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Method of measuring activity of scutellaria baicalensis endogenous beta-D-glucuronidase

A technology of aldolidase and glucuronide, which is applied in the field of determination of endogenous β-D-glucuronidase activity of Scutellaria baicalensis, can solve the problems of cumbersome operation, high detection cost, expensive reagents, etc., and achieve simple operation and high detection efficiency. The effect of low cost and high sensitivity

Inactive Publication Date: 2019-12-20
HUAIYIN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, ultraviolet spectrophotometry is the most common method for the determination of enzyme activity. There are reports that baicalin is used as a substrate to measure enzyme activity. This method will generate baicalein precipitates, which need to be dissolved in methanol and then measured at 450 nm. The operation is cumbersome. Reagents are expensive and costly
In addition, there is also a chromatographic method to detect the activity of baicalein β-D-glucuronidase by measuring the baicalein produced by the reaction, but the chromatographic method is time-consuming and the detection cost is high

Method used

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  • Method of measuring activity of scutellaria baicalensis endogenous beta-D-glucuronidase

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Effect test

Embodiment 1

[0049] Scutellaria baicalensis crude enzyme solution

[0050] After the Scutellaria baicalensis is dried in the sun, take an appropriate amount and add it to a pulverizer to pulverize, turn off the switch every 10 s to prevent excessive high temperature from denaturing and inactivating the enzyme, and pulverize about 5 times to make the sample uniform powder. Weigh 0.5 g of Scutellaria baicalensis powder, add the same amount of polyvinylpyrrolidone, 10 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution pre-cooled to 4 °C, soak overnight in the refrigerator at 4 °C, at 4 °C, 11000 r / min Centrifuge for 10 min, and take the supernatant to obtain the crude enzyme solution. The enzyme solution was stored at 4°C for future use.

[0051] Determination of β-D-glucuronidase activity

[0052]Take two 10 mL centrifuge tubes, tube 1 is used as a control, and tube 2 is used as a sample for measurement. Add 0.3 mL of inactivated enzyme solution treated in a boi...

Embodiment 2

[0055] Scutellaria baicalensis crude enzyme solution

[0056] After the Scutellaria baicalensis is dried in the sun, take an appropriate amount and add it to a pulverizer to pulverize, turn off the switch every 10 s to prevent excessive high temperature from denaturing and inactivating the enzyme, and pulverize about 5 times to make the sample uniform powder. Weigh 0.5 g of Scutellaria baicalensis powder, add 0.4 g of polyvinylpyrrolidone, 8 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution pre-cooled to 3°C, soak in the refrigerator at 3°C ​​for 8 hours, and centrifuge at 10,000 r / min at 3°C After 8 min, the supernatant was taken to obtain the crude enzyme solution. The enzyme solution was stored at 3°C ​​for future use.

[0057] Determination of β-D-glucuronidase activity

[0058] Take two 10 mL centrifuge tubes, tube 1 is used as a control, and tube 2 is used as a sample for measurement. Add 0.3 mL of inactivated enzyme solution treated in a b...

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Abstract

The invention discloses a method of measuring activity of scutellaria baicalensis endogenous beta-D-glucuronidase. The method comprises following steps of (1) preparing a buffer, a sodium carbonate solution and a paranitrophenol standard solution; (2) extracting scutellaria baicalensis crude enzyme; (3) drawing a standard curve with concentration of paranitrophenol as a horizontal axis and absorbance at absorbing peak as a longitudinal axis; (4) mixing the buffer, the crude enzyme and P-nitrobenzene-beta-D-glucuronide solution, heating and reacting, after reaction, and measuring absorbance ofa reaction solution by using a UV spectrophotometer; and (5) substituting obtained absorbance through measurement into the standard curve, calculating molar concentration of paranitrophenol produced in reaction, and calculating activity of beta-D-glucuronidase by using a formula. The method measures quantity of paranitrophenol hydrolyzed from a certain amount of substrate by beta-D-glucuronidase within a certain time period by using UV spectrophotometry, so as to determine enzyme activity.

Description

technical field [0001] The invention belongs to the field of biochemistry, relates to an enzyme activity assay technology, in particular to a method for assaying endogenous β-D-glucuronidase activity of Scutellaria baicalensis. Background technique [0002] Scutellaria baicalensis is a plant of the Labiatae Scutellaria baicalensis ( Scutellaria baicalensis Georgi), as a traditional Chinese medicine, Scutellaria baicalensis has anti-bacterial, anti-inflammatory, anti-viral, anti-tumor and hemostasis and anti-abortion effects. Baicalin and baicalein are the main active ingredients in Scutellaria baicalensis. Baicalin has poor absorption and low conversion rate in the human body. The bioavailability and pharmacological activity of baicalein are higher than those of baicalin, but its content in Scutellaria baicalensis is low and it is easily absorbed by oxygen. And alkali damage, high production costs, so that its application has been greatly limited. Baicalin belongs to gluc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33G01N1/44
CPCG01N1/44G01N21/33
Inventor 喻春皓邹雯燕陈汝珺胡涛夏尧干钱婷婷闵志迪刘娜周跃刘星靳洋烨付晨伟纪香峰庹群
Owner HUAIYIN INSTITUTE OF TECHNOLOGY
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