Study On Separation, Purification And Bioactivities Of Flavoncoids In Phellodendron Amurense Leaf

Phellodendron amurense is the main plant source of Chinese traditionalmedicine-Cortex Phellodendri, and the main medicine parts are the bark ofPhellodendron amurense. While the other parts of Phellodendron amurense such asleaves have not been efficiently utilized. Study demonstrates that the deciduous leavesof Phellodendron amurense have allelopathy and can also inhibit germination of itsown seeds, which sets barriers to self-renewal. Besides, the market demand is veryhuge because of Phellodendron amurense’s medicine value. The relation betweensupply and demand is extremely unbalanced and benefit and interest drive people toplunder wild Phellodendron amurense. As a result, Phellodendron amurense sourcesuffers from severe damage. In this study, we extract, separate and purify the flavonefrom Phellodendron amurense leaves and study the activity, on the one hand, whichcan help to relieve the barrier of self-renewal and contribute to the ecological diversity.On the other hand, the study aims to make full and effective use of the endangeredPhellodendron amurense so as to protect them.In the study, the flavone was extracted by organic solvent extraction fromdeciduous leaves of Phellodendron amurense. The single factor experiment showedthe influence of alcohol concentration, liquid-solid ratio, extraction temperature,extraction time and extraction times on flavone yield. In order to optimize theextraction condition, we deviced the orthogonal tests of four factors(alcoholconcentration, liquid-solid ratio, extraction temperature, extraction time) at threedifferent levels. The optimum extraction condition was1:20liquid-solid ratio,60%alcohol,70℃extraction temperature,2.5h extraction time and2times. UVspectrophotometry and color reaction experiments demonstrated that the flavoneextract mainly contain dihydroflavonol.The flavone extracted by the optimum condition was separated and purified.First, Petroleum ether was used to remove pigment and esters, then the flavone wasfurther extracted by ethyl acetate to get the purified falvone extract, which was then separated by silica gel column chromatography. Gradient elution of methanol andchloroform was used as mobile phase. TLC and HPLC were used to detect the eluent.The eluent had antioxidant activity was further separated by PreparativeLiquid Chromatography. Finally, we obtained one flavonoid compound. Themolecular structure of the compound was phellamurin by HPLC-MS and NMR.We measured and compared the activity of flavone extract, purified flavoneextract and flavonoid(phellamurin) by FRAP assay, DPPH free radical assay andeliminating superoxide anion free radical method. We found that all of them hadantioxidant activity and the antioxidant activity of purified flavone was higher thanflavone extract. To some extent, the flavonoid(phellamurin) also had antioxidantactivity, and the scavenging DPPH free radical rate was92.87%at4mg/mL. Theantioxidant activity increased according with the increase of concentrations by FRAPmethod. We also found that the flavone extracted from Phellodendron amurenseleaves could inhibit activity of DNA topoisomeraseⅠof tumor cells, which suggestedthe flavone had potential antitumor activity.