Study On The Nucleosides Of Bee Pollens

This paper used bee pollens as the experiment material to explore the type andcontent of the nucleosides of them. A preliminary study on the composition of rapebee pollen by using thin-layer chromatography (TLC) and discussing the TLC expandconditions; Extraction time, extraction temperature and the solid-liquid ratio were thethree influence factors of the extraction of total nucleosides content, so the paper hereexplored the three factors and optimized the extraction conditions. To establish anavailable high performance liquid chromatography (HPLC) method for thesimultaneous determination of several nucleosides, and analysis the type and contentof nucleosides of nine kinds of different bee pollens. The compounds of bee pollenswere purified and seprateed by macroporous resin column chromatography, organicsolvent extraction, sephadex LH-20gel resin column chromatography, TLC, andHPLC. The monomers were identifided by HPLC, TLC and liquidchromatography-quadrupole-time of flight mass spectrometer(LC-Q-TOF) method.The results was as follows:1. Preliminary Determination of nucleosides of rape bee pollen by TLCIn this study, using15%ethanol water to extract the material and studying thenucleosides of rape bee pollen for preliminary qualitative analysis by TLC, and theTLC condition was: developer of chloroform-methanol-ammonia(6.5:8.0:1.5),expanded disdance of8cm,1cm between two spots, saturation time of30min,viewedin254nm UV wavelength. To control with the standards, there might be at least twonucleosides of rape bee pollen including uridine and guanosine.2. Optimization of extraction of bee pollen nucleosidesUsing pure water as extracting solvent, and ultrasonic wave helped to extract thethe four nucleosides of rape bee pollen, the cotent of cytidine, uridine, guanosine andadenine were as the total one. It would be most favorable for extraction when theextraction time, extraction temperature and solid-liquid ratio was in the range of40~50min,45~65℃and1:60~1:80g/mL respectively which based on single factor experiment. The final optimal extraction procedure was extraction time of53min,extraction temperature of60℃, material-liquid ratio of1:70g/mL, and the the totalcontent of nucleosides was705.2±0.006mg/100g rape bee pollen.3. Determination of content of nucleosides of bee Pollens by HPLCIn this study, the chromatographic separation was achieved on Agilent1200HPLC chromatograph with Utimate AQ-C18column (4.6mm×250mm,5μm) using amobile phase consisting of a mixture of water(A) and methanol(B) gradient elutionprogram was (0~3min8%B;3~15min,8~15%B;15~20min,15~20%B;20~30min,20~40%B;30~35min,40~8%B;35~50min,8%B)at a flow rate of1mL/min, andthe analytes were detected at254nm, column temperature of30℃and injectionvolume of5mL. Inosine and thymidine were not detected in any of the nine kinds ofbee pollens, but all of them contained cytidine, uridine, guanosine and adenine, andshowed some difference in the content of the different kinds of bee pollens, The totalcontent of the four nucleosides in9kinds of bee pollens was rape> Corn Poppy>lotus> camellia> Chrysanthemum> corn> buckwheat> Leonurus> rose, and therape bee pollen was significantly higher than the other bee pollens.4. Separation and purification conditionsD101macroporous resin column chromatography:3BV ultrapure water tobalance the chromatography material, sample flow rate was1.0BV/h, desorption flowrate was3BV/h of0%and5%ethanol water.Sephadex LH-20gel resin column chromatography:1mL sample/time, flow rateof0.6mL/min, collected eluting sample per min, detection wavelength was254nm.5. Detection conditions of monomersHPLC: chromatographic separation was achieved on Shimadzu LH-20chromatography with AQ-C18column(250mm×4.6mm,5μm); mobile phase was amixture of methanol(A) and water(B); gradient elution:0~5min,5%~9%A;5~24min,9%A;24~25min,9%~10%A;25~27min,10%~40%A;27~37min,40%A;37~40min,40%~5%A;40~50min,5%A; flow rate:1.0mL/min; column temperature:30℃; detection wavelength:254nm; injection volume:5μL.LC-Q-TOF condition: LC: Agilent6510Q-TOF, the SB C18(2.1mm×150mm,3.5μm) column; mobilephase:0.1%formic acid-acetonitrile(70:30); flow rate:0.2mL/min; columntemperature:30℃; detection wavelength:254nm; injection volume:5μL.Mass spectrometer conditions: ionization mode: the ESI+; atomization gaspressure:30psi; dry gas flow rate:9L/min; drying temperature:350℃; capillaryvoltage:4000V; fragmentation voltage:135V; scan mode: full scan; scan qualityrange:60~1000; acquisition rate:1spectra/s.6. The detection of monomersCompounds H, I and J were isolated from rape bee pollen extraction By D101macroporous resin column chromatography, organic extraction and LH-20gel resincolumn chromatography, HPLC and TLC methods.The compound H has maximum UV absorption at213nm and259nm byHPLC-Q-TOF detection for adenine. I, J has maximum UV absorption at264nm and257nm, respectively, they might be nucleosides compounds, but failed to reach aconcrete result for some incomplete information, and needed to be detected again.