Studies On Isolation, Quality Control And Pharmacodynamics Of Active Ingredients Of Radix Et Rhizoma Dysosmae Difformis

Traditional Chinese medicines modernization has been a hot topic in recent years, and the main task is quality control, extraction and isolation and pharmaceutical efficiency. Dysosmae Difformis is one of Berberidaceae Dysosma plants and distributes widely in Hunan Province. Radix and Rhizoma of Dysosmae Difformis are usually used as drug. It has qingrejiedu and sanjiequyu efficacy, and can treat lymph nodes swollen, lumps, pneumonia, etc. To our best knowledge, the research about Dysosmae Difformis is very less. For utilizing the resources of Dysosmae Difformis fully in Hunan province, we focus on the chemical components, quality control, extraction and isolation of active components, pharmacodynamics, etc. The results provide scientific basis for the industrialization of Dysosmae Difformis.1. Research about the main chemical constituents of Dysosmae Difformis for the first time.Six compounds including two lignans and four flavonoids have been isolated from the ethanol extract of Dysosmae difformis using various modern chromatographic techniques, and the structures have been identified as podophyllotoxin,4'-demethylepipodophyllotoxin, kaempferol, quercetin, quercitrin and rutin, by classical chemical methods and modern spectrometric approaches such as UV, IR, EI-MS, FAB-MS, 1H-NMR and 13C-NMR, etc. All the six compounds were isolated from Dysosmae difformis for the first time.2. Systematical research on quality control of Rhizoma Dysosmae difformis, and formulating the control standard.The sources, characteristics, identification, test and determination of Rhizoma Dysosmae difformis have been investigated by requests of ChP 2005, volumeⅠ. The source and the characteristics have been determined. The method for microscopic identification of the powder characteristics has been established and the TLC chromatogram identification of podophyllotoxin and quercetin have also been optimized. The impurity is less than 2%. The the moisture content is less than 15%. The heavy metal content is less than 5 ppm, and the arsenic level is less than 2 ppm. HPLC method for determination of Podophyllotoxin has been established. Separation was performed using Kromasil-C18 column (250mm×4.6mm, i.d.) with 5μm particle size. Mobile phase was consisting of methanol: 0.1%H3PO4= 60:40 (v/v). All separations were operated at room temperature and a flow-rate of 1.0 ml/min. The detective wavelength was at 292 nm. There was a good linear relationship over the range of 0.20-2.00μg for podophyllotoxin (r= 0.9999). The average recovery rate was 100.57%, and RSD was 0.99%. On those basis, we worked out the Control Standard Draft of Radix et Rhizoma Dysosmae difformis.3. Studies on chromatographic fingerprinting of Dysosmae Difformis for the first time.The HPLC fingerprints of Radix et Rhizoma Dysosmae difformis were systematically studied. Samples are prepared by ultrasonic extraction technology with methanol. The detective wavelength was at 254 nm, while the other chromatographic conditions were as the same with those in the determination sections. The method had high precision, good repeatability and all of the components were separated well, so it can be used as the means to assess the fingerprints of Radix et Rhizoma Dysosmae difformis.The paper analyzed chromatograms of 131 samples from seven habitants of China, such as Hunan, Hubei, Guizhou etc. Thirteen five peaks were identified as the characteristic fingerprints of Radix et Rhizoma Dysosmae difformis. Compared with reference substances, we can identify that the first, third, seventh, tenth and twelfth peak are rutin, quercitrin, quercetin, podophyllotoxin and Kaempferol, respectively. The fingerprints of different samples were compared with similarity evaluation software development by central south university. Average similarity of samples from different places was 0.965±0.018. It is obvious that the similarity is correlated with the habitats of herb. The closer the habitat is, the higher the similarity is.4,The extraction and isolation of podophyllotoxin were studied, and the new technology of extraction and isolation of podophyllotoxin were also established.The extraction technology of podophyllotoxin were studied by alcohol reflux and SFE-CO2. The experimental result indicated that the yield of podophyllotoxin extracted by alcohol reflux was more than that by SFE-CO2. The optimum extraction technology is as follows:Firstly,9 time of quantity 80%alcohol extracts for 60 min, and secondly,7 time of quantity 80%alcohol extracts for 45 mins.The isolation process of podophyllotoxin was studied by macroporous resin. The optimum isolation process is as follows: HPD-100^ LSA-5B and HPD-400 are the best resin to separate and purify podophyllotoxin, and with 4.81mg/ml podophyllotoxin solution to adsorb. First, the macroporous resin was eluted by distilled water and 10% alcohol to wipe off impurities. Second, the macroporous resin was eluted by 80%alcohol, and the eluting solvent was collected. The Content of podophyllotoxin is more than 80%.5. Investigating the effects of podophyllotoxin on human gastric cancer cell line SGC7901 cells in vitro and in vivo, and providing its the basis for clinical therapy of gastric cancer.SGC7901 cells were treated by podophyllotoxin at various concentrations for different times. The effects of podophyllotoxin on the growth and apoptosis of SGC7901 cells in vitro has been determined by MTT assay, colony formation assay and flow cytometry, respectively. Furthermore, xenograft experiment was performed to detect the effects of podophyllotoxin on the in vivo tumorigenicity of SGC7901 cells.The results were as the followings:podophyllotoxin inhibited the growth of SGC7901 cells in a concentration-and time-dependent manner, and inhibited the colony formation rate of the cells. Podophyllotoxin induced SGC7901 cell apoptosis and arrested SGC7901 cells at G2/M phases of cell cycle. Podophyllotoxin decrease the volume and weight of SGC7901 cells xenografts in nude mice.Podophyllotoxin could inhibit the growth of human gastric cancer SGC7901 cells, induce the apoptosis of SGC7901 cells in vitro, and inhibit the in vivo tumorigenicity of SGC7901 cells, which provide theoretical basis for clinical therapy of gastric cancer using podophyllotoxin.