Analytical techniques for discovery of bioactive compounds from marine fungi

https://doi.org/10.1016/j.trac.2011.10.014Get rights and content

Abstract

Marine fungi are a promising source of novel bioactive compounds as lead structures for medicine and plant protection. We review current analytical techniques and future perspectives of analytical methodologies from the point of view of the discovery and the characterization of bioactive compounds isolated from marine fungi.

This critical overview also includes a general assessment of sampling and preparation of extracts, and compares different methods used for separation and isolation, and different strategies used for structural characterization of the bioactive compounds. We also cover the evolution of the application of bioassays for discovery of bioactive compounds.

Finally, this review addresses the advantages and the disadvantages of such techniques, and comments on future applications and potential research interest within this field.

Highlights

► Marine fungi are a promising source of novel bioactive compounds. ► We review the current analytical techniques for the discovery of bioactive compounds. ► We also assess the application of bioassays to the discovery of bioactive compounds.

Introduction

Oceans cover more than 70% of the world’s surface and have a wide diversity of marine organisms (e.g., fungi), which offer a rich source of natural compounds with biological activity (bioactive compounds) [1], [2]. Mayer et al. [3] used a modification of Schmitz’s chemical classification to assign marine natural-product structures to six major chemical classes: polyketides, terpenes, peptides, alkaloids, shikimates and sugars (Table 1). Marine fungi have been widely studied [4], [5], [6] for their bioactive metabolites, and these organisms have proved to be a rich, promising source of novel anticancer, antibacterial, antiplasmodial, anti-inflammatory and antiviral agents. Bioactive compounds produced by marine fungi are of interest as new lead structures for medicine as well as for plant protection. In addition, they play an important role in balancing the community and in mediating interactions between micro-organisms and their hosts. In contrast to other microorganisms (e.g., bacteria), basic knowledge on marine fungi (e.g., their distribution and their ecological role) is still scarce [7], [8], [9], [10], [11]. Between 2000 and 2005, approximately 100 marine fungal metabolites were described [12], and, between 2006 and 2010, a total of 690 natural products were reported [13] as being isolated from fungi in marine habitats. From these studies, the majority of the compounds (almost 50%) belonged to polyketides and their isoprene hybrids, followed by alkaloids, terpenes, and peptides, which contributed 14–20%. These new compounds were produced mostly from members of the fungal genera Penicillium and Aspergillus. Representatives of other genera that were less common were Acremonium, Emericella, Epicoccum, Exophiala, Paraphaeospaeria, Phomopsis, and Halarosellinia.

Compounds isolated from marine organisms comprising fungi were recently reviewed [3], [4], [5], [6], [13], [14], [15] and several interesting examples of compounds isolated from marine fungi with antibacterial activity could be mentioned:

  • (1)

    macrolide phomolide B isolated from the marine-derived fungus Phomopsis sp., which inhibits growth of Escherichia coli [16];

  • (2)

    spirodioxynaphthalene ascochytatin isolated from the marine-derived fungus Ascochyta sp. NGB4, which inhibits growth of Bacillus subtilis; and,

  • (3)

    sulfoalkylresorcinol isolated from the marine-derived fungus Zygosporium sp. KNC52, which showed inhibition of methicillin-resistant Staphylococcus aureus [17], [18].

Examples of compounds with anticancer activity can also be mentioned:

  • (1)

    ester-substituted sesquiterpenoid cryptosphaerolide isolated from the ascomycete fungal strain CNL-523 (Cryptosphaeria sp.) [19] exerts cytotoxicity upon a colon carcinoma cell line;

  • (2)

    diketopiperazine halimide that is a natural product from Aspergillus sp. marine-derived fungi from the green alga Halimeda copiosa [11] exerts cytotoxicity upon lung cancer; and,

  • (3)

    plinabulin, synthetic analogue of diketopiperazine halimide is on a Phase II clinical trial for use in patients with non-small cell lung cancer [11].

Finally, as examples of antiviral activity can be mentioned the following compounds:

  • (1)

    stachyflin, a terpenoid isolated from the fungus Stachybotrys sp. RF-7260, which shows activity against influenza A virus (H1N1) and is significantly better than other anti-H1N1 drugs (e.g., amantadine and zanamivir) [15]; and,

  • (2)

    halovirs A–E, which are hexapeptides obtained from Scytalidium sp., a fungus derived from the seagrass Halodule wrightii and exhibiting inhibition of herpes simplex virus 1 (HSV-1) [11].

During the screening of bioactive compounds, they must be extracted from marine fungi and characterized for chemical structure and composition, meaning a step-by-step separation of extracted components based on differences in their physicochemical properties. From an analytical point of view, this process is very difficult due to problems related to the solubility of the bioactive compounds with long hydrophobic chains, the extraordinary complexity of the samples, and often the non-availability of traceable standards.

In this review, we present an overview from an analytical point of view of the different methods for sample treatment and separation, and the different strategies used for structural characterization of the bioactive compounds.

Section snippets

General approaches to screening bioactive compounds

There are two main approaches leading to the discovery of bioactive compounds from the extracts: bioassay-guided fractionation and pure-compound screening [20]. Fig. 1 shows a comparative scheme of the essential steps in the search for bioactive compounds by these approaches.

Bioassay-guided fractionation (Fig. 1A) is the standard, most used procedure, and typically involves the following steps:

  • (1)

    assessment of the potential bioactivity using a batch biological assay;

  • (2)

    extraction using different

Collection and preservation of samples

In the different regions of the ocean there are distinct groups of microorganisms (e.g., fungi) living:

  • (1)

    in free suspension;

  • (2)

    attached to flocculated material;

  • (3)

    in the sediment;

  • (4)

    on living and non-living surfaces; and,

  • (5)

    as partners in symbiosis or in commensalism [24].

Marine fungi isolated from sponges are responsible for the production of a considerable proportion (28%) of new compounds, and are followed by those obtained from algae (27%) [10]. Different sampling strategies, depending on the origin,

Fractionation and separation of pure bioactive compounds

Chemically, bioactive compounds are classified into six classes – polyketides, terpenes, peptides, alkaloids, shikimates, and sugars. The extracts from marine fungi exhibiting bioactivity are mostly mixtures of several classes of compounds, and, since the chemical nature of the bioactive compounds in the complex mixture is unknown, it is impossible to follow a general technique to separate the compounds in the extract. The presence of multiple bioactive compounds, which are closely related and

Bioassays for discovery of bioactive compounds

Bioactivity establishes the potential application of natural products, so the nature and the design of the bioassay are crucial for the detection of bioactivities. It is essential that screening systems for natural products comprise a broad range of bioassays to unravel possible activities related to the substance [36]. During biological screening, the extracts, fractions and pure compounds are screened for their bioactivities in vitro and/or in vivo. The primary screens can be applied to large

Structural characterization of bioactive compounds

After isolation of a pure substance, elucidation of its chemical structure can be addressed, and characterization of the structure of bioactive compounds from marine origin is also a challenging task. Structural elucidation is to ascertain what the skeleton of the molecule is, and this can often be narrowed down by reference to literature on related genera and species. Different techniques based on high-resolution 1D and 2D nuclear magnetic resonance (NMR) spectroscopy [59] are applied for the

On-line combination

The on-line combination of bioassays with chemical and structural characterization enables rapid screening and identification of individual bioactive compounds with several bioactivities without prior purification and bioactivity assays. Bugni et al. [68] developed an efficient HTS approach with potential to eliminate bioassay-guided fractionation, for searching marine natural compounds and rapid drug discovery. A strategy that involved an automated HPLC-MS fractionation protocol to generate

Quantification of bioactive compounds

In addition to identification, the quantification of pure bioactive compounds in the extract is also important, mainly for pharmaceutical purposes. In bioactive compound discovery, quantification is needed to know how much sample (e.g., fermentation volume or fungus material) has to be produced and extracted to obtain sufficient amounts of the targeted compound. The lack of standards for most of the new products of marine fungi origin makes quantification a formidable task. Sun et al. [70] used

Conclusions and future trends

The results of screening new bioactive compounds depend on the quality of the sample, collection and storage of fungi, and cultivation, extraction, and separation and preparation of test samples [38]. There is no specific technique that could be followed for the separation of the constituents of the complex mixture present in the extract of the marine fungi, since the chemical nature of the bioactive compounds is not known a priori. The presence of multiple bioactive compounds, which are

Acknowledgements

This work was supported by the Portuguese Science Foundation (Fundação para a Ciência e Tecnologia) through individual research grants (SFRH/BPD/73781/2010 and SFRH/BPD/65410/2009) under QREN-POPH funds, co-financed by the European Social Fund and Portuguese National Funds from MCTES.

References (72)

  • A.M.S. Mayer et al.

    Comp. Biochem. Phys. C

    (2011)
  • D. Leary et al.

    Mar. Policy

    (2009)
  • J.F. Imhoff et al.

    Biotechnol. Adv.

    (2011)
  • M. Schumacher et al.

    Biotechnol. Adv.

    (2011)
  • J. Yasuhara-Bell et al.

    Antivir. Res.

    (2010)
  • K.R. Watts et al.

    Bioorgan. Med. Chem.

    (2010)
  • S.-Y. Shi et al.

    Trends Anal. Chem.

    (2009)
  • Y. Zhang et al.

    Biotechnol. Adv.

    (2011)
  • A. Aneiros et al.

    J. Chromatogr., B

    (2004)
  • K.B. Glaser et al.

    Biochem. Pharmacol.

    (2009)
  • C. Kasettrathat et al.

    Phytochemistry

    (2008)
  • A.M. Gamal-Eldeen et al.

    Environ. Toxicol. Pharm.

    (2009)
  • A. Abdel-Lateff

    Tetrahedron Lett.

    (2008)
  • J. Meenupriya et al.

    Asian Pac. J. Trop. Med.

    (2010)
  • S.-S. Gao et al.

    Bioorg. Med. Chem. Lett.

    (2011)
  • C.H. Gao et al.

    Chinese Chem. Lett.

    (2010)
  • C.-L. Shao et al.

    Bioorg. Med. Chem. Lett.

    (2011)
  • H. Xiong et al.

    J. Hydro-Environ. Res.

    (2009)
  • K.P. Mishra et al.

    Biomed. Pharmacother.

    (2008)
  • Y. Picó et al.

    Trends Anal. Chem.

    (2008)
  • N.G. Davey et al.

    Trends Anal. Chem.

    (2011)
  • W.F. Smyth et al.

    Trends Anal. Chem.

    (2006)
  • C. Hao et al.

    Trends Anal. Chem.

    (2007)
  • M. Brunati et al.

    Mar. Genomics

    (2009)
  • M. Farré et al.

    Trends Anal. Chem.

    (2010)
  • P. Proksch et al.

    Mar. Drugs

    (2003)
  • A. Penesyan et al.

    Mar. Drugs

    (2010)
  • M.E. Rateb et al.

    Nat. Prod. Rep.

    (2011)
  • I. Bhatnagar et al.

    Mar. Drugs

    (2010)
  • T. Le Calvez et al.

    Appl. Environ. Microb.

    (2009)
  • Y. Zhang et al.

    Mar. Drugs

    (2009)
  • Z. Paz et al.

    Fungal Divers.

    (2010)
  • J. Wiese et al.

    Mar. Drugs

    (2011)
  • M. Saleem et al.

    Nat. Prod. Rep.

    (2007)
  • R. Ebel

    Mar. Drugs

    (2010)
  • X. Du et al.

    J. Antibiot.

    (2008)
  • Cited by (0)

    View full text