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Method for preparing acacetin by enzymatic hydrolysis of buddleoside

A technology for preparing gold and acacetin from anthocyanin, which is applied in the field of bioengineering, can solve the problems such as the lack of examples of enzymatic hydrolysis of anthocyanin, and achieve the effects of high product purity, strong reaction specificity, and reduced separation process

Inactive Publication Date: 2017-05-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are relatively few examples of enzymatic hydrolysis of mongoside. The main reason is the limitation of enzyme sources. It is still worth exploring to obtain higher content and high activity monoglycosides or aglycones by screening suitable tool enzymes and reaction conditions. field

Method used

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  • Method for preparing acacetin by enzymatic hydrolysis of buddleoside
  • Method for preparing acacetin by enzymatic hydrolysis of buddleoside
  • Method for preparing acacetin by enzymatic hydrolysis of buddleoside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Take a 100ml Erlenmeyer flask and add 38.5ml Tris-HCl buffer (pH7.6), 7.5ml crude enzyme solution, 3.6ml HP-β-CD solution, 1.25ml 20mM mongoside solution, 0.4ml 1M CaCl2 solution, after closing the bottle mouth, reacted at 200rpm rotary shaker at 35°C for 1h, and carried out HPLC analysis after the reaction (detection wavelength 330nm, mobile phase: acetonitrile (B) / pure water (A), gradient is, 0.0-4.0min, 30%-42%B; 4.0-4.5min, 42%-70%B; 4.5-12min, 70%-90%B; 12-16min, 90%-90%B; 16.0-16.5, 90%-30% B). Such as figure 1 It was shown that after 1 hour of reaction, the conversion rate of mongoside was above 85%, and the product was acacetin. It can be seen that the enzyme can efficiently convert mongoside.

Embodiment 2

[0025] Take a 100ml Erlenmeyer flask and add 41ml Tris-HCl buffer solution (pH7.6), 5ml crude enzyme solution, 3.6ml HP-β-CD solution, 1.25ml concentration of 20mM mongoside solution, 0.4ml concentration of 1M CaCl2 Solution, after closing the bottle mouth, reacted at 200rpm rotary shaker at 35°C for 2h, and carried out HPLC analysis after the reaction (detection wavelength 330nm, mobile phase: acetonitrile (B) / pure water (A), gradient: 0.0- 4.0min, 30%-42%B; 4.0-4.5min, 42%-70%B; 4.5-12min, 70%-90%B; 12-16min, 90%-90%B; 16.0-16.5, 90% -30% B). Such as figure 2 As shown, after 2 hours of reaction, the conversion rate of mongoside is above 96%, and the product is acacetin, which shows the potential advantage of the enzyme in efficiently preparing acacetin.

Embodiment 3

[0027] Take a 100ml Erlenmeyer flask and add 43.5ml Tris-HCl buffer (pH7.6), 2.5ml crude enzyme solution, 3.6ml HP-β-CD solution, 1.25ml 20mM mongoside solution, 0.4ml 1M CaCl2 solution, after closing the bottle mouth, reacted at 200rpm rotary shaker at 35°C for 6h, and carried out HPLC analysis after the reaction (detection wavelength 330nm, mobile phase: acetonitrile (B) / pure water (A), gradient is, 0.0-3.0min, 30%-42%B; 4.0-4.5min, 42%-70%B; 4.5-12min, 70%-90%B; 12-16min, 90%-90%B; 16.0-16.5, 90%-30% B). Such as image 3 It can be seen that after 6 hours of reaction, mongoside is almost completely converted, and the product is acacetin. It can be seen that the crude enzyme solution can be used as a tool for efficiently preparing acacetin.

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Abstract

The invention discloses a method for preparing corresponding aglycone acacetin by enzymatic hydrolysis of buddleoside, and belongs to the field of bioengineering. The method comprises crude enzyme fluid preparation, enzymatic hydrolysis and purification steps, specifically comprises the following steps: (1) preparation of a crude enzyme fluid: inoculating cellulosimicrobium cellulans into a liquid culture medium, performing culturing for 40h at 30 DEG C, performing 9,000g centrifugation for 10min, extracting supernatant, precipitating ammonium sulfate, collecting 20 to 40 percent of components, and performing re-dissolving with a buffer solution; (2) enzymatic hydrolysis: converting the buddleoside serving as a raw material with the dissolved crude enzyme fluid to hydrolyze beta-D-glucoside bonds of 7 loci to obtain the acacetin; and (3) preparation of the acacetin: after a reaction is completed, mixing the conversion solution and methanol in a double volume, heating to 60 DEG C, repeatedly extracting for 3 to 5 times, and collecting, cooling and crystalizing an extracting solution to obtain the acacetin. The method has the advantages of mild reaction condition, high specificity, high efficiency, low cost and the like, and the purity of the obtained acacetin is over 98 percent.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for preparing acacetin by enzymatically hydrolyzing mongoside. Background technique [0002] The traditional medicinal plant Chrysanthemum chrysanthemum has the functions of dispelling wind, clearing away heat, improving eyesight, detoxifying, etc. It can treat coronary heart disease, lower blood pressure, prevent hyperlipidemia, antibacterial, antiviral, anti-inflammatory, anti-aging and other pharmacological activities. The chemical components in wild chrysanthemum are relatively complex, including flavonoids, triterpenoids, etc., which are the main active ingredients. At present, diosmin and its glycosides, apigenin and its glycosides, luteolin and Its glycosides, quercetin and its glycosides, and flavonoids such as acacetin and its glycosides. Among them, acacetin and its glycosides have significant pharmacological activities, and acacetin has been recogniz...

Claims

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Application Information

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IPC IPC(8): C12P17/06
Inventor 杨凌王平葛广波窦同意崔攀
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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