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

Significance of the Topic

Tetrastigma hemsleyanum is a traditional Chinese medicinal plant valued for its anti‐tumor, antioxidant and anti‐inflammatory properties. Flavonoids and phenolic acids are key active compounds in this species, and their precise spatial localization within tuber tissues can inform quality control, phytochemical research and insights into plant defense mechanisms.

Objectives and Study Overview

This study aimed to map the in situ distribution of multiple flavonoids and phenolic acids in the tuber root of Tetrastigma hemsleyanum using atmospheric‐pressure matrix‐assisted laser desorption/ionization mass spectrometry imaging. Regions of interest including epidermis, phloem and three concentric xylem zones were defined to compare relative compound abundance across tissue layers.

Methodology

Sample sections of 40 µm thickness were prepared by cryosectioning embedded tuber roots. Matrix deposition employed vapor‐deposited CHCA followed by solution spray and airbrush application of 1,5‐DAN on adjacent sections. Imaging mass spectrometry data were acquired at m/z 100–800 in both positive and negative ion modes, using a 10 µm laser spot and 20 µm step size. Data analysis and ROI signal extraction were performed with dedicated imaging software and compound databases.

Used Instrumentation

• iMScope QT imaging mass microscope equipped with built‐in optical microscope and atmospheric‐pressure MALDI source
• Quadruple‐time-of-flight mass analyzer
• CM1950 cryomicrotome for sectioning
• Shimadzu iMLayer vapor deposition system for CHCA
• Airbrush PS-270 for 1,5-DAN application

Main Results and Discussion

Matrix comparison indicated CHCA as optimal for positive‐mode detection of flavonoids and 1,5-DAN for negative‐mode detection of salicylic acid. Key flavonoids such as quercetin, kaempferide, catechin and rutin showed highest intensity in the epidermis, decreased in the phloem and exhibited moderate levels in xylem regions closest to phloem, with declining abundance toward the inner xylem. Phenolic acids including salicylic acid, protocatechuic acid, quinic acid and 5-p-coumaroylquinic acid also peaked in the epidermis; salicylic acid displayed additional enrichment in the phloem, while other acids formed gradients from outer to inner xylem.

Benefits and Practical Applications of the Method

This AP-MALDI-MSI approach enables direct, label-free visualization of endogenous metabolites without extensive sample extraction. The spatial maps support standardized herbal quality evaluation, reveal compound localization linked to plant stress responses and guide targeted harvesting or processing strategies based on tissue‐specific metabolite distribution.

Future Trends and Opportunities

Potential developments include integration with complementary imaging techniques, expansion to other medicinal species, further improvements in quantitation and spatial resolution, application of machine learning for pattern recognition and coupling MSI data with orthogonal LC-MS profiling to enrich metabolomic insights.

Conclusion

The combination of atmospheric‐pressure MALDI imaging and high-resolution optical microscopy in a single instrument provides a powerful platform for spatial metabolomics in plant tissues. The observed distribution patterns of flavonoids and phenolic acids in Tetrastigma hemsleyanum offer valuable references for herb quality control and investigations into secondary metabolite function.

Reference

No external literature list was provided in the source text.