Xanthophyll Isomers:Rapid Preparation,Purification,Metabolic Fate And Their Effects On The Intestinal Function

Xanthophylls are natural yellow carotenoids containing oxygen atoms with antioxidant and physiological activities.Astaxanthin,lutein and zeaxanthin are common xanthophylls.The predominant geometrical isomer of naturally occurring astaxanthin and lutein is all-trans（all-E）-isomer.However,recent study found that 9Z-and 13Z-astaxanthin（AST）isomers were selectively absorbed into human plasma over all-E-AST after oral administration with algae rich of all-E-AST.More interestingly,Z-AST isomers,especially 9Z-AST,had been found to exhibit higher antioxidant potential in vitro than all-E-AST in chemical-based assays.However,despite these findings,there was little information of the physicochemical properties,stability,and functional properties such as anti-oxidative stress and anti-inflammatory effects of Z-AST isomers.It is not clear how and where the Z-isomers of AST are formed in vivo,and whether or not the higher antioxidant activity of Z-ASTs observed in vitro will also occur in vivo.Besides,their uptake mechanism inside human gastrointestinal and the metabolic systems are still unknown.The presence of Z-luteins in human plasma and retina had prompted particular interest.Meanwhile,the formation of Z-luteins in human body,the physiological roles of Z-luteins in humans including the metabolism and the various protective effects such as antioxidant and anti-macular degeneration activities also remained unknown.In order to solve the above-mentioned problems,we first develop an efficient method to isomerize all-E-xanthophyll to Z-isomers,and then detect the antioxidant activity,stability,cellular uptake mechanism,bioaccessibility and further the anti-oxidative stress and anti-inflammatory effects of different AST isomers.The results were shown below.1.Rapid conversion of all?E?AST and the assessment of the stability and antioxidant activities of Z-ASTs.An optimized isomerization method was developed by heating all-E-AST in ethyl acetate（70°C）with I-TiO₂ catalyst,yielding 22.9%and 16.9%of 9Z-and 13Z-AST,respectively in 2 h.Fractions were collected after semi-preparative HPLC.Z-ASTs showed higher antioxidant activity than all-E-AST in oxygen radical absorbing capacity assay for lipophilic compounds（ORAC-L）,photochemiluminescence（PCL）and DPPH radical-scavenging assays.However,there was no significant difference on antioxidant activity between three isomers in CAA assay.13Z-and 9Z-ASTs were isomerized to all-E-AST at pH2.0,common metal ions(Fe³⁺,Cu²⁺,Ca²⁺,Mg²⁺,Na⁺,K⁺)did not significantly（p<0.05）affect the stability or induce the isomerization of three isomers.2.The bioaccessibility,cellular uptake mechanism of three AST geometric isomers were investigated using an in vitro digestion model and human intestinal Caco-2 cells.The result demonstrated that the trans-cis isomerization of all-E-AST and the cis-trans isomerization of Z-ASTs could happen both during in vitro gastrointestinal（GI）digestion and cellular uptake processes.13Z-AST（37.1%）showed higher bioaccessibility than 9Z-AST（28.6%）and all-E-AST（19.7%）during in vitro digestion.The absorption rate of all-E-AST（8.3%）was higher than those of 9Z-and 13Z-ASTs（≤7.12%）,whereas 9Z-AST exhibited higher transport efficiency（0.68%）than 13Z-AST（0.28%）and all-E-AST（0.21%）.These might explain why13Z-and 9Z-ASTs were found at higher concentrations in human plasma in previously reported studies i.e.all-E-AST would isomerize to Z-ASTs during digestion,while the Z-ASTs have higher bioaccessibility and secretion rate from cells.3.The protective effects of different AST isomers against oxidative stress.Except for reacting with free radicals,all the three AST isomers were effective in maintaining cellular redox homoeostasis as seen in the antioxidant enzyme（CAT,SOD）activities studies in H₂O₂stimulated Caco-2 cells.Besides,different AST isomers might have potential anti-inflammatory effects as shown in lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells.Furthermore,9Z-and 13Z-ASTs exhibited higher protective effect than all-E-AST against oxidative stress as demonstrated in lower cellular uptake of Z-ASTs in Caco-2 cells treated with H₂O₂.This work indicated that Z-AST isomers may play a more important role in preventing oxidative stress induced intestinal diseases.4.The anti-inflammatory effects of different AST isomers.Both of all-E-and Z-ASTs were effective to reduce the damage from TNF-αto Caco-2 monolayer and maintain the permeability property and integrity of monolayer in TNF-αinduced Caco-2 monolayer.Additionally,all the three isomers could significantly down regulate the gene expression of pro-inflammatory cytokines and inhibit the secretion of IL-8.Z-ASTs exhibited better inhibition effect than all-E-AST on reducing the expression of COX-2,TNF-αand IL-8.On the other hand,same as all-E-AST,Z-ASTs could also inhibit the activation of NF-κB signaling pathway,and further prevent inflammatory bowel disease（IBD）and other diseases.To sum up,9Z-and13Z-AST could exhibit same or better effects than all-E-AST on anti-oxidative stress and anti-inflammatory activities.The results could clear up the confusion about the functional properties of Z-ASTs.5.Bioaccessibility,cellular uptake and transport of luteins and assessment of their antioxidant activities.An efficient method for producing 9Z-and 13’Z-luteins from all-E-lutein was also developed using I-TiO₂ as catalyst yielding 20.6%and 13.8%of 9Z-and 13’Z-luteins,respectively in 1 h.In a simulated in vitro gastrointestinal digestion model,both trans-cis isomerization of all-E-lutein and cis-trans isomerization of Z-luteins occurred during the intestinal digestion phase.The bioaccessibility of all isomers was in the range of 13.6%–23.3%,and it was significantly higher for Z-luteins（≥23.0%）than all-E-AST（13.6%）.In a Caco-2 cell monolayer model,all isomers were relatively stable during cellular uptake and transport across the membrane as no significant isomerization was detected,but all-E-lutein exhibited significantly higher cellular uptake and transport efficiencies.These results suggest that Z-luteins found in human plasma may likely be absorbed directly from food or supplements or formed during the digestion of all-E-lutein in the upper digestive tract.13’Z-Lutein also exhibited highest antioxidant activity in FRAP,DPPH and ORAC-L assays,but no significant difference in cell-based antioxidant assay compared with other isomers because of its lower cellular uptake.6.Generally,both the antioxidant activity by DPPH and ORAC-L and cellular absorption rate of all the three AST isomers were higher than those of the lutein isomers,the difference on structure,solubility and polarity between lutein and astaxanthin could explain these results.To sum up,an efficient method for the production of Z-xanthophylls was provided,results of the current study will also provide fundamental and useful information for the production and use of Z-xanthophylls as functional food,cosmetics and even pharmaceutical ingredients,broaden the research content of Z-xanthophylls and offer technical support for the further study of other carotenoid isomers.