Preparation And Process Optimization Of Polyphenol Extract From Roxburghii Seeds And Its Anti-aging Effec

Object and significance:The seeds of Rosa roxburghii are often classified as wastes together with the residual pulp and pomace during the development and production of Rosa roxburghii.However,studies have shown that the seeds of Rosa roxburghii contain fatty acids,flavonoids,triterpenoids and many essential trace elements,which have antioxidant,hepatoprotective and anticancer pharmacological activities.As a whole,there are still some gaps in the study of chemical composition and activity mechanism of the seeds of Rosa roxburghii.The aim of this study was to prepare the polyphenol extract from the seeds of Rosa roxburghii for its outstanding antioxidant activity,and to explore its anti-aging effect and possible mechanism at the organism level by using model organisms,so as to lay a theoretical foundation for the rational utilization of the seeds of Rosa roxburghii as a green resource.Methods:1.Preparation and process optimization of Rosa roxburghii seed polyphenol extract:The ethanol extract of Rosa roxburghii seed was prepared by reflux extraction method and purified by macroporous adsorption resin.Folin-Ciocalteu method was used to determine the content of total phenols in the extract.A UV method for the determination of polyphenols in Rosa roxburghii seed extract was established by single factor investigation.The effects of the type of macroporous adsorption resin and the concentration of eluent on the total phenol content in the polyphenol extract were investigated to determine the optimum preparation process of Rosa roxburghii seed polyphenol extract.2.Chemical composition analysis of Rosa roxburghii seed polyphenol extract:Based on UPLC-Q-Exactive-Orbitrap-MS technology,the chemical composition of prickly pear seed polyphenol extract was preliminarily identified.3.The in vitro and in vivo activity research of Rosa roxburghii seed polyphenol extract:Using Vc as a positive control,the in vitro antioxidant activity of Rosa roxburghii seed polyphenol extract was evaluated through DPPH free radical scavenging experiment.Taking Caenorhabditis elegans as an experimental model,the effects of polyphenol extracts from Rosa roxburghii seed on the life span,athletic ability,body length,lipofuscin content level,and anti stress ability of Caenorhabditis elegans were measured to explore the anti-aging effect of polyphenol extracts from Rosa roxburghii seeds.4.Exploration of the anti-aging mechanism of Rosa roxburghii seed polyphenol extract:By measuring the effects of Rosa roxburghii seed polyphenol extract on DAF-16 nuclear translocation,SOD content level,and the lifespan of several DAF-16 upstream and downstream related gene deficient Caenorhabditis elegans under oxidative stress,we aimd to explore the sites of action related to its anti-aging effect.Results:1.The optimal process for preparing Rosa roxburghii seed polyphenol extract was as follows:D101 macroporous adsorption resin was used,after preliminary impurity removal with water and 5%ethanol solution,elution was carried out with 35%ethanol aqueous solution.After purification with macroporous adsorption resin,the total phenolic content in the polyphenol extract of Rosa roxburghii seed increased from 15.52%in the alcohol extract to 38.28%.2.The mass spectrum data of polyphenol extracts from Rosa roxburghii seed was collected by liquid chromatography-mass spectrometry.The quasi-molecular ion peaks,fragment ion peaks and fragmentation patterns of the compounds in the data were compared with the compound information in literature or pubchem database,and 33 compounds were preliminarily identified.3.The DPPH radical scavenging test showed that the polyphenol extract of Rosa roxburghii seed had antioxidant activity in vitro,but the effect was weaker than that of Vc.In the Caenorhabditis elegans experiment,the optimal concentration of polyphenol extract was 50μg/mL,which could increase the length of C.elegans,decrease the content of lipofuscin and prolong the survival time of C.elegans under oxidative stress.Follow-up experiments also showed that 50 μg/mL of Rosa roxburghii seed polyphenol extract could significantly prolong the lifespan of C.elegans and possibly improve their locomotor ability to some extent,but had no significant effect on the survival time of C.elegans under heat stress.It can be concluded that the polyphenol extract of Rosa roxburghii Tratt seed has certain anti-aging effect.4.In the mechanism study,it was found that the Rosa roxburghii seed polyphenol extract could significantly reduce the ROS content level in C.elegans,and it was found by fluorescence photography that when the administration concentration was 50 μg/mL,it could promote the entry of DAF-16 into the nucleus of C.elegans and increase the SOD-3 content level in vivo.Experimental results showed that the polyphenol extract of Rosa roxburghii seed could not prolong the survival time of daf-2,age-1,daf-16 and hsp-16.2 gene deletion strains of C.elegans under oxidative stress,which indicated that these genes were the targets of the polyphenol extract of Rosa roxburghii seed to play an anti-aging role.Conclusion:In this study,the optimal preparation process of polyphenol extract from Rosa roxburghii seed was determined as follows:D101 macroporous resin was used and eluted with 35%ethanol aqueous solution.Finally,the polyphenol extract of Rosa roxburghii seed with total phenol content of 38.28%was obtained.Subsequently,UPLC-Q-Exactive-Orbitrap-MS technique was used to rapidly identify 33 compounds.The antioxidant activity of polyphenol extracts from Rosa roxburghii seed was preliminarily evaluated by DPPH radical scavenging assay in vitro.And then the anti-aging activity of polyphenol extracts from Rosa roxburghii seed was determined for the first time by using Caenorhabditis elegans as an experimental model.Through the relevant experiments of transgenic nematodes and gene-deficient nematodes,it was proved that the anti-aging mechanism of polyphenol extracts from Rosa roxburghii seed may be:reducing ROS level,down-regulating the expression of daf-2 and age-1 genes to relieve the negative regulation of DAF-16,promoting the transcription of DAF-16 into the nucleus,and simultaneously increasing the expression of SOD-3 and HSP-16.2 proteins,thus playing an anti-aging role together.