Untargeted Metabolomics Analysis for Studying Differences in High-Quality Colombian Cocoa Beans.

Colombia is a producer of fine cocoa, according to the International Cocoa Organization; however, most of its exports are in the ordinary cocoa category. To remedy this situation, several national organizations are working to create technological platforms for small producers to certify the quality of their beans. The objective of this study was to identify differential chemical markers in 36 cocoa bean samples from five Colombian departments and associate them with cocoa quality properties. For this purpose, a non-targeted metabolomics approach was performed using UHPLC-HRMS, along with sensory and physicochemical analyses. The 36 samples did not differ in sensory quality, polyphenol content, and theobromine/caffeine ratio. However, the multivariate statistical analysis allowed us to differentiate the samples into four clusters. In addition, a similar grouping of the samples was also observed in the physical analyses. The metabolites responsible for such clustering were investigated with univariate statistical analysis and presumptively identified by comparison of experimental mass spectra with those reported in databases. Alkaloids, flavonoids, terpenoids, peptides, quinolines, and sulfur compounds were identified as discriminants between sample groups. Here, it was presented the metabolic profiles as an important chemical feature for further studies in quality control and more specific characterization of fine cocoa.

Synthesis of Positional Isomeric Phenylphenalenones.

A series of isomeric phenylphenalenones in which the phenyl ring is located at all possible peripheral positions of the phenalenone nuclei was synthesized. The structural characteristics of the series, in which topological variation is permitted with minimal electronic disturbance, could, in principle, allow for easy pharmacophore recognition when the compounds are aligned in steroidomimetic conformations.

Application of the Crystalline Sponge Method to Revise the Structure of the Phenalenone Fuliginone.

The structure of fuliginone was revised from a phenyl substituted phenalenone to a hydroxyl substituted phenalenone as a result of its re-purification via HPLC with subsequent NMR analysis together with an independent synthesis and analysis of the crystal structure, which was secured via the crystalline sponge method. On-flow High Performance Liquid Chromatography coupled to Nuclear Magnetic Resonance spectroscopy (HPLC-NMR) was employed to confirm the presence of the natural product in the plant extract and to monitor for any possible degradation or conversion of the compound.

Phenylphenalenones protect banana plants from infection by Mycosphaerella fijiensis and are deactivated by metabolic conversion.

Phenylphenalenones, polycyclic aromatic natural products from some monocotyledonous plants, are known as phytoalexins in banana (Musa spp.). In this study, (1) H nuclear magnetic resonance (NMR)-based metabolomics along with liquid chromatography and mass spectrometry were used to explore the chemical responses of the susceptible 'Williams' and the resistant 'Khai Thong Ruang' Musa varieties to the ascomycete fungus Mycosphaerella fijiensis, the agent of the black leaf Sigatoka disease. Principal component analysis discriminated strongly between infected and non-infected plant tissue, mainly because of specialized metabolism induced in response to the fungus. Phenylphenalenones are among the major induced compounds, and the resistance level of the plants was correlated with the progress of the disease. However, a virulent strain of M. fijiensis was able to overcome plant resistance by converting phenylphenalenones to sulfate conjugates. Here, we report the first metabolic detoxification of fungitoxic phenylphenalenones to evade the chemical defence of Musa plants.

Synthesis of 8-Phenylphenalenones: 2-Hydroxy-8-(4-hydroxyphenyl)-1H-phenalen-1-one from Eichhornia crassipes.

2-Hydroxy-8-(4-hydroxyphenyl)-1H-phenalen-1-one (1), the first reported 8-phenylphenalenone from the roots of Eichhornia crassipes (water hyacinth), was synthesized starting from 2-methoxynaphthalene in 11 steps and with an overall yield of 2%. A cascade Friedel-Crafts/Michael annulation reaction between acryloyl chloride and 2-methoxynaphthalene afforded 9-methoxyperinaphthanone that, after transformation to 9-methoxy-2-(4-methoxyphenyl)-1H-phenalen-1-one by means of standard Suzuki-Miyaura methodology, was subjected to a reductive carbonyl transposition to afford 8-(4-methoxyphenyl)perinaphthanone. Dehydrogenation, epoxidation, and demethylation of the latter afforded 1.

Metabolomic Analysis of the Effect of Lippia origanoides Essential Oil on the Inhibition of Quorum Sensing in Chromobacterium violaceum.

Bacteria can communicate through quorum sensing, allowing them to develop different survival or virulence traits that lead to increased bacterial resistance against conventional antibiotic therapy. Here, fifteen essential oils (EOs) were investigated for their antimicrobial and anti-quorum-sensing activities using Chromobacterium violaceum CV026 as a model. All EOs were isolated from plant material via hydrodistillation and analyzed using GC/MS. In vitro antimicrobial activity was determined using the microdilution technique. Subinhibitory concentrations were used to determine anti-quorum-sensing activity by inhibition of violacein production. Finally, a possible mechanism of action for most bioactive EOs was determined using a metabolomic approach. Among the EOs evaluated, the EO from Lippia origanoides exhibited antimicrobial and anti-quorum activities at 0.37 and 0.15 mg/mL, respectively. Based on the experimental results, the antibiofilm activity of EO can be attributed to the blockage of tryptophan metabolism in the metabolic pathway of violacein synthesis. The metabolomic analyses made it possible to see effects mainly at the levels of tryptophan metabolism, nucleotide biosynthesis, arginine metabolism and vitamin biosynthesis. This allows us to highlight the EO of L. origanoides as a promising candidate for further studies in the design of antimicrobial compounds against bacterial resistance.

Untargeted Metabolomics for Unraveling the Metabolic Changes in Planktonic and Sessile Cells of Salmonella Enteritidis ATCC 13076 after Treatment with Lippia origanoides Essential Oil.

Nontyphoidal Salmonella species are one of the main bacterial causes of foodborne diseases, causing a public health problem. In addition, the ability to form biofilms, multiresistance to traditional drugs, and the absence of effective therapies against these microorganisms are some of the principal reasons for the increase in bacterial diseases. In this study, the anti-biofilm activity of twenty essential oils (EOs) on Salmonella enterica serovar Enteritidis ATCC 13076 was evaluated, as well as the metabolic changes caused by Lippia origanoides thymol chemotype EO (LOT-II) on planktonic and sessile cells. The anti-biofilm effect was evaluated by the crystal violet staining method, and cell viability was evaluated through the XTT method. The effect of EOs was observed by scanning electron microscopy (SEM) analysis. Untargeted metabolomics analyses were conducted to determine the effect of LOT-II EO on the cellular metabolome. LOT-II EO inhibited S. Enteritidis biofilm formation by more than 60%, without decreasing metabolic activity. Metabolic profile analysis identified changes in the modulation of metabolites in planktonic and sessile cells after LOT-II EO treatment. These changes showed alterations in different metabolic pathways, mainly in central carbon metabolism and nucleotide and amino acid metabolism. Finally, the possible mechanism of action of L. origanoides EO is proposed based on a metabolomics approach. Further studies are required to advance at the molecular level on the cellular targets affected by EOs, which are promising natural products for developing new therapeutic agents against Salmonella sp. strains.

Antibacterial, Antibiofilm and Anti-Virulence Activity of Biactive Fractions from Mucus Secretion of Giant African Snail Achatina fulica against Staphylococcus aureus Strains.

Staphylococcus aureus is an important etiological agent that causes skin infections, and has the propensity to form biofilms, leading to significant mortality and morbidity in patients with wounds. Mucus secretion from the Giant African snail Achatina fulica is a potential source of biologically active substances that might be an important source for new drugs to treat resistant and biofilm-forming bacteria such as S. aureus. This study evaluated the effect of semi-purified fractions from the mucus secretion of A. fulica on the growth, biofilm formation and virulence factors of S. aureus. Two fractions: FMA30 (Mw >30 kDa) and FME30 (Mw 30-10 kDa) exhibited antimicrobial activity against S. aureus with a MIC50 of 25 and 125 µg/mL, respectively. An inhibition of biofilm formation higher than 80% was observed at 9 µg/mL with FMA30 and 120 µg/mL with FME30. Furthermore, inhibition of hemolytic and protease activity was determined using a concentration of MIC20, and FME30 showed a strong inhibitory effect in the formation of clots. We report for the first time the effect of semi-purified fractions of mucus secretion of A. fulica on biofilm formation and activity of virulence factors such as α-hemolysin, coagulase and proteases produced by S. aureus strains.

Essential Oils of Aromatic Plants with Antibacterial, Anti-Biofilm and Anti-Quorum Sensing Activities against Pathogenic Bacteria.

Both the ability of bacteria to form biofilms and communicate through quorum sensing allows them to develop different survival or virulence traits that lead to increased bacterial resistance against conventional antibiotic therapy. Here, seventeen essential oils (EOs) were investigated for the antimicrobial, antibiofilm, and anti-quorum sensing activities on Escherichia. coli O157:H7, Escherichia coli O33, and Staphylococcus epidermidis ATCC 12228. All essential oils were isolated from plant material by using hydrodistillation and analyzed by GC-MS. The antimicrobial activity was performed by using the microdilution technique. Subinhibitory concentrations of each EO were assayed for biofilm inhibition in both bacterial strains. Quantification of violacein in Chromobacterium violaceum CV026 was performed for the anti-quorum sensing activity. The cytotoxicity activity of the EOs was evaluated on Vero cell line by using MTT method. Thymol-carvacrol-chemotype (I and II) oils from Lippia origanoides and Thymus vulgaris oil exhibited the higher antimicrobial activity with MIC values of 0.37-0.75 mg/mL. In addition, these EOs strongly inhibited the biofilm formation and violacein (QS) production in a concentration-dependent manner, highlighting thymol-carvacrol-chemotype (II) oil as the best candidate for further studies in antibiotic design and development against bacterial resistance.

Antimicrobial and Antibiofilm Activities of Essential Oils against Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus (MRSA).

The emergence of multidrug resistant microorganisms represents a global challenge due to the lack of new effective antimicrobial agents. In this sense, essential oils (EOs) are an alternative to be considered because of their anti-inflammatory, antiviral, antibacterial, and antibiofilm biological activities. Therefore, multiple efforts have been made to consider the potential use of EOs in the treatment of infections which are caused by resistant microorganisms. In this study, 15 EOs of both Colombian and introduced aromatic plants were evaluated against pathogenic strains of E. coli O157:H7 and methicillin resistant Staphylococcus aureus (MRSA) in planktonic and sessile states in order to identify relevant and promising alternatives for the treatment of microbial infections. Forty different compounds were identified in the 15 EO with nine of them constituted mainly by oxygenated monoterpenes (OM). EOs from Lippia origanoides, chemotypes thymol, and carvacrol, displayed the highest antibacterial activity against E. coli O157:H7 (MIC50 = 0.9 and 0.3 mg/mL, respectively) and MRSA (MIC50 = 1.2 and 0.6 mg/mL, respectively). These compounds from EOs had also the highest antibiofilm activity (inhibition percentage > 70.3%). Using scanning electron microscopy (SEM), changes in the size and morphology of both bacteria were observed when they were exposed to sub-inhibitory concentrations of L. origanoides EO carvacrol chemotype. EOs from L. origanoides, thymol, and carvacrol chemotypes represented a viable alternative for the treatment of microbial infections; however, the Selectivity Index (SI ≤ 3) indicated that it was necessary to study alternatives to reduce its in vitro cytotoxicity.

4-Phenylphenalenones as a template for new photodynamic compounds against Mycosphaerella fijiensis.

BACKGROUND: Evaluation of 4-phenylphenalenones and structural analogues against the fungal pathogen Mycosphaerella fijiensis (causal agent of black sigatoka disease in bananas) under light-controlled conditions uncovered some key structural features for the design of photodynamic compounds. RESULTS: Structure-activity relationship analysis revealed the importance of a chromophoric aryl-ketone and a steroidomimetic structural motif in the activity of the assayed compounds. The results pointed to 1,2-dihydro-3H-naphtho[2',1':3,4]cyclohepta[1,2-b]furan-3-one, which displayed an activity in the range of propiconazole but with photodynamic behaviour. CONCLUSION: The present work demonstrates that 1,2-dihydro-3H-naphtho[2',1':3,4]cyclohepta[1,2-b]heterocyclic-3-one derivatives can be used as potential lead compounds for the development of fungicides, relying on a dual mode of action. © 2015 Society of Chemical Industry.

4-Methoxycinnamic acid--An unusual phenylpropanoid involved in phenylphenalenone biosynthesis in Anigozanthos preissii.

In vitro root cultures of Anigozanthos preissii and Wachendorfia thyrsiflora (Haemodoraceae) are suitable biological systems for studying the biosynthesis of phenylphenalenones. Here we report how we used these root cultures to investigate precursor-product relationships between phenylpropanoids and phenylphenalenones whose phenyl rings share identical substitution patterns. Four phenylpropanoic acids, including ferulic acid and the unusual 4-methoxycinnamic acid, were used in (13)C-labeled form as substrates to study their incorporation into phenylphenalenones. In addition to the previously reported 2-hydroxy-9-(4'-hydroxy-3'-methoxyphenyl)-1H-phenalen-1-one (trivial name musanolone F), 2-hydroxy-9-(4'-methoxyphenyl)-1H-phenalen-1-one (proposed trivial name 4'-methoxyanigorufone) was found as a biosynthetic product in A. preissii. The carbon skeleton of 4'-methoxycinnamic acid was biosynthetically incorporated as an intact unit including its 4'-O-methyl substituent at the lateral phenyl ring. 4'-Methoxyanigorufone is reported here for the first time as a natural product.

Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora.

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with (13)C-labelled precursors in an (18)O2 atmosphere to observe incorporation of the two isotopes at defined biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composition of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosynthetic stages in the sequence O-1→O-2→O-5. In addition, the incubation of W. thyrsiflora root cultures with (13)C-labelled lachnanthocarpone established a direct biosynthetic precursor-product relationship with 1,2,5,6-tetraoxygenated phenylphenalenones.

Tissue-specific distribution of secondary metabolites in rapeseed (Brassica napus L.).

Four different parts, hypocotyl and radicle (HR), inner cotyledon (IC), outer cotyledon (OC), seed coat and endosperm (SE), were sampled from mature rapeseed (Brassica napus L.) by laser microdissection. Subsequently, major secondary metabolites, glucosinolates and sinapine, as well as three minor ones, a cyclic spermidine conjugate and two flavonoids, representing different compound categories, were qualified and quantified in dissected samples by high-performance liquid chromatography with diode array detection and mass spectrometry. No qualitative and quantitative difference of glucosinolates and sinapine was detected in embryo tissues (HR, IC and OC). On the other hand, the three minor compounds were observed to be distributed unevenly in different rapeseed tissues. The hypothetic biological functions of the distribution patterns of different secondary metabolites in rapeseed are discussed.

Structure-activity relationship in the interaction of substituted perinaphthenones with Mycosphaerella fijiensis.

The levels of native fungitoxic perinaphthenone phytoalexins in susceptible Musa varieties (banana), which are commercially grown in large plantations, are too low to provide plants with long-lasting protection against highly pathogenic fungi. Novel strategies for plant protection are necessary to reduce crop losses and to prevent the development of resistant fungal strains. The synthesis of novel fungicides based on the structures of perinaphthenone natural products is considered to be a promising strategy. Thirteen substituted perinaphthenones, among them two known natural products (1, 2) and 11 synthetics (3-13), were evaluated for their activity against Mycosphaerella fijiensis , and their half-maximal inhibitory concentrations (IC(50)) were calculated to establish structure-activity relationships (SAR). A SAR trend was hypothesized, leading to the design of a new compound, 4-methoxy-2-nitro-1H-phenalen-1-one (14); the new compound displayed significantly enhanced in vitro activity against M. fijiensis compared to other perinaphthenone derivatives. The activity of 14 was comparable to that of two commercial fungicides.