Method of preparing esculentoside B through enzymatic hydrolysis of esculentoside A

A technology of pokeweed saponin A and pokeweed saponin B, applied in the field of bioengineering, can solve problems such as lack of examples, achieve high conversion efficiency, strong reaction specificity, and reduce the effect of separation process

Inactive Publication Date: 2016-11-23
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 pokeweed saponin A. The main reason is the limitation of enzyme sources. It is still worthwhile to obtain higher content and high activity monoglycosides or aglycones by screening suitable tool enzymes and reaction conditions. areas to explore

Method used

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  • Method of preparing esculentoside B through enzymatic hydrolysis of esculentoside A
  • Method of preparing esculentoside B through enzymatic hydrolysis of esculentoside A
  • Method of preparing esculentoside B through enzymatic hydrolysis of esculentoside A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Get 100ml Erlenmeyer flask and add 11ml phosphate buffer solution (PH6.0), 1.4ml concentration is 100mg / ml beta-glucosidase solution (make the final concentration of enzyme be 10mg / ml), 1.2ml concentration is 20mM pokeweed Saponin A solution, 400ul of 20mM CaCl 2 Solution, after closing the bottle mouth, reacted at 200rpm rotary shaker at 37°C for 12h, took samples every 1h and carried out TLC (such as figure 1 As shown, it is the TLC figure of Phyloside A and its reaction supernatant under the developing agent of different proportions), or HPLC analysis (detection wavelength 203nm, mobile phase is: acetonitrile (B) / pure water (A), mobile phase The ratio of A is, 0.01-8.0min, 60%-50%; 8.0-10.0min, 50%-10%; 10.0-13.0min, 10%-10%; 13.0-13.5min, 10%-60%; 13.5 -17.0min, 60%-60%), the conversion rate of pokeweed saponin A was 82.65% in 1 hour, and the conversion rate reached above 90.13% after 12 hours of reaction. like figure 2 Shown is the conversion diagram of pokewee...

Embodiment 2

[0027] Get 100ml Erlenmeyer flask and add 9.8ml phosphate buffer solution (PH6.0) successively, 2.8ml concentration is the beta-glucosidase solution of 100mg / ml (making the final concentration of enzyme be 20mg / ml), 1.2ml concentration is the quotient of 20mM Lusaponin A solution, 400ul concentration of 20mM CaCl2 solution, after closing the bottle mouth, reacted on a rotary shaker at 200rpm at 37°C for 12h, and took samples every 1h for TLC (such as figure 1 As shown, it is the TLC figure of Phyloside A and its reaction supernatant under the developing agent of different proportions), or HPLC analysis (detection wavelength 203nm, mobile phase is: acetonitrile (B) / pure water (A), mobile phase The ratio of A is, 0.01-8.0min, 60%-50%; 8.0-10.0min, 50%-10%; 10.0-13.0min, 10%-10%; 13.0-13.5min, 10%-60%; 13.5 -17.0min, 60%-60%), the conversion rate of pokeweed saponin A was 87.89% in 1 hour, and the conversion rate reached more than 93% after 12 hours of reaction. like image 3 S...

Embodiment 3

[0029] Take 100ml Erlenmeyer flask and add 5.4ml phosphate buffer solution (PH6.0), 7ml concentration is 100mg / ml β-glucosidase solution, 1.2ml concentration is 20mM pokeweed saponin A solution, 400ul concentration is 20mM CaCl 2 Solution, after closing the bottle mouth, reacted at 200rpm rotary shaker at 37°C for 12h, took samples every 1h and carried out TLC (such as figure 1 As shown, it is the TLC figure of pokeweed saponin A and its reaction supernatant under different developing agents), or HPLC analysis (detection wavelength 203nm, mobile phase is: acetonitrile (B) / pure water (A), gradient is , 0.01-8.0min, 60%-50%A; 8.0-10.0min, 50%-10%A; 10.0-13.0min, 10%-10%A; 13.0-13.5min, 10%-60%A; 13.5 -17.0min, 60%-60%A), the conversion rate of pokeweed saponin A was 95.59% in 1 hour, and the conversion rate reached more than 98% after 12 hours of reaction. like Figure 4 Shown is the transformation diagram of pokeweed saponin A under the enzyme concentration of 50mg / ml.

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Abstract

The invention discloses a method of preparing esculentoside B through enzymatic hydrolysis of esculentoside A. Beta-glucosidase is used for selectively hydrolyzing the beta-1,4-glucoside key in esculentoside A to prepare corresponding esculentoside B. The method includes the steps that a solid beta-glucosidase preparation is dissolved to prepare a corresponding enzyme solution, and by adjusting the solution concentration and reaction time, esculentoside A can be selectively hydrolyzed to be converted into esculentoside B but does not continues to be converted into aglycone. Conversion liquid is extracted with organic solvent, and esculentoside B is obtained through HPLC separation and purification. The defects that a traditional chemical method for hydrating esculentoside A is poor in selectivity, serious in pollution and low in efficiency, due to microbial fermentation, operation is tedious, and products are not single and are difficult to purify are overcome through the method.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for preparing phykroside B by enzymatically hydrolyzing phykroside A. Background technique [0002] The medicinal plant pokeweed has the functions of dispelling water and reducing swelling, promoting diuresis, detoxifying and dispelling stagnation, etc. The main active ingredients in pokeweed are triterpene saponins, also known as pokeweed total saponins, which have various pharmacological effects such as expectorant, cough suppressant, asthma, anti-inflammatory, antibacterial and antiviral. The total saponins of pokeweed are complex. At present, 21 triterpenoid saponins have been isolated from pokeweed, all of which are oleanane type, including pokeweed acid, pokeweed acid-30-methyl ester, pokeweed saponin, 5 types of mother nuclei, such as Gallic acid, Phytophthora G, etc. For each class of sapogenins, there are also significant differences in activity due to the differen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/20
Inventor 杨凌葛广波崔攀王平窦同意
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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