The Effect Of Storage Conditions On The Main Active Components In Olea Europaea L.Leaves And The Anti-Aging Activity Of Oleuropein

Olive（Olea europaea L.）,the family of Oleaceae,is a well-known woody oil crop and economic tree.As one of the by-products,olive leaves have a huge annual output and are rich in phenolic compounds.They are an important plant resource with high edible and use value.In this study,olive leaves were used to evaluate the effects of four drying methods of air-dried at room temperature,dried in reezing vacuum dryer,dried in oven at45℃ and dried in oven at 70℃ on the contents of total polyphenols,total flavonoids,oleuropein and hydroxytyrosol.And also studied the storage conditions and storage time on the stability of oleuropein.The model animal Caenorhabditis elegans were used to initially explore the antioxidant,anti-aging properties and preliminary mechanism of oleuropein in vivo.It is expected to provide theoretical and technical support for the application of olive leaves in the functional food and cosmetic industries.The main research results are as follows:（1）The effects of four drying methods of dried in oven at 70℃,dried in oven at45℃,air-dried at room temperature（25℃）and dried in reezing vacuum dryer on the content of total polyphenols in olive leaves were 17.58±1.14、40.90±1.91、51.48±1.58and 51.07±1.60 mg GAE/g;the total flavonoid content were 2.91±0.41、16.12±0.39、28.59±3.20和25.59±1.58 mg GE/g;the content of oleuropein were 1.32±0.11、40.75±1.02、75.59±0.74 and 69.00±0.61 mg/g dw;no hydroxytyrosol was detected in leaves of dried in oven at 70℃,the effects of the above three drying methods on the content of hydroxytyrosol were 0.15±0.02、0.27±0.01和0.25±0.01 mg/g dw.The effects of the four drying methods on the phenolic components in olive leaves generally show a trend that the higher the temperature,the more serious the loss of components.The main astringent component of oleuropein is prone to degradation during the drying and dehydration of leaves.（2）After the olive leaves are dried,under different storage conditions（-20,4,and25℃）,during the 1^st to 9^th weeks,the oleuropein content fluctuates in the range of 74.50±0.52～76.86±0.13、74.51±0.76～76.61±0.54 and 74.48±0.90～76.58±0.63 mg/g dw,the fluctuation range of hydroxytyrosol content is 0.26±0.02～0.29±0.01,0.27±0.01～0.29±0.01 and 0.24±0.03～0.27±0.04 mg/g dw,indicating that the storage conditions has no significant effect on the content of oleuropein and hydroxytyrosol.Therefore,the olive leaves were suggested to preserve at room temperature（25℃）after drying.（3）A rapid HPLC detection method for oleuropein and hydroxytyrosol was established.The detection wavelength was 280 nm,flow rate was 0.8 m L/min;mobile phases are water（A）and acetonitrile（B）;the elution program is set to 0～10 min,90%A,10%B;10～14 min,90～80A,10～20B;14～30 min,80%A,20%B;30～33 min,80～90A,20～10B.The chromatographic method could detect the content of oleuropein and hydroxytyrosol rapidly with simple elution procedure,wide usability and good stability.（4）Under heat stress environment,oleuropein could reduce the MDA content of Caenorhabditis elegans by 38.43%,increase the activities of SOD and CAT enzymes by38.00%and 33.84%,respectively,and increase the mean lifespan by 35.40%.The results indicated that oleuropein has strong antioxidant property in vivo.（5）Under normal culture conditions,various concentrations of oleuropein could prolong the lifespan of wild-type nematodes.The lifespan extension rate increased with the concentration increases.Oleuropein has a significant anti-aging effect with the highest lifespan extension rate of Caenorhabditis elegans by 22.29%.Oleuropein significantly down-regulated the transcription levels of daf-2 and age-1 in the insulin/insulin-like growth factor（IGF）-1 signalling（IIS）pathway by 0.53-fold and0.35-fold.Oleuropein also could activate the transcription factor DAF-16 transfer from the cytoplasm into the nucleus and up-regulate the expression of sod-3,hsp-16.2 ctl-1 and ctl-2 by 2.16-fold,3.14-fold,1,34-fold and 1.55-fold,respectively.Oleuropein increase the expression of sod-3::GFP and hsp-16.2::GFP by 2.90-fold and 2.99-fold,respectively.The heat stress verification test showed that oleuropein could not extend the lifespan of daf-2,age-1 and daf-16 loss-of-function mutants.This indicated that oleuropein extend the lifespan of Caenorhabditis elegans through the IIS signaling pathway.Additionally,the results also showed that oleuropein could increase the expression of skn-1 in wild-type nematode,but could not prolong the mean lifespan of skn-1 loss-of-function mutants under heat stress.This indicated that the anti-aging effect of oleuropein may also depend on skn-1.