Determination And Comparison Of Sialic Acid Content In Bird's Nest And Foods Based On HPLC

Sialic acid is an important component of glycoproteins,oligosaccharides and glycolipids.It is widely distributed in nature including human brain,nerve tissue,blood and emulsion,etc.It owns many different bioactivities,i.e.anti-inflammation,anti-virus,anti-tumor,and effects on nervous system.Therefore,sialic acid is widely used in the diagnosis and treatment of diseases,synthesis of drug precursors and diagnostic indicator of diseases.It comes from bird's nest,milk,milk powder and eggs.However,the precise determination of sialic acid's content is difficult due to the various ingredients existed in food and bird's nest.In this paper we develop an effective method for the extraction and content determination of N-acetylneuraminic acid(NeuAc)in bird's nest and food(eggs and dairy products)based on the recent study of sialic acid.The structure of sialic acid derivative was also elucidated and analyzed based on the LR-MS results.Thus,our study provided experimental basis for judging the quality and nutritional value of bird's nest and food,i.e.,eggs and dairy products,and extraction methods of sialic acid from natural products and food.In the 1st Chapter,we described the distribution of sialic acid in bird's nest and food(eggs and dairy products),the physical and chemical properties,the bioactivities and metabolism of sialic acid in vivo,the roles of sialic acid in antiinflammation,anti-virus,anti-tumor and effects on brain nerve and other physiological diseases.We summarized foreign literatures and domestic literatures and compared four methods(natural products extraction,microbial fermentation,chemical synthesis and enzymatic synthesis)for the extraction and preparation,and four methods(ion exchange filtration,ultrafiltration membrane,gel filtration and crystallization)for the purification and separation of sialic acid.We also compared the advantages and disadvantages of five detection method(UV,FTIR,GC,HPLC and ELISA)for sialic acid in bird's nest based on papers in recent 15 years.Compared with bird's nest,few methods were reported for sialic acid's content detection in foods(i.e.,eggs and dairy products)except UV and HPLC.We retrieved related literatures in recent 15 years and compared the advantages and disadvantages of UV and HPLC.In the 2nd Chapter,we studied the pre-column derivatized HPLC method for sialic acid's content determination by reacting o-phenylenediamine hydrochloride as derivative reagent with the acid hydrolyzed product of sialic acid in each sample.The HPLC condition was as follow: ZORBAX SB-C18 column(4.6 mm x 250 mm,5 micron),acetonitrile and water were used as mobile phase at the ration of 10:90,the flow rate was 1.0 mL/min,detection wavelength was 230 nm,and injection volume was 10 ?L.We also explored the effects of temperature and time of acid hydrolysis and pre-column derivatization on sialic acid detection and optimized the ratio of mobile phase and detection wavelength in HPLC,which was further verified by methodology.The results showed that no obvious difference between the two kinds of bird's nest from different habitats was observed and no characteristic peak of sialic acid was found in pigskin and tremella.In the 3rd Chapter,we studied the distribution of sialic acid in one of the food source eggs.We selected five different kinds including eggs,duck eggs,black bone eggs,quail eggs and pigeon eggs as samples and reacted o-phenylenediamine hydrochloride,with the acid hydrolyzed product of sialic acid in each sample.In the pre-column derivatized process,emulsion was obtained instead of clear solution.Thus,we compared three different kinds of filtration methods(qualitative filter paper filtration,centrifugal precipitation and microporous membrane filtration)and selected microporous membrane filtration as the optimum extraction method,which ensured the minimum loss of sialic acid in the process.The results showed that the sialic acid's content in the five kinds of eggs was different with the content in pigeon eggs as the highest one.There is less sialic acid in eggs than in bird's nests.In the 4th Chapter,we studied the sialic acid' content in one of the food source dairy products with pre-column derivatized HPLC method and selected goat milk powder,infant milk powder,calcium milk powder for elderly,whole milk powder,skim milk and whole milk as the samples.Although there is some fat existed in the dairy products which would interfere with the derivatized product of sialic acid with direct injection,the current extraction and detection methods can still accurately detect the content of sialic acid in the milk by HPLC.The results showed that there was some difference of sialic acid's content existed in the six dairy products with the content in goat milk powder as the highest one.There is less sialic acid in dairy than in bird's nests,which is similar to eggs.In the 5th Chapter,we analysized and elucidated the structure of sialic acid derivative based on the TOF-MS results and the reaction involved in the pre-column derivatization was also elucidated.In the 6th Chapter,we summarized the prospects and trends of sialic acid study in the future.