Fluorescence Characteristics Of Alfalfa Seeds And Their Chemical Basis

Alfalfa（Medicago sativa）is a high-quality species,which is widely planted and utilized in China,and is a high-quality forage indispensable for livestock development.Sweet clover（Melilotus spp.）and alfalfa seeds are morphologically similar and difficult to distinguish after mixing,and seed authenticity cannot be guaranteed.Thus reducing the utilization value of alfalfa and causing economic losses.As a method to identify alfalfa and sweet clover seeds the fluorescence method has the characteristics of rapidity and accuracy,but its identification principle has not been reported.In this study,we used alfalfa seeds as the material to study the specific location of alfalfa seed fluorescent substances,the duration of fluorescence and the fluorescence of alfalfa seeds at different maturity periods,and extracted the fluorescent substances from alfalfa seeds by soaking.The fluorescent substances obtained from the extraction were isolated,purified and identified.This study can lay the foundation for revealing the technical principles of the fluorometric method to identify alfalfa and grass miscanthus seeds.The main research results are as follows.1.Water-absorbing and swollen alfalfa seeds fluoresced under UV lamp.Hard-set seeds of alfalfa that could not absorb water and seeds of sweet clover did not fluoresce.Fluorescence of alfalfa hard solid grains after cutting through the seed coat treatment,but sweet clover seeds never fluoresce.After 24 hours of seed swelling,the photophores were still present.After 3 days,the petri dishes were moved to the UV lamp for observation,the light clusters spread in a continuous manner,which showing that fluorescent substances can be relatively stable.2.For the determination of the location of the presence of fluorescent substances in alfalfa seeds,the results after separating the seed coat showed when observed under UV lamp fluorophores already appeared around the seed coat at only 0.5 h,fluorescence rate of 86%,light cluster diameter of 2 mm,the fluorophore then gradually expanded and reached 5mm in diameter,The fluorescence rate reached98.6%after 4 h,while no blue-green fluorescence appeared around the seeds,fluorescent substance is present in the seed coat area.3.Alfalfa seeds at different maturity stages showed different fluorescent colors.The fluoresce was blue in 16 days after flowering;20 days after flowering showed green fluorescence;on the 24th day,the fluorescence color was blue-green.28 days after flowering,the seed coat color deepened and the fluorescence rate reaches 76%.As the seed ripened to maturity,the seed coat turned dark to yellowish brown.The fluorescence rate of the collected seeds reached 100%40 days after flowering.The result indicated that there was not a single fluorescent substance in the seeds,but multiple fluorescent compounds were present,probably with superimposed fluorescent color of each compound with the change of seed coat color.4.The fluorescent substances in alfalfa seeds were soaked in 10%ethanol,followed by n-butanol extraction,and then the compounds were separated and purified by thin-layer chromatography,dextran gel column chromatography,silica gel column chromatography,and semi-preparative HPLC.In total,10 compounds were obtained.The purified substances were also identified and analyzed by ¹H NMR,¹³C NMR,DEPT and other spectroscopic techniques to determine the structure of the compounds.The structures of nine compounds were identified.Among them,（5,3’-dimethoxy-quercetin-3-O-β-D-glucopyranoside）,（5,7-dimethoxy-quercetin-3-O-β-D-glucopyranoside）,Ferulic acid,（Luteolin 7-methyl ether）,（Luteolin-5,3’-dimethyl ether）showed fluorescence at 365 nm.In this study,the fluorescence characteristics of alfalfa seeds were determined.The chemical structure and type of fluorescent substance in seed coat of alfalfa seed were identified by separation and purification.The material basis of species identification based on fluorescence method was revealed.