Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massa.

Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back ~100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 µM against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

Hydrophilic Species Are the Most Biodegradable Components of Freshwater Dissolved Organic Matter.

Aquatic dissolved organic matter (DOM) is a crucial component of the global carbon cycle, and the extent to which DOM escapes mineralization is important for the transport of organic carbon from the continents to the ocean. DOM persistence strongly depends on its molecular properties, but little is known about which specific properties cause the continuum in reactivity among different dissolved molecules. We investigated how DOM fractions, separated according to their hydrophobicity, differ in biodegradability across three different inland water systems. We found a strong negative relationship between hydrophobicity and biodegradability, consistent for the three systems. The most hydrophilic fraction was poorly recovered by solid-phase extraction (SPE) (3-28% DOC recovery) and was thus selectively missed by mass spectrometry analysis during SPE. The change in DOM composition after incubation was very low according to SPE-ESI (electrospray ionization)-mass spectrometry (14% change, while replicates had 11% change), revealing that this method is sub-optimal to assess DOM biodegradability, regardless of fraction hydrophobicity. Our results demonstrate that SPE-ESI mass spectrometry does not detect the most hydrophilic and most biodegradable species. Hence, they question our current understanding of the relationships between DOM biodegradability and its molecular composition, which is built on the use of this method.

On the structures of the penduflorines from Tabernaemontana penduliflora.

The structures of the recently published monoterpene indole alkaloids penduflorines A and B (1a and 1b), isolated from Tabernaemontana penduliflora (Apocynaceae), have been revised. Rather than an inseparable mixture of two compounds, they appear to be the known alkaloid vobasine (2). Although we could not comprehensively revise the structures of penduflorines C-E due to lacking spectral data, since their structural elucidations were based on that of 1a and 1b, their structures should also be treated with caution.

Antibacterial eremophilane sesquiterpenoids from Xylaria feejeensis, an endophytic fungi of the medicinal plant Geophila repens.

Geophila repens (L.) I.M. Johnst (Rubiaceae) is a traditional medicinal plant used in Sri Lanka for the treatment of bacterial infections. Due to its rich endophytic fungi content, it was postulated that endophytically-produced specialized metabolites may be responsible for its purported antibacterial effects. To test this hypothesis, eight pure endophytic fungal cultures were isolated from G. repens then extracted and screened for antibacterial activity in a disc diffusion assay against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Large scale culturing, extraction, and purification of the most active fungal extract, obtained from Xylaria feejeensis, led to the isolation of 6',7'-didehydrointegric acid (1), 13-carboxyintegric acid (2), and four known compounds including integric acid (3). Compound 3 was isolated as the key antibacterial component (MIC = 16 µg/mL against Bacillus subtilis, 64 µg/mL against Methicillin-Resistant S. aureus). Compound 3 and its analogues were devoid of hemolytic activity up to the highest tested concentration of 45 µg/mL. This study demonstrates that specialized metabolites produced by endophytic fungi may contribute to the biological activity of some medicinal plants. Endophytic fungi should be evaluated as a potential source of antibiotics, especially from unexplored medicinal plants traditionally used for the treatment of bacterial infections.

α-Synuclein Aggregation Inhibitory Prunolides and a Dibrominated ß-Carboline Sulfamate from the Ascidian Synoicum prunum.

Seven new polyaromatic bis-spiroketal-containing butenolides, the prunolides D-I (4-9) and cis-prunolide C (10), a new dibrominated ß-carboline sulfamate named pityriacitrin C (11), alongside the known prunolides A-C (1-3) were isolated from the Australian colonial ascidian Synoicum prunum. The prunolides D-G (4-7) represent the first asymmetrically brominated prunolides, while cis-prunolide C (10) is the first reported with a cis-configuration about the prunolide's bis-spiroketal core. The prunolides displayed binding activities with the Parkinson's disease-implicated amyloid protein α-synuclein in a mass spectrometry binding assay, while the prunolides (1-5 and 10) were found to significantly inhibit the aggregation (>89.0%) of α-synuclein in a ThT amyloid dye assay. The prunolides A-C (1-3) were also tested for inhibition of pSyn aggregate formation in a primary embryonic mouse midbrain dopamine neuron model with prunolide B (2) displaying statistically significant inhibitory activity at 0.5 µM. The antiplasmodial and antibacterial activities of the isolates were also examined with prunolide C (3) displaying only weak activity against the 3D7 parasite strain of Plasmodium falciparum. Our findings reported herein suggest that the prunolides could provide a novel scaffold for the exploration of future therapeutics aimed at inhibiting amyloid protein aggregation and the treatment of numerous neurodegenerative diseases.

Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Citronamine A, an Antiplasmodial Isoquinoline Alkaloid from the Australian Marine Sponge Citronia astra.

Citronamine A (1), an isoquinoline alkaloid containing an unprecedented pentacyclic ring system, was isolated from the Australian marine sponge Citronia astra. Based on the combination of MS and NMR analyses and comparison of experimental and TDDFT calculated ECD spectra, the absolute structure of 1 was determined. Compound 1 displayed moderate activity against drug sensitive (3D7) and drug resistant (Dd2) strains of the parasite, Plasmodium falciparum, responsible for malaria.

Sulfadiazine Masquerading as a Natural Product from Scilla madeirensis (Scilloideae).

The structure of 2,4-(4'-aminobenzenamine)pyrimidine (1), a pyrimidine alkaloid previously isolated from the bulbs of Scilla madeirensis (Asparagaceae, synonym Autonoë madeirensis), has been revised. These conclusions were met via comparison of reported NMR and EIMS data with those obtained from synthetic standards. The corrected structure is the antibiotic sulfadiazine (2), which has likely been isolated as a contaminant from the site of collection. The reported bioactivity of 1 as an α1-adrenoceptor antagonist should instead be ascribed to sulfadiazine. Our findings appear to show another example of an anthropogenic contaminant being identified as a natural product and emphasize the importance of considering the biosynthetic origins of isolated compounds within a phylogenetic context.

Antiplasmodial Bis-Indole Alkaloids from the Bark of Flindersia pimenteliana.

Three new (1: -3: ) and 2 known (4: -5: ) bis-indole alkaloids were identified from the bark of Flindersia pimenteliana (Rutaceae). The structures of 1: -3: were elucidated on the basis of their (+)-HRESESIMS and 2D NMR spectroscopic data. Antiplasmodial activity for 1: -3: against chloroquine sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum is also reported, with IC50 values ranging from 0.96 to 2.41 µg/mL. These results expand our knowledge of the structure-activity relationships of potently antiplasmodial isoborreverine-type alkaloids, the bioactivity of which have recently attracted significant attention in the literature.

Anti-prion Butenolides and Diphenylpropanones from the Australian Ascidian Polycarpa procera.

A library of 500 Australian marine invertebrate extracts was screened for anti-prion activity using a yeast-based assay, and this resulted in an extract from the ascidian Polycarpa procera showing potent activity. Purification of this extract led to the isolation of six new butenolide metabolites, the procerolides 1-4 and two related diphenylpropanones, the procerones 5 and 6, as the bioactive components. The structures of 1-6 were elucidated from the analysis of 1D/2D NMR and MS data, and their absolute configurations determined from comparison of experimental and computed ECD data. Compounds 1-6 were tested for anti-prion activity in a yeast-based assay, and 1 and 5 displayed potent bioactivity (EC50 of 23 and 29 µM, respectively) comparable to the potently active anti-prion compound guanabenz. The procerolides and procerones are the first anti-prion compounds to be reported from ascidians, indicating that ascidians may be an untapped source of new lead anti-prion compounds.

Lamellarin Sulfates from the Pacific Tunicate Didemnum ternerratum.

Six new lamellarin sulfates (1-6) were isolated from the methanolic extract of the Pacific tunicate Didemnum ternerratum, collected from the Kingdom of Tonga. Mass spectrometric molecular networking through the GNPS platform was used to target the isolation of 1-6. Planar structures were elucidated through a combination of NMR and MS experiments. Through comparison of experimental and calculated ECD spectra, the absolute configurations of atropisomers 2-5 were determined, with their energetic barriers to racemization also determined computationally. The cytotoxicity of the compounds was tested against the human colon carcinoma cell line HCT-116, where lamellarin D-8-sulfate (5) exhibited moderate activity with an IC50 of 9.7 µM.

Acrotrione: An Oxidized Xanthene from the Roots of Acronychia pubescens.

A new oxidized xanthene, acrotrione (1), and two known acetophenones (2 and 3) were isolated from a methanol extract of the roots of Acronychia pubescens. The structure of 1 was elucidated on the basis of its (+)-HRESIMS, 2D NMR, and ECD data. Acritrione (1) contains an unusual oxidized furo[2,3- c]xanthene moiety that has not been previously reported. Moderate antiplasmodial activity for these natural products against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum was determined with IC50 values ranging from 1.7 to 4.7 µM.

ß-Triketone-Monoterpene Hybrids from the Flowers of the Australian Tree Corymbia intermedia.

Four new ß-triketone monoterpene hybrids, intermediones A-D (1-4), have been identified from the flowers of the Australian eucalypt tree Corymbia intermedia. Intermediones A-D are ß-triketones that incorporate a pinene moiety attached via a benzyl group to a syncarpic acid. The structures of 1-4, including relative configurations, were elucidated from the analysis of 1D/2D NMR and MS data. The absolute configurations of intermediones A and B were determined by comparison of experimental and predicted ECD spectra. Intermedione D possesses a tetracyclic ring system that is related to that found in the meroterpenes, guadials B and C. Low to moderate antiplasmodial activity toward the chloroquine-sensitive (3D7) strain of Plasmodium falciparum, with IC50 values ranging from 9.9 to 20.8 µM, was observed for intermediones A, B, and D.

HSQC-TOCSY Fingerprinting-Directed Discovery of Antiplasmodial Polyketides from the Marine Ascidian-Derived Streptomyces sp. (USC-16018).

Chemical investigations on the fermentation extract obtained from an ascidian-derived Streptomyces sp. (USC-16018) yielded a new ansamycin polyketide, herbimycin G (1), as well as a known macrocyclic polyketide, elaiophylin (2), and four known diketopiperazines (3⁻6). The structures of the compounds were elucidated based on 1D/2D NMR and MS data. The absolute configuration of 1 was established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Antiplasmodial activities were tested for the natural products against chloroquine sensitive (3D7) and chloroquine resistant (Dd2) Plasmodium falciparum strains; the two polyketides (1⁻2) demonstrated an inhibition of >75% against both parasite strains and while 2 was highly cytotoxic, herbimycin G (1) showed no cytotoxicity and good predicted water solubility.

Alkaloid diversity in the leaves of Australian Flindersia (Rutaceae) species driven by adaptation to aridity.

The genus Flindersia (Rutaceae) comprises 17 species of mostly Australian endemic trees. Although most species are restricted to rainforests, four have evolved to grow in semi-arid and arid environments. In this study, the leaf alkaloid diversity of rainforest and semi-arid/arid zone adapted Australian Flindersia were compared by LC/MS-MS and NMR spectroscopy. Contrary to expectations, Flindersia alkaloid diversity was strongly correlated with environmental aridity, where species predominating in drier regions produced more alkaloids than their wet rainforest congenerics. Rainforest species were also more chemically similar to each other than were the four semi-arid/arid zone species. There was a significant relationship between the presence of alkaloid structural classes and phylogenetic distance, suggesting that alkaloid profiles are influenced by both genetic and environmental factors. The results suggest that the radiation of Flindersia species out of the rainforest and into drier environments has promoted the evolution of unique alkaloid diversity. Plants growing in arid and semi-arid regions of Australia may represent an untapped source of undescribed specialised metabolites.

Pimentelamines A-C, Indole Alkaloids Isolated from the Leaves of the Australian Tree Flindersia pimenteliana.

Three members of a new class of ascorbic acid-adduct indole alkaloids (1-3), a new prenylated indole alkaloid (4), and five known compounds (5-9) were isolated from the leaves of Flindersia pimenteliana. The structures of 1-4 were elucidated on the basis of their (+)-HRESIMS and 2D NMR spectroscopic data. Antiplasmodial activity was also reported for the natural products against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum with IC50 values ranging from 0.19 to 3.6 µM.