Study On Extraction, Purification, Structural Identification And Its Bioactivities Of Flavonoids In Lotus Leaf

The technologies of the extraction and purification of the total flavonoids from lotus leaf were studied in this thesis.The chemical, spectral, chromatographic and mass spectrographic analysis were used to research the structure and bioactivities of flavonoids in lotus leaf. The main results were as follows:1. Optimization for extraction process of flavonoids in lotus leavfEffects of extraction solvent,the surfactant concentration,time, temperature and solidsolvent ratio on extraction yields of flavonoids from lotus leaf were determined individually. The quadratic regression rotational combination design with three factors was used to optimize the extract process and the extraction rate of flavonoids from lotus leaf was chosen as the evaluating index. The equation was Y=67.03+2.68x₁+0.16x₂+2.94x₃-2.06x₁x₂-0.43x₁x₃+0.41x₂x₃-4.74x₁²-4.33x₂²-4.93x₃²,After optimizing the combination different factors by computer, the results showed that the optimum extraction parameters were : temperature(74.8-6.5℃),time(38-43min) and proportion of material and liquid(1:25-1:28). The gain ratio was 59.5%2. Study on methods for purification of flavonoids in lotus leafFive types of macroporous resin were compared in their ability of adsorbing and desorbing flavonoids in lotus leaf. The dynamic absorbing behavior of XAD16 resin was observed. The optimum conditions were as follows: the elution solution concentration 1.1mg/mL, the flow rate of absorption solution 1BV/h, , 50% ethanol as eluent,flow rate in 2BV/h,the elution peak was symmetrical in the optimum conditions.The flavonoids in lotus leaf was further purified by recrystallization and the purity was 92%.3. Preliminary study on the structure of flavonoids in lotus leafThe structure of flavonoids in lotus leaf were characterized by UV-VIS, FT-IR,HPLC-ESI/MS,HPLC and GC respectively. The structure was estimated as Isorhamnetin-3 -O-β-D-glucoside4. Bioactivities of flavonoids in lotus leaf(1)Antioxidation activity of flavonoids in lotus leaf in vitroEffects of flavonoids in lotus leaf on removal of superoxide anion, hydroxide radical and H₂O₂ was determined by Pyrogallol-Luminol system,CuSO₄-Phen-VC-H₂O₂ system and Luminol-H₂O₂ systems respectively. The effect of preventing DNA damage by flavonoids in lotus leaf was determined by CuSO₄-Phen-VC-H₂O₂-DNA chemilum inescence's system.The results showed that flavonoids in lotus leaf possessed a high scavenging potency on ROS and preventing DNA damage in different systems Effects of flavonoids in lotus leaf on antioxidation of zoogenic tissue in vitro were determined by chromatometry.The results showed that flavonoids in lotus leaf could eliminate OH effectively , inhibit lipid peroxidation induced by OH., protect the biomembrane, reduce RBC hemolysis and lighten the degree of liver mitochondria swelling. The results suggested that flavonoids in lotus leaf possessed remarkable antioxidation activities.(2) Effects of flavonoids in Lotus leaf on activities of amylase and lipase in vitroEffects of the flavonoids concentration,action time and dosage of enzyme on activities of amylase and lipase were determined by chromatometry.The results were as follows: (a)flavonoids in lotus leaf blocked the enzyme-substrate contact by modifying the emulsion of the lipid substrate, and the lipase inhibitor demonstrated uncompetitive inhibition for lipase; (b) The intensive inhibiting effect of crude flavonoids in lotus leaf on the amylase activity was higher than that of purified flavonoids.The results suggested that inhibiting effect of flavonoids in lotus leaf on amylase activity might be synergistic effect between flavonoids and other compounds in lotus leaf.