Development Of A New Method For Detecting DL-amino Acids In Natto Based On Fluorescence Derivation And Research On The Effective Material Basis Of Korean Medicine Lurong Dabu Decoction

Natto is a kind of functional health food.Natto can be thrombolytic and lipid-lowering,soften blood vessels,prevent arteriosclerosis,so it has attracted much attention.Under the action of Bacillus subtilis,natto was formed from steamed soybean and released free amino acids.In recent years,D-glutamate has been found in the mucus of natto.However,the existence and function of other chiral amino acids in natto have not been reported.Therefore,a new method for the simultaneous determination of DL amino acids in natto by UPLC-FL with pre-column fluorescence derivatization was developed using DBD-trans-2-methyl-L-proline(DBD-M-Pro).This method was applied to the analysis of natto and mucus from different manufacturers.DBD-M-Pro reacted with DL amino acids at 60°C for 60 min.to form diastereomers.The diastereomers were detected on a BEH C18(2.1 mm×100 mm,1.7μm)column at excitation wavelength(Ex):450 nm and fluorescence wavelength(EM):560 nm.The results showed that 4 types of DL amino acids(Gln,Asp,Glu,Ala)achieved completely separation with R_S1.65-3.50.The limits of detection was0.25-2.5 pmol.Four kinds of DL amino acids were detected in the bean and mucus of three different manufacturers,and the contents of DL-Gln,DL-Glu and DL-Ala in the mucus of natto were significantly higher than those in the bean.In addition,a simple method for ratios of D/L-Gln and D/L-Asp peak areas can be used to identify different manufactures.Chinese Korean medicine is a theory that Korean people have been fighting against diseases for a long time."Lurong Dabu Decoction"(LRDBT)is the classic prescription of Korean medicine,which is consisted of eight herbs,namely,Cartialgenous;Ephedrae;Almond;Ophiopogon japonicas,Asparagus cochinchinensis,Schisandra chinensis,Dioscorea oppositifolia L,Semen coicis.It has obvious improvement and therapeutic effect on asthma of Taiyin people.However,due to the unclear pharmacodynamic components and mechanism of action in vivo of"Lurong Dabu Decoction",the pharmacodynamic components of"Lurong Dabu Decoction"was studied by UHPLC-QE-Orbitrap-MS methods,combined with compound discoverer and standards.CD analysis was based on the comparison of retention time and MS/MS fragmentation mode,and 86 compounds were identified,including alkaloids,flavonoids,amides,amino acids and so on.12 compounds were identified in Lurong Dabu decoction,including Amygdalin,Malic acid,Naringenin,Catechin,Adenosine,Betaine,Guanine,Proline,Histidine,DL-Pipecolic Acid,Uridine.In order to explain the source of effective components,the basic components of single drug were detected,compared and screened.In this study,two new detection methods were established.Firstly,a new method for the simultaneous determination of four kinds of DL-chiral amino acids in natto was developed based on pre-column derivatization of DBD-M-Pro.Four free DL-amino acids were found in natto and mucus for the first time.In addition,in this study,an UHPLC-MS/MS method was established to analyze the chemical constituents of Lurong Dabu decoction for the first time,which provides an effective method and basic data for further study of metabolic pathways and drug targets in vivo.