Histochemical Study Of Alkaloids In The Roots Of Boluo Back Based On LMD-LC-MS/MS Technology

In the present study, laser microdissection together with fluorescence microscopy and LC-MS/MS were used to histochemically study alkaloids in the main and lateral roots of Macleaya cordata(Willd.) R. Br. from different growth stages.The technique of fluorescence microscopy was applied to identify exactly various tissues(including cork, cortex, phloem, xylem vascular bundles, xylem rays, etc.),which then were dissected accurately by laser. UHPLC-Q/TOF MS was used to qualitatively analyze dissected tissues. Finally, 49 alkaloids were deduced. Structural assignments were confirmed for 13 compounds by comparing with standards.Twenty-four chemicals were detected for the first time in M. Cordata. The present deduced the biosynthesis for alkaloids identified in the root of M. cordata by combining the reported metabolic pathways of isoquinoline alkaloids in other plant.The biosynthetic pathways for sanguinarine and chelerythrine exist in M. cordata root.Then, 49 compounds were profiled on tissue samples. The results revealed that total numbers of alkaloid in tissues from young roots were less than in tissues from old roots. And corks from lateral roots contained a bit of more alkaloids than it from main roots. The cork and xylem vascular bundles were found to accumulate more alkaloids than other tissues. Characteristic components in the cork usually belonged to the hydrophobic alkaloids(benzophenanthridine- and dihydrobenzophenanthridine-type).According to the findings of tissue-specific qualitative analysis of alkaloids, contents of protopine, allocryptopine, sanguinarine, chelerythrine, dihydrochelerythrine and dihydrosanguinarine were further determine in specified microdissected tissues using UHPLC-QqQ MS. It was shown that tissues from young roots have lower amounts of six kinds of alkaloid than those from old roots, but the ratio of sanguinarine and chelerythrine contents account for the six alkaloids in young and old sample tissues presented the opposite trend. The contents of six compounds in cork from lateral root were higher than in cork from main root. Cork and xylem vascular bundles ordinarily had larger quantities of alkaloids than others.Therefore, the integrated method is high-resolution, specific, sensitive and reliable for histochemical study. The identified alkaloids and its distribution and accumulation patterns in the study would provide basis for the illumination of mechanism of alkaloid production, transportation and storage in the root of M. cordata. In addition,it was also significant for the quality assessment and reasonable harvest of raw plant materials.