Chemical and Antiplasmodial Investigations on Eremophila-Derived Alkaloids and Semisynthetic Ether Analogues.

Microthecaline A (1), the known antiplasmodial quinoline serrulatane alkaloid from the roots of Eremophila microtheca F. Muell. ex Benth. (Scrophulariaceae), was targeted for isolation and subsequent use in the generation of a semisynthetic ether library. A large-scale extraction and isolation yielded the previously undescribed quinoline serrulatane microthecaline B (2), along with crystalline 1 that enabled the first X-ray crystallographic analysis to be undertaken on this rare alkaloid structure class. The X-ray diffraction analysis of 1 supported the absolute configuration assignment of microthecaline A, which was originally assigned by ECD data analysis. Microthecaline A (1) was converted into 10 new semisynthetic ether derivatives (3-12) using a diverse series of commercially available alkyl halides. Chemical structures of the new serrulatane alkaloid and semisynthetic ether analogues were assigned by spectroscopic and spectrometric analyses. Antiplasmodial evaluations of 1-12 showed that the semisynthetic derivative 5 elicited the most potent activity with an IC50 value of 7.2 µM against Plasmodium falciparum 3D7 (drug-sensitive) strain.

Reversal of ABCG2/BCRP-Mediated Multidrug Resistance by 5,3',5'-Trihydroxy-3,6,7,4'-Tetramethoxyflavone Isolated from the Australian Desert Plant Eremophila galeata Chinnock.

Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata, a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29par) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemotherapeutic SN-38 (i.e., HT29SN38 cells). This identified a fraction, eluted with 40% acetonitrile on a solid-phase extraction column, which showed weak growth-inhibitory activity on HT29SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3',5'-trihydroxy-3,6,7,4'-tetramethoxyflavone (2), which at a concentration of 25 µg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 µM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results indicate that 2 acts synergistically with SN-38, with 2 being a BCRP efflux pump inhibitor while SN-38 inhibits topoisomerase-1.

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.

The biosynthesis of the anti-microbial diterpenoid leubethanol in Leucophyllum frutescens proceeds via an all-cis prenyl intermediate.

Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti-microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage (Leucophyllum frutescens) has demonstrated activity against multi-drug-resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L. frutescens and compare tissue-specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short-chain cis-prenyl transferase (LfCPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase (LfTPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short-chain cis-prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila. LfTPS1 represents neofunctionalization of a compartment-switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti-microbial therapeutics in heterologous systems.

Nerylneryl diphosphate is the precursor of serrulatane, viscidane and cembrane-type diterpenoids in Eremophila species.

BACKGROUND: Eremophila R.Br. (Scrophulariaceae) is a diverse genus of plants with species distributed across semi-arid and arid Australia. It is an ecologically important genus that also holds cultural significance for many Indigenous Australians who traditionally use several species as sources of medicines. Structurally unusual diterpenoids, particularly serrulatane and viscidane-types, feature prominently in the chemical profile of many species and recent studies indicate that these compounds are responsible for much of the reported bioactivity. We have investigated the biosynthesis of diterpenoids in three species: Eremophila lucida, Eremophila drummondii and Eremophila denticulata subsp. trisulcata. RESULTS: In all studied species diterpenoids were localised to the leaf surface and associated with the occurrence of glandular trichomes. Trichome-enriched transcriptome databases were generated and mined for candidate terpene synthases (TPS). Four TPSs with diterpene biosynthesis activity were identified: ElTPS31 and ElTPS3 from E. lucida were found to produce (3Z,7Z,11Z)-cembratrien-15-ol and 5-hydroxyviscidane, respectively, and EdTPS22 and EdtTPS4, from E. drummondii and E. denticulata subsp. trisulcata, respectively, were found to produce 8,9-dihydroserrulat-14-ene which readily aromatized to serrulat-14-ene. In all cases, the identified TPSs used the cisoid substrate, nerylneryl diphosphate (NNPP), to form the observed products. Subsequently, cis-prenyl transferases (CPTs) capable of making NNPP were identified in each species. CONCLUSIONS: We have elucidated two biosynthetic steps towards three of the major diterpene backbones found in this genus. Serrulatane and viscidane-type diterpenoids are promising candidates for new drug leads. The identification of an enzymatic route to their synthesis opens up the possibility of biotechnological production, making accessible a ready source of scaffolds for further modification and bioactivity testing.

The plant natural product 2-methoxy-1,4-naphthoquinone stimulates therapeutic neural repair properties of olfactory ensheathing cells.

Olfactory ensheathing cells (OECs) are crucial for promoting the regeneration of the primary olfactory nervous system that occurs throughout life. Transplantation of OECs has emerged as a promising therapy for nervous system injuries, in particular for spinal cord injury repair. Functional outcomes in both animals and humans are, however, highly variable, primarily because it is difficult to rapidly obtain enough OECs for transplantation. Compounds which can stimulate OEC proliferation without changing the phenotype of the cells are therefore highly sought after. Additionally, compounds which can stimulate favourable cell behaviours such as migration and phagocytic activity are desirable. We conducted a medium-throughput screen testing the Davis open access natural product-based library (472 compounds) and subsequently identified the known plant natural product 2-methoxy-1,4-naphthoquinone as a stimulant of OEC viability. We showed that 2-methoxy-1,4-naphthoquinone: (i) strongly stimulates proliferation over several weeks in culture whilst maintaining the OEC phenotype; (ii) stimulates the phagocytic activity of OECs, and (iii) modulates the cell cycle. We also identified the transcription factor Nrf2 as the compound's potential molecular target. From these extensive investigations we conclude that 2-methoxy-1,4-naphthoquinone may enhance the therapeutic potential of OECs by stimulating proliferation prior to transplantation.

Biomimetic Synthesis of Mitchellenes B-H from the Abundant Biological Precursor 14-Hydroxy-6,12-muuroloadien-15-oic Acid.

A step-economic biomimetic synthesis of mitchellenes B-H found in Eremophila sturtii has been achieved. Starting from the putative muurolane biological precursor, redox isomerization of the allylic alcohol gave an epimeric mixture of aldehydes, which could be used as a handle for cyclization onto the C6 position, using Bu3SnH-mediated radical cyclization or NHC-catalyzed Stetter reaction. The NHC-mediated approach was superior as the epimeric mixture underwent a dynamic kinetic resolution during the reaction, and reduction of the mixture with NaBH4 selectively formed the mitchellene ring system in 56% yield for the three steps. In the campaign to obtain the acid-starting material, two new natural products, mitchellene H and a muurolane aldehyde, were isolated. Synthetic procedures to access this family of natural products will enable further studies on their biological properties.

The serrulatane diterpenoid natural products RAD288 and RAD289 stimulate properties of olfactory ensheathing cells useful for neural repair therapies.

Olfactory ensheathing cells (OECs) are being trialled for cell transplantation therapies for neural repair as they have unique properties which can enhance neuron regeneration. However, improvements in cell viability, proliferation and migration are needed to enhance therapeutic outcomes. Growth factors can enhance cell activity, but they can also induce side effects as they can act on numerous cell types. An alternative approach is to identify natural products (NPs) that more selectively activate specific cell functions. We have examined two pure NPs, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid (RAD288) and 3,7,8-trihydroxyserrulat-14-en-19-oic acid (RAD289) isolated from the Australian plant Eremophila microtheca. We determined that RAD288 and RAD289 stimulated the viability and proliferation of OECs in two-dimensional cultures and increased cell viability in three-dimensional spheroids. Both compounds also enhanced OEC-mediated phagocytosis of neural debris. However, only RAD288 stimulated migration of OECs, demonstrating that key structural changes to the compound can dramatically affect the resultant cellular action. In addition, cell-type specific action is highlighted by the result that neither compound stimulated the viability of Schwann cells which are a closely-related glial cell type. Therefore, these small molecules may have high potential for selective activation of specific therapeutically-useful activities of OECs for transplantation therapies to repair the nervous system.

Microthecaline A, a Quinoline Serrulatane Alkaloid from the Roots of the Australian Desert Plant Eremophila microtheca.

Chemical investigation of the roots of the Australian desert plant Eremophila microtheca yielded microthecaline A (1), a novel quinoline-serrulatane natural product. The structure of 1 was determined by spectroscopic analysis, and the absolute configuration was assigned by ECD. Compound 1 exhibited moderate antimalarial activity against Plasmodium falciparum (3D7 strain), with an IC50 of 7.7 µM. Microthecaline A represents the first quinoline-serrulatane alkaloid to be isolated from Nature.

Metabolites of endophytic fungi from Australian native plants as potential anticancer agents.

Interest in endophytes as natural sources for new medicines was inspired by the discovery of paclitaxel-producing endophytic fungi. This study investigated the anti-cancer activity of extracts of endophytes isolated from two Australian plants, Eremophila longifolia (EL) and Eremophila maculata (EM). Endophytes were isolated from surface-sterilised leaf tissue, grown as pure cultures and identified by sequencing of Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. To determine cytotoxicity, two leukaemic (MOLT-4, T-cell leukaemia; PreB-697, Pre-B leukaemia), a lung cancer cell line (A549) and a normal human fibroblast cell line were treated with endophyte extracts to assess cytotoxicity in relation to alternariol monomethyl ether (AME) and alternariol (AOH). Endophyte extracts that showed cell cytotoxicity were analysed by UV-HPLC to determine the metabolites. Pure AME and AOH, three extracts form Alternaria sp. (EM-6, EM-7 and EM-9) and one from Preussia minima (EL-14) were cytotoxic to the cancer cell lines. All cytotoxic endophytes contained AME and AOH, the most cytotoxic endophyte EM-6 also contained two unique peaks. These data indicate that these four endophyte extracts may have anti-cancer properties due to the presence of AME and AOH; however, the unique compounds found in the EM-6 extract may be exclusively cytotoxic and warrant further investigation.

Revision of the Phytochemistry of Eremophila sturtii and E. mitchellii.

Eremophila sturtii and E. mitchellii are found in the arid and temperate regions of Australia and, because of their similar appearances, are often confused. Previous phytochemical investigations have described mitchellene sesquiterpenes (1-5) reported from E. mitchellii but are here demonstrated to be from E. sturtii. A previous study that described serrulatic acids (16 and 17) from a species reported as E. sturtii actually used E. mitchellii. In addition, two new C-15 modified analogues, mitchellenes F (14) and G (15), were isolated from E. sturtii. The absolute configuration of 14 was determined with the first X-ray structure of a compound with the mitchellene skeleton.

5,6,7,3',4',5'-Hexamethoxyflavone from the Australian plant Eremophila debilis (Myoporaceae).

The aerial parts of the endemic Australian plant Eremophila debilis (Myoporaceae) contain 3% dry weight of the biologically active 5,6,7,3',4',5'-hexamethoxyflavone, which had its structured confirmed using X-ray crystal crystallography. The presence of significant levels of the polypharmacologically active 5,6,7,3',4',5'-hexamethoxyflavone in the edible parts of the plant has potential implications for its use as a food and bush medicine.

Population assignment in autopolyploids.

Understanding the patterns of contemporary gene dispersal within and among populations is of critical importance to population genetics and in managing populations for conservation. In contrast to diploids, there are few studies of gene dispersal in autopolyploids, in part due to complex polysomic inheritance and genotype ambiguity. Here we develop a novel approach for population assignment for codominant markers for autotetraploids and autohexaploids. This method accounts for polysomic inheritance, unreduced gametes and unknown allele dosage. It can also utilise information regarding the origin and genotype of one parent for population assignment of maternal or paternal parents. Using simulations, we demonstrate that our approach achieves high levels of accuracy for assignment even when population divergence is low (FST~0.06) and with only 12 microsatellite loci. We also show that substantially higher accuracy is achieved when known maternal information is utilised, regardless of whether allele dosage is known. Although this novel method exhibited near identical levels of accuracy to Structure when population divergence was high, it performed substantially better for most parameters at moderate (FST=0.06) to low levels of divergence (FST=0.03). These methods fill an important gap in the toolset for autopolyploids and pave the way for investigating contemporary gene dispersal in a widespread group of organisms.

Synthesis of antimalarial amide analogues based on the plant serrulatane diterpenoid 3,7,8-trihydroxyserrulat-14-en-19-oic acid.

A plant-derived natural product scaffold, 3,7,8-trihydroxyserrulat-14-en-19-oic acid (1) was isolated in high yield from the aerial parts of the endemic Australian desert plant Eremophila microtheca. This scaffold (1) was subsequently used in the generation of a series of new amide analogues via a one-pot mixed anhydride amidation using pivaloyl chloride. The structures of all analogues were characterized using MS, NMR, and UV data. The major serrulatane natural products (1-3), isolated from the plant extract, and all amide analogues (6-15) together with several pivaloylated derivatives of 3,7,8-trihydroxyserrulat-14-en-19-oic acid (16-18) were evaluated for their antimalarial activity against 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant) Plasmodium falciparum strains, and preliminary cytotoxicity data were also acquired using the human embryonic kidney cell line HEK293. The natural product scaffold (1) did not display any antimalarial activity at 10µM. Replacing the carboxylic acid of 1 with various amides resulted in moderate activity against the P. falciparum 3D7 strain with IC50 values ranging from 1.25 to 5.65µM.

Antibacterial Nerol Cinnamates from the Australian Plant Eremophila longifolia.

Two new antimicrobial agents, neryl ferulate (1) and neryl p-coumarate (2), were identified using bioassay-guided isolation from the leaves of Eremophila longifolia, which is a medicinal plant used by some Australian Aboriginal communities. Although gradual autoxidation of the nerol subunit hindered the initial attempts to purify and characterize 1 and 2, it was found that the autoxidation could be stopped through storage under argon at -20 °C. Biological evaluation showed that neryl ferulate (1) had moderate activity against various Gram-positive bacteria, while neryl p-coumarate (2) was active only against Enterococcus faecium.

Transcriptome profiling of the Australian arid-land plant Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) for the identification of monoterpene synthases.

Plant terpenoids are a large and highly diverse class of metabolites with an important role in the immune defense. They find wide industrial application as active pharmaceutical ingredients, aroma and fragrance compounds. Several Eremophila sp. derived terpenoids have been documented. To elucidate the terpenoid metabolism, the transcriptome of juvenile and mature Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) leaves was sequenced and a transcript library was generated. We report on the first transcriptomic dataset of an Eremophila plant. IlluminaMiSeq sequencing (2 × 300 bp) revealed 7,093,266 paired reads, which could be assembled to 34,505 isogroups. To enable detection of terpene biosynthetic genes, leaves were separately treated with methyl jasmonate, a well-documented inducer of plant secondary metabolites. In total, 21 putative terpene synthase genes were detected in the transcriptome data. Two terpene synthase isoenzymatic genes, termed ES01 and ES02, were successfully expressed in E. coli. The resulting proteins catalyzed the conversion of geranyl pyrophosphate, the universal substrate of monoterpene synthases to myrcene and Z-(b)-ocimene, respectively. The transcriptomic data and the discovery of the first terpene synthases from Eremophila serrulata are the initial step for the understanding of the terpene metabolism in this medicinally important plant genus.

A new genus and twenty new species of Australian jumping plant-lice (Psylloidea: Triozidae) from Eremophila and Myoporum (Scrophulariaceae: Myoporeae).

The Triozidae is a diverse, cosmopolitan family of jumping plant-lice (Hemiptera: Psylloidea) from an exceptionally diverse range of plant families, but with few described Australian species. As a direct outcome of the Australian Biological Resources Study Bush Blitz species discovery program, many new Psylloidea from novel host plants in remote localities have been revealed. In this study a new genus Myotrioza Taylor gen. nov. and 20 new species are described from southern and central Australia which also establishes the first host plant records from Eremophila and Myoporum (Scrophulariaceae: Myoporeae). New species, delineated using a combination of morphological and mitochondrial COI sequence data, are: Myotrioza clementsiana sp. nov., M. darwinensis sp. nov., M. desertorum sp. nov., M. eremi sp. nov., M. eremophili sp. nov., M. flindersiana sp.nov., M. gawlerensis sp. nov., M. insularis sp. nov., M. interioris sp. nov., M. interstantis sp. nov., M. longifoliae sp. nov., M. markmitchelli sp. nov., M. myopori sp. nov., M. oppositifoliae sp. nov., M. pantonii sp. nov., M. platycarpi sp. nov., M. remota sp. nov., M. scopariae sp. nov., M. serrulatae sp. nov., and M. telowiensis sp. nov. Genetic divergence data, host associations, biogeographic data, diagnoses and a key to species are presented. Myotrioza appears to be particularly diverse in ephemeral southern Australia, especially in inland Western Australia and South Australia, matching regions of high diversity of the host genera; some species are likely to be short range endemics.

Serrulatane Diterpenoid from Eremophila neglecta Exhibits Bacterial Biofilm Dispersion and Inhibits Release of Pro-inflammatory Cytokines from Activated Macrophages.

The purpose of this study was to assess the biofilm-removing efficacy and inflammatory activity of a serrulatane diterpenoid, 8-hydroxyserrulat-14-en-19-oic acid (1), isolated from the Australian medicinal plant Eremophila neglecta. Biofilm breakup activity of compound 1 on established Staphylococcus epidermidis and Staphylococcus aureus biofilms was compared to the antiseptic chlorhexidine and antibiotic levofloxacin. In a time-course study, 1 was deposited onto polypropylene mesh to mimic a wound dressing and tested for biofilm removal. The ex-vivo cytotoxicity and effect on lipopolysaccharide-induced pro-inflammatory cytokine release were studied in mouse primary bone-marrow-derived macrophage (BMDM) cells. Compound 1 was effective in dispersing 12 h pre-established biofilms with a 7 log10 reduction of viable bacterial cell counts, but was less active against 24 h biofilms (approximately 2 log10 reduction). Compound-loaded mesh showed dosage-dependent biofilm-removing capability. In addition, compound 1 displayed a significant inhibitory effect on tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion from BMDM cells, but interleukin-1 beta (IL-1ß) secretion was not significant. The compound was not cytotoxic to BMDM cells at concentrations effective in removing biofilm and lowering cytokine release. These findings highlight the potential of this serrulatane diterpenoid to be further developed for applications in wound management.

An ethnopharmacological approach to the preliminary screening of native Australian herbal medicines for anticancer activity.

BACKGROUND: Five plants used traditionally by Australian Aboriginals and two edible native Australian fruits have been investigated for anticancer activity. The aim was to identify native Australian herbal medicines which displayed anticancer activity, with cytotoxicity to cancer cells but sparing or even proliferating normal immunological cells, and subsequently provide potentially new anticancer drug leads. METHODS: Extracts and derived fractions were assayed for cell viability against a multiple myeloma cell line, RPMI-8226, in comparison to the peripheral blood mononuclear cells (PBMC) representing normal human immunological cells. RESULTS: None of the crude extracts exhibited the desirable differential activity; however, following further fractionation of the Eremophila duttonii F. Muell. (Myoporaceae) extract, one fraction (termed F01) exhibited a greater cytotoxicity to the cancer cell line than to the normal cells. CONCLUSIONS: One fraction may potentially contain valuable compounds which may be useful for further investigation. This may focus on the identification of the bioavailable purified compounds present within these fractions or by detailed delineation of the related mechanisms of action.

Effects of feeding plant-derived agents on the colonization of Campylobacter jejuni in broiler chickens.

The aim of this work was to test the potential use of plant-derived extracts and compounds to control Campylobacter jejuni in broiler chickens. Over a 7-wk feeding period, birds were fed a commercial diet with or without plant extracts (Acacia decurrens, Eremophila glabra), essential oil [lemon myrtle oil (LMO)], plant secondary compounds [terpinene-4-ol and α-tops (including α-terpineol, cineole, and terpinene-4-ol)], and the antibiotic virginiamycin. Traditional culture and real-time quantitative PCR techniques were used to enumerate the numbers of C. jejuni in chicken fecal and cecal samples. In addition, BW and feed intake were recorded weekly for the calculation of BW gain and feed conversion ratio. The mean log10 counts of C. jejuni were similar (P > 0.05) across treatments. However, significantly lower levels of fecal Campylobacter counts (P < 0.05) were recorded at d 41 for the α-tops treatment by culture methods. No differences (P > 0.05) in BW gain were obtained for dietary supplementation, except for the E. glabra extract, which had a negative impact (P < 0.001) on BW, resulting in sporadic death. Results from this study suggest that supplemental natural compounds used in the current study did not reduce the shedding of C. jejuni to desired levels.