Studies On Biotransformation Mechanism Of Mistletoe By Photosynthetic Bacteria And The Anti-tumor Fractionations

Objective:The changes of enzymes from photosynthetic bacteria were selected as factors to explore the biotransformation mechanism of mistletoe by photosynthetic bacteria.To study on protein and total triterpenes of photosynthetic bacteria transforming mistletoe culture fluid.Methods:Experiment 1:(1) The pH of photosynthetic bacteria transforming mistletoe culture fluid (PSBT) were detected by pH meter every day; (2) The PSB's electrophoretogram of protein, esterase(EST), peroxidases(POD) and superoxide dismutase(SOD) were established by gel electrophoresis; (3) Indole was added to mistletoe culture fluid, then the indigo of PSBT was determined by HPLC after a period of time.Experiment 2:After centrifugalization of PSBT, the supernatants and biomass precipitation were collected. Biomass precipitation were broken by ultrasonic assisted freezing and thawing, then centrifuged by hydroextractor, the supernatants were biomass extracts. The PSBT's supernatants and biomass extracts were precipitated with ammonium sulphate and dialyzed, the solutions were protein fractions. The anti-tumor activities of protein fractions on human tumor cell in vivor were estimated by MTT method.The protein fractions of PSBT's supernatants were purified by Hitrap Desalting column and CM FF column with AKTA purifier 10 chromatographic system in different buffer systems.Experiment 3:Total triterpenes of PSBT and extracts of mistletoe were determined by UV spectrophotometery. Oleanic acid was identified by thin layer chromatography,and the contents of oleanic acid was determined by HPLC. The chromatography column was Diamonsil C18 column (200mm×4.6mm, i.d.5μm). The mobile phase was methanol-phosphoric acid-water (86:0.18:14) at a flow rate of 1mL/min. The detection wavelength was set at 210 nm. The column temperature was 25℃Results:Experiment 1:The pH of PSBT decreased rapidly, while the pH of pure PSB culture fluid rose slowly; The PSB's electrophoretograms of protein, EST and POD were different between PSBT and pure PSB culture fluid; There weren't PSB's electrophoretograms of SOD in every culture fluid; After the adding of indole, indigo were generated in PSBT, while there wasn't indigo in pure PSB culture fluid.Experiment 2:The 50% inhibiting concentration (IC50) of the protein fractions as follows. (1) The IC50 values of PSBT supernatants to HO-8910(mg/mL):①IC50<200mg/mL:R. palustris transforming mistletoe culture fluid containing water extracts(JZ) was 0.18, mistletoe culture fluid containing water extracts(JD) was 20, R. sphaeroides transforming mistletoe culture fluid containing water extracts(JQ) was 60;②200mg/mL 2000mg/mL:R. sphaeroides transforming mistletoe culture fluid containing ethanol extracts(CQ), R. palustris transforming mistletoe culture fluid containing ethanol extracts(CZ), mistletoe culture fluid containing ethanol extracts (CD).(2) The IC50 values of PSBT supernatants to SGC-7901 (mg/mL):①IC50<200mg/ mL:JD,JZ,JQ,CQ;②200mg/mL2000mg/mL:pure R. sphaeroides culture fluid (Q),pure R. palustris culture fluid (Z),SQ,CQ;(4) The IC50 values of PSB to SGC-7901 (IC50, mL/mL):①IC50<200mg/mL:JZ was 14,JQ was 18;②200mg/mL