Phyllobilins (PBs) are linear tetrapyrroles and represent the only known bilin family originating from chlorophyll rather than heme. Although colorless phylloleucobilins have long been regarded as the predominant chlorophyll metabolites, recent evidence indicates that colored phylloxanthobilins (PxBs) and phylloroseobilins (PrBs) are more widespread and structurally diverse than previously assumed. Here, we employed UHPLC-VWD-HRMS2 in combination with feature-based molecular networking (FBMN) to systematically search for PrBs in plant extracts known to contain PxBs, revealing that both PB-types co-occur in several plant extracts. Functionally, we show that a PrB from Cercidiphyllum japonicum inhibits directed migration of T24 cancer cells in a Boyden chamber assay and disrupts endothelial tube stability in HUVEC while leaving extracellular matrix remodeling largely unaffected. These findings establish PrB as an actin-targeting bilin and highlight PBs as a previously overlooked natural-product scaffold for modulating the actin cytoskeleton in the context of cancer progression and angiogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE:Leaf preparations of Carica papaya L. (Caricaceae) are widely used in traditional medicine across Africa, Asia, the Caribbean, and Australia for the management of inflammatory conditions, dengue fever, asthma, infections, wound healing, and, in some traditions, cancer. Ethnopharmacological reports further indicate that aged or senescent leaves are sometimes preferred over fresh material for extract preparation, yet the scientific basis for this practice remains largely unexplored. AIM OF THE STUDY:This study aims to investigate how leaf senescence influences the phytochemical composition and biological activities of C. papaya leaf extracts (PLE) and to evaluate whether these changes support the traditional preference for aged leaf material. MATERIAL AND METHODS:Methanolic extracts and traditionally inspired aqueous leaf juice and decoction preparations were characterized by HPLC-DAD-MS and UHPLC-VWD-HRMS/MS. Feature-based molecular networking was applied to visualize compositional differences. Antioxidative activity was evaluated using ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl assays and by measuring intracellular reactive oxygen species levels. Wound-healing potential was assessed in scratch assays using rat epithelial and alveolar cells. Immunomodulatory effects were investigated using a THP-1-Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter system and pro-inflammatory cytokine quantification. Lipid mediator profiling in polarized macrophages was performed using a UHPLC-QTRAP setup. Antiviral activity was assessed in dengue virus serotype 2-infected Huh7 cells and chikungunya virus-infected Vero cells using plaque assay. Antifungal activity was evaluated against five pathogenic fungi. RESULTS:Senescent extracts showed enrichment of flavonoid-associated compounds, while extracts derived from senescent and withered leaves additionally contained phyllobilins (PBs). Senescent PLE exhibited superior antioxidative effects. Withered extracts demonstrated the most pronounced wound-healing activity. Green PLE significantly reduced NF-κB activation and modestly decreased interleukin 6, interleukin 8, and tumor necrosis factor α release, whereas senescent and withered extracts more strongly reduced the formation of specific 5-lipoxygenase-derived lipid mediators by macrophages. Only minimal antiviral effects and no antifungal effects were observed under the tested conditions. CONCLUSION:Leaf senescence substantially reshapes the phytochemical composition and bioactivity of PLE. Whereas fresh and aged leaves differentially modulate inflammatory pathways, aged leaves display superior antioxidant and wound-healing properties. These findings provide experimental support for the ethnopharmacological preference for aged leaf material and highlights the importance of plant developmental stage in ethnopharmacological research and standardization of herbal preparations.

Aromatic amino acids are precursors of neurotransmitters and immunomodulatory molecules, and their catabolism is dysregulated in various disorders associated with inflammation. This dysregulation often correlates with disease stage, symptom severity, comorbidities, quality of life, and cognitive performance, making its measurement valuable for research, diagnostics, and personalized monitoring. We developed a rapid, reliable, and cost-effective HPLC method for the simultaneous quantification of phenylalanine, tyrosine, tryptophan, and kynurenine in human serum and plasma. After protein precipitation, analytes were separated on a reversed-phase C18 column under isocratic conditions. Detection was performed based on intrinsic fluorescence for tryptophan, phenylalanine, and tyrosine and on UV absorption for kynurenine and the internal standard nitrotyrosine. The method showed linearity (R2 > 0.99) over 0.31-20 μM for kynurenine, 1.56-200 μM for phenylalanine, 0.08-200 μM for tryptophan, and 0.78-200 μM tyrosine. Limits of detection were 0.01 μM for tryptophan, 0.08 μM for tyrosine and kynurenine, and 0.39 μM for phenylalanine. Precision and accuracy were within 15%, and recovery rates ranged from 98 to 100%. Samples remained stable after processing and after three freeze-thaw cycles. Interlaboratory testing confirmed the reproducibility of the results. This validated method enables sensitive, accurate, and simultaneous quantification of key aromatic amino acids, providing a practical alternative to LC-MS/MS for routine diagnostics and biomarker studies.

Apatococcus ammoniophilus (Trebouxiophyceae) is a recently identified green alga forming green biofilms on the bark and twigs of coniferous trees. It contains high amounts of an unknown mycosporine-like amino acid (MAA), a compound class known for its photoprotective, antioxidant, and wound-healing properties. To identify this MAA, a workflow combining UHPLC-VWD-HRMS2 analysis, feature-based molecular networking, and metabolite annotation using a rationally designed combinatorial database was developed. By leveraging the limited set of known MAA core structures and amino acid-related substituents, a theoretical compound library was created in silico and implemented into the bioinformatics platform SIRIUS. Applying the workflow to various extracts of A. ammoniophilus and the basidiomycete Stereum gausapatum, included as an MAA-producing reference to test whether both organisms share the same unknown MAA, enabled the putative annotation of several MAA candidates and led to the isolation, structure elucidation, and biological activity evaluation of three compounds: mycosporine-serinol, gadusol, and the novel algasporine-glycine. Overall, this study provides valuable insights into the variable MAA profile of an aerophytic microalga, highlighting its potential for future biotechnological applications in studying photoprotection in terrestrial organisms, while also establishing a broadly applicable workflow for the discovery and annotation of MAAs in natural extracts.

Chlorophyll (Chl) metabolism is pivotal to both photosynthesis and plant senescence and represents one of the most fundamental biological processes on Earth with an estimated annual turnover of 1 billion tons. During Chl degradation, only early catabolites and corresponding enzymes are well characterized, whereas for late-stage degradation products it remains often unclear if their formation involves specific enzymes. Here, we report that the ubiquitous YUCCA10 enzymes from the YUCCA flavin-containing monooxygenase (FMOs) family in land plants, normally implicated in the biosynthesis of indole-3-acetic acid (IAA) as the primary form of auxin, surprisingly catalyze the production of several predominant Chl catabolites via mechanistically distinct Baeyer-Villiger oxidation and subsequent hydrolytic γ-lactam-forming deformylation reactions. These historically postulated but hitherto undiscovered Chl degradation steps on several high molecular weight chl catabolites were verified for YUCCA10 from Vitis vinifera and Coffea arabica, while YUCCA10 from Arabidopsis thaliana lacked this activity. In contrast, all three homologs were able to catalyze the rate-limiting key step in IAA biosynthesis, akin to other YUCCA enzymes. Interestingly, Chl catabolites at physiological concentrations impaired IAA formation by YUCCA10 in vitro, suggesting a key role in leaf senescence through enzymatic feedback regulation of auxin levels.

Many fish species achieve ultraviolet radiation (UVR) protection by accumulating mycosporine-like amino acids (MAAs) from their diet. High concentrations are often found in the eyes, but the localization of MAAs in specific fish eye tissues is unknown. In the present study we dissected the eyes of six fish species, and prepared cornea, lens, vitreous body and a mixed tissue sample consisting of retina, choroidea and sclera (RCS). Furthermore, to test if MAAs are transferred further up the food web, we investigated the occurrence of MAAs also in eyes of warm-blooded piscine predators. Most, but not all, fish eye tissue samples contained the MAAs palythine, asterina-330, usujirene and palythene, but in different quantitative patterns. The highest MAA concentrations were found in the cornea, lens and vitreous body, while the innermost RCS layer showed a much smaller content. All data indicate that most MAAs are mainly located in the cornea and/or lens and vitreous body, thereby providing an improved photoprotection of the RCS layer, and in particular of the innermost, UVR-sensitive retina. No MAAs were detected in the eyes of cormorants and piscivorous mammals, suggesting that the trophic transfer of MAAs from the primary producers via zooplankton apparently ends in the fish.

Hops is of high relevance to the food sector, and increasingly valued as medicinal plant. Its complex phytochemistry includes phenolic compounds and bitter prenylated polyketides, but phyllobilins─bioactive linear tetrapyrroles from chlorophyll catabolism─remain underexplored. In this work, several dioxobilin-type phylloleucobilins (DPleBs) and phylloxanthobilins (DPxBs) were identified in yellowish leaves of common hops (Humulus lupulus). Isolation from 107 g of leaves yielded 0.24 mg of Hl-DPleB-28 and 0.80 mg of Hl-DPxB-31. Structural elucidation via UV/vis, HR-MS2, and NMR confirmed those as new phyllobilins, featuring an unusual hydroxylation motif, indicating an uncharacterized metabolic pathway. Hl-DPxB constituted about 40% of HPLC peak areas at 420 nm in yellow leaves, suggesting its significant role in the visual senescence of hops. Hl-DPxB-31 possessed high antioxidative activity, comparable to quercetin. A virtual tool predicted over 60% bitterness probability. These findings expand the phytochemical profile of hops and highlight potential for upcycling leaf waste.

Coastal marine organisms are often exposed to high levels of biologically harmful ultraviolet radiation (UVR), the most photochemically reactive waveband of sunlight. It is well known that marine organisms at higher trophic levels, such as fish, enhance their UV protection by accumulating UV-protective metabolites from their diet, as primary producers can effectively synthesize these compounds. Among the best studied natural UV-sunscreens in marine organisms are mycosporine-like amino acids (MAAs). They are known for their high molar extinction coefficients in the UVR-region along with pronounced photo- and thermal stability. In the present study we investigated the qualitative and quantitative MAA distribution in organs (eyes, gills, heart, intestine, kidneys, liver, skin, and stomach) of three flatfish species from the Baltic Sea, the benthivorous European flounder (Platichthys flesus) and European plaice (Pleuronectes platessa), and the piscivorous turbot (Scophthalmus maximus), using state-of-the-art analytical methods. Most of the analyzed organ samples contained the MAAs palythine, asterina-330, porphyra-334, usujirene, and palythene at concentrations sufficient for reliable detection and quantification using an established HPLC-UV method. Additionally, in a few samples also shinorine and mycosporine-methylamine-threonine were found. The highest MAA contents (0.04 to 0.25 mg g-1 dry weight) occurred in the eyes of the three fish species, while the other organs exhibited much lower but still detectable concentrations. Our data support the assumed trophic transfer of MAAs from primary producers via the food web to fish. For the first time we show that MAAs are not only found in the eyes but also in internal organs that possibly represent transfer points from the digestive tract to UV-sensitive tissues. The underlying mechanisms are, however, still unknown.

Polyamines, particularly spermidine, play a vital role in regulating cellular functions and influencing processes such as proliferation, ageing, and immune modulation. This study explores the effects of spermidine on the immunoregulatory pathway of tryptophan breakdown, which is mediated by indoleamine 2,3-dioxygenase 1 (IDO-1), as well as on neopterin synthesis, in peripheral blood mononuclear cells. Spermidine treatment was found to suppress IDO-1 activity in mitogen-stimulated cells in a dose-dependent manner without affecting neopterin synthesis. This suppressive effect on tryptophan breakdown contributes to the debate surrounding the health-promoting antioxidant properties of spermidine. However, the divergent regulation of the two immunobiochemical pathways investigated, which share common upstream signalling events, requires further exploration.

Phyllobilins are chlorophyll-derived metabolites that represent a distinct class of bioactive natural products. Although widespread in nature, they remain largely underexplored in phytochemical research. In this study, we present a workflow for the dereplication and identification of phyllobilins in complex plant extracts. The approach combines RP-MPLC fractionation, UHPLC-VWD-HRMS2 analysis, antioxidant activity testing, and data analysis using feature-based molecular networking. Methanolic extracts of senescent Echinacea purpurea leaves, known to be rich in structurally diverse chlorophyll metabolites, were used to generate experimental MS2 spectra for a set of phylloxanthobilins, which were subsequently uploaded to the GNPS community database. The workflow was then applied to senescent Salvia officinalis leaves, a pharmaceutically important plant with an unknown phyllobilin profile, leading to the annotation of several phylloxanthobilins. One metabolite featuring a malonic acid ester moiety was isolated and structurally confirmed using 1D- and 2D-NMR spectroscopy. Future applications of this workflow may enable the systematic annotation and prioritization of previously overlooked phyllobilins across diverse plant species, addressing a critical gap in the analytical profiling of bioactive plant extracts. By leveraging public MS2 spectral libraries and network-based data analysis, it offers a scalable tool to address a long-standing blind spot of natural product research.

Background Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.Results ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.Conclusion ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

Abstract Phyllobilins are open-chain products of the biological degradation of chlorophyll a in higher plants. Recent studies reveal that phyllobilins exert anti-oxidative and anti-inflammatory properties, as well as activities against cancer cells, that contribute to the human health benefits of numerous plants. In general, phyllobilins have been overlooked in phytochemical analyses, and – more importantly – in the analyses of medicinal plant extracts. Nevertheless, over the past three decades, > 70 phyllobilins have been identified upon examination of more than 30 plant species. Eight distinct chromophoric classes of phyllobilins are known: phyllolumibilins (PluBs), phylloleucobilins (PleBs), phylloxanthobilins (PxBs), and phylloroseobilins (PrBs)–each in type-I or type-II groups. Here, we present a database of absorption and fluorescence spectra that has been compiled of 73 phyllobilins to facilitate identification in phytochemical analyses. The spectra are provided in digital form and can be viewed and downloaded at www.photochemcad.com. The present review describes the plant origin, molecular structure, and absorption and fluorescence features of the 73 phyllobilins, along with an overview of key medicinal properties. The review should provide an enabling tool for the community for the straightforward identification of phyllobilins in plant extracts, and the foundation for deeper understanding of these ubiquitous but underexamined plant-derived micronutrients for human health.

Since the first report on a yeast three-hybrid system, several approaches have successfully utilized different setups for discovering targets of small molecule drugs. Compared to broadly applied MS based target identification approaches, the yeast three-hybrid system represents a complementary method that allows for the straightforward identification of direct protein binders of selected small molecules. One major drawback of this system, however, is that the drug has to be taken up by the yeast cells in sufficient concentrations. Here, we report the establishment of a yeast three-hybrid screen in the deletion strain ABC9Δ, which is characterized by being highly permeable to small molecules. We used this system to screen for protein binding partners of ethinylestradiol, a widely used drug mainly for contraception and hormone replacement therapy. We identified procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2 or lysyl hydroxylase, LH2) as a novel direct target and were able to confirm the interaction identified with the yeast three-hybrid system by a complementary method, affinity chromatography, to prove the validity of the hit. Furthermore, we provide evidence for an interaction between the drug and PLOD2 in vitro and in cellulo.

AbstractBeim Chlorophyllabbau entstehen Phyllobiline. Sie galten lange als Abfallprodukte eines Entgiftungsprozesses, zeigen sich aber nun als pharmakologisch aktiv. Diese Pflanzeninhaltsstoffe können wichtig für die Lebensmittelindustrie sein.

Consumers' tendency to waste suboptimal food poses challenges for sustainable food systems. In the case of aging produce, consumers often experience a conflict between the goals of not wanting to waste food and food safety. In addition to safety, perceived healthfulness can influence the decision between consumption and disposal of (suboptimal) food items. Using an online information experiment on the safety and healthfulness of aging produce, this study investigates consumers' willingness to consume aging produce. Results show that providing information on safety or healthfulness results in increased safety and health perception, which in turn increases willingness to consume aging produce. In addition, consumers with lower food disgust sensitivity, a decision style characterized by less reliance on expiration dates, and a preference for vegetarian dishes are more willing to consume aging produce. This study adds to the growing body of literature discussing how to increase the consumption of suboptimal foods. The results imply that informing consumers about safety and healthfulness can be a viable path to increase the consumption of aging produce at home.

Phyllobilins are natural products derived from the degradation of chlorophyll, which proceeds via a common and strictly controlled pathway in higher plants. The resulting tetrapyrrolic catabolites-the phyllobilins-are ubiquitous in nature; despite their high abundance, there is still a lack of knowledge about their physiological properties. Phyllobilins are part of human nutrition and were shown to be potent antioxidants accounting with interesting physiological properties. Three different naturally occurring types of phyllobilins-a phylloleucobilin, a dioxobilin-type phylloleucobilin and a phylloxanthobilin (PxB)-were compared regarding potential antioxidative properties in a cell-free and in a cell-based antioxidant activity test system, demonstrating the strongest effect for the PxB. Moreover, the PxB was investigated for its capacity to interfere with immunoregulatory metabolic pathways of tryptophan breakdown in human blood peripheral mononuclear cells. A dose-dependent inhibition of tryptophan catabolism to kynurenine was observed, suggesting a suppressive effect on pathways of cellular immune activation. Although the exact mechanisms of immunomodulatory effects are yet unknown, these prominent bioactivities point towards health-relevant effects, which warrant further mechanistic investigations and the assessment of the in vivo extrapolatability of results. Thus, phyllobilins are a still surprisingly unexplored family of natural products that merit further investigation.

Phyllobilins are a group of chlorophyll-derived bilin-type linear tetrapyrroles, generated in the process of chlorophyll breakdown. Since the first phyllobilin was isolated and characterized in 1991, more and more structures of these chlorophyll catabolites were identified alongside the biochemical players involved in chlorophyll breakdown. In the meantime, phyllobilins are known to occur in a large natural structural variety, and new modifications are still being discovered. Phyllobilins have been regarded as products of chlorophyll detoxification for a very long time, hence they have been completely overlooked as a natural product class in terms of their biological role or pharmacological activity. A change of this paradigm, however, is long overdue. Here, we review the current knowledge of the pharmacological activities of phyllobilins and give an overview of the diverse structural modifications, laying the groundwork for analyzing their role(s) as active components in medicinal plants.

AbstractChlorophyll und Häm sind sogenannte “Pigmente des Lebens”, tetrapyrrolische Strukturen, ohne die das Leben auf der Erde nicht möglich wäre. Ihre Abbauprodukte, die Phyllobiline bzw. die Biline, teilen nicht nur strukturelle Merkmale, sondern auch eine ähnliche Geschichte: Lange Zeit wurden sie nur als Abfallprodukte von Entgiftungsprozessen angesehen; inzwischen konnten für beide Klassen wichtige Bioaktivitäten nachgewiesen werden. Für die Phyllobiline gibt es jedoch nur wenige Erkenntnisse über physiologische Rollen. Hier stellen wir mit Aktin, dem Hauptbestandteil des Zytoskeletts, die erste entdeckte Zielstruktur von Phyllobilinen und eine neue Zielstruktur von Bilinen vor. Wir zeigen die Hemmung der Aktindynamik in vitro und Auswirkungen auf Aktin und Aktin‐abhängige Prozesse in Krebszellen. Eine direkte Interaktion mit G‐Aktin wird durch in silico‐Studien dargelegt und durch Affinitätschromatographie bestätigt. Unsere Ergebnisse eröffnen ein neues Kapitel über Bioaktivitäten von Tetrapyrrolen – insbesondere der Phyllobiline – und bilden die Grundlage für weitreichende Anwendungen in der Pflanzenwissenschaft, Ökologie und Physiologie.

Chlorophyll and heme are among the "pigments of life", tetrapyrrolic structures, without which life on Earth would not be possible. Their catabolites, the phyllobilins and the bilins, respectively, share not only structural features, but also a similar story: Long considered waste products of detoxification processes, important bioactivities for both classes have now been demonstrated. For phyllobilins, however, research on physiological roles is sparse. Here, we introduce actin, the major component of the cytoskeleton, as the first discovered target of phyllobilins and as a novel target of bilins. We demonstrate the inhibition of actin dynamics in vitro and effects on actin and related processes in cancer cells. A direct interaction with G-actin is shown by in silico studies and confirmed by affinity chromatography. Our findings open a new chapter in bioactivities of tetrapyrroles-especially phyllobilins-for which they form the basis for broad implications in plant science, ecology, and physiology.