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Published online: 8 November 2019

Enlisting plants in the battle for new antibacterial compounds

Dane Lyddiard A and Ben W Greatrex B

A Biomedical Science
School of Science and Technology
University of New England
Armidale, NSW 2351, Australia
Tel: +61 2 6773 4050
Fax: +61 2 6773 3267
Email: dlyddia2@une.edu.au

B Pharmacy
School of Rural Medicine
University of New England
Armidale, NSW 2351, Australia
Tel: +61 2 6773 2402
Fax: +61 2 6773 3267
Email: ben.greatrex@une.edu.au

A rise in antibacterial drug resistance comes at a time when our once reliable sources of antibacterial natural products, bacteria and fungi, are failing us. The search for new drugs to fight pathogens has led to a range of innovative approaches and includes screening organisms which have developed evolutionary adaptions to prevent bacterial attack. The discovery of antibacterial phytochemicals from plants can be achieved using an activity-guided platform involving biological and chemical pre-screening, compound isolation, structure elucidation, and the direct testing of isolated compounds. Challenges include the clean isolation of natural products, avoiding the rediscovery of known compounds, toxicity, and poor levels of activity.

For a good part of the 20th century, humans had the upper hand against bacterial pathogens thanks to the pioneering work of Alexander Fleming, René Dubos and Selman Waksman et al. who demonstrated the value of mining antibacterial natural products from bacteria and fungi. For a few decades this approach (which yielded the likes of penicillin, streptomycin and tetracycline) seemed to be an impenetrable fortress against bacterial pathogens, until the walls began to strain under the force of growing antimicrobial resistance and a dearth of new antibiotic classes1. The ‘old’ approach eventually failed to produce significantly new clinical agents against the background of known compounds1. Researchers have responded in a range of novel ways: running large compound libraries through high throughput screenings2, mining the natural products in previously unculturable organisms3, screening the chemicals hidden away on the shelves of chemistry labs4, disarming bacteria of their virulence factors5, and developing phage therapies6; each approach with its merits and limitations. Another approach is screening botanical natural products.

The plant world as a whole is estimated to produce over 100 000 secondary metabolites with low molecular mass, generally derived from isoprenoid, phenylpropanoid, alkaloid and fatty acid or polyketide pathways7. While plants and animals have some common antibacterial defences such as apoptosis of infected tissue, antibacterial peptides (purothionins from Triticum aestivum8 are a noteworthy plant-based example) and the targeted exploitation of reactive oxygen species, they do not produce antibodies, relying instead on a limited number of receptors to recognize pathogens along with a diverse armoury of small molecules with antibacterial activity9. Compounds with known specific antibacterial targets are not common in plants, although there are examples such as coumarins with comparable action to the DNA-gyrase inhibitor novobiocin9. While activity is usually weak, it is possible that plants target virulence rather than growth or that relatively weak antibacterial agents work in synergy with each other to create potent activity as seen with the antibacterial compound berberine from Berberis fremontii together with the multi drug resistance (MDR) pump inhibitor 5’-methoxyhydnocarpin9,10.

Important phytochemical groups include phenolics and polyphenols, quinones, coumarins, flavonoids, terpenoids and alkaloids11 (Figure 1). Phenolics and polyphenols include the simple phenols, phenolic acids and tannins. Antibacterial examples are found in the tea plant Camellia sinensis and include gallic acid, a phenolic acid which disrupts cell membranes12,13, and the tannin tannic acid which reduces Staphylococcus aureus biofilm formation14. A representative of the quinones is juglone found in the black walnut tree Juglans nigra15, while the coumarins include osthole found in Arracacia tolucensis var. multifida16. Flavonoids include myricetin, found in the sweet potato plant Ipomoea batatas and which appears to affect protein synthesis17,18.

Figure 1.  Examples of antibacterial phytochemicals and the chemical classes to which they belong.
Click to zoom

Terpenoids are common phytochemicals based on the isoprene structure and include terpinen-4-ol, an antibacterial terpene found abundantly in Melaleuca alternifolia tea tree oil19, which evidence suggests leads to damage to the cell membrane and loss of cytoplasmic material20. Alkaloids are another common phytochemical group and include berberine (previously discussed) found in Coptis chinensis and Berberis fremontii. The mode of action of berberine may be through binding with double helical DNA21 and/or the inhibition of the bacterial division protein FtsZ22.

There are many ways to decipher a plant’s defences to find these antibacterial small molecules and these are accessible to a microbiologist who has support from a multidisciplinary team that includes a chemist and botanist. A simple schema developed in our lab is shown in Figure 2 and involves plant collection, secondary metabolite extraction, antimicrobial disk diffusion screen (adapted from the EUCAST23 method), GC-MS analysis coupled with databases (e.g. NIST Mass Spectral Libraries) for dereplication, TLC bioautography24,25, compound isolation by flash chromatography (or prep-HPLC) guided by testing fractions by TLC bioautography, elucidating new compound structures by NMR and/or crystallography and undertaking MICs on isolated compounds (adapting the CLSI methods26). Additionally, screening phytochemicals against specific virulence factors could uncover a trove of treasures, but there are diverse targets5 and each target requires a suitable assay: ultimately lots of work which may result in few if any hits. Inclusion in a compound library for high throughput screening is a possible solution. If good activity is seen during crude extract screening but is poor in the isolated compounds, combinations of compounds suspected to potentiate each other can be tested in a checkerboard assay27.

Figure 2.  Schema for discovering antibacterial phytochemicals.
Click to zoom

While the potential of antimicrobial phytochemicals is clear, there is a dearth of examples that have made it into the clinic. Many reasons for this exist including the differences in human and plant biology and physiology giving rise to toxicity concerns. An isolated compound with promising MIC activity needs to demonstrate low toxicity with preliminary tests such as in vitro cytotoxicity assays28 presenting a hurdle. Other factors for the lack of plant-based antibacterial agents include plants making diverse antimicrobial compounds but each with relatively poor activity9, and their production of a range of structurally similar compounds making isolation difficult and resource intensive. Compounding these problems, often the researcher spends time and resources to simply discover a known compound.

Attacking drug resistant bacteria from multiple fronts gives us the best chance for success. Screening phytochemicals as one of those approaches makes sense given the reliance of flora on secondary metabolites for antibacterial protection, and the incredible diversity of structures present across an enormous number of plant species. While plants have thus far generated few clinical candidates, successes in other anti-infective classes such as that of the antimalarial drug artemisinin from Artemisia annua29 allow for optimism. In Australia, only a limited number of researchers have looked at our unique flora as a potential solution and research has tended to focus on a limited number of genera, notably, Acacia, Melaleuca, Eucalyptus and Eremophila, leaving most species still to be screened.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

Our research has been supported by an Australian Government Research Training Program (RTP) Scholarship.



Biographies

Dane Lyddiard is a PhD candidate at the University of New England. His research interests include the phytochemistry of Australian native plants and its application to antimicrobial compound discovery.

Dr Ben Greatrex is a synthetic organic chemist in the Pharmacy discipline at the University of New England. His research interests include the discovery of bioactive natural products in Australian native plant species, and the synthesis of advanced functional materials and pharmaceuticals from waste lignocellulosic biomass.

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