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Separation and purification method of pneumocandin B0 serine analog

A technology for separation and purification of pneumocidine, applied in the separation and purification of pneumocidine B0 serine analogues, pneumocidine B0 crude product separation and purification of pneumocidine B0 serine analogues, can solve the problem of not carrying out the separation and preparation of single compounds And other issues

Pending Publication Date: 2020-01-07
ZHEJIANG MEDICINE CO LTD XINCHANG PHAMACEUTICAL FACTORY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] These three papers respectively introduced the discovery, detection and structural identification of several structural analogues in the fermented broth of pneumocantine B0, but did not conduct research on the isolation and preparation of a single compound

Method used

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  • Separation and purification method of pneumocandin B0 serine analog
  • Separation and purification method of pneumocandin B0 serine analog
  • Separation and purification method of pneumocandin B0 serine analog

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: the preparation of pneumocantine B0 fermented liquid

[0047] With reference to the Chinese patent No. 201210066411.1 submitted by the applicant on March 14, 2012, the fermented liquid of pneumocidine B0 was prepared. The specific preparation method is as follows.

[0048] Strain: Zalerion arboricola (HCCB30111, CGMCC No.5412);

[0049] Incline medium: PDA;

[0050] Incline culture conditions: 28°C, 5 days;

[0051] Seed Medium (%): Glucose 2.5, KH 2 PO 4 0.9, yeast powder 0.5, drug vehicle 1.0, 85% lactic acid 0.2ml, trace elements 0.1ml, pH 6.0;

[0052] Seed culture conditions: Digging and inoculation, 25°C, 220rpm, 3 days;

[0053] Fermentation medium (%): sucrose 12.5, sodium glutamate 0.8, yeast powder 0.8, proline 1.5, KH 2 PO 4 0.15, magnesium sulfate heptahydrate 0.04, trace elements 1ml, MES buffer salt 1.5, pH 5.3;

[0054] Fermentation conditions: 10% seed transfer, 25°C, 220rpm, 7 days of fermentation;

[0055] Trace element compos...

Embodiment 2

[0057] Embodiment 2: the preparation of the crude product of pneumocidine B0 containing serine analog

[0058] With reference to the Chinese patent No. 201210066411.1 submitted by the applicant on March 14, 2012, the crude product of pneumocidine B0 containing serine analogs was prepared. The specific preparation method is as follows.

[0059] Washing and filtering: adopt ceramic membrane microfiltration equipment, carry out cross-flow filtration to 30L of pneumocantine B0 fermented liquid prepared by embodiment 1, obtain concentrated mycelium suspension; use 10% phosphoric acid in 0.05 μm ceramic membrane Adjust the pH to 3.0 to wash and filter the mycelia suspension.

[0060] Extraction and filtration: After washing with water, filter until about 15L of feed liquid containing mycelia is extracted directly with methanol-water solution (methanol volume ratio is 20%) ceramic membrane, and the temperature is controlled at 10-15°C for extraction and filtration operate.

[0061...

Embodiment 3

[0068] Embodiment 3: Separation and preparation of nemocontin B0 serine analog

[0069] a) The first chromatographic enrichment of nemocontin B0 serine analogs

[0070] The crude product of neomocontin B0 containing serine analogs is enriched by the first chromatographic enrichment with a polymer matrix chromatographic filler, such as UniPS from Navitas, with a particle size of 10 μm and a pore size of 300 A. The mobile phase is aqueous methanol, methanol The ratio is 75% (volume ratio), isocratic elution, and fractional collection to obtain the first chromatographic enrichment solution containing neumeridine B0 serine analogue with a purity > 30%.

[0071] b) The second chromatographic purification of nemocontin B0 serine analog

[0072] Concentrate under reduced pressure to remove the solvent from the first chromatographic enrichment liquid containing nemocontin B0 serine analogs with a purity > 30%, and then use a hydrophilic interaction chromatography filler, such as XAmi...

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Abstract

The invention relates to a separation and purification method of a pneumocandin B0 serine analog. The separation and purification method comprises the following steps of (a) performing chromatographyenrichment for the first time through polymer substrate chromatogram fillings, performing isocratic elution, and collecting parts of which the purity is greater than 30%; (b) performing chromatographyenrichment for the second time through chromatogram fillings having hydrophilic effects, performing isocratic elution, and collecting parts of which the purity is greater than 60%; (c) performing chromatography enrichment for the third time through antiphase silica gel substrate chromatogram fillings, performing isocratic elution, and collecting parts of which the purity is greater than 80%; and(d) after purifying for the third time, performing decompressed concentration on chromatography liquid of which the pneumocandin B0 serine analog purity is greater than 80%, removing a solvent, and performing freeze-drying so as to obtain the pneumocandin B0 serine analog. Through the adoption of the preparation method disclosed by the invention, the pneumocandin B0 serine analog of which the purity is greater than 80% is subjected to structure identification, and a base is established for further performing biological activity screening or performing biological activity screening after structure modification.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for separating and purifying pneumocidine B0 serine analogues, in particular to a method for separating and purifying pneumocidine B0 serine analogues from pneumocidine B0 crude products containing serine analogues method. Background technique [0002] Fungi are conditional pathogens for healthy humans and are harmless to the human body under normal circumstances; however, when the body's resistance decreases, it may cause local or systemic fungal infections. In recent years, due to the clinical abuse of cytotoxic drugs, immunosuppressants, broad-spectrum antibiotics and hormonal drugs, not only the immunity of the patient's body has been suppressed, but also the micro-ecological balance in the body has been destroyed, making the originally non-pathogenic fungi dominant bacteria, thereby inducing fungal infection; coupled with the development of open treatment methods su...

Claims

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Application Information

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IPC IPC(8): C07K7/56C07K1/20C07K1/16
CPCC07K7/56
Inventor 张磊
Owner ZHEJIANG MEDICINE CO LTD XINCHANG PHAMACEUTICAL FACTORY
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