Antioxidative Effect Of Tyrosol Acyl Ester On Mussel Oil And Its Mechanisms Of Absorption And Metabolism

Lipophenols,can be manufactured by linking of fatty acids with phenols through ester bonds.Compared to phenols themselves,their lipophilic derivatives have several advantages such as combining the health-related benefits of both omega-3-polyunsaturated fatty acids(?-3 PUFA)and polyphenols in a single lipophenolic molecule,and increasing the protective effects on lipidic food matrices(mainly edible oils)from oxidation.In 2014,palmitoyl esters of green tea polyphenols were approved for food use in China by China Food and Drug Administration with the maximum-tolerated dose of 600 mg/kg.However,there is little information about the anti-oxidation effect of lipophenols on ?-3 PUFA in the PL form,and some ambiguity regarding the mechanisms of body digestion,absorption and metabolism still remains.First,taking tyrosol as polyphenol donor and lauric acid(C12:0),myristic acid(C14:0),palmitic acid(C16:0),stearic acid(C18:0),oleic acid(C18:1),linoleic acid(C18:2)and docosahexaenoic acid(DHA)as fatty acid donors,series of tyrosol acyl esters with various chain length and degree of unsaturation(TYr-Es,i.e.T-C12:0,T-C14:0,T-C16:0,T-C18:0,T-C18:1,T-C18:2 and T-DHA)were prepared by an enzymatic synthesis and a saponification purification procedure,and their antioxidant protective activities in mussel(Mytilus edulis)oil were tested.Taking the mussel oil extracted by methyl-tert-butyl ether(MTBE)as an example,thin layer chromatography-flame ionization detection(TLC-FID)analysis indicated that the oil was rich in phospholipid(PL)(61.68% of total lipid).GC-MS analysis indicated that the mussel oil contained high proportion of PUFA(26.51% of total FAs),especially EPA(10.74% of total FAs)and DHA(5.08% of total FAs).High-performance liquid chromatography electrospray ionization-tandem mass spectrometry(HPLC-ESI-MS/MS)analysis indicated that the mussel oil contained 85 species of glycerophosphocholine(GPCho),83 species of glycerophosphoethanolamine(GPEtn),and 38 species of glycerophosphoserine(GPSer).Most of the predominant glycerophospholipids(GP)molecular species contained PUFA,especially EPA and DHA.Therefore,mussel oil is a type of ?-3 LC-PUFA in the PL form.Effects of T-C14:0,T-C16:0,T-C18:0,butylated hydroxytoluene(BHT)and tyrosol on stability of mussel oil during accelerated oxidation experiment were studied.Results indicated that T-C14:0,T-C16:0 and T-C18:0 could inhibit the production of primary oxidation product(peroxide value)and secondary oxidation product(thiobarbituric acid reactive substances).Compared to TYr,TYr-Es were more effective to inhibit production of primary oxidation product,but less effective to inhibit production of secondary oxidation product.Second,the digestive stability of T-C12:0,T-C14:0,T-C16:0,T-C18:0,T-C18:1,T-C18:2 and T-DHA was evaluated using in vitro simulated gastrointestinal tract models containing various digestive enzymes(pancreatin,pancreatic lipase and phospholipase A2).HPLC-UV analysis demonstrated that TYr-Es were stable in artificial saliva and simulated gastric fluid(SGF),but were hydrolyzed to free TYr in simulated intestinal fluid(SIF).In SIF,pancreatin and pancreatic lipase,but not phospholipase A2,could hydrolyze TYr-Es to free TYr.The degree of TYr-E hydrolysis negatively correlated with the chain length but positively correlated with the degree of unsaturation of their lipid moiety.In addition,the fact that TYr in fatty acid ester forms could be absorbed by the intestinal lumen,at least partially in the form of free TYr,may explain a sustained release behavior of TYr-Es to TYr during the time-course following the digestion process.Third,the characteristics of digestion and absorption of tyrosol acyl esters with various chain length and degree of unsaturation was evaluated using mice model and everted rat gut sacs model.After intragastric administration of TYr,T-C14:0,T-C16:0 and T-C18:0 to mice,the concentrations of the aforementioned compounds in contents from different segments of the gastrointestinal tract were detected.HPLC-UV analysis demonstrated that TYr could be detected within 2h in both gastric contents and small intestinal contents after administration of TYr,but could not be detected in contents from large intestine and cecum even within 6h.This indicated that TYr was rapidly absorbed in rat stomach and small intestine.In contrast,the concentrations of TYr-Es in gastric contents and small intestinal contents decreased slowly,and still could be detected in gastric contents and intestinal contents even after 6h.Moreover,all of the TYr-Es could reach the large intestine and cecum,and the concentrations initially increased but then decreased.It is noteworthy that no free TYr was detected in all of the contents after intragastric administration of TYr-Es.Furthermore,the hydrolysis and transport characteristics of TYr,T-C12:0,T-C14:0,T-C16:0,T-C18:0,T-C18:1,T-C18:2 and T-DHA were evaluated using an everted rat gut sacs model.HPLC-UV analysis demonstrated that TYr-Es were hydrolyzed to TYr,and the hydrolysis rate correlated negatively with the chain length of their lipid moiety,but showed positive correlation with the degree of unsaturation.Meanwhile,TYr in fatty acid ester forms could be transported across the sacs in the form of free TYr,and the transport rate also negatively correlated with the chain length but positively correlated with the degree of unsaturation of their lipid moiety.Above results indicated that the hydrolysis of TYr-Es contributed significantly to the transport of TYr across intestinal mucosa.Finally,the characteristics of in vivo absorption and metabolism of TYr,T-C12:0,T-C18:0 and T-C18:2 were evaluated through plasma kinetic assay rat models.After intragastric administration of TYr and TYr-Es,only two metabolites of TYr,tyrosol-glucuronide and tyrosol-sulfate,not TYr itself,were identified in rat plasma by HPLC-MS/MS analysis,indicating that TYr was rapidly metabolized in enterocyte and(or)liver.After administration of TYr,T-C12:0,T-C18:0 and T-C18:2,the time to peak concentration of tyrosol-sulfate in rat plasma were 10,10,45 and 15 min,respectively,and the corresponding MS peak areas were 5.82,1.70,1.03 and 0.87×107,respectively.While the corresponding time to peak concentration of tyrosol-glucuronide were also 10,10,45 and 15 min,respectively,and the corresponding MS peak areas were 1.24,0.37,0.34 and 0.31×107,respectively.In contrast,TYr exhibited faster absorption rate than TYr-Es.After reaching the peak values,the concentrations of the both metabolites in TYr group decreased rapidly,but the concentrations of the both metabolites in T-C12:0,T-C18:0 and T-C18:2 groups initially decreased but then increased to the second peak values at 6,8 and 4h post intragastric administration,respectively.In addition,after 4h of intragastric administration,the concentrations of tyrosol-sulfate and tyrosol-glucuronide in TYr-Es groups were higher than those in TYr groups.The above results reflected the slow metabolism and excretion of TYr-Es compared to TYr,and a long duration of in vivo action.Given this,TYr-Es shows certain sustained-release behavior,which may be a useful method to improve bioactivities of TYr due to prolonged duration of action.In summary,TYr-Es show antioxidant protective effect in ?-3 PUFA in the PL form.TYr-Es are digestible and absorbable in the intestine tract,and also exhibit certain sustained-release behavior and prolong the in vivo duration of action.Therefore,TYr-Es could be a novel source of ingredients for functional foods.