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A method for extracting FR901464 from Burkholderia fermented liquid

A technology of Burkholderia and extraction method, applied in the direction of resistance to vector-borne diseases, organic chemistry, etc., can solve the problem of not being able to obtain high purity, and achieve the effect of simple operation steps

Active Publication Date: 2022-05-20
ZHEJIANG HUIDA BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although Nakajima H etc. disclosed the purification method in "New antitumor substances, FR901463, FR901464 and FR901465 I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities", this method needs to consume a large amount of ethyl acetate for extraction, and the applicant Tried and couldn't get high-purity FR901464

Method used

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  • A method for extracting FR901464 from Burkholderia fermented liquid
  • A method for extracting FR901464 from Burkholderia fermented liquid
  • A method for extracting FR901464 from Burkholderia fermented liquid

Examples

Experimental program
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Effect test

preparation example Construction

[0042] (1) Preparation and cultivation of slant strains:

[0043] Slant medium formula (g / L): yeast extract powder 4.0g / L, malt extract 10.0g / L, glucose 4.0g / L, agar 20.0g / L, pH before elimination 7.2~7.4, test tube 30× 200mm, filling volume 15mL, sterilized at 121°C for 20min, cooled to about 55-60°C and placed on a slope, after cooling and solidification, inoculated to the slope, cultured at 28±1°C for 3 days, the strains matured.

[0044] (2) Preparation and cultivation of seed solution:

[0045] Seed medium formula (g / L): glucose 30g / L, sorbitol 5g / L, cottonseed cake powder 10g / L, yeast extract powder 20g / L, calcium chloride 10g / L, magnesium sulfate 10g / L, phosphoric acid Potassium dihydrogen 1g / L, pH 7.0 before disinfection; 250mL Erlenmeyer shaker flask, filled with 50mL, sterilized at 121°C for 20min. Vaccination 10 7 ~10 8 cfu / mL into the seed medium, 28±1°C, 250rpm shaking culture for 24 hours, at this time the pH of the culture solution is 6.8-7.2, and the OD600 ...

Embodiment 1

[0048] Embodiment 1 macroporous resin adsorption

[0049] Take four groups of fermentation broths, add macroporous resins of different specifications according to the table below, stir and absorb, take the fermentation supernatant, and detect the titer of FR901464 in the supernatant by HPLC.

[0050]

[0051] Adsorption rate (%) = 100% - supernatant titer / fermentation pot titer * 100%

Embodiment 2

[0052] Embodiment 2 solvent extraction

[0053]Obtain the mixture of fermented liquid and macroporous resin LX-30 according to the adsorption method of the first group in the above-mentioned Example 1, and obtain the macroporous resin LX-30 through vibrating sieve separation, and add the extraction solvent to the macroporous resin according to the following table After stirring in LX-30 for 2h, separate and collect the extract. The potency of FR901464 in the extract was detected by HPLC.

[0054]

[0055]

[0056] Extraction yield = extraction solution titer * extraction solution volume / (resin weight * resin titer) * 100%

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Abstract

The invention provides a method for extracting FR901464 from a fermented liquid obtained by fermentation of Burkholderia. The fermentation method includes: adding the macroporous resin to the fermentation liquid, mixing and absorbing, and filtering; soaking and separating the filtered macroporous resin with an organic solvent, and concentrating the separated organic solvent; mixing the concentrated product with n-hexane and ethyl acetate The solvent is dissolved, and after chromatographic separation with silica gel, the eluent is obtained by eluting with ethyl acetate; It is a C18 filler, which is eluted with a liquid phase eluent to obtain an eluate; the eluate is extracted and the organic phase is collected and evaporated to dryness to form a solid powder.

Description

technical field [0001] The invention relates to the technical field of fermentation broth purification, in particular to a method for extracting FR901464 from Burkholderia fermentation broth. Background technique [0002] FR901464 is a natural compound with high anti-tumor activity. It was originally isolated and purified from Pseudomonas sp. by fermentation in 1996 by Nakajima et al. (Nakajima, H.; Sato, B.; Fujita, T. ; Takase, S.; Terano, H.; Okuhara, M. Antitumor substances, FR901463, FR901464 and FR901465. I. taxonomy, fermentation, isolation, physico-chemical properties and biological activities [J]. J. Antibiot., 1996, 49: 1196 -1203.), wherein, the Pseudomonas sp.No.2663 fermentation output described in the text is about 360mg / L. Its chemical structural formula is shown in Formula 1. [0003] [0004] As a splicing inhibitor of precursor mRNA in eukaryotic cells, FR901464 has a strong inhibitory effect on the growth of tumor cells. Activity tests show that FR901...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D493/10
CPCC07D493/10Y02A50/30
Inventor 朱进伟陈世敏郑玲辉彭湘屏张敏石磊高祥汪超
Owner ZHEJIANG HUIDA BIOTECHNOLOGY CO LTD
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