Comprehensive metabolomics of Philippine Stichopus cf. horrens reveals diverse classes of valuable small molecules for biomedical applications.

Stichopus cf. horrens is an economically important sea cucumber species in Southeast Asia due to their presumed nutritional and medicinal benefits. However, compared to other sea cucumbers such as Apostichopus japonicus, there are no biochemical studies on which compounds contribute to the purported bioactivities of S. cf. horrens. To address this, a high-throughput characterization of the global metabolite profile of the species was performed through LC-MS/MS experiments and utilizing open-access platforms such as GNPS, XCMS, and metaboAnalyst. Bioinformatics-based molecular networking and chemometrics revealed the abundance of phospholipids such as phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and phosphatidylserines (PSs) in the crude samples. Body wall extracts were observed to have higher levels of structural, diacylated PCs, while the viscera have higher relative abundance of single-tail PCs and PEs that could be involved in digestion via nutrient absorption and transport for sea cucumbers. PEs and sphingolipids could also be implicated in the ecological response and morphological transformations of S. cf. horrens in the presence of predatory and other environmental stress. Interestingly, terpenoid glycosides and saponins with reported anti-cancer benefits were significantly localized in the body wall. The sulfated alkanes and sterols present in S. cf. horrens bear similarity to known kairomones and other signaling molecules. All in all, the results provide a baseline metabolomic profile of S. cf. horrens that may further be used for comparative and exploratory studies and suggest the untapped potential of S. cf. horrens as a source of bioactive molecules.

Mining Small Molecules from Teredinibacter turnerae Strains Isolated from Philippine Teredinidae.

Endosymbiotic relationship has played a significant role in the evolution of marine species, allowing for the development of biochemical machinery for the synthesis of diverse metabolites. In this work, we explore the chemical space of exogenous compounds from shipworm endosymbionts using LC-MS-based metabolomics. Priority T. turnerae strains (1022X.S.1B.7A, 991H.S.0A.06B, 1675L.S.0A.01) that displayed antimicrobial activity, isolated from shipworms collected from several sites in the Philippines were cultured, and fractionated extracts were subjected for profiling using ultrahigh-performance liquid chromatography with high-resolution mass spectrometry quadrupole time-of-flight mass analyzer (UHPLC-HRMS QTOF). T. turnerae T7901 was used as a reference microorganism for dereplication analysis. Tandem MS data were analyzed through the Global Natural Products Social (GNPS) molecular networking, which resulted to 93 clusters with more than two nodes, leading to four putatively annotated clusters: lipids, lysophosphatidylethanolamines, cyclic dipeptides, and rhamnolipids. Additional clusters were also annotated through molecular networking with cross-reference to previous publications. Tartrolon D cluster with analogues, turnercyclamycins A and B; teredinibactin A, dechloroteredinibactin, and two other possible teredinibactin analogues; and oxylipin (E)-11-oxooctadec-12-enoic acid were putatively identified as described. Molecular networking also revealed two additional metabolite clusters, annotated as lyso-ornithine lipids and polyethers. Manual fragmentation analysis corroborated the putative identification generated from GNPS. However, some of the clusters remained unclassified due to the limited structural information on marine natural products in the public database. The result of this study, nonetheless, showed the diversity in the chemical space occupied by shipworm endosymbionts. This study also affirms the use of bioinformatics, molecular networking, and fragmentation mechanisms analysis as tools for the dereplication of high-throughput data to aid the prioritization of strains for further analysis.

Sustainable Hues: Exploring the Molecular Palette of Biowaste Dyes through LC-MS Metabolomics.

Underutilized biowaste materials are investigated for their potential as sustainable textile colorants through an approach based on mass spectrometry, bioinformatics, and chemometrics. In this study, colorful decoctions were prepared from the outer bark of Eucalyptus deglupta and fruit peels of Syzygium samarangense, Syzygium malaccense, Diospyros discolor, and Dillenia philippinensis. Textile dyeing was performed along with liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics to determine the small molecules responsible for the observed colors. Global Natural Products Social Molecular Networking (GNPS) guided the annotation of black-producing proanthocyanidins in D. philippinensis and E. deglupta through complexation with FeSO4 mordant. Flavonoids from the yellow-colored D. philippinensis extracts were found to be similar to those in Terminalia catappa, a known traditional dye source. A higher intensity of epicatechin in E. deglupta produced a red-brown color in the presence of Cu2+. Furthermore, Syzygium fruit peels have poor wash-fastness in cotton fibers, but bioactive chalcone unique to S. samarangense samples may be a potential nutritional food colorant. Unsupervised PCA and supervised OPLS-DA chemometrics distinguished chemical features that affect dyeing properties beyond the observed color. These findings, along with growing data on natural dyes, could guide future research on sustainable colorants.

Small in size, big on taste: Metabolomics analysis of flavor compounds from Philippine garlic.

Philippine garlic (Allium sativum L.) is arguably known to pack flavor and aroma in smaller bulbs compared to imported varieties saturating the local market. In this study, ethanolic extracts of Philippine garlic cultivars were profiled using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF). γ-Glu dipeptides, oligosaccharides and lipids were determined in Philippine garlic cultivars through bioinformatics analysis in GNPS Molecular Networking Platform and fragmentation analysis. Multivariate statistical analysis using XCMS Online showed the abundance of γ-Glu allyl cysteine in Batanes-sourced garlic while γ-Glu propenyl cysteine, γ-Glu methyl cysteine, and alliin are enriched in the Ilocos cultivar. Principal component analysis showed that the γ-Glu dipeptides found in local garlic influenced their distinct separation across PC1 from imported varieties. This presence of high levels of γ-Glu dipeptides and probiotic oligosaccharides may potentially contribute to the superior flavor and nutritional benefits of Philippine garlic.

Profiling the Philippine Blue: Liquid chromatography/mass spectrometry-based metabolomics study on Philippine Indigofera.

RATIONALE: High-throughput liquid chromatography/mass spectrometry (LC/MS) analysis presents an interesting platform for natural dyes research. A particular example is the assessment of the dynamic changes in fermentation mixtures of Philippine Indigofera, and in the investigation of commercially available indigo prepared using traditional and optimized methods. METHODS: Leaves from Indigofera tinctoria and Indigofera suffruticosa were subjected to methanolic extraction and aqueous fermentation for 48 h. Indigo powders prepared following 2-day and 15-day fermentation were also subjected to profiling using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS). MS2 spectra were annotated through a library search in the community-curated Global Natural Products Social Molecular Networking (GNPS). Spectra with no library hits in GNPS were annotated by analysis of their fragmentation pathways. RESULTS: UHPLC/MS-based detection and fragmentation analysis led to characterization of leucoindigo and the unreported tryptanthrin intermediate, 5a-hydroxy-5,5a-dihydroindolo[2,1-b]quinazoline-6,12-dione, in the fermentation extract of I. tinctoria leaves. Indigo-associated metabolites were absent in an Indigofera specimen in Laguna Province, which explained why it did not produce blue dye. Locally produced indigo was abundant in indigotin and indirubin, differentiated based on product ions with the corresponding predicted fragmentation pattern. The relative intensity of indigotin, however, decreased with the traditional process of extended fermentation to produce indigo. CONCLUSIONS: The study is the first to demonstrate simultaneous MS-based analysis of reaction intermediates, indigotin dye, side products, and catabolites on actively transforming fermentation extracts of I. tinctoria. New results include annotated mass spectra for leucoindigo, and for the unreported 5a-hydroxy-5,5a-dihydroindolo[2,1-b]quinazoline-6,12-dione, which is probably an intermediate in tryptranthrin synthesis. The proposed fragmentation schemes could guide the annotation of analogous compounds in complex mixtures.

Molecular Affinity of Mabolo Extracts to an Octopamine Receptor of a Fruit Fly.

Essential oils extracted from plants are composed of volatile organic compounds that can affect insect behavior. Identifying the active components of the essential oils to their biochemical target is necessary to design novel biopesticides. In this study, essential oils extracted from Diospyros discolor (Willd.) were analyzed using gas chromatography mass spectroscopy (GC-MS) to create an untargeted metabolite profile. Subsequently, a conformational ensemble of the Drosophila melanogaster octopamine receptor in mushroom bodies (OAMB) was created from a molecular dynamics simulation to resemble a flexible receptor for docking studies. GC-MS analysis revealed the presence of several metabolites, i.e. mostly aromatic esters. Interestingly, these aromatic esters were found to exhibit relatively higher binding affinities to OAMB than the receptor's natural agonist, octopamine. The molecular origin of this observed enhanced affinity is the π -stacking interaction between the aromatic moieties of the residues and ligands. This strategy, computational inspection in tandem with untargeted metabolomics, may provide insights in screening the essential oils as potential OAMB inhibitors.

Quantitative analysis of autoinducing peptide I (AIP-I) from Staphylococcus aureus cultures using ultrahigh performance liquid chromatography-high resolving power mass spectrometry.

Staphylococcus aureus infections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant S. aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25µM and a linear dynamic range of 2.6 to 63µM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16h. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.

Sarothrin from Alkanna orientalis is an antimicrobial agent and efflux pump inhibitor.

An Alkanna orientalis leaf and flower extract inhibited the growth of Staphylococcus aureus, a pathogen that causes an estimated 478 000 hospitalizations in the US annually. Bioassay-guided fractionation of A. orientalis resulted in isolation of the flavonoid sarothrin (5,7,4'-trihydroxy-3,6,8-trimethoxyflavone), which inhibited the growth of Mycobacterium smegmatis (MIC 75 µM) and S. aureus (MIC > 800 µM), and possessed efflux pump inhibitory activity. This is the first report of antimicrobial or efflux pump inhibitory activity of sarothrin, and of its presence in A. orientalis. Our findings suggest that the effectiveness of A. orientalis extracts is due to a combination of multiple constituents, including sarothrin.

Quorum quenching and antimicrobial activity of goldenseal (Hydrastis canadensis) against methicillin-resistant Staphylococcus aureus (MRSA).

The popular herbal remedy goldenseal (Hydrastis canadensis L.) is traditionally used to treat skin infections. With this study, we show activity of H. canadensis extracts in vitro against methicillin-resistant Staphylococcus aureus (MRSA). An extract from H. canadensis leaves demonstrated more potent antimicrobial activity than the alkaloid berberine alone (MICs of 75 µg/mL and 150 µg/mL, respectively). LC-MS detected alkaloids and efflux-pump inhibitory flavonoids in the extract, and the latter may explain the enhanced efficacy of the extract compared to berberine alone. We also show evidence of anti-virulence activity as a second mechanism by which H. canadensis acts against S. aureus. The H. canadensis leaf extract (but not the isolated alkaloids berberine, hydrastine, and canadine) demonstrated quorum quenching activity against several clinically relevant MRSA isolates (USA300 strains). Our data suggest that this occurs by attenuation of signal transduction through the AgrCA two-component system. Consistent with this observation, the extract inhibited toxin production by MRSA and prevented damage by MRSA to keratinocyte cells in vitro. Collectively, our results show that H. canadensis leaf extracts possess a mixture of constituents that act against MRSA via several different mechanisms. These findings lend support for the traditional application of crude H. canadensis extracts in the prevention of infection.

Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis).

It is often argued that the efficacy of herbal medicines is a result of the combined action of multiple constituents that work synergistically or additively. Determining the bioactive constituents in these mixtures poses a significant challenge. We have developed an approach to address this challenge, synergy-directed fractionation, which combines comprehensive mass spectrometry profiling with synergy assays and natural products isolation. The applicability of synergy-directed fractionation was demonstrated using the botanical medicine goldenseal (Hydrastis canadensis) as a case study. Three synergists from goldenseal were identified, sideroxylin, 8-desmethyl-sideroxylin, and 6-desmethyl-sideroxylin. These flavonoids synergistically enhance the antimicrobial activity of the alkaloid berberine (also a constituent of H. canadensis) against Staphylococcus aureus by inhibition of the NorA multidrug resistance pump. The flavonoids possess no inherent antimicrobial activity against S. aureus; therefore, they could have been missed using traditional bioactivity-directed fractionation. The flavonoid synergists are present at higher concentration in extracts from H. canadensis leaves, while the antimicrobial alkaloid berberine is present at higher levels in H. canadensis roots. Thus, it may be possible to produce an extract with optimal activity against S. aureus using a combination of goldenseal roots and leaves.

Goldenseal (Hydrastis canadensis L.) extracts synergistically enhance the antibacterial activity of berberine via efflux pump inhibition.

Goldenseal (Hydrastis canadensis L.) is used to combat inflammation and infection. Its antibacterial activity in vitRO has been attributed to its alkaloids, the most abundant of which is berberine. The goal of these studies was to compare the composition, antibacterial activity, and efflux pump inhibitory activity of ethanolic extracts prepared from roots and aerial portions of H. canadensis. Ethanolic extracts were prepared separately from roots and aerial portions of six H. canadensis plants. Extracts were analyzed for alkaloid concentration using LC-MS and tested for antimicrobial activity against Staphylococcus aureus (NCTC 8325-4) and for inhibition of ethidium bromide efflux. Synergistic antibacterial activity was observed between the aerial extract (FIC 0.375) and to a lesser extent the root extract (FIC 0.750) and berberine. The aerial extract inhibited ethidium bromide efflux from wild-type S. aureus but had no effect on the expulsion of this compound from an isogenic derivative deleted for norA. Our studies indicate that the roots of H. canadensis contain higher levels of alkaloids than the aerial portions, but the aerial portions synergize with berberine more significantly than the roots. Furthermore, extracts from the aerial portions of H. canadensis contain efflux pump inhibitors, while efflux pump inhibitory activity was not observed for the root extract. The three most abundant H. canadensis alkaloids, berberine, hydrastine, and canadine, are not responsible for the efflux pump inhibitory activity of the extracts from H. canadensis aerial portions.