The Extract Of The Flavonoids From Ginkgo Biloba Leaf And The Effect On Human Hepatoma SMMC-7721Cell

Objective：To prepare the crude flavonoids extracted from Ginkgo biloba leaf and to detectits inhibition on human hepatoma SMMC-7721cell growth.Explored the mechanismof the inhibition of proliferation and the promotion of apoptosison on humanhepatoma SMMC-7721cell by observing the morphological changes and detected theexpression of gene Caspase-3、P21, to provide a theoretical and experimental basisfor Ginkgo leaves flavonoids used for anti-tumor therapy in clinical.Methods：Extracted the flavonoids from Ginkgo biloba leaf by the microwave aidedmethod with the optimum extraction parameters（ethanol concentration of90%、microwave time of3min、material-to-liquid ratio of1∶25）.CCK8assay was used todetect the growth inhibition of the extracts on SMMC-7721cell in differentconcentrations. Cultured human hepatoma cells SMMC-7721and seeded in96-wellplates.Divided into five groups by drug concentration(Drug concentrations:100μg/mL、200μg/mL、300μg/mL、400μg/mL、500μg/mL)and set a cell-free control groupanother.set up four wells each group and separated cells without drugs blankwells.The different concentration of flavonoids was added into the wells for24hours、48hours respectively.Detected the proliferation o f SMMC-7721cells withvarious centrations of Ginkgo leaves flavonoids by using the CCK-8assay andobserved the morphological changes of SMMC-7721cells by phase-contrastmicroscopy.seeded SMMC-7721cells in6-well plates and cultured24hours.Extracted RNA from SMMC-7721cells after adding flavonoids with differentconcentrations into the wells for24hours.Reverse Transcriptase-Tolymerase chainreaction (RT-PCR) was used to measure the mRNA levels of gene Caspase-3and P21.Results：The flavonoids yield of microwave assisted ethanol warm immersion is1.52%extracted with the optimum extraction parameters（ethanol concentration of90%、 microwave power of600W、microwave time of3min、material-to-liquid ratio of1∶25）.CCK8assay indicated that the inhibition rates of SMMC-7721treated withdifferent concentrations of Ginkgo biloba leaf Flavonoids were significantlyincreased compared with the control group incubated in vitro for24h、48h.Theconcentration of inhibition rate(%)respectively are16.2+2.5、26.9+2.9、42.4+3.1、60.6+3.8、67.2+4.6in the experimental group for24h and25.1+2.1、37.3+3.0、57.1+3.1、73.4+4.6、82.3+5.2for48h respectively.They all have significantdifferences compared with the blank control group(P<0.01). Ginkgo biloba leafFlavonoids significantly inhibited the proliferation of SMMC-7721in adose—dependent and time—dependent manner．After stimulation of SMMC-7721byGinkgo biloba leaf Flavonoids with the concentrations of100μg/mL、200μg/mL、300μg/mL、400μg/mL、500μg/mL for24h，phase-contrast microscopy showed themorphological changes of SMMC-7721，including cells shrinkage,separated fromtheir neighboring cells，Cytoplasm concentrated,Nuclear concentrated,the number ofcells reduced and other changes. The changes were mo re significant as theconcentration increased．The expression level of Caspase-3、P21gene increased as thedose of Ginkgo leaves flavonoids increased detected by RT-PCR.The formation ofDNA ladder Can be seen clearly by agarose gel electrophoresis．The relative gray ofCaspase-3for different concentration groups are respectively0.337+0.02、0.446+0.03、0.611+0.028、0.754+0.021、0.858+0.032and They all have significantdifferences compared with the blank control group （0.137+0.017）(P<0.01).Therelative gray of P21for different concentration groups are respectively0.380+0.031、0.522+0.031、0.690+0.030、0.829+0.035、0.893+0.035and They all have significantdifferences compared with the blank control group（0.166+0.019）(P<0.01)Conclusion：1、 In this study, the optimum extraction conditions screened are ethanolconcentration of90%、microwave time of5minutes、material-to-liquid ratio of1∶25.The yield of microwave assisted ethanol warm immersion is1.52%.2、 Human hepatoma SMMC-7721cells can be inhibited by differentconcentrations of flavonoides in a dosage and time dependent manner.3、Apoptosis of hepatoma cells SMMC-7721could be induced by Ginkgo bilobaleaves flavonoids, whose action strength was in direct ratio to its concentration。The effects were dose-dependent。The mechanism to prompte the apoptosis of hepatomacells SMMC-7721may be due to the gene modulation.