Screening And Purification Of Inhibitors Of Advanced Glycation Endproduct Formation From Microalgae And Their Anti-neuroinflammation Activity

With dramatic rise in lifespan of human and rapidly aging of population,neurodegenerative diseases,especially alzheimer's disease(AD),have become a major threat to people's health and longevity.It is a hot topic in the field of food science to explore the functional components that prevent AD and reveal their mechanisms.In particular,finding food components with an intervention on glycation and neuroinflammation relating to AD is attracting more and more attention.The diet is one of the most effective ways to keep healthy.Serving as a huge food and bioactive products,microalgae have many biomodulatory effects such as antioxidation,anti-inflammation and anti-obesity,etc.However,there have been very few studies on the antiglycation and anti-neuroinflammatory of microalgae.The aim of the present work was to fill in the gap.In this paper,the inhibitory effects of the extracts from nine microalgae on the formation of AGEs by using in vitro models were investigated and the inhibitors of AGE formation from microalgal extracts were screened.The method for the analysis of key carotenoids in acetone extracts was established,and for the first time,fucoxanthinol and fucoxanthin from the diotam Nitzschia laevis were identified.Then the key antiglycation constituents of the microalgae were confirmed as fucoxanthinol and fucoxanthin by pure fucoxanthinol and fucoxanthin standards.The nonlinear dynamic models about the cell growth,the synthesis of fucoxanthinol and fucoxanthin and the consumption of substrates in N.laevis were established under the photoautrophic,heterotrophic ad mixotrophic conditions,elaborating the correlation among the several kinetic models for the synthesis of desirable products.Then,under the mixotrophic condition,the large-scale production of fucoxanthinol and fucoxanthin was obtained for further study.The solid phase extraction and flash column chromatography and thin layer chromatography,are selected to separate and purify the fucoxanthinol and fucoxanthin of N.laevis.In order to study the antiglycation mechanisms of purified fucoxanthinol and fucoxanthin,the scavenging of free radicals and dicarbonyl compounds was investigated.Moreover,the inhibition effects of purified fucoxanthinol and fucoxanthin on protein oxidative damage were evaluated.Furthermore,LPS-induced BV2 microglia inflammation as a cell model is chosen to illustrate the effects of purified fucoxanthinol and fucoxanthin on neuroinflammation.The present study lays a solid foundation for AD intervention of fucoxanthinol and fucoxanthin,and provides innovative approaches and strategies for the development of functional ingredients of microalgae.Our major findings are summarized as follows:(1)The results of BSA-glucose and BSA-MGO models showed that the aqueous acetone extracts exhibited stronger antiglycation activity than the other extracts(ethyl acetate and dichloromethane)and that the marine microalgal extracts were generally more effective than the freshwater ones.The AGE inhibitory effects depended on the concentration of extracts.The active ingredients in these extracts were tentatively identified as carotenoids.(2)To analyze the composition of carotenoids in the five marine microlagl extracts and vertify the key inhibitors of AGE formation,the methods of HPLC-PDA and UPLC-PDA-IMSTOFMS were established.The qualitative and quantitative analyses were conducted for fucoxanthinol and fucoxanthin.The methods were proved to be sensitive,stable,and reliable.By the correlation analysis and the vertification of standards,fucoxanthinol and fucoxanthin showed inhibitory effects on the formation of AGEs.(3)The above results for the first time were reported that N.laevis could synthesize and accumulate the fucoxanthinol and fucoxanthin,particularly fucoxanthinol.Thus,it is important to compare and assess the desirable products of N.laevis under the different cultivation modes.The nonlinear dynamic models for the cell growth,the synthesis of fucoxanthinol and fucoxanthin,and the consumptionof substrates in N.laevis were thus established under the photoautrophic,heterotrophic and mixotrophic conditions.The results revealed that the established kinetic equations could well fit with the experimental data of cell growth,the synthesis of products and the consumption of substrates in all cultivation modes.Moreover,the mixotrophic culture was the most suitable for the accumulation of these two compounds,and the consumption of glucose and sodium nitrate affected their contents during the cultivation.(4)To obtain high purity fucoxanthinol and fucoxanthin,the solid phase extraction and flash column chromatography and thin layer chromatography,are selected to separate and purify the fucoxanthinol and fucoxanthin of N.laevis from the mixotrophic condition.The results showed that the purity of both fucoxanthinol and fucoxanthin was approximately 98%.(5)To illustrate the antiglycation mechanisms of fucoxanthinol and fucoxanthin,the scavenging of free radicals and dicarbonyl compounds was investigated.The results showed that fucoxanthinol and fucoxanthin demonstrated the free radical and dicarbonyl compounds scavenging abilities and the inhibition effects on protein oxidative damage.They play an important role in inhibiting AGE formation.(6)Finally,the intervention of fucoxanthinol and fucoxanthin on LPS-induced BV2 microglia inflammation response was investigated.The results demonstrated that fucoxanthinol and fucoxanthin significantly suppressed the formation of LPS-induced production of reactive oxygen species(ROS).Moreover,fucoxanthinol and fucoxanthin significantly reduced the levels of inflammatory mediators such as IL-1?,IL-6,TNF-?,COX-2 and iNOS.In addition,fucoxanthinol and fucoxanthin remarkably decreased the overexpression of COX-2 in BV2 cells induced by LPS.Therefore,the inflammation response of LPS-induced BV2 cells was interfered.