A method for quality detection of Danshen medicinal material based on multi-target enzymes
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A quality detection method and target enzyme technology, which can be used in the measurement of color/spectral characteristics, etc., can solve the problems of difficult quality and cannot reflect the biological activity of Salvia miltiorrhiza, and achieve the effects of reducing analysis costs, reducing detection loss, and shortening the analysis process.
Active Publication Date: 2019-12-27
ZHEJIANG UNIV
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Problems solved by technology
However, the components detected by these methods only account for a small part of the medicinal ingredients of Danshen, and it is difficult to fully evaluate its quality, nor can it reflect the biological activity of Danshen
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Embodiment 1
[0039] 1. The quality detection method of Salvia miltiorrhiza based on thrombin, the specific steps are as follows:
[0040] (1) Take 5 different batches of Danshen herbal medicine 20g, add methanol-water (volume ratio: 7:3) mixed solution, ultrasonically extract for 30min, and dry with a centrifugal concentrator to obtain Danshen extract powder.
[0041] (2) Take the Salvia miltiorrhiza extract powder, dissolve the Salvia miltiorrhiza extract powder with a phosphate (PB) buffer solution of pH=6.5, and prepare a salvia miltiorrhiza sample solution; then take thrombin, dissolve the thrombin with a PB buffer solution of pH=6.5, Prepare a 0.2U / mL enzyme solution.
[0042] (3) Add the sample solution and enzyme solution obtained in step (2) to a 96 microwell plate and incubate for 1 hour, then add the thrombin substrate S-2238 to obtain a reaction solution, in which the sample solution, thrombin solution and S- The final concentrations of 2238 were 0.5mg / mL, 0.4U / mL and 0.4mM, re...
Embodiment 2
[0055] The reaction conditions involved in embodiment 1 and the optimization process of parameter are as follows:
[0056] 1. pH optimization of thrombin buffer and ACE buffer
[0057] (1) Take thrombin, dissolve it in PB buffer with pH of 6.0, 6.5, 7.0 and 7.5 respectively, add substrate S-2238 and react for 30 minutes, measure the absorbance, the higher the absorbance, the stronger the enzyme activity.
[0058] The results showed that when the thrombin was dissolved in the PB buffer of pH=6.5, the finally measured absorbance was the highest, so the PB buffer of pH=6.5 was used.
[0059] (2) Take ACE and dissolve it with Tris-HCl buffer solution with pH of 7.5, 7.8, 8.0, 8.3 and 8.5 respectively. After adding the substrate FAPGG, measure the absorbance immediately. After reacting for 30 minutes, measure the absorbance again. The greater the difference in absorbance indicating higher enzyme activity.
[0060] The results showed that when ACE was dissolved in the Tris-HCl buf...
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Abstract
The invention discloses a method for detecting the quality of radix salviae miltiorrhizae medicinal materials on the basis of multiple target enzymes. The method includes extracting radix salviae miltiorrhizae extract from radix salviae miltiorrhizae and dissolving the radix salviae miltiorrhizae extract by the aid of buffer solution to obtain sample solution; acquiring buffer solution and dissolving the target enzymes with the activity capable of being inhibited by the radix salviae miltiorrhizae so as to obtain enzyme solution; mixing the sample solution and the enzyme solution with each other to obtain mixed solution, incubating the mixed solution, adding target enzyme substrates into the mixed solution and carrying out reaction to obtain reaction solution used as sample groups; replacing the sample solution by the aid of the same quantity of buffer solution to be used as control groups, measuring the absorbance of the reaction solution of the sample groups and the absorbance of reaction solution of the control groups and computing the inhibition ratios of radix salviae miltiorrhizae samples for at least two types of target enzymes to judge the quality of the radix salviae miltiorrhizae medicinal materials. The method has the advantages that the inhibition degrees of the radix salviae miltiorrhizae for the diversified target enzymes are measured, the target enzyme inhibition ratios are computed, the inhibition ratios for the target enzymes are used as quality control indexes for reflecting the quality of the radix salviae miltiorrhizae medicinal materials, accordingly, the quality control indexes are related to clinical curative effects of medicines, and the quality of the radix salviae miltiorrhizae medicinal materials can be quickly, economically, efficiently and comprehensively evaluated.
Description
technical field [0001] The invention relates to the technical field of quality evaluation of traditional Chinese medicines, in particular to a method for detecting the quality of Danshen medicinal materials based on multi-target enzymes. Background technique [0002] Quality control of traditional Chinese medicine is a key factor restricting the modernization and internationalization of traditional Chinese medicine, and it is also one of the hot spots and difficulties of research. Quality control of traditional Chinese medicine is of great significance to ensure the safety, effectiveness and consistency of medicinal materials and their products. [0003] From appearance identification to microscopic identification, from index components to medicinal components, and then from thin-layer chromatography to fingerprints, the quality evaluation technology of traditional Chinese medicine has experienced a development process from scratch, from coarse to fine, and is gradually beco...
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