Extraction, Separation And Identification Of Glucosinolates And Its Enzymatic Products In Hainan Mustard Seed

Mustard seed is the ripe seed of Brassica juncea (Brassicaeae family). It contains richly glucosinolates which could produce isothiocyanates after enzymatic hydrolysis with Root-Knot nematicidal activity.Methods of qualitative and quantitative analysis for glucosinolates and Allyl isothiocyanate were established in this research. Optimum conditions of extraction, resin separation and enzymatic hydrolysis of glucosinolates, from mustard seed planted in Hainan province, were investigated. Chemical constituents of isothiocyanates were purified by Pre-HPLC for further spectral analysis of chemical structure. Followed is the main researches.1. Quantitative analysis of glucosinolates by LC-ESI-MSSeparation of the individual glucosinolates was achieved using a reversed-phased C18column with5mM aqueous ammonium acetate+0.1%formic acid/methanol gradient, coupled with ESI/MS. The calibration curve was linear in the range of0.5-50mg/L, with a correlation coefficient R>0.998. The LOD was0.5-1.0ng.2. ESI-MS/MS screening method for glucosinolates and its splitting fragmentsThe negative ion ESI-MS/MS behavior of the different glucosinolates were investigated and revealed three groups of typical fragments. For example, all investigated glucosinolate anions fragment to produce product ions at m/z291and259with the neutral loss of RCN and RCNS. The ion at m/z275was formed after H-rearrangement with the loss of RCNO, which further dissociates neutral complex (H2S and SO3) to give two peaks at m/z241and195. Altogether, the characteristic R groups in the form of the R-C(SH)=N-O or R=C=N-OSO3ion could be traced to their corresponding glucosinoaltes in the MS/MS spectra by the loss of C6H10O8Sor C6H12O5S after rearrangement reactions. The structures of glucosinoaltes could be confirmed by the characteristic product ions at m/z195,241,259,275,291and R-C(SH)=N-O" or R=C=N-OSO3.3. Quantitative analysis of Allyl isothiocyanate by LC-APCI-MSSeparation was performed on a C18column with0.05%formic acid-methanol,3:7(V/V) as mobile phase. Detection and quantitation were performed by APCI/MS. The linear concentration range was0.1-100mg/L, with a correlation coefficient R=0.998. The LOD was0.1ng.4. Optimized the glucosinolates extraction condition through the single factor experiment and LC-ESI-MS quantitaion for Sinigrin. Optimum condition was achieved in70%methanol, at a solid/liquid ratio1:10, at a temperature of water bath80℃for1.5h.5. Purified condition of glucosinolates on a DEAE Sephadex A-25resin was also discussed. Optimum condition was at a resin/samle ratio2:35(g/mL), adsorption at45癈for20min, at a minimum elution amount of35mL of0.1mol/L K2SO4.6. Optimum enzymatic hydrolysis condition was determined through orthogonal experiment with the established LC-APCI-MS method. Optimum condition was at pH7.0, with the mixed of0.5mg myrosinase for reacting40min at40℃7. At optimum conditions, glucosinolates were extracted, followed by resin purification, Pre-HPLC isolation with ESI-MS/MS determination, and only unidentified glucosinolates could be hydrolysized for characterization. With the utilization of HRMS, EA, IR and NMR, the structures of five isolated compounds were identified. They were elucidated as follows:methallyl isothiocyanate(I),3-cyanophenyl isothiocyanate(Ⅱ).4-cyanophenyl isothiocyanate(III),3-(methylthio)phenyl isothiocyanate(Ⅳ) and phenethyl isothiocyanate(V). Compound V had been reported in Mustard, while the others were isolated firstly in its enzymatic products.All the results indicated that this process was very simple, reliable, and effective in extraction, separation and identification of glucosinolates and isothiocyanates. This study would provide a good foundation for further research, development and application of glucosinolates and isothiocynates.