Barley-derived beer brewing by-products contain a high diversity of hydroxycinnamoylagmatines and their dimers.

To aid valorisation of beer brewing by-products, more insight into their composition is essential. We have analysed the phenolic compound composition of four brewing by-products, namely barley rootlets, spent grain, hot trub, and cold trub. The main phenolics detected were hydroxycinnamoylagmatines and dimers thereof. Barley rootlets contained the highest hydroxycinnamoylagmatine content and cold trub the highest dimer content. Additionally, variations in (dimeric) hydroxycinnamoylagmatine composition and content were observed in fourteen barley rootlet samples. The most abundant compound in all rootlets was the glycosylated 4-O-7'/3-8'-linked heterodimer of coumaroylagmatine and feruloylagmatine, i.e. CouAgm-4-O-7'/3-8'-(4'Hex)-DFerAgm. Structures of glycosylated and hydroxylated derivatives of coumaroylagmatine were elucidated by NMR spectroscopy after their purification from a rootlet extract. An MS-based decision tree was developed, which aids in identifying hydroxycinnamoylagmatine dimers in complex mixtures. In conclusion, this study shows that the diversity of phenolamides and (neo)lignanamides in barley-derived by-products is larger than previously reported.

Comment on "Three New Dimers and Two Monomers of Phenolic Amides from the Fruits of Lycium barbarum and Their Antioxidant Activities".

This Comment critically addresses the article by Gao et al. (Gao, K., et al. J. Agric. Food Chem. 2015, 63, 1067-1075), providing the structural elucidation of three phenolamide dimers (neolignanamides) from the fruits of Lycium barbarum. A more recent article published by Chen et al. (Chen, H., et al. J. Agric. Food Chem. 2023, 71, 11080-11093) incorporates these structures into further research on the bioactivity of these compounds. Although the analytical techniques used by Gao et al. are adequate, in our opinion, the nuclear magnetic resonance (NMR) spectroscopic data have not been interpreted correctly, resulting in incorrect structures for three neolignanamides from the fruits of L. barbarum. In this Comment, an alternative interpretation of the NMR spectroscopic data and the corresponding structures are proposed. The proposed structures feature linkage types that are much more common for neolignanamides than the linkage types in the originally reported structures of these compounds.

Exploring the formation of heterodimers of barley hydroxycinnamoylagmatines by oxidative enzymes.

Dimers of hydroxycinnamoylagmatines are phenolic compounds found in barley and beer. Although they are bioactive and sensory-active compounds, systematic reports on their structure-property relationships are missing. This is partly due to lack of protocols to obtain a diverse set of hydroxycinnamoylagmatine homo- and heterodimers. To better understand dimer formation in complex systems, combinations of the monomers coumaroylagmatine (CouAgm), feruloylagmatine (FerAgm), and sinapoylagmatine (SinAgm) were incubated with horseradish peroxidase. For all combinations, the main oxidative coupling products were homodimers. Additionally, minor amounts of heterodimers were formed, except for the combination of FerAgm and CouAgm. Oxidative coupling was also performed with laccases from Agaricus bisporus and Trametes versicolor, resulting in formation of the same coupling products and no formation of CouAgm-FerAgm heterodimers. Our protocol for oxidative coupling combinations of hydroxycinnamoylagmatines yielded a structurally diverse set of coupling products, facilitating production of dimers for future research on their structure-property relationships.

Presence of free gallic acid and gallate moieties reduces auto-oxidative browning of epicatechin (EC) and epicatechin gallate (ECg).

Auto-oxidation of flavan-3-ols leads to browning and consequently loss of product quality during storage of ready-to-drink (RTD) green tea. The mechanisms and products of auto-oxidation of galloylated catechins, the major flavan-3-ols in green tea, are still largely unknown. Therefore, we investigated auto-oxidation of epicatechin gallate (ECg) in aqueous model systems. Oxidation products tentatively identified based on MS included δ- or γ-type dehydrodicatechins (DhC2s) as the main contributors to browning. Additionally, various colourless products were detected, including epicatechin (EC) and gallic acid (GA) from degalloylation, ether-linked ε-type DhC2s, and 6 new coupling products of ECg and GA possessing a lactone interflavanic linkage. Supported by density function theory (DFT) calculations, we provide a mechanistic explanation on how presence of gallate moieties (D-ring) and GA affect the reaction pathway. Overall, presence of gallate moieties and GA resulted in a different product profile and less intense auto-oxidative browning of ECg compared to EC.

Biomimetic Enzymatic Oxidative Coupling of Barley Phenolamides: Hydroxycinnamoylagmatines.

Oxidative coupling of hydroxycinnamoylagmatines in barley (Hordeum vulgare) and related Hordeum species is part of the plant defense mechanism. Three linkage types have been reported for hydroxycinnamoylagmatine dimers, but knowledge on oxidative coupling reactions underlying their formation is limited. In this study, the monomers coumaroylagmatine, feruloylagmatine, and sinapoylagmatine were each incubated with horseradish peroxidase. Their coupling reactivity was in line with the order of peak potentials measured: sinapoylagmatine (245 mV) > feruloylagmatine (341 mV) > coumaroylagmatine (506 mV). Structure elucidation of fourteen in vitro coupling products by NMR and MS revealed that the three main linkage types were identical to those naturally present in Hordeum species, namely, 4-O-7'/3-8', 2-7'/8-8', and 8-8'/9-N-7'. Furthermore, we identified two linkage types that were not previously reported for hydroxycinnamoylagmatine dimers, namely, 8-8' and 4-O-8'. We conclude that oxidative coupling by horseradish peroxidase can be used for biomimetic formation of natural antifungal hydroxycinnamoylagmatine dimers from barley.

Facile Amidation of Non-Protected Hydroxycinnamic Acids for the Synthesis of Natural Phenol Amides.

Phenol amides are bioactive compounds naturally present in many plants. This class of compounds is known for antioxidant, anti-inflammatory, and anticancer activities. To better understand the reactivity and structure-bioactivity relationships of phenol amides, a large set of structurally diverse pure compounds are needed, however purification from plants is inefficient and laborious. Existing syntheses require multiple steps, including protection of functional groups and are generally overly complicated and only suitable for specific combinations of hydroxycinnamic acid and amine. Thus, to facilitate further studies on these promising compounds, we aimed to develop a facile general synthetic route to obtain phenol amides with a wide structural diversity. The result is a protocol for straightforward one-pot synthesis of phenol amides at room temperature within 25 h using equimolar amounts of N,N'-dicyclohexylcarbodiimide (DCC), amine, hydroxycinnamic acid, and sodium bicarbonate in aqueous acetone. Eight structurally diverse phenol amides were synthesized and fully chemically characterized. The facile synthetic route described in this work is suitable for a wide variety of biologically relevant phenol amides, consisting of different hydroxycinnamic acid subunits (coumaric acid, ferulic acid, and sinapic acid) and amine subunits (agmatine, anthranilic acid, putrescine, serotonin, tyramine, and tryptamine) with yields ranging between 14% and 24%.

Toward a Systematic Nomenclature for (Neo)Lignanamides.

Phenylalkenoic acid amides, often referred to as phenol amides or hydroxycinnamic acid amides, are bioactive phytochemicals, whose bioactivity can be enhanced by coupling to form dimers or oligomers. Phenylalkenoic acid amides consist of a (hydroxy)cinnamic acid derivative (i.e., the phenylalkenoic acid subunit) linked to an amine-containing compound (i.e., the amine subunit) via an amide bond. The phenylalkenoic acid moiety can undergo oxidative coupling, either catalyzed by oxidative enzymes or due to autoxidation, which leads to the formation of (neo)lignanamides. Dimers described in the literature are often named after the species in which the compound was first discovered; however, the naming of these compounds lacks a systematic approach. We propose a new nomenclature, inspired by the existing system used for hydroxycinnamic acid dimers and lignin. In the proposed systematic nomenclature for (neo)lignanamides, compound names will be composed of three-letter codes and prefixes denoting the subunits, and numbers that indicate the carbon atoms involved in the linkage between the monomeric precursors. The proposed nomenclature is consistent, future-proof, and systematic.

Browning of Epicatechin (EC) and Epigallocatechin (EGC) by Auto-Oxidation.

Green tea catechins are well known for their health benefits. However, these compounds can easily be oxidized, resulting in brown color formation, even in the absence of active oxidative enzymes. Browning of catechin-rich beverages, such as green tea, during their shelf life is undesired. The mechanisms of auto-oxidation of catechins and the brown products formed are still largely unknown. Therefore, we studied auto-oxidative browning of epicatechin (EC) and epigallocatechin (EGC) in model systems. Products of EC and EGC auto-oxidation were analyzed by reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled to mass spectrometry (RP-UHPLC-PDA-MS). In the EC model system, 11 δ-type dehydrodicatechins (DhC2s) and 18 δ-type dehydrotricatechins (DhC3s) that were related to browning could be tentatively identified by their MS2 signature fragments. In the EGC model system, auto-oxidation led to the formation of 13 dihydro-indene-carboxylic acid derivatives and 2 theaflagallins that were related to browning. Based on the products formed, we propose mechanisms for the auto-oxidative browning of EC and EGC. Furthermore, our results indicate that dimers and oligomers that possess a combination of an extended conjugated system, fused rings, and carbonyl groups are responsible for the brown color formation in the absence of oxidative enzymes.