New cinnamid and rosmarinic acid derived compounds biosynthesized in Escherichia coli as Leishmania amazonensis arginase inhibitors

Arginase is a metalloenzyme that plays a central role in Leishmania infection. Previously, rosmarinic and caffeic acids were described as antileishmanial and as a Leishmania amazonensis arginase inhibitor and now, we describe the inhibition of arginase in L. amazonensis by rosmarinic acid analogs (1-7) and new caffeic acids derived amides (8-10). Caffeic acid esters and amides were produced by means of the engineered synthesis in E. coli and tested against L. amazonensis arginase. New amides (8-10) were biosynthesized in E. coli cultured with 2 mM of different combinations of feeding substrates. The most potent arginase inhibitors showed Ki(s) between 2 - 5.7 μM. Compounds 2-4 and 7 inhibited L-ARG through a noncompetitive mechanism, and 9 showed a competitive inhibition. By applying an in silico protocol we determined the binding mode of compound 9. The competitive inhibitor of L-ARG targets key residues within the binding site of the enzyme establishing a metal coordination bond with the metal ions as well as a series of hydrophobic and polar contacts supporting its micromolar inhibition of L-ARG. These results highlight that the dihydroxycinnamic-derived compounds can be used as the basis for the development of new drugs using a powerful tool based on the biosynthesis of arginase inhibitors.

Feature-Based Molecular Networking to Target the Isolation of New Caffeic Acid Esters from Yacon (Smallanthus sonchifolius, Asteraceae)

Smallanthus sonchifolius (yacon) is an edible tuberous Andean shrub that has been included in the diet of indigenous people since before recorded history. The nutraceutical and medicinal properties of yacon are widely recognized, especially for the improvement of hyperglycemic disorders. However, the chemical diversity of the main bioactive series of caffeic acid esters has not been explored in detail. In this metabolomics study, we applied the latest tools to facilitate the targeted isolation of new caffeic acid esters. Using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we analyzed extracts from different organs (roots, vascular tissues of the stems, stem epidermis, leaves, bracts, and ray flowers) and followed a feature-based molecular networking approach to characterize the structural diversity of caffeic acid esters and recognize new compounds. The analysis identified three potentially new metabolites, one of them confirmed by isolation and full spectroscopic/spectrometric assignment using nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), and MS/MS. This metabolite (5-O-caffeoyl-2,7-anhydro-d-glycero-β-d-galacto-oct-2-ulopyranosonic acid), along with eight known caffeic acid esters, was isolated from the roots and stems. Furthermore, based on detailed tandem MS analyses, we suggest that the two isomeric monocaffeoyl-2,7-anhydro-2-octulopyranosonic acids found in yacon can be reliably distinguished based on their characteristic MS2 and MS3 spectra. The outcome of the current study confirms the utility of feature-based molecular networking as a tool for targeted isolation of previously undescribed metabolites and reveals the full diversity of potentially bioactive metabolites from S. sonchifolius.

Metabolomic and Gene Expression Studies Reveal the Diversity, Distribution and Spatial Regulation of the Specialized Metabolism of Yacón (Smallanthus sonchifolius, Asteraceae)

Smallanthus sonchifolius, also known as yacón, is an Andean crop species commercialized for its nutraceutical and medicinal properties. The tuberous roots of yacón accumulate a diverse array of probiotic and bioactive metabolites including fructooligosaccharides and caffeic acid esters. However, the metabolic diversity of yacón remains unexplored, including the site of biosynthesis and accumulation of key metabolite classes. We report herein a multidisciplinary approach involving metabolomics, gene expression and scanning electron microscopy, to provide a comprehensive analysis of the diversity, distribution and spatial regulation of the specialized metabolism in yacón. Our results demonstrate that different metabolic fingerprints and gene expression patterns characterize specific tissues, organs and cultivars of yacón. Manual inspection of mass spectrometry data and molecular networking allowed the tentative identification of 71 metabolites, including undescribed structural analogues of known bioactive compounds. Imaging by scanning electron microscopy revealed the presence of a new type of glandular trichome in yacón bracts, with a distinctive metabolite profile. Furthermore, the high concentration of sesquiterpene lactones in capitate glandular trichomes and the restricted presence of certain flavonoids and caffeic acid esters in underground organs and internal tissues suggests that these metabolites could be involved in protective and ecological functions. This study demonstrates that individual organs and tissues make specific contributions to the highly diverse and specialized metabolome of yacón, which is proving to be a reservoir of previously undescribed molecules of potential significance in human health.

Caffeic Acid Esters Are Effective Bactericidal Compounds Against Paenibacillus larvae by Altering Intracellular Oxidant and Antioxidant Levels

American Foulbrood (AFB) is a deadly bacterial disease affecting pupal and larval honey bees. AFB is caused by the endospore-forming bacterium Paenibacillus larvae (PL). Propolis, which contains a variety of organic compounds, is a product of bee foraging and is a resinous substance derived from botanical substances found primarily in trees. Several compounds from the class of caffeic acid esters, which are commonly found in propolis, have been shown to have antibacterial activity against PL. In this study, six different caffeic acid esters were synthesized, purified, spectroscopically analyzed, and tested for their activity against PL to determine the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). Caffeic acid isopropenyl ester (CAIE), caffeic acid benzyl ester (CABE), and caffeic acid phenethyl ester (CAPE) were the most effective in inhibiting PL growth and killing PL cell with MICs and MBCs of 125 µg/mL when used individually, and a MIC and MBC of 31.25 µg/mL for each compound alone when CAIE, CABE, and CAPE are used in combination against PL. These compounds inhibited bacterial growth through a bactericidal effect, which revealed cell killing but no lysis of PL cells after 18 h. Incubation with CAIE, CABE, and CAPE at their MICs significantly increased reactive oxygen species levels and significantly changed glutathione levels within PL cells. Caffeic acid esters are potent bactericidal compounds against PL and eliminate bacterial growth through an oxidative stress mechanism.

Modulation of ERK1/2 and Akt Pathways Involved in the Neurotrophic Action of Caffeic Acid Alkyl Esters

Neurodegenerative diseases affect millions of human lives all over the world. The number of afflicted patients is rapidly growing, and disease-modifying agents are urgently needed. Caffeic acid, an important member of the hydroxycinnamic acid family of polyphenols, has considerable neurotrophic effects. We have previously shown how caffeate alkyl ester derivatives significantly promote survival and differentiation in neuronal cells. In this study, the mechanisms by which these ester derivatives exert their neurotrophic effects are examined. A series of eight caffeic acid esters with different alkyl chain lengths, ranging from methyl (CAF1) to dodecyl esters (CAF8), were synthesized and studied for their influence on neurotrophic signaling pathways. Caffeate esters did not induce tropomyosin-receptor kinase A (TrkA) phosphorylation, which was assessed by immunoblotting up to a concentration of 25 µM. NIH/3T3 cells overexpressing TrkA were generated to further examine phosphorylation of this receptor tyrosine kinase. None of the esters induced TrkA phosphorylation in these cells either. Assessment of the effect of caffeate derivatives on downstream neurotrophic pathways by immunoblotting showed that the most potent esters, decyl caffeate (CAF7) and dodecyl caffeate (CAF8) caused extracellular signal-regulated kinase (ERK1/2) and Akt serine threonine kinase phosphorylation in PC12 cells at 5 and 25 µM concentrations. In conclusion, this study shows that caffeate esters exert their neurotrophic action by modulation of ERK1/2 and Akt signaling pathways in neuronal cells, and further demonstrates the potential therapeutic implications of these derivatives for neurodegenerative diseases.

Novel Phenolic Constituents of Pulmonaria officinalis L. LC-MS/MS Comparison of Spring and Autumn Metabolite Profiles

Lungwort (Pulmonaria officinalis L., Boraginaceae) is considered to possess therapeutic properties and it has been traditionally used as a remedy against various lung disorders in many countries. Nevertheless, very few data concerning its phytochemical composition are available. This research aims to provide a detailed description of specialized metabolites from the aerial parts of lungwort. Nine previously undescribed and 36 known phenolic compounds were detected in the 50% methanolic extract. Following multistep preparative procedures, structures of newly discovered compounds were determined using one- and two-dimensional techniques of NMR spectroscopy. Among the identified compounds were caffeic acid esters with aliphatic hydroxycarboxylic acids, conjugates of dicaffeic acid with rosmarinic acid, and previously unknown isomers of isosalvianolic acid A and yunnaneic acid E, as well as other lignans. Concentrations of all identified phenolic derivatives in the investigated herbal material were estimated using a method based on liquid chromatography with high-resolution mass spectrometry detection. Seasonal changes in the concentration of metabolites were also investigated using targeted and untargeted metabolomics techniques.

Caffeic Acid Esters Affect Intracellular Oxidation and Vitality of Yeast Saccharomyces cerevisiae Cells

The effect of four esters of caffeic acid, caffeic acid methanol ester (CAME), caffeic acid ethanol ester (CAEE), caffeic acid isopropyl ester (CAIPE) and caffeic acid phenethyl ester (CAPE) on intracellular oxidation, vitality and viability of the yeast Saccharomyces cerevisiae as a model eukaryotic organism was investigated. Results showed that each ester showed its own behavior at the concentrations tested. For CAPE, CAIPE and CAEE decreased intracellular oxidation and simultaneously increased cellular vitality with no changes in cellular viability compared to the control were determined. Additionally, a combination of CAIPE or CAPE with ethanolic propolis extract was observed to be more effective in decreasing intracellular oxidation compared to propolis extract alone.