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A novel calcifediol (25-hydroxyvitamin D3) separation and purification method

A hydroxyvitamin and calcifediol technology, applied in the field of bioengineering, can solve the problems of less elaboration of the calcifediol extraction and purification process, difficulty in industrialized scale-up production, high equipment cost and high cost, and achieves strong repeatable operation and equipment. Low cost and high throughput

Active Publication Date: 2019-11-01
山东惠仕莱生物科技有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] At present, the biotransformation of calcifediol is mostly focused on the screening of enzyme-producing bacteria, the optimization of fermentation enzyme production conditions and the research of enzyme conversion technology, such as: Patent CN 103898004 B Pseudonocardia and its fermentation method to produce calcifediol Alcohol method and patent CN201210112509.6 A method for microbial transformation and production of 25-hydroxyvitamin D, a method for producing calcifediol by microbial transformation is mentioned, and several hydroxylase-producing bacteria are proposed species and fermentation enzyme production method and enzyme catalysis method, but the extraction and purification process of calcifediol is described less
Zeng Zhigang et al. in the paper "Actinomycetes SIIA243 on Vitamin D 3 Hydroxylation Research” mentioned that silica gel column and preparative chromatography can be used to purify calcifediol. However, due to the high cost of equipment and complicated operation, this method cannot be mass-produced, so it is only suitable for analysis and detection of product components in laboratories. It is difficult to carry out industrial scale-up production

Method used

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  • A novel calcifediol (25-hydroxyvitamin D3) separation and purification method

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Experimental program
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Effect test

Embodiment 1

[0032] (1) Vitamin D is catalyzed by microbial enzymes 3 Generate calcifediol (25-hydroxyvitamin D 3 ) into the conversion solution, ethyl acetate was added at a volume ratio of 1:1, extracted by shaking for 1 h, and the organic solvent in the upper layer was collected to obtain calcifediol extract a.

[0033] (2) Concentrate the above calcifediol extract a under reduced pressure, recover ethyl acetate, redissolve the concentrate with methanol, adjust the concentration of calcifediol to 0.5 mg / L, and obtain column solution b.

[0034] (3) After the agarose gel filtration chromatography is activated, the column is packed by a wet method, and the upper column solution b is passed through the agarose gel filtration chromatography column at a flow rate of 2BV / h, and the chromatographic molecular weight is selected to be 400Da. 1BV of ethyl acetate was used as the eluent, and it was eluted at a flow rate of 2BV / h. The eluate was collected, concentrated under reduced pressure at 60...

Embodiment 2

[0036] (1) Vitamin D is catalyzed by microbial enzymes 3 Generate calcifediol (25-hydroxyvitamin D 3 ) into the conversion solution, add ethyl hexanoate at a volume ratio of 1:2, shake and extract for 0.5 h, collect the organic solvent in the upper layer, and obtain calcifediol extract a.

[0037] (2) Concentrate the above calcifediol extract a under reduced pressure, recover ethyl caproate, redissolve the concentrate with methanol, adjust the concentration of calcifediol to 0.8 mg / L, and obtain column solution b.

[0038] (3) After the dextran gel filtration chromatography is activated, the column is packed by a wet method, and the upper column solution b is passed through the dextran gel filtration chromatography column at a flow rate of 2BV / h, and the chromatography molecular weight is selected as 500Da, using 1BV of ethyl acetate as the eluent, eluted it at a flow rate of 2BV / h, and collected the eluate.

[0039] (4), the above-mentioned eluent was concentrated under red...

Embodiment 3

[0042] (1) Vitamin D is catalyzed by microbial enzymes3 Generate calcifediol (25-hydroxyvitamin D 3 ) into the transformation solution, petroleum ether was added at a volume ratio of 1:5, extracted by shaking for 0.5 h, and the organic solvent in the upper layer was collected to obtain calcifediol extract a.

[0043] (2) Concentrate the above calcifediol extract a under reduced pressure, recover petroleum ether, redissolve the concentrate with methanol, adjust the concentration of calcifediol to 1 mg / L, and obtain column solution b.

[0044] (3) After the agarose gel filtration chromatography is activated, the column is packed by a wet method, and the upper column solution b is passed through the agarose gel filtration chromatography column at a flow rate of 2BV / h, and the molecular weight of the chromatography is selected to be 700Da. 1BV of ethyl acetate was used as the eluent, and it was eluted at a flow rate of 2BV / h, and the eluate was collected.

[0045] (4), the above-...

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Abstract

A novel calcifediol (25-hydroxyvitamin D3) separation and purification method is provided. The method mainly includes subjecting calcifediol to extraction and separation with an organic solvent; concentrating an extract liquid; dissolving the concentrate again to obtain a calcifediol stock solution for column chromatography; preliminarily purifying the calcifediol product by utilizing a gel chromatographic column; then further purifying and separating the calcifediol product by utilizing macroporous adsorption resin; finally preparing a high-purity calcifediol product through column chromatography on silica gel; subjecting an eluate to concentration and crystallization to obtain a calcifediol crystal product. The method is advantageous in that 1) the calcifediol product obtained by separation and purification through the method is high in purity and high in yield; 2) gel column chromatography and macroporous resin adsorption chromatography are applied for separation and purification ofthe calcifediol product for the first time; 3) the method is simple in process and convenient to operate, a plurality of times of cyclic operation can be performed, the equipment cost is low and themethod is particularly suitable for industrial promotion; 4) organic solvents adopted in the method can be recycled and reused, so that the production cost is reduced, and the method is economical andenvironmentally friendly.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a novel calcifediol (25-hydroxyvitamin D 3 ) Separation and purification method. Background technique [0002] Calcifediol, also known as 25-hydroxyvitamin D 3 [25(OH)VD 3 ], is vitamin D 3 A biologically active derivative of vitamin D 3 It has no activity in the human body and must be converted into active biological components by biological enzymes to play a role. Animal experiments have shown that calcifediol has obvious effects on metabolic bone diseases such as osteoporosis, rickets, and osteomalacia, and can also be used to treat hypocalcemia caused by hemodialysis. It can also be used as a health food raw material and a feed additive, and has a wide range of applications in health food, medicine and feed additives. [0003] Calcifediol can be synthesized by chemical method, but the chemical synthesis method has disadvantages such as many reaction steps, low conversion r...

Claims

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Application Information

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IPC IPC(8): C07C401/00
CPCC07C401/00C07C2601/14C07C2602/24
Inventor 孙荣刘建民任明张雷
Owner 山东惠仕莱生物科技有限公司
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