Compound Specific Trends of Chemical Defences in Ficus Along an Elevational Gradient Reflect a Complex Selective Landscape.

Elevational gradients affect the production of plant secondary metabolites through changes in both biotic and abiotic conditions. Previous studies have suggested both elevational increases and decreases in host-plant chemical defences. We analysed the correlation of alkaloids and polyphenols with elevation in a community of nine Ficus species along a continuously forested elevational gradient in Papua New Guinea. We sampled 204 insect species feeding on the leaves of these hosts and correlated their community structure to the focal compounds. Additionally, we explored species richness of folivorous mammals along the gradient. When we accounted for Ficus species identity, we found a general elevational increase in flavonoids and alkaloids. Elevational trends in non-flavonol polyphenols were less pronounced or showed non-linear correlations with elevation. Polyphenols responded more strongly to changes in temperature and humidity than alkaloids. The abundance of insect herbivores decreased with elevation, while the species richness of folivorous mammals showed an elevational increase. Insect community structure was affected mainly by alkaloid concentration and diversity. Although our results show an elevational increase in several groups of metabolites, the drivers behind these trends likely differ. Flavonoids may provide figs with protection against abiotic stressors. In contrast, alkaloids affect insect herbivores and may provide protection against mammalian herbivores and pathogens. Concurrent analysis of multiple compound groups alongside ecological data is an important approach for understanding the selective landscape that shapes plant defences.

Distribution of Protein Precipitation Capacity within Variable Proanthocyanidin Fingerprints.

Proanthocyanidins (PAs) are highly bioactive plant specialized metabolites. One of their most characteristic features is their ability to precipitate proteins. In this study, eleven plant species were used to study the structure-activity patterns between PAs and their protein precipitation capacity (PPC) with bovine serum albumin. To obtain a comprehensive selection of PAs with highly variable procyanidin to prodelphinidin ratios and mean degree of polymerizations, nearly 350 subfractions were produced from the eleven plant species by semi-preparative liquid chromatography. Their PA composition was defined by tandem mass spectrometry and high-resolution mass spectrometry, and their PPC was measured with a turbidimetry-based well-plate reader assay. The distribution of the PPC within plant species varied significantly. The mean degree of polymerization of the PAs had a strong correlation with the PPC (r = 0.79). The other structural features were significant from the PPC point of view as well, but they contributed to the PPC in different ways in different plant species. Retention time, prodelphinidin proportion, and mean degree of polymerization explained 64% of the measured variance of the PPC.

Community structure of insect herbivores is driven by conservatism, escalation and divergence of defensive traits in Ficus.

Escalation (macroevolutionary increase) or divergence (disparity between relatives) in trait values are two frequent outcomes of the plant-herbivore arms race. We studied the defences and caterpillars associated with 21 sympatric New Guinean figs. Herbivore generalists were concentrated on hosts with low protease and oxidative activity. The distribution of specialists correlated with phylogeny, protease and trichomes. Additionally, highly specialised Asota moths used alkaloid rich plants. The evolution of proteases was conserved, alkaloid diversity has escalated across the studied species, oxidative activity has escalated within one clade, and trichomes have diverged across the phylogeny. Herbivore specificity correlated with their response to host defences: escalating traits largely affected generalists and divergent traits specialists; but the effect of escalating traits on extreme specialists was positive. In turn, the evolution of defences in Ficus can be driven towards both escalation and divergence in individual traits, in combination providing protection against a broad spectrum of herbivores.

Quantitative and qualitative composition of proanthocyanidins and other polyphenols in commercial red wines and their contribution to sensorially evaluated tannicity.

The analysis of proanthocyanidins (PA) in red wine has typically been conducted using few key methods, such as phloroglucinolysis or precipitation assays. Here, the content of PAs and other common polyphenol groups in commercial red wines were analyzed with a group-specific liquid chromatography-tandem mass spectrometry method. Besides concentrations, the method provides qualitative information about the detected compound groups in the form of two-dimensional (2D) chromatographic fingerprints. The 2D fingerprints of PAs have not been utilized in analysis of red wine before. For instance, 2D chromatographic fingerprints revealed that the complex PA compositions were qualitatively notably similar between many wine types, even when there were considerable differences in concentrations. Finally, 201 commercial red wines had been categorized as either tannic or medium tannic based on their sensorial evaluations. The content of PAs and three different groups of oligomeric adducts of malvidin glycosides and PAs were measured from these wines. The compositional features of the PAs and PA-malvidin glycoside adducts were more important than concentrations in explaining the perceived tannicity.

Red Wine Inspired Chemistry: Hemisynthesis of Procyanidin Analogs and Determination of Their Protein Precipitation Capacity, Octanol-Water Partition, and Stability in Phosphate-Buffered Saline.

Ten dimeric procyanidin (PC) analogs were hemisynthesized from catechin or epicatechin and from five different aldehydes using the same mechanism that produces the important acetaldehyde-mediated adducts of proanthocyanidins (PAs) and anthocyanins in red wine. Protein precipitation capacity (PPC), octanol-water partition coefficient (log P) and stability of the PC analogs were determined. The emphasis was on the PPC because it has been shown to correlate with anthelmintic activity against gastrointestinal nematodes in ruminants and with other beneficial bioactivities in animals, as well. The PPC of PC analogs was greatly improved compared to natural PC dimers, but the capacity was not as great as that of a PC trimer or epigallocatechin gallate. The log P of PC analogs varied from hydrophobic to hydrophilic depending on the intramolecular linkage. Great variation was observed in stabilities of PC analogs in phosphate buffered saline, and the mixtures of degradation products were characterized using high-resolution mass spectrometry.

Composition and evolution of oligomeric proanthocyanidin-malvidin glycoside adducts in commercial red wines.

Studies regarding composition and evolution of oligomeric proanthocyanidin-anthocyanin adducts in red wines have often focused only on a limited number of small dimers. Now, a group-specific liquid chromatography-tandem mass spectrometry method was utilized to measure two-dimensional (2D) chromatographic fingerprints of three different types of oligomeric adducts in commercial red wines. A new protocol was developed to visualize and summarize the chromatographic data. The 2D fingerprints showed how the compositions of the oligomeric adducts had typically only minor differences between wine varieties in young wines, excluding the Pinot Noir wines. Major quantitative differences were found between the wine varieties despite the lack of major compositional differences. The evolution of the concentrations differed between the three structural sub-groups, while similar general patterns were observed in the compositional evolution. Via statistical modelling, several characteristics in the polyphenolic starting material composition were tentatively suggested to affect the formation of the oligomeric adducts.

Relevance of the Concentrations and Sizes of Oligomeric Red Wine Pigments to the Color Intensity of Commercial Red Wines.

Color is a major sensorial characteristic of red wines. Numerous monomeric and some small oligomeric pigments have been characterized from red wines but the contribution of larger oligomeric pigments to the color intensity has not been established by direct measurements. We measured the color intensity of 317 commercial red wines and semiquantified the malvidin glycosides and eight different adduct groups derived from the malvidin glycosides by ultra-performance liquid chromatography-tandem mass spectrometry. Two of these groups were oligomeric pigments consisting of proanthocyanidins and malvidin glycosides with either direct or methylmethine linkages. The carboxypyranomalvidins and the oligomeric pigments were found to be major contributors to the color intensity. Besides the concentrations, the sizes of the oligomeric pigments had a positive and significant connection to the color intensity. The 1-year-old wines were studied separately and, even in the youngest of wines, the adducts of the malvidin glycosides were the major contributors to the color intensity.

Liquid chromatography-tandem mass spectrometry reveals detailed chromatographic fingerprints of anthocyanins and anthocyanin adducts in red wine.

A method was developed with ultra-high performance liquid chromatography-tandem mass spectrometry for the qualitative and semi-quantitative analysis of anthocyanins and anthocyanin-derived adducts in red wine. The method utilized in-source collision induced dissociation in conjunction with multiple reaction monitoring to achieve group-specific detection of the targeted compound groups. The method detected quantification marker ions instead of intact molecules and, as a result, 2D chromatographic fingerprints were produced. Altogether we could detect 18 groups of wine polyphenols, including up to 50 individual monomeric pigments together with fingerprints of three different types oligomeric proanthocyanidin-malvidin adducts. Importantly, the method was able to separate small, medium-sized and large oligomeric adducts. The quantitative and qualitative function of the method was tested with 10 model wines. Ultimately, we were able to obtain a comprehensive picture of the main pigment composition of any model wine with only a single UPLC-MS/MS analysis.