CN101503368B - Chemical separation and purification method for myriocin crude product - Google Patents

Abstract

The invention provides a method for chemical separation and purification of crude myriocin. In the presence of a chromatographic medium, an acid catalyst and one or more of ethyl acetate, acetone, absolute ethanol, methanol, and n-butanol according to the Under the conditions of any proportion of solvents, the chemical reaction and the separation process are coupled, and the myriocin in the crude product of myriocin is derivatized into myriocin lactone, and then the myriocin is alkalized Purified myriocin monomers were obtained from steroid lactones. The chemical reaction and separation are coupled together, and the two are carried out at the same time, which greatly simplifies the subsequent purification process of the product, improves the yield, reduces energy consumption, reduces investment, and is suitable for industrial production.

Description

Chemical separation and purification method of myriocin crude product

technical field

The invention relates to a separation and purification method for crude myriocin, in particular to a chemical separation and purification method for crude myriocin.

Background technique

Myriocin has a variety of pharmacological effects. Studies by Fujita et al. have shown that myriocin has a significant two-way immunoregulatory effect, can block the pathway below the interleukin-2 receptor, inhibit the activity of serine palmitoyltransferase, and thus specifically inhibit the proliferation of T cells. In addition, Hojjati et al. reported that myriocin can inhibit the formation of atherosclerosis, using the mechanism of myriocin to inhibit the key enzyme of sphingolipid synthesis - serine palmitoyltransferase, 60 days of continuous treatment of 8-week-old Rats with atherosclerosis were injected with a concentration of 0.3 mg/kg myriocin every other day, and the results showed that myriocin had a significant inhibitory effect on the synthesis of sphingolipids, and the area of arteriosclerotic damage in the rats was obvious Reduction, therefore, myriocin inhibition of serine palmitoyltransferase activity may serve as an option for the treatment of atherosclerosis. Based on the pharmacological value and clinical application prospect of myriocin, it is of great significance to prepare high-purity myriocin required by the pharmaceutical industry. Myriocin lactone also has similar pharmacological effects as myriocin.

At present, none of the reports on the separation and purification methods of myriocins adopts chemical separation and purification methods. Fujita et al. (Fungal metabolites. 47 (2): 208-215) tracked the activity of immunosuppressive cell model, and obtained myriocin by combining macroporous adsorption resin and preparative thin-layer chromatography for separation and purification. Kluepfel et al. (Myriocin, a new antifungal antibiotic from Myriococcum albomyces. Kluepfel D, et al. Journal of Antibiotics, 25: 109-115, 1972) used water-methanol-dichloromethane to purify myriocin by liquid-liquid extraction and crystallization; The method has a simple process, but the yield is low, the repeatability is poor, and the operation technique is difficult. The disadvantage of these methods is that they are not suitable for industrialization. In the current report on the preparation method of myriocin lactone, the literature (Fungal metabolites. Part 12. Potent immuno-suppressant, 14-deoxomyriocin, (2S, 3R, 4R)-(E)-2-amino-3, 4-dihydroxy-2-hydroxymethyleicos-6-enoic acid and structure-activity relationships of myriocin derivatives.Fujita et al.Journal of Antibiotics, 47:216-224, 1994) provides a kind of myriocin methanol solution and hydrogen chloride Methanol solution reaction, the reactant uses silica gel as chromatographic medium, chloroform-methanol as eluent for eluting, and then crystallizes with chloroform-petroleum ether to prepare the method of myriocin lactone monomer. It is not feasible to use this method for the separation and purification of crude myriocin, because the method uses myriocin monomers to prepare myriocin lactones, and the myriocin monomers need to be prepared first . At present, there is no report on the separation and purification of crude myriocin using chemical methods.

Contents of the invention

The purpose of the present invention is to provide a chemical separation and purification method of crude myriocin which can simplify the subsequent purification process of the product, increase the yield, reduce energy consumption and is suitable for industrialization.

The technical scheme adopted in the present invention is as follows: the separation and purification method of myriocin crude product comprises the following steps:

(1) Take the myriocin crude product with a weight content of 2-50% and dissolve it with absolute ethanol, then separate it by silica gel column chromatography, elute with a mixed solution of solvent A and an acid catalyst, and collect the myriocin Bacteroid lactone fraction; wherein the volume ratio of the solvent A to the acid catalyst is 6-12.5: 0.2-1.5; the solvent A is selected from ethyl acetate, acetone, absolute ethanol, methanol, n-butanol One or more solvents composed in any proportion, and the acid catalyst is one of formic acid or acetic acid;

(2) The myriocin lactone fraction obtained in step (1) is evaporated to dryness, the residue is crystallized with solvent B, and the crystals are collected, and the myriocin lactone monomer is obtained; wherein the solvent B is selected from One or more solvents composed of ethanol, methanol, and water in any proportion;

(3) Get the myriocin monomer and place it in alkali and water to form a reaction system, and the myriocin monomer reacts completely with water under alkaline conditions to obtain a myriocin solution, and then The residue obtained by evaporating the solution to dryness is the isolated and purified myriocin monomer; wherein the base is one of pyridine or ammonia water.

Wherein step (1) described myriocin weight content is the myriocin crude product of 2～50%, refers to all kinds of products that contain myriocin, wherein the myriocin monomer The range of percentage content (weight) is 2-50%. For example, the ISP extract of the effective part of the immunosuppressive activity of Cicada flower is prepared by the method provided by the Chinese invention patent "Extraction and separation method of the ISP extract of the effective part of the immunosuppressive activity of Cicada flower" with application number 2007100786688. The separation and purification method of the present invention is suitable for crude products with a content of 2-50% myriocin, so a preliminary analysis and estimation of the content of myriocin in the sample should be carried out before separation. The preliminary analysis method of the content of myriocin in the crude product of myriocin can adopt literature (solid-phase extraction combined with HPLC to measure the content of mycelium of cicadae cicadae, Yu Jiawen etc., Chinese Journal of Pharmaceutical Sciences, 43: 1191-1193, 2008) provided high performance liquid chromatography for determination.

Wherein step (1) the preferred preparation of the mixed solution of solvent A and acid catalyst is: ethyl acetate, dehydrated alcohol, formic acid, prepare by volume ratio 5.5～8.5: 0.5～4.0: 0.5～1.5, preferably Ethyl acetate, absolute ethanol, and formic acid were prepared in a volume ratio of 7:3:1.

Wherein the solvent B in step (2) is preferably prepared with ethanol and water at a volume ratio of 10-20:1-5.

Wherein the alkali and water added in step (3) must be sufficient, the amount of added alkali needs to dissolve myriocin lactone completely, the mol ratio of added water and myriocin lactone must be greater than 3 times, Make the myriocin lactone monomer react completely, otherwise the yield and product purity will be affected.

Wherein the base added in the step (3) is preferably pyridine.

In order to confirm that the myriocin crude product obtained after the technical scheme of the present invention is processed is the myriocin monomer, the following experiment has been done:

1. Weigh about 5 mg of the residue obtained in step (3), dissolve it with 10 mL of methanol, and perform HPLC analysis. Adopt 2695 high performance liquid chromatographs (U.S. Waters Company), comprise 2996 diode array detectors, online degasser, autosampler, column thermostat; Chromatographic column is InertsilODS-3 (GL Sciences Inc., 4.6mm * 250mm , 5μm), the column temperature is 35℃, the mobile phase is acetonitrile/water (75:25, V/V, filtered through a 0.45μm microporous membrane), the flow rate is 1.0mL/min, the injection volume is 10μL, and the detection wavelength is 201nm . Result is shown in accompanying drawing 1, and as seen at retention time is that a peak occurs at 10.1min, and peak shape is good (the peak of about 3min of retention time is solvent peak). Spectral analysis of this peak with a diode array detector and a purity check with Waters chromatographic workstation software showed that the peak was a single peak with terminal absorption.

2. Weigh about 2 mg of the residue obtained in step (3), then add about 200 mg of dry KBr powder, grind it thoroughly in an agate mortar, put it into a tablet machine and press it into a transparent sheet, and then put it into an infrared spectrophotometer Scanning, the scanning range is 400 ~ 4000cm ^-1 , the obtained infrared spectrogram has strong absorption at 3440.8, 2958.6, 2929.7, 1666.4, 1537.2, 1450.4, 1384.8, 1082.0cm ^-1 , which is consistent with the literature (Myriocin, a new antifungal antibiotic from Myriococcumalbomyces. Kluepfel D, et al. Journal of Antibiotics, 25: 109-115, 1972) reported the same infrared spectrum of myriocin monomer.

3. Weigh about 5 mg of the residue obtained in step (3), dissolve it with 10 mL of methanol, and inject 2 μL into a mass spectrometer for mass spectrometry analysis. ABI Q-TRAP mass spectrometer (Applied Biosystems, USA) is selected, and the scanning range m/z is 100-800, in the ESI negative ion full-scan mode, the [MH] ^- quasi-molecular ion peak is mainly generated, and its (MH) is 400.3, and its molecular weight is 401, which is the same as the theoretical value of myriocin monomer.

Based on the above high-performance liquid chromatography analysis results, infrared spectrum analysis results, mass spectrometry analysis results and other data, it can be determined that the residue obtained in step (3) of the analyzed component is the myriocin monomer.

The present invention is realized through reaction separation coupling technology: the crude product of myriocin to be separated and purified is coupled with the chemical reaction and separation process under the condition of chromatographic medium, acid catalyst and solvent to obtain myriocin lactone, and then alkalization of myriocin lactone to obtain purified myriocin monomer. The schematic diagram of the chemical reaction occurring in the whole process is shown in Figure 2.

Utilizing the principle of the present invention, the crude myriocin can also be used to prepare the myriocin lactone monomer, while the prior art conventionally uses the myriocin monomer to prepare the myriocin lactone monomer.

The beneficial effect of the present invention is that the crude myriocin is separated and purified in a chemical manner, and the chemical reaction and separation are coupled together and performed simultaneously. Derivatize the target compound in the mixture with similar properties into new substances with different properties, and at the same time separate and purify the new substances from the complex system. This greatly simplifies the subsequent purification process of the product, improves the yield, reduces energy consumption, reduces investment, and is suitable for industrial production.

Description of drawings

Figure 1 HPLC chart of myriocin monomer

The schematic diagram of the chemical reaction involved in Fig. 2 of the present invention

Specific embodiment (total 2 examples)

Example 1

(1) Take 150 grams of 100-150 mesh silica gel, mix well with ethyl acetate and load the column, rinse with ethyl acetate/absolute ethanol/formic acid (7:3:1, V/V) and balance the chromatographic column, spare. Take 500 mg of crude product with about 10% myriocin content and dissolve it with absolute ethanol, stir evenly so that the sample is fully adsorbed on 5 grams of silica gel, then evaporate the solvent and put it on the column; use ethyl acetate/absolute ethanol /formic acid (7:3:1, V/V) elution, followed by thin-layer chromatography, and collected myriocin lactone fractions.

Thin-layer chromatography is to apply the eluent of step (1) on a silica gel GF ₂₅₄ thin-layer plate with a capillary tube with an inner diameter of 0.5 mm. After the sample is dried, it is placed in a closed chromatography cylinder for development. The developing agent is chloroform/methanol /water (65:30:5, V/V). After development, the solvent is evaporated to dryness, sprayed with ninhydrin ethanol solution with a mass concentration of 2%, and then heated in an oven at 105-110°C to develop color.

(2) The myriocin lactone fraction obtained by the above steps was evaporated to dryness, and ethanol at a temperature of 75° C. was added to prepare a saturated solution of myriocin lactone, cooled to 0° C. for crystallization, and the crystals were collected. Polycinolide monomer is about 10mg.

(3) Take the obtained myriocin lactone monomer, add pyridine and water to a volume of 10 mL and 2 mL respectively, reflux for 2 hours, evaporate the solution to dryness, and the obtained residue is the myriocin monomer.

Example 2

(1) Take 1.5 kg of 80-100 mesh silica gel, pack it into a column, wash it with ethyl acetate/formic acid (6:0.2, V/V) and equilibrate the chromatographic column, and set aside. Get about 3.5 grams of crude product of myriocin content about 5% and make it dissolve with dehydrated alcohol, stir to make sample fully adsorbed on 30 grams of silica gel, after evaporating solvent, upper column; Use ethyl acetate/formic acid (6 : 0.2, V/V) elution, followed by thin-layer chromatography, and collected myriocin lactone fractions.

Thin-layer chromatography is to apply the eluent of step (1) on a silica gel GF ₂₅₄ thin-layer plate with a capillary tube with an inner diameter of 0.5 mm. After the sample is dried, it is placed in a closed chromatography cylinder for development. The developing agent is chloroform/methanol /water (65:30:5, V/V). After development, the solvent is evaporated to dryness, sprayed with ninhydrin ethanol solution with a mass concentration of 1%, and then heated in an oven at 105-110°C to develop color.

(2) The myriocin lactone fraction obtained by the above steps is evaporated to dryness, and a mixed solution prepared by volume ratio of 1:1 by volume ratio of 50% and 30% ethanol and methanol is added at 80° C. Prepare a saturated solution of myriocin lactone, cool to 4° C. for crystallization, collect crystals, and the monomer of myriocin lactone is about 40 mg.

(3) Take the obtained myriocin lactone monomer, add pyridine and water, the volumes are 20mL and 60mL respectively, reflux for 2 hours, evaporate the pyridine and the aqueous solution to dryness, and the residue obtained is the myriocin monomer .

Example 3

(1) Take 3 kg of 80-100 mesh silica gel, pack it into a column, rinse and balance the column with ethyl acetate/absolute ethanol/n-butanol/acetic acid (8:2:2.5:1.5, V/V), and set aside . Take 4 grams of crude product with a myriocin content of about 20% and dissolve it with absolute ethanol, stir evenly so that the sample is fully adsorbed on 50 grams of silica gel, then evaporate the solvent and put it on the column; use ethyl acetate/dehydrated alcohol /n-butanol/acetic acid (8:2:2.5:1.5, V/V) was eluted, tracked by thin layer chromatography, and the myriocin lactone fraction was collected.

Thin-layer chromatography is to apply the eluent of step (1) on a silica gel GF ₂₅₄ thin-layer plate with a capillary tube with an inner diameter of 0.5 mm. After the sample is dried, it is placed in a closed chromatography cylinder for development. The developing agent is chloroform/methanol /water (65:30:5, V/V). After development, the solvent is evaporated to dryness, sprayed with ninhydrin ethanol solution with a mass concentration of 1%, and then heated in an oven at 105-110°C to develop color.

(2) The myriocin lactone fraction obtained through the above steps, evaporate the solvent to dryness, add a methanol solution with a temperature of 80° C. and a concentration of 30% by mass to prepare a saturated solution of myriocin lactone, and cool Crystallize at room temperature, collect crystals, about 300 mg of polycinolide monomer.

(3) Take the obtained myriocin lactone monomer, add pyridine and water, the volumes are 5mL and 50mL respectively, reflux for 2 hours, evaporate the pyridine and the aqueous solution to dryness, and the residue obtained is the myriocin monomer .

Claims (4)

1. the chemical separation purification process of a myriocin bullion may further comprise the steps:

(1) get the myriocin weight content be 2～50% myriocin bullion with anhydrous alcohol solution after silica gel column chromatography separate, with the mixing solutions wash-out of solvent orange 2 A and acid catalyst, collection myriocin lactone cut; The volume ratio of wherein said solvent orange 2 A and acid catalyst is 6～12.5: 0.2～1.5; Said solvent orange 2 A is one or more solvents of forming by arbitrary proportion that are selected from ETHYLE ACETATE, acetone, absolute ethyl alcohol, methyl alcohol, the propyl carbinol, and said acid catalyst is a kind of in formic acid or the acetate;

(2) with the myriocin lactone cut evaporate to dryness of step (1) gained, residuum is collected crystal with solvent B crystallization, gets the myriocin internal ester monomer; Wherein said solvent B is one or more solvents of forming by arbitrary proportion that are selected from ethanol, methyl alcohol, the water;

(3) getting many spherical shells bacterium internal ester monomer places alkali and water to form reaction system; The myriocin internal ester monomer under alkaline condition with the water complete reaction; Obtain myriocin solution, then this solution evaporate to dryness is got the myriocin monomer that residuum is separation and purification; Wherein said alkali is pyridine.

2. the chemical separation purification process of myriocin bullion as claimed in claim 1, wherein the mixing solutions of said solvent orange 2 A of step (1) and acid catalyst is ETHYLE ACETATE, absolute ethyl alcohol, a formic acid by volume 5.5～8.5: 0.5～4.0: 0.5～1.5 prepares.

3. the chemical separation purification process of myriocin bullion as claimed in claim 2, wherein the mixing solutions of said solvent orange 2 A of step (1) and acid catalyst is that ETHYLE ACETATE, absolute ethyl alcohol, formic acid were prepared in 7: 3: 1 by volume.

4. the chemical separation purification process of myriocin bullion as claimed in claim 1, wherein step (2) said solvent B is prepared by the second alcohol and water by volume at 10～20: 1～5.

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