Study On The Proteins Formed By Self-assembly From Gegen-qin-lian Decoction

Gegen-qin-lian decoction is a classic traditional Chinese medicine which comes from Shang Han Lun. This decoction composes of puerarin, baicalin, berberine, and licorice root. It can relieve the heat and quench in body. The researches are more concentrated in puerarin, berberine and other active ingredients from Gegen-qin-lian decoction, but it has few studies in the decoction multiphase system, especially colloidal system for association studies. In recent years, some literature shown that traditional Chinese medicine decoction appeared colloidal aggregates, this finding provided the mechanism of traditional Chinese medicine decoction of a new direction.This subject is selecting the protein as a subject to examine the self-assembly of supramolecular aggregates by heating up the protein separated from Gegen-qin-lian decoction. This subject is a preliminary exploratory experiment for understanding the mechanism of Gegen-qin-lian decoction in the future, first there is separation and purification of the major proteins of pueraria and coptis chinensis, moreover, it quantitatively analyzed the proteins, then it primarily research purified proteins for the optimum condition of forming nano-particles, property, cytotoxicity, and its capacity as earner.The experiment obtained the Pueraria protein (named PP) and the Coptis protein (named CP), After a series of studies in protein such as N-terminal sequencing, mass spectrometry, sequence comparison, experimental verification, it had confirme the character of Pueraria protein and Coptis protein finally. Pueraria protein is a kind of trypsin inhibitor, and Coptis protein is a kind of plant glycoprotein. This kind of trypsin inhibitor could inhibited the activity of natue Pueraria protein completely with the concentration of 1/2, but lose the activity of inhibit at 90℃, the optimum condition is pH 8, while pH did few influence on Pueraria trypsin inhibitor.Used the laser particle analyzer to realize the character of two kinds of proteins nanoparticles formed by self-assembly. It selected the optimum conditions for forming nano-particles, the Pueraria protein nanoparticles formed in conditions:pH 6.05,100℃, keeping 60 min, the protein concentration of 0.089 mg/mL; the Coptis protein nanoparticles formed conditions:pH 7.15,80℃, keeping 60 min, the protein concentration of 0.109 mg/mL.The result of experimental drug carrying capacity shows that the efficiency of Pueraria protein particles embedded puerarin is 33.8%, the efficiency of Coptis protein nanoparticles embedded berberine is 44.2%. This result explained that the nanoparticles could combine with on the active ingredients. Cytotoxicity used L-02 hepatic cells and Hep-G2 hepatoma cells, the results show that Pueraria protein inhibited the L-02 cells and Hep-G2 cells growth, while conducted no influence from Coptis protein studied. Nanoparticles contained with Pueraria and berberine all could inhibit Hep-G2 cells growth.