Targeted Isolation of Antibiotic Brominated Alkaloids from the Marine Sponge Pseudoceratina durissima Using Virtual Screening and Molecular Networking.

Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8).

Phytochemical Profiling and Biological Testing of the Constituents of the Australian Plant Haemodorum brevisepalum.

Phytochemical profiling was undertaken on the crude extracts of the bulbs, stems, and the fruits of Haemodorum brevisepalum, to determine the nature of the chemical constituents present. This represents the first study to investigate the fruits of a species of Haemodorum. In total, 13 new and 17 previously reported compounds were isolated and identified. The new compounds were of the phenylphenalenone-type class, with a representative of a novel structural form, named tentatively "oxabenzochromenone" (1), a compound akin to an intermediate in a recently proposed phenylphenalenone metabolic network (2), seven new phenylphenalenones (4-10), four new phenylbenzoisochromenones (11-14), and a new phenylbenzoisochromenone derivative (18). The previously reported compounds identified were of the following structure classes: oxabenzochrysenone (3, 23-26), flavonol (15, 16), phenylbenzoisochromenone (17, 21, 22, 27-30), and phenylphenalenone (19, 20). Compounds 2-4, 6-9, 15-18, 21, 22, and 26 were subjected to antimicrobial evaluation with moderate activity observed against Staphylococcus aureus MRSA and slight activity against Pseudomonas aeruginosa and Candida albicans. Compounds 4, 6-9, 17, and 21 were also evaluated for anthelminthic activity against larvae of the blood-feeding parasitic nematode Haemonchus contortus.

Phytochemical Profiling and Biological Activity of the Australian Carnivorous Plant, Drosera magna.

Phytochemical profiling was undertaken on the crude extracts of Drosera magna to determine the nature of the chemical constituents present. In total, three new flavonol diglycosides (1-3), one new flavan-3-ol glycoside (4), and 12 previously reported compounds of the flavonol (5, 9), flavan-3-ol (15), flavanone (8), 1,4-napthoquinone (6, 7, 13, 14), 2,3-dehydroxynapthalene-1,4-dione (10-12), and phenolic acid (16) structure classes were isolated and identified. Compounds 1-9, 13, 17, and 18 were assessed for antimicrobial activity, with compounds 6, 7, 8, and 9 showing significant activity. Compounds 1, 2, and 6 were also evaluated for anthelmintic activity against larval forms of Hemonchus contortus, with compound 6 being active.

Application of Networking Approaches to Assess the Chemical Diversity, Biogeography, and Pharmaceutical Potential of Verongiida Natural Products.

This study provides a review of all isolated natural products (NPs) reported for sponges within the order Verongiida (1960 to May 2020) and includes a comprehensive compilation of their geographic and physico-chemical parameters. Physico-chemical parameters were used in this study to infer pharmacokinetic properties as well as the potential pharmaceutical potential of NPs from this order of marine sponge. In addition, a network analysis for the NPs produced by the Verongiida sponges was applied to systematically explore the chemical space relationships between taxonomy, secondary metabolite and drug score variables, allowing for the identification of differences and correlations within a dataset. The use of scaffold networks as well as bipartite relationship networks provided a platform to explore chemical diversity as well as the use of chemical similarity networks to link pharmacokinetic properties with structural similarity. This study paves the way for future applications of network analysis procedures in the field of natural products for any order or family.

Natural Products of Marine Macroalgae from South Eastern Australia, with Emphasis on the Port Phillip Bay and Heads Regions of Victoria.

Marine macroalgae occurring in the south eastern region of Victoria, Australia, consistingof Port Phillip Bay and the heads entering the bay, is the focus of this review. This area is home toapproximately 200 different species of macroalgae, representing the three major phyla of the greenalgae (Chlorophyta), brown algae (Ochrophyta) and the red algae (Rhodophyta), respectively. Overalmost 50 years, the species of macroalgae associated and occurring within this area have resultedin the identification of a number of different types of secondary metabolites including terpenoids,sterols/steroids, phenolic acids, phenols, lipids/polyenes, pheromones, xanthophylls andphloroglucinols. Many of these compounds have subsequently displayed a variety of bioactivities.A systematic description of the compound classes and their associated bioactivities from marinemacroalgae found within this region is presented.

Distribution, biosynthesis, and biological activity of phenylphenalenone-type compounds derived from the family of plants, Haemodoraceae.

Covering: up to 2018 The Haemodoraceae family is a monocotyledonous family in the order Commelinales consisting of 14 genera. Many species from the family are endemic to Australia and their use by the Aboriginal People of Australia as both pigments or remedies has been ethnobotanically documented. Phenylphenalenones are phenolic specialised metabolites consisting of a tricyclic phenalene nucleus with a ketone moiety and a lateral phenyl ring. Depending on their structural variance, four classes can be distinguished including the phenylphenalenones, oxabenzochrysenones, phenylbenzoisochromenones and phenylbenzoisoquinolinediones. The phenylphenalenone class has become the order's chemotaxonomic marker with a documented range of biological activities. This biological activity arises from the phototoxic properties of their ring system, a phenomenon most comprehensively observed amongst a widely cultivated family of the Commelinales order, Musaceae (banana). Within the family Haemodoraceae, the formation of the phenylphenalenone-class phytoanticipins is an intrinsic function of their growth, whereas within the family Musaceae these compounds are formed as phytoalexins in response to pathogenic attack or stress. The compounds produced within these two families differ in their substitution, with Musaceae-derived phytoalexins tending to be the more phototoxic 4-phenylphenalenones and the Haemodoraceae-derived phytoanticipins being of the more inert 9-phenylphenalenone type structure. Various other substitution patterns have been documented across the class, yet their biosynthetic mechanism is consistent, proceeding from simple phenylpropanoids through a diarylheptanoid intermediate, which cyclises to form the phenylphenalenone nucleus. Phenylphenalenone-related compounds have also been observed within the fungal kingdom, yet their biosynthetic route is based upon an alternative polymalonate pathway. This review focuses on Haemodoraceae-derived phenylphenalenone-type compounds, their distribution amongst species, throughout the plant organism, their biological activity and their biosynthesis.

Bromophenolics from the Red Alga Polysiphonia decipiens.

The isolation and the structure determination of a new bromophenolic compound, polysiphonol (10), as well as five previously reported compounds, (4-8), from the red alga Polysiphonia decipiens is reported. In addition, the absolute configuration of the natural product rhodomelol (8) could be unequivocally confirmed for the first time, and on biosynthetic grounds, the absolute configuration of polysiphonol (10) was tentatively suggested. Compounds 4-8 were evaluated for their antibacterial activity against both Gram-positive and Gram-negative bacteria, but none of the compounds showed any appreciable activity.

Absolute Configuration Determination of Retroflexanone Using the Advanced Mosher Method and Application of HPLC-NMR.

The absolute configuration of retroflexanone (1) and a closely related phlorogluinol (2) was established using the advanced Mosher method and by application of HPLC-NMR. HPLC-NMR permitted a small scale Mosher method analysis to be carried out on these unstable phloroglucinols.

A Brn2-Zic1 axis specifies the neuronal fate of retinoic-acid-treated embryonic stem cells.

Mouse embryonic stem cells (ESCs) treated with all-trans retinoic acid differentiate into a homogenous population of glutamatergic neurons. Although differentiation is initiated through activation of target genes by the retinoic acid receptors, the downstream transcription factors specifying neuronal fate are less well characterised. Here, we show that the transcription factor Brn2 (also known as Pou3f2) is essential for the neuronal differentiation programme. By integrating results from RNA-seq following Brn2 silencing with results from Brn2 ChIP-seq, we identify a set of Brn2 target genes required for the neurogenic programme. Further integration of Brn2 ChIP-seq data from retinoic-acid-treated ESCs and P19 cells with data from ESCs differentiated into neuronal precursors by Fgf2 treatment and that from fibroblasts trans-differentiated into neurons by ectopic Brn2 expression showed that Brn2 occupied a distinct but overlapping set of genomic loci in these differing conditions. However, a set of common binding sites and target genes defined the core of the Brn2-regulated neuronal programme, among which was that encoding the transcription factor Zic1. Small hairpin RNA (shRNA)-mediated silencing of Zic1 prevented ESCs from differentiating into neuronal precursors, thus defining a hierarchical Brn2-Zic1 axis that is essential to specify neural fate in retinoic-acid-treated ESCs.

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.

Comparative analysis of carotenoid content in Momordica cochinchinensis (Cucurbitaceae) collected from Australia, Thailand and Vietnam.

Momordica cochinchinensis (Cucurbitaceae) is the richest source of lycopene and ß-carotene of all known fruits but the influences of collection sites, variety and environment on carotenoid accumulation is unknown. This study analysed the carotenoid content of 44 M. cochinchinensis aril samples collected from Australia, Thailand and Vietnam using HPLC, UV-visible spectrophotometry and compared with the colorimetry method. The highest lycopene content was observed in samples collected from Ha Noi (7.76 mg/g) of Northern Vietnam and Lam Ha (6.45 mg/g) and Lam Dong (6.64 mg/g) provinces of Central Vietnam. The highest ß-carotene content was observed in a sample from Nam Dinh (9.60 mg/g) in Northern Vietnam while a variety from Hoa Binh province in Northern Vietnam had high contents of both lycopene (5.17 mg/g) and ß-carotene (5.66 mg/g). Lycopene content was higher in samples collected from low temperatures (<14 °C) and higher elevations whilst ß-carotene content was greatest at temperatures between 27 and 33 °C. Crop improvement for increased lycopene and ß-carotene requires rapid and accurate methods of quantification. All three analytical methods utilised were in agreement for lycopene quantification. The (a*/b*)2 transformed colour value resulted in more linear relationship for lycopene indicating that colorimetry method could potentially be developed to select lycopene rich fruits in the field.

Phosphorylation of the retinoic acid receptor RARγ2 is crucial for the neuronal differentiation of mouse embryonic stem cells.

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest by activating nuclear RA receptors (RARs) (α, ß and γ), which are ligand-dependent transcription factors. RARs are also phosphorylated in response to RA. Here, we investigated the in vivo relevance of the phosphorylation of RARs during RA-induced neuronal differentiation of mouse embryonic stem cells (mESCs). Using ESCs where the genes encoding each RAR subtype had been inactivated, and stable rescue lines expressing RARs mutated in phospho-acceptor sites, we show that RA-induced neuronal differentiation involves RARγ2 and requires RARγ2 phosphorylation. By gene expression profiling, we found that the phosphorylated form of RARγ2 regulates a small subset of genes through binding an unusual RA response element consisting of two direct repeats with a seven-base-pair spacer. These new findings suggest an important role for RARγ phosphorylation during cell differentiation and pave the way for further investigations during embryonic development.

Comparison of cytotoxicity between extracts of Clinacanthus nutans (Burm. f.) Lindau leaves from different locations and the induction of apoptosis by the crude methanol leaf extract in D24 human melanoma cells.

BACKGROUND: Clinacanthus nutans (Burm. f.) Lindau leaves are widely used by cancer patients and the leaf extracts possess cytotoxic and antiproliferative effects on several human cancer cell lines. However, the effect of C. nutans leaf extract on human melanoma, which is the least common but most fatal form of skin cancer and one of the most common cancers diagnosed in both sexes worldwide, is unknown. There is also limited information on whether the bioactivity of extracts differs between C. nutans leaves grown in different geographical locations with varying environmental conditions. METHODS: The present study, for the first time, compared and demonstrated the cytotoxicity of the crude methanol extracts of C. nutans leaves from 11 different locations in Malaysia, Thailand and Vietnam, with diverse environmental conditions against D24 melanoma cells through WST-8 assay. The percentage of apoptotic cells following treatment with the most active extract was determined in a dose- and time-dependent manner by a cytofluorometric double staining technique. Biochemical and morphological changes in the treated and untreated cells were examined by fluorescence and transmission electron microscopy techniques, respectively, to further affirm the induction of apoptosis. RESULTS: The leaves of plants grown at higher elevations and lower air temperatures were more cytotoxic to the D24 melanoma cells than those grown at lower elevations and higher air temperatures, with the leaf extract from Chiang Dao, Chiang Mai, Thailand exhibited the highest cytotoxicity (24 h EC50: 0.95 mg/mL and 72 h EC50: 0.77 mg/mL). This most active crude extract induced apoptotic cell death in the D24 cells in a dose- and time-dependent manner. Typical biochemical and morphological characteristics of apoptosis were also observed in the treated D24 cells. CONCLUSIONS: The results, showing the cytotoxicity of C. nutans and the induction of apoptosis in D24 cells, are significant and useful to facilitate the development of C. nutans as a potential novel chemotherapeutic agent for the management of skin melanoma.

Determination of the Absolute Configuration of the Pseudo-Symmetric Natural Product Elatenyne by the Crystalline Sponge Method.

Elatenyne is a marine natural product that was isolated in 1986. Despite its simple 2,2'-bifuranyl backbone, its relative structure was only recently determined. The absolute configuration of elatenyne has still not been unequivocally confirmed because of its pseudo-meso core structure, which results in a specific rotation, [α]D , of almost zero. In this work, the structure of natural elatenyne was determined by the crystalline sponge method and the use of a porous coordination network (a crystalline sponge) capable of absorbing organic guests; in the sponge, the absorbed guests are ordered and crystallographically observable. The crystalline sponge could differentiate between the two very similar alkyl side chains, and the absolute structure of elatenyne was thus reliably determined. The total amount required for the experiments was only approximately 100 µg, and the majority (95 µg) could be recovered after the experiments.

HPLC-NMR and HPLC-MS Profiling and Bioassay-Guided Identification of Secondary Metabolites from the Australian Plant Haemodorum spicatum.

Phytochemical dereplication was undertaken on the bioactive crude CH2Cl2 extract of the bulbs of the Australian plant Haemodorum spicatum employing HPLC-NMR and HPLC-MS methodologies. Subsequent bioassay-guided isolation resulted in the identification of two new phenylphenalenones [haemoxiphidone (8) and haemodoronol (17)] and two new chromenes [haemodordione (13) and haemodordiol (16)], together with seven previously described compounds. Antimicrobial testing showed that the compounds displayed selective antibacterial activity. Most noteworthy were the activities displayed by several of the compounds against multi-drug-resistant Pseudomonas aeruginosa.

Phytochemical Investigation of the Constituents Derived from the Australian Plant Macropidia fuliginosa.

A phytochemical study of the flowers and bulbs derived from the Australian plant Macropidia fuliginosa, involving hyphenated spectroscopic methodologies (HPLC-NMR and HPLC-MS), together with conventional isolation strategies, resulted in the identification of 16 constituents (1, 2, 4-17) representative of six different structural classes. Six new compounds (12-17) were identified from the bulbs of the plant. The isolated compounds were assessed for antimicrobial activity, and compound 8 was found to be more potent against P. aeruginosa than ampicillin.

Dereplication and chemotaxonomical studies of marine algae of the Ochrophyta and Rhodophyta phyla.

Dereplication and chemotaxonomic studies of six marine algae of the Ochrophyta and one of the Rhodophyta phyla resulted in the detection of 22 separate compounds. All 16 secondary metabolites, including four new compounds (16-19), could be rapidly dereplicated using HPLC-NMR and HPLC-MS methodologies in conjunction with the MarinLit database. This study highlights the advantages of using NMR data (acquired via HPLC-NMR) for database searching and for the overall dereplication of natural products.

Chemical profiling (HPLC-NMR & HPLC-MS), isolation, and identification of bioactive meroditerpenoids from the southern Australian marine brown alga Sargassum paradoxum.

A phytochemical investigation of a southern Australian marine brown alga, Sargassum paradoxum, resulted in the isolation and identification of four new (5, 9, 10, and 15) and nine previously reported (1, 2, 6-8, and 11-14) bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight (1, 2, 5-7, and 10-12) other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 µL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration.

A Review of the Ethnobotanical Use, Chemistry and Pharmacological Activities of Constituents Derived from the Plant Genus Geijera (Rutaceae).

Geijera Schott is a plant genus of the Rutaceae Juss. (rue and citrus) family, comprising six species which are all native to Oceania. Of the plants belonging to this genus, the most significant species that has a customary use is Geijera parviflora, which was used by Indigenous Australians, primarily as a pain reliever. Herein, a comprehensive review of the literature published on the genus Geijera from 1930 to 2023 was conducted. This is the first review for this plant genus, and it highlights the chemical constituents reported to date, together with the range of pharmacological properties described from the various species and different parts of the plant. These properties include anti-inflammatory, anti-microbial, anti-parasitic, insect repellent, analgesic, neuroactive, and anti-cancer activities. Finally, a reflection on some of the important areas for future focused studies of this plant genus is provided.