Visualizing the spatial distribution of flavonoids and phenolic acids in the tuber root of Tetrastigma hemsleyanum using AP-MALDI-MSI (ASMS)

Importance of the Topic

This study focuses on Tetrastigma hemsleyanum, a traditional herbal medicine prized for its heat-clearing, detoxifying, antioxidant, and anti-inflammatory properties. Flavonoids and phenolic acids are the primary bioactive constituents and serve as key quality markers in plant-based therapeutics.

Study Objectives and Overview

The main goal was to simultaneously visualize the spatial distribution of multiple flavonoids and phenolic acids within the tuber root of T. hemsleyanum. Atmospheric-pressure MALDI mass spectrometry imaging (AP-MALDI-MSI) was applied to generate detailed in situ maps across the epidermis, phloem, and xylem tissues.

Methodology and Instrumentation

Root sections (40 µm) were prepared via gelatin embedding, cryosectioning at –20 °C, and mounting on ITO slides. CHCA matrix was applied for positive-ion mode and 1,5-DAN for negative-ion mode. AP-MALDI-MSI data (m/z 100–800) were acquired with a 10 µm laser spot and 20 µm step size. IMAGEREVEAL MS software enabled image reconstruction and ROI-based signal quantification.

• iMScope QT imaging mass spectrometer (Shimadzu, AP-MALDI source with Q-TOF analyzer)
• iMLayer™ matrix vapor deposition system (Shimadzu)
• CM1950 cryomicrotome (Leica)
• Airbrush PS-270 (GSI Creos) for matrix spraying

Main Results and Discussion

Flavonoids including quercetin, kaempferide, catechin, rutin, kaempferol, procyanidin dimer, hyperoside, apigenin, daidzein, and biochanin A concentrated predominantly in the epidermis, with decreasing intensity toward the phloem and inner xylem regions. Phenolic acids such as salicylic acid, protocatechuic acid, quinic acid, and 5-p-coumaroylquinic acid also peaked in the epidermis; salicylic acid showed additional enrichment in the phloem. These localization patterns likely reflect protective roles against biotic and abiotic stressors.

Benefits and Practical Applications

• Enables direct in situ mapping of endogenous metabolites without extraction steps.
• Offers high spatial resolution to resolve tissue-specific localization.
• Supports quality assessment of T. hemsleyanum based on metabolite distribution profiles.
• Provides a foundation for pharmacological research and plant defense mechanism studies.

Future Trends and Opportunities

Future developments may integrate AP-MALDI-MSI with quantitative LC-MS, transcriptomic correlations, and expand applications to other medicinal plants. Advances in spatial resolution, multivariate data analysis, and imaging workflows will further enhance metabolomic visualization and functional characterization of bioactive compounds.

Conclusion

The AP-MALDI-MSI approach on the iMScope QT instrument effectively reveals spatial distributions of flavonoids and phenolic acids in T. hemsleyanum tuber. This method offers a powerful tool for quality control and deeper insights into plant secondary metabolism.