Studies On The Antimutageic Functional Factor And Its Mechanism Of Codonopsis Lanceolata

Codonopsis lanceolata is a climbing herb belonging to campanulaceae codonopsis perennation and used for both food and medicine. As the effect of strengthening with tonics, it is regarded as high grade health food. This paper researched antimutagenic effect of Codonopsis lanceolata ethanol extract using mouse bone marrow cell micronucleus test, mouse teratospermia test and single cell gel electrophoresis assay, researched mechanism of anti-mutagenesis by determining the activity of SOD, content of MDA and test of cleaning free radical in vitro. The results showed that Codonopsis lanceolata ethanol extract significantly inhibit the micronucleus rate in cyclophosphamide(CP) treated mouse bone marrow cells, DNA damage in CP-treated rat lymphocyte and sperm abnormal rate in CP-treated mice. Codonopsis lanceolata ethanol extract significantly increased the activity of SOD and decreased the content of MDA in CP-treated mouse liver. Codonopsis lanceolata ethanol extract obviously cleared the hydroxy radical in vitro with dose dependent. In addition, Codonopsis lanceolata ethanol extract was able to reduce the frequency of micronuclei induced by CP using pretreatment, co-treatment and post-treatment protocol, but when the concentration of Codonopsis lanceolata ethanol extract was lower, the antimutagenic effect was the best using pretreatment protocol. The results of cytokinesis block micronucleus assay suggested that Codonopsis lanceolata ethanol extract able to protect intracellular genetic material as bio-antimutagen; meanwhile it was acting as a desmutagen to alter the rates of mutagen absorption and uptake or deactivate extracellularly the toxicity.Using Ames test to screen the antimutagenic active fraction of Codonopsis lanceolata demonstrated that the antimutagenic effect of acetoacetate fraction and petroleum benzin fraction were the most powerful. Six monomer compounds were obtained by using isolated methods of gel column chromatography from above two effective fractions and identified their structural formula by using the analysis techniques of physical and chemical constant and optical spectrum(MS, EI-MS, GC/MS, ¹H-NMR, ¹³C-NMR). These six compounds respectively were oleanolic acid, a-spinasterol, taraxerol, heptacosanic acid, ethyl hexadecanoate and 9-octadecyne. Among these compounds ethyl hexadecanoate and 9-octadecyne were found first time in Codonopsis lanceolata. Antimutagenic test carriedout with oleanolic acid, a-spinasterol, taraxerol and heptacosanic, which were higher yield compounds. The results showed that the first three compounds displayed stronger antimutagenic effect on the direct mutagen of sodium azide and indirect mutagen of B(a)P, and the most strong anti-mutagen among these three compounds was oleanolic acid. Thereby it was demonstrated that the main antimutagenic functional factor in Codonopsis lanceolata was oleanolic acid, but it was not singular material. The antimutagenic effect of Codonopsis lanceolata may be due to synergistic effect of multiple compounds.Using Ames test, cytokinesis block micronucleus assay and DNA lysis assay, the antimutagenic dose rage as well as preventive effect and mechanism of oleanolic acid isolated from Codonopsis lanceolata against to DNA damage were researched. The results showed that in the range of 25²00ug/plate oleanolic acid displayed a strong antimutagenic effect with dose-dependent increasing.Oleanolic acid significantly inhibit the CP-induced micronucleus frequency in rat lymphocyte in vitro. After pre-treated to rat spleen cells with oleanolic acid, the cells were irradiated by ultraviolet radiation can significantly reduce DNA breaking damage, and promoting DNA repair process.Taken together, our findings indicate that oleanolic acid possesses anti-frameshift mutation, base replacement mutation and chromosomal aberration, protect genetic material free from UV-damage. The major mechanism is antioxidation and free radical clearing and promoting damaged DNA repair process.The best extracting condition of oleanolic acid was researched using uniformity design. The results showed that when concentration of ethanol was 50%, volume of ethanol was 18 time of Codonopsis lanceolata, time of recirculation was 1.5h, temperature of recirculation was 95 °C, the yield of oleanolic acid was the highest. This condition can use to processing of production of Codonopsis lanceolata antimutagenic health foodvConclusion:1. The ethanol extract of Codonopsis lanceolata showed powerful antimutagenic effect. Its main active components are oleanolic acid, taraxerol and a-spinasterol. The effect of oleanolic acid is the most powerful, so it is the main antimutagenic functional factor in Codonopsis lanceolata.2. Main mechanism of antimutagenesis of Codonopsis lanceolata extract is cleaning active oxygen radical, protecting activity of antixoidase, quickly entering to intra-cellular and protecting DNA againt damage, as well as promoting DNA repair process.3. Optimization condition of oleanolic acid extraction: concentration of ethanol is 50%, usage amount of ethanol is 18 time of herb, the temperature of refluxing extract is 95 °C, time of extract is 1.5h.