid were respectively obtained. 3. the method for separating and purifying tacrolimus from mycelia according to claim 1, characterized in that: The cation in the hydrophilic ionic liquid described in step (2) is 1-methyl-3-methylimidazole, 1-methoxy-3-methylimidazole, 1-carboxymethyl-3-methyl Imidazole, 1-ethyl-3-methylimidazole, 1-vinyl-3-methylimidazole, 1-hydroxyethyl-3-methylimidazole, 1-ethoxy-3-methylimidazole, 1- Carboxyethyl-3-methylimidazole, 1-acetate ethyl-3-methylimidazole, 1-aminoethyl-3-methylimidazole, 1-propyl-3-methylimidazole, 1-aminopropyl Base-3-methylimidazole, 1-cyanopropyl-3-methylimidazole, 1-allyl-3-methylimidazole, 1-butyl-3-methylimidazole, 1-pentyl-3- Methylimidazole, 1-hexyl-3-methylimidazole, 1-octyl-3-methylimidazole, 1-methyl-2,3-dimethylimidazole, 1-methoxy-2,3-di Methylimidazole, 1-carboxymethyl-2, 3-dimethylimidazole, 1-ethyl-2, 3-dimethylimidazole, 1-vinyl-2, 3-dimethylimidazole, 1-hydroxy Ethyl-2, 3-dimethylimidazole, 1-ethoxy-2, 3-dimethylimidazole, 1-carboxyethyl-2, 3-dimethylimidazole, 1-acetate ethyl-2 , 3-dimethylimidazole, 1-aminoethyl-2, 3-dimethylimidazole, 1-propyl-2, 3-dimethylimidazole, 1-aminopropyl-2, 3-dimethyl Imidazole, 1-cyanopropyl-2, 3-dimethylimidazole, 1-allyl-2, 3-dimethylimidazole, 1-butyl-2, 3-dimethylimidazole, 1-pentyl - one or at least two of 2,3-dimethylimidazole, 1-hexyl-2,3-dimethylimidazole and 1-octyl-2,3-dimethylimidazole; The anion in the hydrophilic ionic liquid described in step (2) is chlorine, bromine, iodine, tetrafluoroboric acid, acetic acid, nitric acid, perchloric acid, hydrogen sulfate, dihydrogen phosphate, trifluoromethanesulfonic acid, trifluoro One or at least two of acetic acid and p-toluenesulfonic acid. 4. the method for separating and purifying tacrolimus from mycelium according to claim 1, is characterized in that: The cations in the hydrophobic ionic liquid described in step (3) are 1-decyl-3-methylimidazole, 1-dodecyl-3-methylimidazole, 1-tetradecyl-3-methylimidazole imidazole, 1-hexadecyl-3-methylimidazole, 1-octadecyl-3-methylimidazole, 1-ethyl-2,3-dimethylimidazole, 1-hexyl-2,3 - one or at least two of dimethylimidazole and 1-hexadecyl-2,3-dimethylimidazole; The anion in the hydrophobic ionic liquid described in step (3) is one or at least two of bromine, sulfonic acid, hexafluorophosphoric acid and bistrifluoromethanesulfonylimide. 5. the method for separating and purifying tacrolimus from mycelium according to claim 1, is characterized in that: The extraction condition of the supercritical _CO2 extraction described in step (5) is: _CO2 consumption and hydrophobic phase are proportioned by mass ratio 3～20:1; The pressure of extraction system is 9～30MPa, and the temperature of extraction is 35～20MPa. 60°C, add entrained solvent in an amount equivalent to 0-5% of the mass of carbon dioxide, the outlet temperature is 35-55°C, and the flow rate is 0.1-1L/min; The reaction conditions for spraying into the reaction chamber by means of atomization in step (6) are as follows: the pressure of the reaction system is 10-50 MPa, the temperature is 40-65° C., and the flow rate is 0.5-2 L/min. 6. the method for separating and purifying tacrolimus from mycelium according to claim 5, is characterized in that: The entrained solvent is one or at least two of methanol, ethanol, acetone, ethyl acetate, chloroform and ether; The extract collection solvent used in the supercritical _CO extraction described in step (5) is one or at least two of methanol, ethanol, acetone, ethyl acetate, chloroform, ether, dimethylsulfoxide and acetonitrile kind. 7. the method for separating and purifying tacrolimus from mycelium according to claim 1, is characterized in that: The amount of the hydrophilic ionic liquid described in step (1) is according to the dry weight of the mycelium: the mass of the hydrophilic ionic liquid=1:2～1:20 ratio; The amount of the hydrophobic ionic liquid described in step (3) is the mixture of the hydrophobic ionic liquid and the extracted mycelia-hydrophilic ionic liquid solution in a mass ratio of 10:1 to 1:10. Compare. 8. The method for separating and purifying tacrolimus from mycelium according to claim 1, characterized in that: The water content of the mycelia described in step (1) is not higher than 50% by mass; The amount of the organic solvent A described in step (2) is the ratio of the mycelium-hydrophilic ionic liquid solution to the organic solvent A in a volume ratio of 2:1 to 1:10; The washing condition described in the step (4) is washing at 20-60°C; The amount of ultrapure water described in step (4) is equivalent to 1 to 10 times the volume of the hydrophobic phase. 9. the method for separating and purifying tacrolimus from mycelium according to claim 1, is characterized in that: The concentration of the extract solution described in step (5) is 0.5% to 30% by mass; The liquid carbon dioxide described in the step (6) and the described extract solution are mixed according to a mass volume ratio of 1-20:1. 10. Application of the method for separating and purifying tacrolimus from mycelia according to any one of claims 1 to 9 in industrial separation and purification of tacrolimus.