Kirsten Benkendorff

Molluscan biological and chemical diversity: secondary metabolites and medicinal resources produced by marine molluscs

The phylum Mollusca represents an enormous diversity of species with eight distinct classes. This review provides a taxonomic breakdown of the published research on marine molluscan natural products and the medicinal products currently derived from molluscs, in order to identify priority targets and strategies for future research. Some marine gastropods and bivalves have been of great interest to natural products chemists, yielding a diversity of chemical classes and several drug leads currently in clinical trials. Molluscs also feature prominently in a broad range of traditional natural medicines, although the active ingredients in the taxa involved are typically unknown. Overall secondary metabolites have only been investigated from a tiny proportion (<1%) of molluscan species. At the class level, the number of species subject to chemical studies mirrors species richness and our relative knowledge of the biology of different taxa. The majority of molluscan natural products research is focused within one of the major groups of gastropods, the opisthobranchs (a subgroup of Heterobranchia), which are primarily comprised of soft‐bodied marine molluscs. Conversely, most molluscan medicines are derived from shelled gastropods and bivalves. The complete disregard for several minor classes of molluscs is unjustified based on their evolutionary history and unique life styles, which may have led to novel pathways for secondary metabolism. The Polyplacophora, in particular, have been identified as worthy of future investigation given their use in traditional South African medicines and their abundance in littoral ecosystems. As bioactive compounds are not always constitutively expressed in molluscs, future research should be targeted towards biosynthetic organs and inducible defence reactions for specific medicinal applications. Given the lack of an acquired immune system, the use of bioactive secondary metabolites is likely to be ubiquitous throughout the Mollusca and broadening the search field may uncover interesting novel chemistry.

Indole derivatives from the egg masses of Muricid molluscs

A range of brominated indole derivatives were found in the egg masses of six species of muricid molluscs. Several non-brominated indoles were also present in the eggs of two Mediterranean Muricidae, although these were not found in the Australian species. Tyrindoleninone (3), was the only compound found in all six species and is likely to be responsible for the observed antimicrobial activity of these muricid egg masses [1,2]. These bioactive indoles appear to be characteristic of muricid egg masses and were not found in the egg masses from 17 species in different families of marine molluscs.

TYRIAN PURPLE PRECURSORS IN THE EGG MASSES OF THE AUSTRALIAN MURICID, Dicathais orbita: A POSSIBLE DEFENSIVE ROLE

We report a putative defensive role for the precursors of Tyrian purple in the egg masses of the Australian muricid, Dicathais orbita. The fresh egg masses contain a high proportion of tyrindoleninone, which reacts to form tyriverdin and subsequently Tyrian purple and 6-bromoisatin as the eggs develop and the larvae hatch. Antimicrobial testing revealed that tyrindoleninone is toxic to both marine and human pathogens at a concentration of 1 mg/ml. Tyriverdin inhibits the growth of two marine pathogens, as well as the yeast Candida albicans at 0.001 mg/ml and was effectively bacteriostatic at 0.0005 mg/ml against three human pathogenic bacteria. Tyriverdin did not appear to significantly lyse the microbial cells. 6-Bromoisatin has mild antimicrobial properties, whereas Tyrian purple exhibited no significant activity. The antimicrobial properties of these compounds and changes in their presence during egg development correlates with ripening in the egg masses of D. orbita. This is the first report of the chemical ripening of eggs in a marine environment.

Influence of elevated temperatures on the immune response of abalone, Haliotis rubra

Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.

Sex-Specific Tyrian Purple Genesis: Precursor and Pigment Distribution in the Reproductive System of the Marine Mollusc, Dicathais orbita

Exploitation of Tyrian purple from muricid molluscs, since antiquity, has prompted much interest in its chemical composition. Nevertheless, there remains a paucity of information on the biosynthetic routes leading to observed sexual differences in pigmentation. A liquid chromatography-mass spectrometry (LQ-MS) method was developed to simultaneously quantify dye pigments and precursors in male and female Dicathais orbita. The prochromogen, tyrindoxyl sulfate, was detected for the first time, by using this method in hypobranchial gland extracts of both sexes. Intermediates tyrindoxyl, tyrindoleninone, and tyriverdin were detected in female hypobranchial glands, along with 6,6′-dibromoindigo, while males contained 6-bromoisatin and 6,6′-dibromoindirubin. Multivariate analysis revealed statistically significant differences in the dye composition of male and female hypobranchial glands (ANOSIM, P = 0.002), thus providing evidence for sex-specific genesis of Tyrian purple in the Muricidae. Dye precursors were also present in male and female gonoduct extracts, establishing a mechanism for the incorporation of bioactive intermediates into muricid egg masses. These findings provide a model for investigating sex-specific chemical divergences in marine invertebrates and support the involvement of Tyrian purple genesis in muricid reproduction.

Natural Product Research in the Australian Marine Invertebrate Dicathais orbita

The predatory marine gastropod Dicathais orbita has been the subject of a significant amount of biological and chemical research over the past five decades. Natural products research on D. orbita includes the isolation and identification of brominated indoles and choline esters as precursors of Tyrian purple, as well as the synthesis of structural analogues, bioactivity testing, biodistributional and biosynthetic studies. Here I also report on how well these compounds conform to Lipinski’s rule of five for druglikeness and their predicted receptor binding and enzyme inhibitor activity. The composition of mycosporine-like amino acids, fatty acids and sterols has also been described in the egg masses of D. orbita. The combination of bioactive compounds produced by D. orbita is of interest for further studies in chemical ecology, as well as for future nutraceutical development. Biological insights into the life history of this species, as well as ongoing research on the gene expression, microbial symbionts and biosynthetic capabilities, should facilitate sustainable production of the bioactive compounds. Knowledge of the phylogeny of D. orbita provides an excellent platform for novel research into the evolution of brominated secondary metabolites in marine molluscs. The range of polarities in the brominated indoles produced by D. orbita has also provided an effective model system used to develop a new method for biodistributional studies. The well characterized suite of chemical reactions that generate Tyrian purple, coupled with an in depth knowledge of the ecology, anatomy and genetics of D. orbita provide a good foundation for ongoing natural products research.

Marine compounds selectively induce apoptosis in female reproductive cancer cells but not in primary-derived human reproductive granulosa cells.

Anticancer properties of tyrindoleninone and 6-bromoisatin from Dicathais orbita were tested against physiologically normal primary human granulosa cells (HGC) and reproductive cancer cell lines. Tyrindoleninone reduced cancer cell viability with IC50 values of 39 µM (KGN; a tumour-derived granulosa cell line), 39 μM (JAr), and 156 μM (OVCAR-3), compared to 3516 μM in HGC. Apoptosis in HGC’s occurred after 4 h at 391 µM tyrindoleninone compared to 20 µM in KGN cells. Differences in apoptosis between HGC and KGN cells were confirmed by TUNEL, with 66 and 31% apoptotic nuclei at 4 h in KGN and HGC, respectively. These marine compounds therefore have potential for development as treatments for female reproductive cancers.

Enhanced acute apoptotic response to azoxymethane-induced DNA damage in the rodent colonic epithelium by Tyrian purple precursors: a potential colorectal cancer chemopreventative.

Colorectal cancer (CRC) is the second most prevalent and deadly cancer worldwide. Due to the mortality and morbidity associated with chemotherapeutic regimes, research is turning to natural product enhancement of the acute apoptotic response to genotoxic carcinogens (AARGC). Although Tyrian purple dye pigments and precursors from muricid molluscs are known for their anti-proliferative and proapoptotic activity, the chemoprotective properties of these edible molluscs has not been assessed. Enhancement of AARGC by oral administration of muricid extract (ME), containing a mixture of the cytotoxins tyrindoleninone and 6-bromoisatin, was assessed in an azoxymethane (AOM) rodent model. A dose-dependent increase in apoptotic index was observed in the distal colon, with a significant increase detected at an ME dose of 1.0 mg/g (P < 0.01). Proliferation (PCNA) index failed to vary significantly at this ME concentration, which confirms the ME-induced increase in apoptotic response to DNA alkylation. ME also appears to confer no major toxic side effects, as all mice consistently gained weight during the trial and colonic crypt height was maintained (P > 0.05) independent of ME dose. Although, this is the first example of AARGC enhancement by indole-based compounds, bioactive precursor degradation in simulated gastric fluid may prevent introduction of muricids as a chemopreventative food. Nevertheless, the protective effect of ME against CRC in vivo clearly substantiates further research into the chemopreventative efficacy of Muricidae natural products.

Bioactivity of the Murex homeopathic remedy and of extracts from an Australian Muricid mollusc against human cancer cells

Marine molluscs from the family Muricidae are the source of a homeopathic remedy Murex, which is used to treat a range of conditions, including cancer. The aim of this study was to evaluate the in vitro bioactivity of egg mass extracts of the Australian muricid Dicathais orbita, in comparison to the Murex remedy, against human carcinoma and lymphoma cells. Liquid chromatography coupled with mass spectrometry (LC-MS) was used to characterize the chemical composition of the extracts and homeopathic remedy, focusing on biologically active brominated indoles. The MTS (tetrazolium salt) colorimetric assay was used to determine effects on cell viability, while necrosis and apoptosis induction were investigated using flow cytometry (propidium iodide and Annexin-V staining, resp.). Cells were treated with varying concentrations (1–0.01 mg/mL) of crude and semi-purified extracts or preparations (dilute 1 M and concentrated 4 mg/mL) from the Murex remedy (4 h). The Murex remedy showed little biological activity against the majority of cell lines tested. In contrast, the D. orbita egg extracts significantly decreased cell viability in the majority of carcinoma cell lines. Flow cytometry revealed these extracts induce necrosis in HT29 colorectal cancer cells, whereas apoptosis was induced in Jurkat cells. These findings highlight the biomedical potential of Muricidae extracts in the development of a natural therapy for the treatment of neoplastic tumors and lymphomas.

Mass spectrometry imaging on porous silicon: investigating the distribution of bioactives in marine mollusc tissues

Desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) is an attractive alternative to conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of low molecular weight compounds. Porous silicon (pSi) chips are also suitable as support for mass spectrometry imaging (MSI). Here, we report an implementation of DIOS-MSI using the biosynthetic organs of a marine mollusc for proof of principle. The tissue section is stamped onto a fluorocarbon-functionalized pSi chip, which extracts and traps small hydrophobic molecules from the tissue under retention of their relative spatial distribution. The section is subsequently removed and the chip is imaged without any remaining tissue. We apply this novel tissue contact printing approach to investigate the distribution of biologically active brominated precursors to Tyrian purple in the hypobranchial gland of the marine mollusc, Dicathais orbita, using DIOS-MSI. The tissue contact printing is also compatible with other types of desorption/ionization surfaces, such as nanoassisted laser desorption/ionization (NALDI) targets.