Identification of Plant-Derived Bioactive Compounds Using Affinity Mass Spectrometry and Molecular Networking.

Affinity selection-mass spectrometry (AS-MS) is a label-free binding assay system that uses UHPLC-MS size-based separation methods to separate target-compound complexes from unbound compounds, identify bound compounds, classify compound binding sites, quantify the dissociation rate constant of compounds, and characterize affinity-extracted ligands. This label-free binding assay, in contrast to conventional biochemical (i.e., high-throughput screening (HTS)) approaches, is applicable to any drug target, and is also concise, accurate, and adaptable. Although AS-MS is an innovative approach for identifying lead compounds, the possibilities of finding bioactive compounds are limited by competitive binding, which occurs during the equilibration of extracts with the target protein(s). Here, we discuss the potential for metabolite profiling complemented with molecular networking to be used alongside AS-MS to improve the identification of bioactive compounds in plant extracts. AS-MS has gained significant prominence in HTS labs and shows potential to emerge as the driving force behind novel drug development in the future.

Ambrafuran (AmbroxTM) Synthesis from Natural Plant Product Precursors.

Ambergris, an excretion product of sperm whales, has been a valued agent in the formulation of perfumes. The composition of ambergris consists of two major components: 40-46% cholestanol type steroids and approximately 25-45% of a triterpenoid known as ambrein. Ambergris undergoes oxidative decomposition in the environment to result in odorous compounds, such as ambraoxide, methylambraoxide, and ambracetal. Its oxidized form, ambrafuran (IUPAC name: 3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1H-benzo[e][1]benzofuran), is a terpene furan with a pleasant odor and unique olfactive and fixative properties. The current state of the fragrance industry uses ambrafuran materials entirely from synthetic or semisynthetic sources. However, natural compounds with the potential to be converted to ambergris-like odorants have been extracted from several different types of plants. Here we review plant terpenoids suitable as starting materials for the semisyntheses of ambrafuran or intermediates, such as ambradiol, that can be used in biocatalytic transformations to yield ambrafuran.

GC-MS based profiling of alkanes in the filamentous yeast Hyphozyma roseoniger (Moesziomyces antarcticus).

OBJECTIVES: Hyphozyma roseoniger, a filamentous yeast, is used as a biocatalyst in the bio-transformation of terpenoids; however, the microorganism's endogenous ability to synthesise and metabolise hydrophobic terpenes and alkanes has not been characterised. RESULTS: When grown in potato dextrose broth the organism reached the stationary phase at 14 d. The non-polar fraction from cells, harvested every second day, were obtained with ethyl acetate extraction and analysed by gas chromatography with mass-spectrometric detection. Principal component-and hierarchical cluster analysis indicated growth-dependent clustering of the sample groups. A total of 26 alkanes were annotated across the different developmental stages. CONCLUSIONS: The major hydrocarbons comprised linear and branched structures. The dominant alkanes were all odd- or even-carbon numbered long-chain n-alkanes, C15 > C18 > C24.

Similar, but different: structurally related azelaic acid and hexanoic acid trigger differential metabolomic and transcriptomic responses in tobacco cells.

BACKGROUND: Plants respond to various stress stimuli by activating an enhanced broad-spectrum defensive ability. The development of novel resistance inducers represents an attractive, alternative crop protection strategy. In this regard, hexanoic acid (Hxa, a chemical elicitor) and azelaic acid (Aza, a natural signaling compound) have been proposed as inducers of plant defense, by means of a priming mechanism. Here, we investigated both the mode of action and the complementarity of Aza and Hxa as priming agents in Nicotiana tabacum cells in support of enhanced defense. RESULTS: Metabolomic analyses identified signatory biomarkers involved in the establishment of a pre-conditioned state following Aza and Hxa treatment. Both inducers affected the metabolomes in a similar manner and generated common biomarkers: caffeoylputrescine glycoside, cis-5-caffeoylquinic acid, feruloylglycoside, feruloyl-3-methoxytyramine glycoside and feruloyl-3-methoxytyramine conjugate. Subsequently, quantitative real time-PCR was used to investigate the expression of inducible defense response genes: phenylalanine ammonia lyase, hydroxycinnamoyl CoA quinate transferase and hydroxycinnamoyl transferase to monitor activation of the early phenylpropanoid pathway and chlorogenic acids metabolism, while ethylene response element-binding protein, small sar1 GTPase, heat shock protein 90, RAR1, SGT1, non-expressor of PR genes 1 and thioredoxin were analyzed to report on signal transduction events. Pathogenesis-related protein 1a and defensin were quantified to investigate the activation of defenses regulated by salicylic acid and jasmonic acid respectively. The qPCR results revealed differential expression kinetics and, in general (except for NPR1, Thionin and PR1a), the relative gene expression ratios observed in the Hxa-treated cells were significantly greater than the expression observed in the cells treated with Aza. CONCLUSIONS: The results indicate that Aza and Hxa have a similar priming effect through activation of genes involved in the establishment of systemic acquired resistance, associated with enhanced synthesis of hydroxycinnamic acids and related conjugates.

Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl-quinic acids and hydroxycinnamoyl-isocitric acids.

BACKGROUND: Plants contain a myriad of metabolites which exhibit diverse biological activities. However, in-depth analyses of these natural products with current analytical platforms remains an undisputed challenge due to the multidimensional chemo-diversity of these molecules, amplified by both isomerization and conjugation. In this study, we looked at molecules such as hydroxyl-cinnamic acids (HCAs), which are known to exist as positional and geometrical isomers conjugated to different organic acids namely quinic- and isocitric acid. OBJECTIVE: The study aimed at providing a more defined distinction between HCA conjugates from Amaranthus viridis and Moringa oleifera, using mass spectrometry (MS) approaches. METHODS: Here, we used a UHPLC-MS/MS targeted approach to analyze isobaric HCA conjugates extracted from the aforementioned plants. RESULTS: Mass spectrometry results showed similar precursor ions and fragmentation pattern; however, distinct differences were seen with ions at m/z 155 and m/z 111 which are associated with isocitric acid conjugates. CONCLUSION: Our results highlight subtle differences between these two classes of compounds based on the MS fingerprints, enabling confidence differentiation of the compounds. Thus, these findings provide a template reference for accurate and confident annotation of such compounds in other plants.

Stimulatory Effects of Acibenzolar-S-Methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells.

Centella asiatica is a perrenial herb that grows in tropical regions with numerous medicinal properties mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs) that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analyzed using a UHPLC-qTOF-MS platform. Data was processed and analyzed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid) and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the effects of precursor molecules on the levels of the CGAs were insignificant. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs.

Analyses of chlorogenic acids and related cinnamic acid derivatives from Nicotiana tabacum tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision-induced dissociation method.

BACKGROUND: Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures. RESULTS: Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof. CONCLUSION: Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.