Mechanisms Of Absorption And Cellular Antioxidant Of Crocetin,derived From Gardenia Jasminoides

In this work,highly purified Crocetin,a composition derived from Gardenia Jasminoide, was successfully prepared trough an alkali hydrolysis reaction. Its physical and chemical properties,absorption mechanism as well as antioxidant activities were systematic investigated. Briefly, the work consisted in:1.An alkali hydrolysis reaction was performed with crocins, which were extracted from Gardenia fruit,for preparing highly purified Crocetin.The optimum preparation conditions were discussed by a quadratic regression rotary unitized design method,combined with UV spectrophotometry and high performance liquid chromatography with mass spectrometry(HPLC-MS).Physical indicators such as solubility and stability of the prepared Crocetin were also evaluated to provide theoretical references for the further researches.Results are shown as following:Crocins were hydrolyzed by 10% of KOH at a ratio of 1:5(g:m L) at 60 oC for 2h,then neutralized by 1mol·L-1HCl. The neutralization product was further centrifuged under freezing condition and repetitive scrubbing with distilled water,and then collected by freeze drying process.The theoretical preparation yield was 57.23% with total purity for 95.2±3.0%. The chemical structures found were isomers.Crocetin showed better solubility in solvents with medium polarity and good stability in acid conditions.2. Caco-2 cells in a monolayer state were successfully established and assessed as the absorption model.The TEER values of the cells monolayer reached values from 230 to 280?·cm2 after 21 days in vitro, as a culture.The mannitol efflux of fluorescein sodium was lower than Papp<10-7cm·s-1.Tight junction between the Caco-2 cells was formed with good permeability as well, which suggested that the established monolayer can be used as an model for absorption studies in vitro.Transport studies showed that the safety absorption concentration was less than 0.1mg·m L-1,in this case, the cell survival rate was more than 80%. The PappAPâ†'BL was determined as PappAPâ†'BL(0.01mg·m L-1,180min)=14.009±0.534>10-6cm·s-1,which implied a fast absorption.The value of PappAPâ†'BL/PappBLâ†'AP=2.346>2 and the influence of the P-gp on the value of PappAPâ†'BL,suggested that Crocetin was absorbed by active uptake across the intestinal epithelial cells trough a passive mechanism. Meanwhile,the absorption may simply be dose-dependent.3.The antioxidant activity of Crocetin was evaluated by using BPLC method and the determination of radical scavenging capacity for DPPH,reducing power assay according to the indicator measurements of the radical scavenging rates.The protective effect of Crocetin on H2O2-induced MRC-5 cell injury was further investigated.The cell morphology,cell survival rates and cell cycles of the oxidant damaged model on MRC-5 cell were evaluated using MTS method and flow cytometry cycle detection technology.