Orthogonal Reversed-Phase C18 and Pentafluorophenyl HPLC Separation for Phytochemical Profiling of Serrulatanes in Eremophila denticulata.

Serrulatanes constitute a class of unique diterpenoids derived from all-Z nerylneryl diphosphate rather than the conventional all-E diterpenoid precursor geranylgeranyl diphosphate and thus provide an intriguing expansion of the chemical space of plant specialized metabolites. Plants of the Australian Eremophila genus are rich sources of structurally diverse serrulatanes. Here, we report the identification of 15 hitherto undescribed serrulatanes (eremoculatanes A-N), together with 16 previously reported compounds, from the EtOAc extract of Eremophila denticulata leaves. Isolation was performed by a combined use of systematic HPLC-PDA-HRMS-based phytochemical profiling and orthogonal reversed-phase C18 and pentafluorophenyl separations. Among the new compounds isolated, eremoculatane A contains a C12 backbone, for which the configuration was established by comparison of experimentally measured and theoretically calculated ECD spectra. The antihyperglycemic and antibacterial activities of the E. denticulata extract were investigated by high-resolution inhibition profiling, and they indicated that major constituents, mainly serrulatanes and flavonoids, contributed to the observed activity of the extract. One flavonoid, eupafolin (4), displayed moderate α-glucosidase inhibitory activity with an IC50 value of 41.3 µM, and four serrulatanes (8, 9, 19g, and 19j) showed more than 50% PTP1B inhibition at 200 µM.

Identification of new PTP1B-inhibiting decipiene diterpenoid esters from Eremophila clarkei by high-resolution PTP1B inhibition profiling, enzyme kinetics analysis, and molecular docking.

In this study, an extract of the leaves of Eremophila clarkei Oldfield & F.Muell. showed protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with an IC50 value of 33.0 µg/mL. The extract was therefore investigated by high-resolution PTP1B inhibition profiling to pinpoint the constituents responsible for the activity. Subsequent isolation and purification using analytical-scale HPLC led to identification of eight previously undescribed decipiene diterpenoids, eremoclarkanes A-H, as well as eremoclarkic acid, a biogenetically related new phenolic acid. In addition, one known decipiene diterpenoid and ten known O-methylated flavonoids were isolated. The structures of the isolated compounds were elucidated by extensive analysis of their HRMS and 1D and 2D NMR spectra. The absolute configuration of decipiene diterpenoids was determined by comparison of experimental and calculated ECD spectra. The flavonoid hispidulin (2b) and the four decipiene diterpenoids 13a, 13b, 13f, and 14b exhibited PTP1B inhibitory activity with IC50 values ranging from 22.8 to 33.6 µM. This is the first report of PTP1B inhibitory activity of decipienes, and enzyme kinetics revealed that 13a and 13b are competitive inhibitors of PTP1B, whereas 13f and 14b displayed mixed-type-mode inhibition of PTP1B. Finally, molecular docking indicated that 13a, 13b, 13f, and 14b showed comparable binding affinity towards the active and/or allosteric site of PTP1B enzyme. Structure-activity relationship (SAR) of the identified O-methylated flavonoids and decipiene diterpenoids towards PTP1B is discussed. Plausible enzymatic and photochemically driven routes for the formation of the decipienes and conversion products thereof are presented and discussed.

Polypharmacology-Labeled Molecular Networking: An Analytical Technology Workflow for Accelerated Identification of Multiple Bioactive Constituents in Complex Extracts.

Discovery of sustainable and benign-by-design drugs to combat emerging health pandemics calls for new analytical technologies to explore the chemical and pharmacological properties of Nature's unique chemical space. Here, we present a new analytical technology workflow, polypharmacology-labeled molecular networking (PLMN), where merged positive and negative ionization tandem mass spectrometry-based molecular networking is linked with data from polypharmacological high-resolution inhibition profiling for easy and fast identification of individual bioactive constituents in complex extracts. The crude extract of Eremophila rugosa was subjected to PLMN analysis for the identification of antihyperglycemic and antibacterial constituents. Visually easy-interpretable polypharmacology scores and polypharmacology pie charts as well as microfractionation variation scores of each node in the molecular network provided direct information about each constituent's activity in the seven assays included in this proof-of-concept study. A total of 27 new non-canonical nerylneryl diphosphate-derived diterpenoids were identified. Serrulatane ferulate esters were shown to be associated with antihyperglycemic and antibacterial activities, including some showing synergistic activity with oxacillin in clinically relevant (epidemic) methicillin-resistant Staphylococcus aureus strains and some showing saddle-shaped binding to the active site of protein-tyrosine phosphatase 1B. PLMN is scalable in the number and types of assays included and thus holds potential for a paradigm shift toward polypharmacological natural-products-based drug discovery.

A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis.

The ginkgo tree (Ginkgo biloba) is considered a living fossil due to its 200 million year's history under morphological stasis. Its resilience is partly attributed to its unique set of specialized metabolites, in particular, ginkgolides and bilobalide, which are chemically complex terpene trilactones. Here, we use a gene cluster-guided mining approach in combination with co-expression analysis to reveal the primary steps in ginkgolide biosynthesis. We show that five multifunctional cytochrome P450s with atypical catalytic activities generate the tert-butyl group and one of the lactone rings, characteristic of all G. biloba trilactone terpenoids. The reactions include scarless C-C bond cleavage as well as carbon skeleton rearrangement (NIH shift) occurring on a previously unsuspected intermediate. The cytochrome P450s belong to CYP families that diversifies in pre-seed plants and gymnosperms, but are not preserved in angiosperms. Our work uncovers the early ginkgolide pathway and offers a glance into the biosynthesis of terpenoids of the Mesozoic Era.

Serrulatane diterpenoids with unusual side chain modifications from root bark of Eremophila longifolia.

The plant genus Eremophila is endemic to Australia and widespread in arid regions. Root bark extract of Eremophila longifolia (R.Br.) F.Muell. (Scrophulariaceae) was investigated by LC-PDA-HRMS, and dereplication suggested the presence of a series of diterpenoids. Using a combination of preparative- and analytical-scale HPLC separation as well as extensive 1D and 2D NMR analysis, the structures of 12 hitherto unreported serrulatane diterpenoids, eremolongine A-L, were established. These structures included serrulatanes with unusual side chain modifications to form hitherto unseen skeletons with, e.g., cyclopentane, oxepane, and bicyclic hexahydro-1H-cyclopenta[c]furan moieties. Serrulatane diterpenoids in Eremophila have recently been shown to originate from a common biosynthetic precursor with conserved stereochemical configuration, and this was used for tentative assignment of the relative and absolute configuration of the isolated compounds. Triple high-resolution α-glucosidase/α-amylase/PTP1B inhibition profiling demonstrated that several of the eremolongines had weak inhibitory activity towards targets important for management of type 2 diabetes.

Serrulatane diterpenoids from the leaves of Eremophila glabra and their potential as antihyperglycemic drug leads.

Eremophila (Scrophulariaceae) is a genus of Australian desert plants, which have been used by Australian Aboriginal people for various medicinal purposes. Crude extracts of the leaf resin of Eremophila glabra (R.Br.) Ostenf. showed α-glucosidase and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC50 values of 19.3 ± 1.2 µg/mL and 11.8 ± 2.1 µg/mL, respectively. Dual α-glucosidase/PTP1B high-resolution inhibition profiling combined with HPLC-PDA-HRMS and NMR were used to isolate and identify the compounds providing these activities. This resulted in isolation of seven undescribed serrulatane diterpenoids, eremoglabrane A-G, together with nine previously identified serrulatane diterpenoids and flavonoids. Three of the serrulatane diterpenoids showed PTP1B inhibitory activities with IC50 values from 63.8 ± 5.8 µM to 104.5 ± 25.9 µM.

Navigating through chemical space and evolutionary time across the Australian continent in plant genus Eremophila.

Eremophila is the largest genus in the plant tribe Myoporeae (Scrophulariaceae) and exhibits incredible morphological diversity across the Australian continent. The Australian Aboriginal Peoples recognize many Eremophila species as important sources of traditional medicine, the most frequently used plant parts being the leaves. Recent phylogenetic studies have revealed complex evolutionary relationships between Eremophila and related genera in the tribe. Unique and structurally diverse metabolites, particularly diterpenoids, are also a feature of plants in this group. To assess the full dimension of the chemical space of the tribe Myoporeae, we investigated the metabolite diversity in a chemo-evolutionary framework applying a combination of molecular phylogenetic and state-of-the-art computational metabolomics tools to build a dataset involving leaf samples from a total of 291 specimens of Eremophila and allied genera. The chemo-evolutionary relationships are expounded into a systematic context by integration of information about leaf morphology (resin and hairiness), environmental factors (pollination and geographical distribution), and medicinal properties (traditional medicinal uses and antibacterial studies), augmenting our understanding of complex interactions in biological systems.

PTP1B-Inhibiting Branched-Chain Fatty Acid Dimers from Eremophila oppositifolia subsp. angustifolia Identified by High-Resolution PTP1B Inhibition Profiling and HPLC-PDA-HRMS-SPE-NMR Analysis.

Ten new branched-chain fatty acid (BCFA) dimers with a substituted cyclohexene structure, five new monomers, and two known monomers, (2E,4Z,6E)-5-(acetoxymethyl)tetradeca-2,4,6-trienoic acid and its 5-hydroxymethyl analogue, were identified in the leaf extract of Eremophila oppositifolia subsp. angustifolia using a combination of HPLC-PDA-HRMS-SPE-NMR analysis and semipreparative-scale HPLC. The dimers could be classified as three types of Diels-Alder reaction products formed between monomers at two different sites of unsaturation of the dienophile. Two of the monomers represent potential biosynthetic intermediates of branched-chain fatty acids. Several compounds were found by high-resolution bioactivity profiling to inhibit PTP1B and were purified subsequently by semipreparative-scale HPLC. The dimers were generally more potent than the monomers with IC50 values ranging from 2 to 66 µM, compared to 38-484 µM for the monomers. The ten fatty acid dimers represent both a novel class of compounds and a novel class of PTP1B inhibitors.

The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia.

Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast.

Biological activity and LC-MS/MS profiling of extracts from the Australian medicinal plant Acacia ligulata (Fabaceae).

Acacia ligulata A.Cunn. ex Benth. (Fabaceae: Mimosoideae) is a native Australian plant used traditionally by Australian Aboriginal groups. This study was undertaken to investigate the bioactivity of A. ligulata extracts and to evaluate their chemical composition. Potential antibacterial, cytotoxic and enzyme inhibitory effects relevant to traditional medicinal and food uses of the species were examined and LC-MS/MS was performed to investigate the chemical composition. Antibacterial activity was observed for bark and leaf extracts with an MIC for the bark extract of 62.5 µg/mL against Streptococcus pyogenes. Pod extracts showed cytotoxic effects against cancer cells, with the highest activity against melanoma SK-MEL28 cells with IC50 values between 40.8 and 80.6 µg/mL. Further, the leaf and pod extracts also inhibited α-amylase EC-3.2.1.1 and α-glucosidase EC-3.2.1.20 with IC50 values between 9.7-34.8 and 12.6-64.3 µg/mL, respectively. The LC-MS/MS profiling indicated that several different saponins were present in the active extracts.

An expression tag toolbox for microbial production of membrane bound plant cytochromes P450.

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here, we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalyzed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than twofold. The developed toolbox serves as a platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals. Biotechnol. Bioeng. 2017;114: 751-760. © 2016 Wiley Periodicals, Inc.

High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy: Proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida.

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 µM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

Homology modeling of the three membrane proteins of the dhurrin metabolon: catalytic sites, membrane surface association and protein-protein interactions.

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein-protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F' and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.

CYP704B1 is a long-chain fatty acid omega-hydroxylase essential for sporopollenin synthesis in pollen of Arabidopsis.

Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes omega-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.

Effect of glucuronosylation on anthocyanin color stability.

The effect of glucuronosylation on the color stability of anthocyanins was investigated using glucuronosylated anthocyanins isolated from the flower petals of the red daisy (Bellis perennis) or obtained by enzymatic in vitro synthesis using heterologously expressed red daisy glucuronosyltransferase BpUGT94B1. Color stability toward light and heat stress was assessed by monitoring CIELAB color coordinates and stability at pH 7.0 by A(550). Cyanidin-3-O-2''-O-glucuronosylglucoside showed improved color stability in response to light compared to both cyanidin 3-O-glucoside and cyanidin 3-O-2''-O-diglucoside. A similar increase in color stability was not observed following heat treatment. Glucuronosylation did not increase the stability of anthocyanins at pH 7.0 as determined by A(550). To test for a possible effect of glucuronosylation on the color stability of anthocyanins in plant extracts used for food coloration, an elderberry (Sambucus nigra) extract was glucuronosylated in vitro. Glucuronosylation of approximately 50% of total anthocyanins proceeded fast and resulted in increased color stability in response to both heat and light. The data show that glucuronosylation may be used to stabilize industrially used extracts of natural colorants.

CYP703 is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen.

CYP703 is a cytochrome P450 family specific to land plants. Typically, each plant species contains a single CYP703. Arabidopsis thaliana CYP703A2 is expressed in the anthers of developing flowers. Expression is initiated at the tetrad stage and restricted to microspores and to the tapetum cell layer. Arabidopsis CYP703A2 knockout lines showed impaired pollen development and a partial male-sterile phenotype. Scanning electron and transmission electron microscopy of pollen from the knockout plants showed impaired pollen wall development with absence of exine. The fluorescent layer around the pollen grains ascribed to the presence of phenylpropanoid units in sporopollenin was absent in the CYP703A2 knockout lines. Heterologous expression of CYP703A2 in yeast cells demonstrated that CYP703 catalyzes the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids, with a preferential hydroxylation of lauric acid at the C-7 position. Incubation of recombinant CYP703 with methanol extracts from developing flowers confirmed that lauric acid and in-chain hydroxy lauric acids are the in planta substrate and product, respectively. These data demonstrate that in-chain hydroxy lauric acids are essential building blocks in sporopollenin synthesis and enable the formation of ester and ether linkages with phenylpropanoid units. This study identifies CYP703 as a P450 family specifically involved in pollen development.

Production of highly phosphorylated glycopolymers by expression of R1 in Escherichia coli.

The possible involvement of the starch bound R1 protein from potato (Solanum tuberosum L.) in the phosphorylation of starch was investigated by functional expression and characterisation of R1 in Escherichia coli. By expression of R1 in E. coli it is shown that it is possible to produce glycopolymers, e.g., glycogen, with an increased degree of phosphate substitution. The expression of R1 in E. coli resulted in a sixfold increase in glycogen bound phosphate and in an increased accumulation of glycogen leading to a glycogen excess (gex) phenotype. There was an overall shift in the unit-chain length of the isolated glycogen towards smaller degrees of polymerisation. The pleiotropic effects on the glycogen biosynthetic and amylolytic enzyme activities was investigated and showed an increase in ADPglucose pyrophosphorylase activity, as well as a decrease in exo-amylolytic activity. These results are discussed in relation to starch phosphorylation and a possible role of R1 in this respect.