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

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.

Description

technical field [0001] The invention relates to a method for measuring the in vitro biological activity of leech medicinal materials, decoction pieces and processed products. It belongs to the field of traditional Chinese medicine. Background technique [0002] Leech is an animal medicinal material, which tastes salty, bitter, flat in nature, and returns to the liver meridian. It has the effect of breaking blood and removing blood stasis. Contained in various editions of "Chinese Pharmacopoeia", its medicinal materials are the dried whole body of leech (Wide-bodied golden leech) Whitmania pigra Whitman, leech (Japanese medical leech) Hirudonipponica Whitman or willow leaf leech Whitmania acranulata Whitman, summer and autumn Captured in the second season, scalded to death with boiling water, dried in the sun or dried at low temperature; the decoction pieces are washed, cut into sections, and dried; the processed product is scalded leeches, which are cleaned leeches and scal...

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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 the results of the biological activity measurement of medicinal materials, and the curative effects of the two in clinical oral preparations are also basically the same. To analyze the reasons, the antithrombin under the medicinal materials of leeches Activity determination is to detect the effect of macromolecular hirudin substances extracted from normal saline, but hirudin mainly exists in the salivary glands of blood-sucking leeches such as Japanese medical leeches, and hirudin has not been found in non-blood-sucking leeches such as broad-bodied golden leeches. There is a big difference between the two in vitro activity tests, and after oral administration, the hirudin and other macromolecular proteins in the Japanese medical leech are degraded into small molecular peptides in the gastrointestinal tract before being absorbed into the blood, and the hirudin structure of the Japanese medical leech is Destruction and anticoagulant activity are weakened, while the broad-bodied golden line leech is less affected, resulting in the antithrombin activity of the two in vivo is basically the same.

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