Anti-Inflammatory Activity of Lauraceae Plant Species and Prediction Models Based on Their Metabolomics Profiling Data.

The Lauraceae is a botanical family known for its anti-inflammatory potential. However, several species have not yet been studied. Thus, this work aimed to screen the anti-inflammatory activity of this plant family and to build statistical prediction models. The methodology was based on the statistical analysis of high-resolution liquid chromatography coupled with mass spectrometry data and the ex vivo anti-inflammatory activity of plant extracts. The ex vivo results demonstrated significant anti-inflammatory activity for several of these plants for the first time. The sample data were applied to build anti-inflammatory activity prediction models, including the partial least square acquired, artificial neural network, and stochastic gradient descent, which showed adequate fitting and predictive performance. Key anti-inflammatory markers, such as aporphine and benzylisoquinoline alkaloids were annotated with confidence level 2. Additionally, the validated prediction models proved to be useful for predicting active extracts using metabolomics data and studying their most bioactive metabolites.

Targeted Isolation of Anti-Trypanosomal Naphthofuran-Quinone Compounds from the Mangrove Plant Avicennia lanata.

The discovery of new secondary metabolites from natural origins has become more challenging in natural products research. Different approaches have been applied to target the isolation of new bioactive metabolites from plant extracts. In this study, bioactive natural products were isolated from the crude organic extract of the mangrove plant Avicennia lanata collected from the east coast of Peninsular Malaysia in the Setiu Wetlands, Terengganu, using HRESI-LCMS-based metabolomics-guided isolation and fractionation. Isolation work on the crude extract A. lanata used high-throughput chromatographic techniques to give two new naphthofuranquinone derivatives, hydroxyavicenol C (1) and glycosemiquinone (2), along with the known compounds avicenol C (3), avicequinone C (4), glycoquinone (5), taraxerone (6), taraxerol (7), ß-sitosterol (8) and stigmasterol (9). The elucidation and identification of the targeted bioactive compounds used 1D and 2D-NMR and mass spectrometry. Except for 6-9, all isolated naphthoquinone compounds (1-5) from the mangrove plant A. lanata showed significant anti-trypanosomal activity on Trypanosoma brucei brucei with MIC values of 3.12-12.5 µM. Preliminary cytotoxicity screening against normal prostate cells (PNT2A) was also performed. All compounds exhibited low cytotoxicity, with compounds 3 and 4 showing moderate cytotoxicity of 78.3% and 68.6% of the control values at 100 µg/mL, respectively.

Isolation of a Novel Flavanonol and an Alkylresorcinol with Highly Potent Anti-Trypanosomal Activity from Libyan propolis.

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4'-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction.

Metabolomic-Guided Isolation of Bioactive Natural Products from Curvularia sp., an Endophytic Fungus of Terminalia laxiflora.

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology. Metabolomic tools were successfully employed to compare the metabolite fingerprints of solid and liquid culture extracts of endophyte Curvularia sp. isolated from the leaves of Terminalia laxiflora. Natural product databases were used to dereplicate metabolites in order to determine known compounds and the presence of new natural products. Multivariate analysis highlighted the putative metabolites responsible for the bioactivity of the fungal extract and its fractions on NF-κB and the myelogenous leukemia cell line K562. Metabolomic tools and dereplication studies using high-resolution electrospray ionization mass spectrometry directed the fractionation and isolation of the bioactive components from the fungal extracts. This resulted in the isolation of N-acetylphenylalanine (1: ) and two linear peptide congeners of 1: : dipeptide N-acetylphenylalanyl-L-phenylalanine (2: ) and tripeptide N-acetylphenylalanyl-L-phenylalanyl-L-leucine (3: ).

Bioactive pyrrole alkaloids isolated from the Red Sea: marine sponge Stylissa carteri.

Fifteen pyrrole alkaloids were isolated from the Red Sea marine sponge Stylissa carteri and investigated for their biological activities. Four of them were dibrominated [(+) dibromophakelline, Z-3-bromohymenialdisine, (±) ageliferin and 3,4-dibromo-1H-pyrrole-2-carbamide], nine compounds were monobrominated [(-) clathramide C, agelongine, (+) manzacidin A, (-) 3-bromomanzacidin D, Z-spongiacidin D, Z-hymenialdisine, 2-debromostevensine, 2-bromoaldisine and 4-bromo-1H-pyrrole-2-carbamide)] and finally, two compounds were non-brominated derivatives viz., E-debromohymenialdisine and aldisine. The structure elucidations of isolated compounds were based on 1D & 2D NMR spectroscopic and MS studies, as well as by comparison with literature. In-vitro, Z-spongiacidin D exhibited a moderate activity on (ARK5, CDK2-CycA, CDK4/CycD1, VEGF-R2, SAK and PDGFR-beta) protein kinases. Moreover, Z-3-bromohymenialdisine showed nearly similar pattern. Furthermore, Z-hymenialdisine displayed a moderate effect on (ARK5 & VEGF-R2) and (-) clathramide C showed a moderate activity on AURORA-A protein kinases. While, agelongine, (+) manzacidin A, E-debromohymenialdisine and 3,4-dibromo-1H-pyrrole-2-carbamide demonstrated only marginal inhibitory activities. The cytotoxicity study was evaluated in two different cell lines. The most effective secondary metabolites were (+) dibromophakelline and Z-3-bromohymenialdisine on L5178Y. Finally, Z-hymenialdisine, Z-3-bromohymenialdisine and (±) ageliferin exhibited the highest cytotoxic activity on HCT116. No report about inhibition of AURORA-A and B by hymenialdisine/hymenialdisine analogs existed and no reported toxicity of ageliferin existed in literature.

Metabolomics-Guided Isolation of Anti-trypanosomal Metabolites from the Endophytic Fungus Lasiodiplodia theobromae.

Fungal endophytes offer diverse and unique secondary metabolites, making these organisms potential sources of promising drug leads. The application of high-resolution-liquid chromatography mass spectrometry and nuclear magnetic resonance-based metabolomics to fungal endophytes is practical in terms of dereplication studies and the mining of bioactive compounds. In this paper, we report the application of metabolomics in parallel with anti-trypanosomal assays to determine the ideal conditions for the medium-scale fermentation of the endophyte Lasiodiplodia theobromae. The 1H NMR comparison between the active versus inactive fractions identified several unique chemical fingerprints belonging to the active fractions. Furthermore, by integrating high-resolution-liquid chromatography mass spectrometry data with multivariate data analysis, such as orthogonal partial least squares-discriminant analysis (OPLS-DA) and the bioactivity results of the fractions of L. theobromae, the anti-trypanosomal agents were easily discerned. With available databases such as Antibase and Dictionary of Natural Products coupled to MZmine through in-house algorithms optimized in our laboratory, the predicted metabolites were readily identified prior to isolation. Fractionation was performed on the active fractions and three known compounds were isolated, namely, cladospirone B, desmethyl-lasiodiplodin, and R-(-)-mellein. Cladospirone B and desmethyl-lasiodiplodin were among the predicted compounds generated by the OPLS-DA S-plot, and these compounds exhibited good activity against Trypanosoma brucei brucei with minimum inhibitory concentrations of 17.8 µM and 22.5 µM, respectively.

Isolation of Petrocidin A, a New Cytotoxic Cyclic Dipeptide from the Marine Sponge-Derived Bacterium Streptomyces sp. SBT348.

A new cyclic dipeptide, petrocidin A (1), along with three known compounds-2,3-dihydroxybenzoic acid (2), 2,3-dihydroxybenzamide (3), and maltol (4)-were isolated from the solid culture of Streptomyces sp. SBT348. The strain Streptomyces sp. SBT348 had been prioritized in a strain collection of 64 sponge-associated actinomycetes based on its distinct metabolomic profile using liquid chromatography/high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). The absolute configuration of all α-amino acids was determined by HPLC analysis after derivatization with Marfey's reagent and comparison with commercially available reference amino acids. Structure elucidation was pursued in the presented study by mass spectrometry and NMR spectral data. Petrocidin A (1) and 2,3-dihydroxybenzamide (3) exhibited significant cytotoxicity towards the human promyelocytic HL-60 and the human colon adenocarcinoma HT-29 cell lines. These results demonstrated the potential of sponge-associated actinomycetes for the discovery of novel and pharmacologically active natural products.

Metabolomic Tools to Assess the Chemistry and Bioactivity of Endophytic Aspergillus Strain.

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology that may find applications as pharmaceutical and agrochemical drugs. In this study, a combination of metabolomics and bioactivity-guided approaches were employed to isolate secondary metabolites with cytotoxicity against cancer cells from an endophytic Aspergillus aculeatus. The endophyte was isolated from the Egyptian medicinal plant Terminalia laxiflora and identified using molecular biological methods. Metabolomics and dereplication studies were accomplished by utilizing the MZmine software coupled with the universal Dictionary of Natural Products database. Metabolic profiling, with aid of multivariate data analysis, was performed at different stages of the growth curve to choose the optimized method suitable for up-scaling. The optimized culture method yielded a crude extract abundant with biologically-active secondary metabolites. Crude extracts were fractionated using different high-throughput chromatographic techniques. Purified compounds were identified by HR-ESI-MS, 1D- and 2D-NMR. This study introduced a new method of dereplication utilizing both high-resolution mass spectrometry and NMR spectroscopy. The metabolites were putatively identified by applying a chemotaxonomic filter. We also present a short review on the diverse chemistry of terrestrial endophytic strains of Aspergillus, which has become a part of our dereplication work and this will be of wide interest to those working in this field.

Prediction of Anti-inflammatory Plants and Discovery of Their Biomarkers by Machine Learning Algorithms and Metabolomic Studies.

Nonsteroidal anti-inflammatory drugs are the most used anti-inflammatory medicines in the world. Side effects still occur, however, and some inflammatory pathologies lack efficient treatment. Cyclooxygenase and lipoxygenase pathways are of utmost importance in inflammatory processes; therefore, novel inhibitors are currently needed for both of them. Dual inhibitors of cyclooxygenase-1 and 5-lipoxygenase are anti-inflammatory drugs with high efficacy and low side effects. In this work, 57 leaf extracts (EtOH-H2O 7 : 3, v/v) from Asteraceae species with in vitro dual inhibition of cyclooxygenase-1 and 5-lipoxygenase were analyzed by high-performance liquid chromatography-high-resolution-ORBITRAP-mass spectrometry analysis and subjected to in silico studies using machine learning algorithms. The data from all samples were processed by employing differential expression analysis software coupled to the Dictionary of Natural Products for dereplication studies. The 6052 chromatographic peaks (ESI positive and negative modes) of the extracts were selected by a genetic algorithm according to their respective anti-inflammatory properties; after this procedure, 1241 of them remained. A study using a decision tree classifier was carried out, and 11 compounds were determined to be biomarkers due to their anti-inflammatory potential. Finally, a model to predict new biologically active extracts from Asteraceae species using liquid chromatography-mass spectrometry information with no prior knowledge of their biological data was built using a multilayer perceptron (artificial neural networks) with the back-propagation algorithm using the biomarker data. As a result, a new and robust artificial neural network model for predicting the anti-inflammatory activity of natural compounds was obtained, resulting in a high percentage of correct predictions (81 %), high precision (100 %) for dual inhibition, and low error values (mean absolute error = 0.3), as also shown in the validation test. Thus, the biomarkers of the Asteraceae extracts were statistically correlated with their anti-inflammatory activities and can therefore be useful to predict new anti-inflammatory extracts and their anti-inflammatory compounds using only liquid chromatography-mass spectrometry data.

Antichlamydial sterol from the Red Sea sponge Callyspongia aff. implexa.

Marine sponges are rich sources of natural products exhibiting diverse biological activities. Bioactivity-guided fractionation of the Red Sea sponge Callyspongia aff. implexa led to the isolation of two new compounds, 26,27-bisnorcholest-5,16-dien-23-yn-3ß,7α-diol, gelliusterol E (1) and C27-polyacetylene, callimplexen A (2), in addition to the known compound ß-sitosterol (3). The structures of the isolated compounds were determined by 1D- and 2D-NMR techniques as well as high-resolution tandem mass spectrometry and by comparison to the literature. The three compounds (1-3) were tested against Chlamydia trachomatis, an obligate intracellular gram-negative bacterium, which is the leading cause of ocular and genital infections worldwide. Only gelliusterol E (1) inhibited the formation and growth of chlamydial inclusions in a dose-dependent manner with an IC50 value of 2.3 µM.

Tumor regression following intravenous administration of lactoferrin- and lactoferricin-bearing dendriplexes.

The possibility of using gene therapy for the treatment of cancer is limited by the lack of safe, intravenously administered delivery systems able to selectively deliver therapeutic genes to tumors. In this study, we investigated if the conjugation of the polypropylenimine dendrimer to lactoferrin and lactoferricin, whose receptors are overexpressed on cancer cells, could result in a selective gene delivery to tumors and a subsequently enhanced therapeutic efficacy. The conjugation of lactoferrin and lactoferricin to the dendrimer significantly increased the gene expression in the tumor while decreasing the non-specific gene expression in the liver. Consequently, the intravenous administration of the targeted dendriplexes encoding TNFα led to the complete suppression of 60% of A431 tumors and up to 50% of B16-F10 tumors over one month. The treatment was well tolerated by the animals. These results suggest that these novel lactoferrin- and lactoferricin-bearing dendrimers are promising gene delivery systems for cancer therapy. FROM THE CLINICAL EDITOR: Specific targeting of cancer cells should enhance the delivery of chemotherapeutic agents. This is especially true for gene delivery. In this article, the authors utilized a dendrimer-based system and conjugated this with lactoferrin and lactoferricin to deliver anti-tumor genes. The positive findings in animal studies should provide the basis for further clinical studies.

The 9th European Conference on Marine Natural Products.

The 9th European Conference on Marine Natural Products (ECMNP) in Glasgow follows its predecessors in La Toja (2013), Tjärnö (2011), Porto (2009), Ischia (2007), Paris (2005), Elmau (2002), Santiago de Compostela (1999), and Athens (1997). [...].

Two new antioxidant actinosporin analogues from the calcium alginate beads culture of sponge-associated Actinokineospora sp. strain EG49.

Marine sponge-associated actinomycetes represent an exciting new resource for the identification of new and novel natural products . Previously, we have reported the isolation and structural elucidation of actinosporins A (1) and B (2) from Actinokineospora sp. strain EG49 isolated from the marine sponge Spheciospongia vagabunda. Herein, by employing different fermentation conditions on the same microorganism, we report on the isolation and antioxidant activity of structurally related metabolites, actinosporins C (3) and D (4). The antioxidant potential of actinosporins C and D was demonstrated using the ferric reducing antioxidant power (FRAP) assay. Additionally, at 1.25 µM, actinosporins C and D showed a significant antioxidant and protective capacity from the genomic damage induced by hydrogen peroxide in the human promyelocytic (HL-60) cell line.

Dereplication strategies for targeted isolation of new antitrypanosomal actinosporins A and B from a marine sponge associated-Actinokineospora sp. EG49.

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC50 value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.

Metabolomic tools for secondary metabolite discovery from marine microbial symbionts.

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR 1H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.

Metabolomic profiling and genomic study of a marine sponge-associated Streptomyces sp.

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.

Isolation and identification of antitrypanosomal and antimycobacterial active steroids from the sponge Haliclona simulans.

The marine sponge Haliclona simulans collected from the Irish Sea yielded two new steroids: 24-vinyl-cholest-9-ene-3ß,24-diol and 20-methyl-pregn-6-en-3ß-ol,5a,8a-epidioxy, along with the widely distributed 24-methylenecholesterol. One of the steroids possesses an unusually short hydrocarbon side chain. The structures were elucidated using nuclear magnetic resonance spectroscopy and confirmed using electron impact- and high resolution electrospray-mass spectrometry. All three steroids possess antitrypanosomal and anti-mycobacterial activity. All the steroids were found to possess low cytotoxicity against Hs27 which was above their detected antitrypanosomal potent concentrations.

The isolation of antiprotozoal compounds from Libyan propolis.

Propolis is increasingly being explored as a source of biologically active compounds. Until now, there has been no study of Libyan propolis. Two samples were collected in North East Libya and tested for their activity against Trypanosoma brucei. Extracts from both samples had quite high activity. One of the samples was fractionated and yielded a number of active fractions. Three of the active fractions contained single compounds, which were found to be 13-epitorulosal, acetyl-13-epi-cupressic acid and 13-epi-cupressic acid, which have been described before in Mediterranean propolis. Two of the compounds had a minimum inhibitory concentration value of 1.56 µg/mL against T. brucei. The active fractions were also tested against macrophages infected with Leishmania donovani, and again moderate to strong activity was observed with the compounds having IC50 values in the range 5.1-21.9 µg/mL.

Targeted Isolation of Antibiofilm Compounds from Halophytic Endophyte Bacillus velezensis 7NPB-3B Using LC-HR-MS-Based Metabolomics.

The discovery of new natural products has become more challenging because of the re-isolation of compounds and the lack of new sources. Microbes dwelling in extreme conditions of high salinity and temperature are huge prospects for interesting natural metabolites. In this study, the endophytic bacteria Bacillus velezensis 7NPB-3B isolated from the halophyte Salicornia brachiata was screened for its biofilm inhibition against methicillin-resistant Staphylococcus aureus (MRSA). The fractionation of the crude extract was guided by bioassay and LC-HRMS-based metabolomics using multivariate analysis. The 37 fractions obtained by high-throughput chromatography were dereplicated using an in-house MS-Excel macro coupled with the Dictionary of Natural Products database. Successive bioactivity-guided separation yielded one novel compound (1), a diketopiperazine (m/z 469.258 [M - H]-) with an attached saturated decanoic acid chain, and four known compounds (2-5). The compounds were identified based on 1D- and 2D-NMR and mass spectrometry. Compounds 1 and 5 exhibited excellent biofilm inhibition properties of >90% against the MRSA pathogen at minimum inhibition concentrations of 25 and 35 µg/mL, respectively. The investigation resulted in the isolation of a novel diketopiperazine from a bacterial endophyte of an untapped plant using an omics approach.

Unveiling the metabolites underlying the skin anti-ageing properties of Cytinus hypocistis (L.) L. through a biochemometric approach.

BACKGROUND: The genus Cytinus, recognised as one of the most enigmatic in the plant kingdom, has garnered attention for its bioactive potential, particularly its skin anti-ageing properties. Despite this recognition, much remains to be accomplished regarding deciphering and isolating its most active compounds. HYPOTHESIS: This study aimed to identify the compounds responsible for C. hypocistis skin anti-ageing potential. METHODS: Using multivariate analysis, a biochemometric approach was applied to identify the discriminant metabolites by integrating extracts' chemical profile (Liquid Chromatography-High-Resolution Mass Spectrometry, LCHRMS) and bioactive properties. The identified bioactive metabolite was structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR). RESULTS: Among the studied bioactivities, the anti-elastase results exhibited a significant variation among the samples from different years. After the biochemometric analysis, the compound 2,3:4,6-bis(hexahydroxydiphenoyl)glucose, with a molecular mass of 784.075 Da, was structurally elucidated as the discriminant feature responsible for the outstanding human neutrophil elastase inhibition. Remarkably, the subfraction containing this compound exhibited a tenfold improvement in neutrophil elastase inhibition efficacy compared to the crude extract; its effectiveness fell within the same range as SPCK, a potent irreversible neutrophil elastase inhibitor. Moreover, this subfraction displayed no cytotoxicity or phototoxicity and excellent efficacy for the tested anti-ageing properties. CONCLUSIONS: Hydrolysable tannins were confirmed as the metabolites behind C. hypocistis skin anti-ageing properties, effectively mitigating critical molecular mechanisms that influence the phenotypically distinct ageing clinical manifestations. Pedunculagin was particularly effective in inhibiting neutrophil elastase, considered one of the most destructive enzymes in skin ageing.