Method for preparing hesperetin, preparation method of hesperetin intermediate and biological enzyme for preparing hesperetin

A technology for hesperetin and hesperidin, applied in the field of biomedicine, can solve the problems of difficult product purification, low yield, high price and the like, and achieve the effects of less further hydrolysis, increased productivity and low cost

Active Publication Date: 2019-02-05
BONTAC BIO ENG SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the acid hydrolysis method is commonly used to hydrolyze neohesperidin (Neohesperidin) or hesperidin to obtain hesperetin. In addition, there are many methods for preparing hesperetin such as the aqueous phase-ethanol method, the methanol method, and the cyclohexanol method. However, these methods are cumbersome in process, serious in pollution, and difficult in product purification, etc.
The enzymatic hydrolysis method developed recently has the characteristics of low hydrolysis temperature and good control of hydrolysis reaction. However, the yield of enzyme produced by fermentation is not high, the price is expensive, and the hydrolysis rate is low. If the enzyme system contains multiple enzymes, The resulting reaction products are still complex, making separation and purification difficult

Method used

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  • Method for preparing hesperetin, preparation method of hesperetin intermediate and biological enzyme for preparing hesperetin
  • Method for preparing hesperetin, preparation method of hesperetin intermediate and biological enzyme for preparing hesperetin
  • Method for preparing hesperetin, preparation method of hesperetin intermediate and biological enzyme for preparing hesperetin

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Embodiment 1

[0071] A preparation method of hesperetin, comprising:

[0072] Suspend 100 g of hesperidin in 300 mL of water, and add 10 mol / L sodium hydroxide solution until the hesperidin is completely dissolved to obtain a substrate preparation.

[0073] The substrate preparation was added to the sodium phosphate buffer (300 mL) containing the crude enzyme solution (400 mL) co-expressed with α-L-rhamnosidase and β-glucosidase at a rate of 1 mL / min by substrate flow addition. , 100mM) to carry out stirring reaction to obtain the reaction solution; feed for about 5h until the substrate preparation solution is added and continue to react for 1h, control the reaction pH to 6.0, and maintain the temperature at 55°C;

[0074] Slowly add 2mol / L HCl to the above reaction solution, add to pH 5.0 and stir for 0.5h, filter to obtain the solid hesperetin, dry to obtain the crude product of hesperetin, ensure that the water content is not more than 5%; dissolve the crude product in 1000mL In methano...

Embodiment 2

[0089] A preparation method of hesperetin, comprising:

[0090] Suspend 100 g of hesperidin in 300 mL of water, and add 10 mol / L sodium hydroxide solution until the hesperidin is completely dissolved to obtain a substrate preparation.

[0091]The substrate preparation was added to the sodium phosphate buffer (300 mL) containing the crude enzyme solution (400 mL) co-expressed with α-L-rhamnosidase and β-glucosidase at a rate of 1 mL / min by substrate flow addition. , 100mM) to carry out stirring reaction to obtain the reaction solution; feed for about 5h until the substrate preparation solution is added and continue to react for 1h, control the reaction pH to 7.0, and maintain the temperature at 55°C;

[0092] Slowly add 2mol / L HCl to the above reaction solution, add to pH 5.0 and stir for 0.5h, filter to obtain the solid hesperetin, dry to obtain the crude product of hesperetin, ensure that the water content is not more than 5%; dissolve the crude product in 1000mL In methanol...

Embodiment 3

[0095] A preparation method of hesperetin, comprising:

[0096] Suspend 100 g of hesperidin in 300 mL of water, and add 10 mol / L sodium hydroxide solution until the hesperidin is completely dissolved to obtain a substrate preparation.

[0097] The substrate preparation was added to the sodium phosphate buffer (300 mL) containing the crude enzyme solution (400 mL) co-expressed with α-L-rhamnosidase and β-glucosidase at a rate of 1 mL / min by substrate flow addition. , 100mM) to carry out stirring reaction to obtain the reaction solution; feed for about 5h until the substrate preparation solution is added and continue to react for 1h, control the reaction pH to 6.5, and maintain the temperature at 55°C;

[0098] Slowly add 2mol / L HCl to the above reaction solution, add to pH 5.0 and stir for 0.5h, filter to obtain the solid hesperetin, dry to obtain the crude product of hesperetin, ensure that the water content is not more than 5%; dissolve the crude product in 1000mL In methano...

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Abstract

The present invention provides a method for preparing hesperetin, comprising: suspending hesperidin or neohesperidin in water, and adding a sodium hydroxide solution until the hesperidin or the new hesperidin is completely dissolved, and btaining a substrate preparation solution; adding the substrate preparation solution to a buffer containing alpha-L-rhamnosidase and beta-glucosidase at a rate of0.1 to 1 mL/min for stirring at a flow rate of 0.1 to 1 mL/min to obtain a reaction solution, and after the completion of the addition, continuing the reaction for 0.5 to 1 h, the pH of the reactionsolution is 6.0 to 7.0, and the reaction temperature is maintained at 45 to 65 DEG C. The alpha-L-rhamnosidase is derived from Streptomyces, and the beta-glucosidase is derived from the genus Thermotoxin; adjusting the pH of the reaction solution to 3.0 to 5.0 to completely precipitate the solid product, and collecting the solid product to obtain the hesperetin. The method is efficient, simple, green and environmentally friendly, and can be applied to industrial mass production.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method of hesperetin, a preparation method of a hesperetin intermediate and a biological enzyme for preparing hesperetin. Background technique [0002] Hesperetin (Hesperetin), the molecular formula is C 16 h 14 o 6 , melting point is 227.5 ℃, its water solubility is very poor, almost insoluble in water, also insoluble in chloroform and stupid, easily soluble in ethanol and so on. It exists mostly in the form of hesperidin in nature. Studies have shown that hesperetin is a kind of traditional Chinese medicine extract, which mainly exists in traditional Chinese medicinal materials such as tangerine peel, green peel, and citrus aurantium. And many other effects. [0003] At present, the acid hydrolysis method is commonly used to hydrolyze neohesperidin (Neohesperidin) or hesperidin to obtain hesperetin. In addition, there are many methods for preparing hesper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16C12P17/06C12N9/42C12N9/24
CPCC12N9/2402C12N9/2445C12P17/06C12P17/162C12Y302/0104C12Y302/01043
Inventor 傅荣昭刘立辉刘滔滔曹磊郭杏林
Owner BONTAC BIO ENG SHENZHEN
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