Effect Of Alkaloids From Rhizoma Coptis On The Expression Of Mitochondrial Complex Ⅲ

Diabetes mellitus (DM) is a metabolic disease resulted from hereditary factors, disorder of immunological fucking, microorganism infection, toxin and paychic factor, etc. Hyperglycemia is the main character of clinical manifestation, polyuria, polyphagia (three polys and one little) can appear in typical cases. If Diabetes mellitus is not in control, hyperglycemia can lead to the failure lesions of kidney, eye, foot and other parts, which cannot be cured. At present there’s no treatment method can completely cure for DM, medical treatment, such as sulfonylureas and other chemicals, leading to the diabetic complications, such as gastrointestinal reaction, nephrotoxicity and hepatotoxicity. As a consequence, the using of natural profucts, Rhizoma Coptisine (Huanglian, RC) for curing DM and diabetic complications is becoming a trend. RC is an important traditional Chinese medicinal herb which has been effectively used for treatment of DM, heat-clearing, damp-drying, relieving fidgetiness and detoxicating, etc. Its major bioactive components consist of five protoberberine alkaloids, which are berberine, palmatine, coptisine, epiberberine and jatrorrhizine. While at the same time, the mechanism of reducing blood sugar by RC has not been studied completely, it is necessary to study the drug’s ability enter the cells. The present study is to establish analytical methods for detecting protoberberine alkaloids of RC in cells, and study the distribution of alkaloids inside and outside cells, and alkaloids’function on the expression of Mitochondrial Complex Ⅲ, which has not been studied by previous research. In present study, a HPLC method was chosen due to Its higher column efficiency, lower solvent consumption and increased detectability to investigate the alkaloids absorbed by HepG2cells. Beyond that, a Western blot was chosen to detect the contents of Mitochondrial Complex Ⅲ.The main focus of the study is:(1) Culture of HepG2cells and detecting protoberberine alkaloids of RC in cells by HPLC.(2) Preparation for Mitochondrial Complex Ⅲ from HepG2cells.(3) Investigating the four alkaloids’function on Mitochondrial Complex Ⅲ by Western blot.(4) Investigating the function of derivatives of protoberberine on Mitochondrial Complex Ⅲ by Western blot.The results of the four experiments are shown as follows:(1) The authentic concentrations of alkaloids in HepG2cells can be detected by HPLC and calculated by formula:coptisine> berberine> epiberberine> palmatine, while jatrorrhizine can hardly be absorbed. In addition, with the increasing coptisine and berberine, the intramural concentrations also increase and show a liner correlation.(2) Preparation for Mitochondrial Complex Ⅲ from HepG2cells:the RIPA Lysis Buffer was chosen to crack cells.(3) To investigate the function of different berberines on Mitochondrial Complex III, Western blot was put into use:after the protoberberine were absorbed by cells, the Mitochondrial Complex Ⅲ protein amounts in HepG2cells showed a clear increase in Mitochondrial Complex Ⅲ protein levels by berberine and coptisine, epiberberine and palmatine almost had no effect.(4) To investigate the function of different derivatives of protoberberine on Mitochondrial Complex Ⅲ, Western blot was put into use, it was found that only C8(0.01) and C12(0.02) could have some certain stimulation on Mitochondrial Complex Ⅲ.