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. 2020 Sep 2;25(17):4006.
doi: 10.3390/molecules25174006.

Comprehensive Metabolome Analysis of Fermented Aqueous Extracts of Viscum album L. by Liquid Chromatography-High Resolution Tandem Mass Spectrometry

Evelyn Peñaloza  1   2 , Carla Holandino  2 , Claudia Scherr  3 , Paula I P de Araujo  1 , Ricardo M Borges  4 , Konrad Urech  3 , Stephan Baumgartner  3   5 , Rafael Garrett  1
Affiliations

Comprehensive Metabolome Analysis of Fermented Aqueous Extracts of Viscum album L. by Liquid Chromatography-High Resolution Tandem Mass Spectrometry

Evelyn Peñaloza et al. Molecules. .

Abstract

Fermented aqueous extracts of Viscum album L. are widely used for cancer treatment in complementary medicine. The high molecular weight compounds viscotoxins and lectins are considered to be the main active substances in the extracts. However, a vast number of small molecules (≤1500 Da) is also expected to be present, and few studies have investigated their identities. In this study, a comprehensive metabolome analysis of samples of fermented aqueous extracts of V. album from two host tree species (Malus domestica and Pinus sylvestris), both prepared by two pharmaceutical manufacturing processes, was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). A total of 212 metabolites were putatively annotated, including primary metabolites (e.g., amino acids, organic acids, etc.) and secondary metabolites (mostly phenolic compounds). A clear separation between V. album samples according to the host tree species, but not due to manufacturing processes, was observed by principal component analysis. The biomarkers responsible for this discrimination were assessed by partial least squares-discriminant analysis. Because V. album extracts from different host trees have different clinical applications, the present work highlights the possibility of characterizing the metabolome for identification and traceability of V. album fermented aqueous extracts.

Keywords: LC-MS; host tree; metabolic profiling; metabolomics; mistletoe; natural products.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Metabolites in the fermented aqueous extracts of V. album putatively annotated by LC-HRMS/MS according to the ionization mode and chemical group.
Figure 2
Figure 2
Typical LC-HRMS chromatograms of fermented aqueous extracts of V. album from the two host trees (M. domestica and P. sylvestris) and manufacturing processes (APVAE and VAE) in positive (ESI+) and negative (ESI-) electrospray ionization modes. Abbreviations: ISCM: M. domestica/APVAE, HGM: M. domestica/VAE, ISCP: P. sylvestris/APVAE; HGP: P. sylvestris/VAE.
Figure 3
Figure 3
PCA (A,B) and PLS-DA (C,D) score plots from data of V. album fermented aqueous extracts analyzed by LC-ESI(±)-HRMS. Abbreviations: QC: pooled quality control samples, ISCM: M. domestica/APVAE, HGM: M. domestica/VAE, ISCP: P. sylvestris/APVAE; HGP: P. sylvestris/VAE.
Figure 4
Figure 4
Box plot graphs of annotated metabolites responsible for the discrimination of P. sylvestris group from M. domestica in the PLS-DA model. Abbreviations: ISCM: M. domestica/APVAE, HGM: M. domestica/VAE, ISCP: P. sylvestris/APVAE; HGP: P. sylvestris/VAE. Tukey’s multiple comparison test was performed for significant difference. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 5
Figure 5
Box plot graphs of annotated metabolites responsible for the discrimination of M. domestica group from P. sylvestris in the PLS-DA model. Abbreviations: ISCM: M. domestica/APVAE, HGM: M. domestica/VAE, ISCP: P. sylvestris/APVAE; HGM: P. sylvestris/VAE. Tukey’s multiple comparison test was performed for significant difference. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. n.s. (not significant): p > 0.05.
Figure 6
Figure 6
Nodes from the molecular network related to flavonoid structures obtained from fermented aqueous extracts of V. album.
Figure 7
Figure 7
Nodes from the molecular network highlighting the compounds with high loading value in PLS-DA analysis and their distribution across different sample groups of fermented aqueous extracts of V. album.
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