Extraction And Biological Activity Of Main Active Components Of Liangshan Introducted Olea Europaea L.levaves

Olea europaea L.is an evergreen tree of the Oleaceae family and is one of the world's four largest woody oil plants.Olive trees were introduced to China in 1956 and now has been cultivated in 67 counties(cities),37 prefecture-level cities,12 provinces.According to statistics,almost 80,000 hm² of total area are distributed in China.A huge quantity of olive leaves is generated as an agricultural byproduct of the olive oil industry and accumulates during pruning,up to 600,000 tons each year.However,these byproducts are burned or directly discarded,causing environmental pollution,waste of a resource,and ecological destruction,even increasing the cost of waste disposal for farmers.Therefore,BBD(Box-Behnken design)was used to optimize the main active ingredients of total polyphenols(TP),total flavonoids(TF)and oleuropein(OE)from olive leaves,then obtained the best extraction process.High performance liquid chromatography(HPLC)was used to determine and quantitatively analyze of phenolic and nutrient components in different varieties and different seasons.Finally,the antioxidant and anticancer activities of phenolic extracts(PE)and OE from olive leaves were evaluated in order to provide theoretical basis and technical support for the application of olive leaves in medicine,cosmetics and functional foods.The main results are as follows:1.BBD was optimized the extraction of total polyphenols from olive leaves by HWD and UAE.The optimum extraction conditions of HWD were as follows:temperature 75?,ethanol concentration 50%,solid-liquid ratio 1:40 g/m L,time 40 min,and TP yield 18.98%.The optimum process of UAE is as follows:solid-liquid ratio 1:20 g/m L,power 240 W,time 20 min,concentration 58%,and TP yield 31.08%.2.BBD was optimized the total flavonoids of HWD and UAE from olive leaves.The optimum process of HWD was methanol concentration 82%,solid-liquid ratio 1:29 g/m L,temperature 90?,time 106 min,and TF yield 3.07%.The optimum process of UAE is as follows:temperature 50?,power 270W,time 50 min,solid-liquid ratio 1:41 g/m L,TF yield 7.50%.3.BBD was optimized the optimal extraction process of oleuropein at 40?and 20min by UAE:ultrasonic power 240 W,solid-liquid ratio 1:20 g/m L,methanol concentration 78%,and OE yield 7.45%.4.There were significant differences in TPC,TFC,OE and six flavonoids in different olive varieties and seasons.Among them,the annual variation range of TPC,TFC and OE is:328.81-124.30 mg GAE/g,275.32-42.20 mg RE/G and 151.74-34.45 mg/g DW,respectively,and the overall performance is increased in spring and winter,but fell summer and autumn.Among them,luteolin and apigenin were the most predominant flavonoid compound,followed by quercetin and rutin,and apigenin and luteolin were the least.The TPC,TFC and OE contents in olive leaves of KLMD,ZL and ABSL were more stable in the 22 olive varieties,but the flavonoids in QMLE,ZL,XPG,GDE and BLT were more abundant.5.FAAC(free amino acid content),SSC(soluble sugar content)and SPC(soluble protein content)of olive leaves in different varieties showed irregular seasonal changes.The annual fluctuation range was 18.30-2.49,18.25-6.31 and 6.47-0.87 mg/g DW.FAAC and SPC increased in the spring,and SSC content was higher in May-July.Among the 22main varieties,FO,BLT and JF have higher FAAC,SSC and SPC than other varieties.6.The antioxidant results showed that the ability of PE to scavenge DPPH,superoxide radicals and total reducing power was dose-dependently dependent on its mass concentration in the range of 20-120 mg/m L.The half-inhibitory concentration IC₅₀(0.14mg/m L)of PE scavenging DPPH free radicals was lower than OE(0.27 mg/m L),but the IC₅₀(0.93 mg/m L)for scavenging superoxide radicals was higher than OE(0.29 mg/m L),and its total reducing power was similar to OE.7.In the concentration range of 40-200?g/m L,the higher the PE,the stronger the proliferation of He La and S180 cells.At 24 and 48 h,the IC₅₀ values of PE inhibited He La cells were 0.83 and 0.53?g/m L,respectively,and the IC₅₀ values of inhibited S180 cells were 2.19 and 0.97?g/m L,respectively,but the IC₅₀values of OE inhibited both cells were lower.When the concentration of PE increased from 160?g/m L to 200?g/m L,the MMP(mitochondrial membrane potential)level of He La cells was lower,and the chromosome breakage was more serious.The MMP and cell morphology of S180 cells also changed significantly,but not sensitive to changes in concentration.PE induced apoptosis in He La and S180 cells by increasing the signaling pathways of caspase-3 and caspase-9 activity.OE inhibited the proliferation of He La and S180 cells more strongly than PE,and the pathways of apoptosis were similar to PE.