Method for measuring activity of beta-glucosidase in propolis

A technology of glucosidase and propolis, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, color/spectral characteristic measurement, etc., to achieve the effect of low detection cost and high sensitivity

Inactive Publication Date: 2009-07-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on the study of β-D-glucosidase in propolis. In order to confirm the existence of this enzyme, a method for the extraction and identification of β-D-glucosidase in propolis must be established.

Method used

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  • Method for measuring activity of beta-glucosidase in propolis
  • Method for measuring activity of beta-glucosidase in propolis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Take 3.0g each of propolis from different origins (Pinghu, Shaoxing, Jiangshan), dissolve it in 25mL pH 6.0 disodium hydrogen phosphate-citric acid buffer, add 0.9g PVPP, a little quartz sand, and grind on an ice bath to a paste, 10000g Centrifuge at 4°C for 15 minutes, transfer the supernatant to another centrifuge tube, and centrifuge at 14000g for 30 minutes at 4°C to take the supernatant to a constant volume of 25 mL. Take two test tubes, add 0.5 mL crude enzyme solution and 0.5 mL of p-nitrophenol β-D-glucoside with a substrate concentration of 25 mmol / L. One of the test tubes is incubated at 37°C for 1.5 hours. Always keep the solution uniform during the reaction, add 2.5mL 1M Na after incubation 2 CO 3 Stop the reaction. The other is a blank control. The reaction mixture is immediately heated in a 100℃ water bath for 5 minutes to inactivate the enzyme, and then 1M Na is added. 2 CO 3 Stop the reaction. Measure the absorbance of p-nitrophenol at 400nm with a spectroph...

Embodiment 2

[0022] Take 3.0g each of propolis from different origins (Pinghu, Shaoxing, Jiangshan), dissolve it in 25mL pH 6.0 disodium hydrogen phosphate-citric acid buffer, add 0.6g PVPP, a little quartz sand, and grind on an ice bath to a paste, 10000g Centrifuge at 4°C for 15 minutes, transfer the supernatant to another centrifuge tube, and centrifuge at 14000g for 30 minutes at 4°C to take the supernatant to a constant volume of 25 mL. Take two test tubes, add 0.5mL crude enzyme solution and 0.5mL p-nitrophenol β-D-glucoside with a substrate concentration of 30mmol / L. One of the test tubes is incubated at 57℃ for 1.5h. Always keep the solution uniform during the reaction, add 2.5mL 1M Na after incubation 2 CO 3 Stop the reaction. The other is a blank control. The reaction mixture is immediately heated in a 100℃ water bath for 5 minutes to inactivate the enzyme, and then 1M Na is added. 2 CO 3 Stop the reaction. Measure the absorbance of p-nitrophenol at 400nm with a spectrophotometer to ...

Embodiment 3

[0025] Take 3.0g each of propolis from different origins (Pinghu, Shaoxing, Jiangshan), dissolve it in 25mL pH 5.0 disodium hydrogen phosphate-citric acid buffer, add 0.6g PVPP, a little quartz sand, and grind it on an ice bath to a paste, 10000g Centrifuge at 4°C for 15 minutes, transfer the supernatant to another centrifuge tube, and centrifuge at 14000g for 30 minutes at 4°C to take the supernatant to a constant volume of 25 mL. Take two test tubes and add 0.5 mL crude enzyme solution and 0.5 mL p-nitrophenol β-D-glucoside with a substrate concentration of 10 mmol / L. One of the test tubes is incubated at 37°C for 1.5 hours. Always keep the solution uniform during the reaction, add 2.5mL 1M Na after incubation 2 CO 3 Stop the reaction. The other is a blank control. The reaction mixture is immediately heated in a 100℃ water bath for 5 minutes to inactivate the enzyme, and then 1M Na is added. 2 CO 3 Stop the reaction. Measure the absorbance of p-nitrophenol at 400nm with a spectr...

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Abstract

The invention provides a method for measuring Beta-D-glucosaccharase activity in propolis. The enzymatic activity is determined by the amount of the Beta-D-glucosaccharase which can hydrolyze a given amount of the substrate into pnitrophenol in a certain time using spectrophotometric determination method. The method can determine the presence of the Beta-D-glucosaccharase in propolis, and distinguish the difference of the propolis from the other propolis plant from the angle of the component and pharmacological activity, and distinguish the true propolis from the false propolis from the angle of the raw material propolis, thereby providing the criterion of the propolis quality standardization. The method has features of simpleness, high sensitivity and low measurement cost.

Description

Technical field [0001] The present invention belongs to the determination of effective components in propolis, and relates to a method for determining β-glucosidase activity in propolis. Background technique [0002] Propolis is a fragrant colloidal solid that is processed by honeybees from plant buds, bark and trunk cracks, and mixes them with mandibular gland secretions and beeswax. The chemical composition of propolis is complex, containing flavonoids, acids, alcohols, phenols, aldehydes, esters, ethers, enes, terpenes, steroids, and a variety of amino acids, fatty acids, enzymes, vitamins and a variety of trace elements. Among these chemical components, due to the high content of flavonoids and strong biological activity, research on the extraction and efficacy of flavonoids in propolis has become a hot spot. [0003] Flavonoids mostly exist in the form of flavonoid glycosides in nature, and only a very small amount exist in the form of free flavonoid aglycones. Although the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31C12Q1/34C12Q1/00
Inventor 胡福良张翠平
Owner ZHEJIANG UNIV
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