| In this paper, antioxidant activity of the olive leaf extract was evaluated by ·OH and DPPH· radical-scavenging assays, a HPLC method for determination of antioxidative compounds which were screened and identified in olive leaves extracts was developed and the urine pharmacokinetics features of urinary excretion investigated in rats after fed olive leaf extracts. This study provides a theoretical basis for the development of functional products with olive leaf extracts as raw materials. Main content as follows:1. Study on antioxidant activity of olive leaf extract(1) Study on antioxidant activity of olive leaf extracts by scavenging free radical assays. oleuropein and ascorbic acid were chosen as control, the phenanthroline-Fe2+oxidation and DPPH assay were used to evaluate the antioxidant activity of the olive leaf extracts. At the optimal conditions, the IC50 of olive leaf extracts, ascorbic acid and oleuropein to ·OH and DPPH· were 0.0791, 0.0457 and 0.0515 mg/ml respectively, 0.0743, 0.0581 and 0.0156 mg/ml respectively. Results showed that olive leaf extracts had certain scavenging activity to free radical.(2) The screening and determination of the four kinds of antioxidant compounds of olive leaf extracts. The chromatographic conditions was improved, to develop a HPLC method for simultaneous determination of four antioxidant compounds(oleuropein, hydroxytyrosol acetate, apigenin-7-O-glucoside, luteolin-7-O-glucoside) of olive leaf extract. The antioxidant compounds in olive leaf extracts were screened by HPLC-DPPH and identified by precision relative molecular mass measured by HPLC-MS, combined with retention time, UV spectrogram and references. HPLC chromatographic conditions, chromatography column was performed on an Sinochrom ODS-BP(250 mm×4.6 mm, 5 μm), the detection wave length were 230 nm and 345 nm, the mobile phase was water: acetonitrile(v:v, 75:25), flow rate of 1 ml/min. The limits of detection were 0.42-1.90 μg/ml, the recoveries were 98.12-107.8%, and the RSD% of precision, stability, repetitive were less than 3%.2. Study on urinary excretion features of olive leaf extracts in rats(1) Study on hydrolysis conditions of oleuropein metabolites. In this research, with the content of hydroxytyrosol as the assessment index, the concentration of hydrochloric acid, hydrolysis time and temperature as investigation factors, the hydrolysis conditions of oleuropein metabolites were optimized by single-factor and L9(34) orthogonal combination experiment. The optimum hydrolysis conditions were selected as follows: the concentration of hydrochloric acid was 2 mol/l, hydrolysis temperature was 90 ℃ and hydrolysis time was 60 min.(2) Urine extraction kinetics research of metabolites in rats based on HPLC. A HPLC method was developed to determine hydroxytyrosol in urine, and the features of urinary excretion in rats after olive leaf extracts and oleuropein ingestion was investigated by the HPLC method. The separation was achieved on an Sinochrom ODS-BP(250 mm×4.6 mm, 5 μm), Jade C18 protection column(12.5 mm×4.6 mm, 5 μm), detection wave length was 230 nm, flow phase was water: methanol(v:v, 75:25), flow rate was 1 ml/min. Linearity relationships of hydroxytyrosol concentration in urine ranges 4.49-575.00 μg/ml, the average recovery rate were 82.98-90.43%, precision, stability RSD% were less than 15%. 16 rats were divided into two groups, ingested 400 mg/kg of olive leaf extracts(oleuropein occupies 100 mg/kg) and 100 mg/kg oleuropein, respectively. After intragastric administration, 0~10 h was the main period of excretion for rats, up to 24 h, the total amount of hydroxytyrosol in urine excretion were(1907.57±302.15) μg and(366.28±99.05) μg respectively. During 2-4 h, Urinary excretion rate was the highest,(233.73± 60.04) μg/h and(69.20±25.19) μg/h, respectively. 24 h urine cumulative excretion/dosage ratio were 27.49% and 5.29% respectively. |
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