In-vitro biological activity determination method of leech medicinal materials, decoction pieces and processed products

A technology of biological activity and measurement method, applied in the field of in vitro biological activity measurement, can solve the problems of weak correlation, undetectable antithrombin activity, weakened anticoagulant activity, etc.

Pending Publication Date: 2021-09-17

山东禹泽药康产业技术研究院有限公司 +1

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AI-Extracted Technical Summary

Problems solved by technology

This test method has certain limitations: first, it can only control the original medicinal materials, and cannot measure the decoction pieces and processed products. Both leech decoction pieces and processed products need to be heat-treated, and the protein has been denatured after treatment (hirudin components It is also a kind of protein), physiological saline cannot be dissolved and extracted, and antithrombin activity cannot be detected; second, the anticoagulant activity characterized by this biological activity assay is not strongly correlated with the in vivo drug effect, and the Pharmacopoeia leech medicinal material item The difference in anticoagulant activity between leeches (Hirucia widebodied) and leeches (Hirucia japonica) in vitro measured by thrombin titration was more than 4 times, and the results of a large number of pharmacodynamic experiments in animals showed that Hirudos chinensis wide-bodied and Japanese medical leeches were different. The medicinal efficacy and activity of leeches in vivo are basically the same, and there is no four-fold difference in effect like t...

Abstract

The invention relates to an in-vitro biological activity determination method for leech medicinal materials, decoction pieces and processed products, which are characterized in that after leeches are subjected to protease enzymolysis by pepsin and trypsin, thrombin titration is carried out to determine the antithrombin activity. In-vitro activity detection can be carried out on leech medicinal materials, decoction pieces and processed products, and the in-vitro activity detection result is basically consistent with an in-vivo drug effect conclusion.

Application Domain

Chemical analysis using titrationBlood disorder +2

Technology Topic

Medicinal herbsProtease +9

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Examples

Experimental program(1)

Example Embodiment

[0052] Example 1

[0053] Determination of anticoagider enzyme activity of different batch wide gold ribs: collecting Rugao City, Jiangsu Province, Jining City, Shandong Province, 3 wide gold lines in Pinghu, Zhejiang Province, China Medicinal material

[0054] Preparation of the test solution: Take the water, pulverize the three sieves, weigh 2.5g, precision weighing, add water 20ml, weighing weight, heating at 85 ° C for 15 minutes, cooling to 40 ° C, using dilute hydrochloric acid to regulate pH 2.0, the addition of the enzyme foundation is 1% of the gastric protease (1: 15000), 40 ° C is enzymatically eoxidin, and the pH is 8.0 with a 20% sodium hydroxide solution, and the enzyme base is 1% trypsin (enzyme). The viability is 2500u / mg), 40 ° C is more than 3 hours, and the dilute hydrochloric acid is adjusted to neutral neutral, and heat for 15 minutes at 85 ° C, to be cooled to room temperature, centrifuge at high speed, the supernatant 25mL volumetric flask, precipitate and add 5ml Wash, high speed centrifugation, supernatant into the bottle, add water to the scale, mix, filter it,

[0055] Measurement method: Precision quantity is taken from 100 μl of the test solution, and the test tube is added to 200 μL, shake well, shaken, shaken, and improving the 37 ° C water bath containing 0.5% fibrinogen (solid object). 5 minutes, add 1 ml of 10 units of thrombin solution (1 time every 4 minutes, 2 μL each time, gently shake the side) to solidification, recording the volume of thrombin solution, according to the formula Calculate antipinease activity:

[0056] U = c 1 V 1 / (C 2 V 2 )

[0057] Where: U - per 1G otter contains anticoagulase active unit, U / g;

[0058] C 1 - Thrombin solution concentration, u / ml;

[0059] C 2 - G / mL of the test solution solution

[0060] V 1 - Volume, μL of the thrombin solution

[0061] V 1 The amount of the test solution, μL.

[0062] The measurement results are shown in the table below.

[0063] Table water vessel external anticoagulase activity measurement results

[0064]

[0065]

[0066] It can be seen from the above table, and the percentage of germplasm is 10u to 14u, and the average value is 11.73U; the Japanese medical zone antithrombin activity is 16U, slightly higher than the wide body gold line, It is basically comparable to the conclusions of the body.

PUM

Property	Measurement	Unit
Vitality	2500.0	U/mg

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