Elsevier

Journal of Ethnopharmacology

Volume 182, 22 April 2016, Pages 1-9
Journal of Ethnopharmacology

Antibacterial constituents of Eremophila alternifolia: An Australian aboriginal traditional medicinal plant

https://doi.org/10.1016/j.jep.2016.02.011Get rights and content

Abstract

Ethnopharmacological relevance

For traditional medicinal purposes Aboriginal Australians have utilised numerous plant species, Eremophila alternifolia is among the most prominent. Traditionally, fresh leaves, leaf-infusions and handmade leaf-pastes have been used as both external and internal preparations to provide relief from a variety of conditions. Preparations of the species have been used to treat various infections of skin, eyes and throat including the treatment of septic wounds. These usages suggest that the plant contains antibacterial compounds; however, to date they have not been isolated and identified.

Aim of the study

The present study aimed to identify antibacterial compounds from this important traditionally recorded medicinal species.

Materials and methods

Bioassay-guided fractionation was used to isolate compounds from the crude leaf-extract. Antibacterial activity of pure compounds was assessed through broth microdilution method by determining both minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). Structure elucidation was performed using spectroscopic techniques such as 1D and 2D nuclear magnetic resonance spectroscopy and high resolution mass spectrometry.

Results

Four compounds have been isolated from the leaf-extract; they include previously known flavanones [pinobanksin (1), pinobanksin-3-acetate (2) and pinobanksin-3-cinnamate (3)] and a serrulatane diterpene, 8-hydroxyserrulat-14-en-19-oic acid (4). While compound 4 had been found in other Eremophilas, flavanones 2 and 3 are identified for the first time from the genus Eremophila. The flavanone 3 is the most promising antibacterial compound with significant activity (10−20 µM) against strains of the Gram-positive bacterium Staphylococcus aureus including methicillin resistant and biofilm forming strains. No activity was observed for any isolated compounds against the Gram-negative bacterium Escherichia coli.

Conclusion

The antibacterial activity of the crude extract of E. alternifolia and of the isolated compounds against Gram-positive bacteria provides a Western scientific explanation of the therapeutic modality of this plant species in traditional Aboriginal medicinal practice.

Introduction

Geographic isolation has resulted in Australia having a unique and distinct flora. Many Australian plant species have developed specific survival strategies that allow them to inhabit the harsh arid environment found in many parts of Australia. Australian Aboriginal people have used a number of these plant species with distinct curative properties for their well-being (Cock, 2011, Jones et al., 2007). Among over 70 plant species that have been used for medicinal purposes by Aboriginal people living in arid, central regions of the country, species of the genus Eremophila (Scrophulariaceae) are considered as of prime importance in their oral cultural lore (Latz, 1995, Richmond, 1993, Singab et al., 2013).

The unique Australian plant genus Eremophila, comprises 217 described species and several yet undescribed species that are widely distributed in the semi-arid and arid regions of Australia (Chinnock, 2007). A number of desert-adapted Eremophila species have featured in the medicinal, cultural and religious aspects of Australian Aboriginal people’s lives (Ghisalberti, 1994, Latz, 1995). Eremophila species were also used for smoke fumigation (Sadgrove et al., 2014). Some Eremophila species have been investigated for the molecular origins of traditional usages, with serrulatanes and o-naphthoquinones so far identified as antibacterial components (Forster et al., 1986, Ndi et al., 2007a, Ndi et al., 2007b).

In general, plants used in Australian Aboriginal medicine have been prepared as skin washes or drinks (infusions or hot water extracts), ointments, poultices, and steam or smoke treatments. A large number of Aboriginal plant remedies have been prepared to use externally to relieve problems associated with skin disorders, microbial infections, fever, rheumatic pains, wounds and burns (Barr, 1988, Latz, 1995). However, a more limited number, approximately 10% of preparations have been prepared to use internally for conditions such as colds, influenza, sore throat, pain and headaches (Clarke, 2008, Ghisalberti, 1994, Latz, 1995). While a number of Eremophila species are highly valued ingredients in externally used preparations, the species Eremophila alternifolia was and still is highly valued for both internal and external use (Barr et al., 1993). In more recent years small-scale businesses owned by Aboriginal communities selling formulations of this species have emerged providing an opportunity for economic development based on this plant (Morse, 2005).

E. alternifolia grows as a small to medium shrub, usually varying in height from 1−4 m. It is found growing mainly in rocky soils of hills and ranges in Central Australia and different arid zones of Western Australia and South Australia. Plants have alternate leaves and tubular flowers with a variety of colours, usually appearing in early winter to early autumn. Other names include poverty bush, narrow-leaf fuchsia bush and native honeysuckle (Barr, 1988, Chinnock, 2007, Pennacchio et al., 1995, Richmond, 1993).

Ethnobotanical reports have provided much information on the traditional medicinal use of E. alternifolia. In a review of the ethnopharmacology and phytochemistry of Eremophila species (Ghisalberti, 1994) Ghisalberti noted of E. alternifolia that “it has been, and still is, considered ‘number one medicine’” with a variety of uses by Aboriginal people, including the treatment of septic wounds. An ethnobotanical project funded by the Northern Territory Government in Australia (Barr et al., 1993), where Aboriginal communities worked with the project team to document traditional knowledge about plants, recorded the use of this species from five different communities in the Northern Territory. Those recorded uses contain a remarkably high proportion of conditions that Western medicine associates with microbial infections. E. alternifolia has also been used as a tea or rubbed on the skin to induce deep, pleasant sleep and general well-being (Latz, 1995, Low, 1990). In addition, decoctions of leaves were made for skin, eye and body washes (Richmond, 1993). Sometimes leaf-paste with water was prepared and used as ‘rubbing medicine’ for the head. Moreover, this was one of few species whose dried leaves were carried by nomadic Aboriginal groups for use in medicinal and cultural practices in case of need (Barr et al., 1993, Ghisalberti, 1994).

Previous phytochemical investigations of E. alternifolia have shown the occurrence of a moderate amount of essential oils including fenchone, limonene and camphor (Ghisalberti, 1993, Ghisalberti, 1994). Furanosesquiterpene β-ketols (Sutherland and Rodwell, 1989) along with flavonoids, galangin-3-methyl ether and pinobanksin have also been isolated from this species (Ghisalberti, 1994, Jefferies et al., 1962). Another study revealed the significant cardioactive effects of E. alternifolia in rodent hearts with the caffeoyl phenylethanoid glycoside verbascoside isolated as the cardioactive compound, while geniposidic acid was also identified but at a lower concentration (Pennacchio et al., 1995, Pennacchio et al., 1996).

Although several ethnobotanical reports have emphasized the pharmacological importance of this species for conditions that would appear to be associated with microbial infections, to our best knowledge, identification of the antimicrobial compounds from this plant has not been studied. However, a few reports of the in vitro antimicrobial activity of crude extracts of E. alternifolia have been published. A study which investigated the antibacterial activity of traditional Australian medicinal plants showed that an ethanolic leaf- extract of E. alternifolia exhibited substantial antibacterial activity against the Gram-positive bacteria, Staphylococcus aureus and Streptococcus pyogenes (Palombo and Semple, 2001). A collaborative project with Aboriginal groups from arid regions of Australia also demonstrated the antibacterial activity of this species (Evans et al., 2010). Another similar study reported the antimycobacterial activity of an extract from this species against Mycobacterium smegmatis and M. fortuitum (Meilak and Palombo, 2008). The reported traditional uses of this plant, the high esteem in which it is held as a medicine in a number of different Aboriginal communities and its previously reported antimicrobial activity led us investigate the antimicrobial activity of this species and to isolate and identify the active antibacterial compounds.

Here we present the activity-guided isolation, structural elucidation and antibacterial activity of four compounds from the leaves of E. alternifolia. These compounds belong to two different classes; flavonoids and serrulatane diterpenoids. They include three known flavanones, pinobanksin (1), pinobanksin-3-acetate (2) and pinobanksin-3-cinnamate (3) and a known serrulatane diterpene, 8-hydroxyserrulat-14-en-19-oic acid (4). Notably, this is the first report of studying antibacterial compounds from E. alternifolia as well as of the occurrence of flavanones 2 and 3 in the genus Eremophila. The identification of these four compounds establishes the scientific basis for the traditional usage of this plant for ailments associated with bacterial infections and provides an interesting initial comparison of structure versus antibacterial activity for pinobanksin type flavanones.

Section snippets

Collection of plant material

Leaves of E. alternifolia R.Br were collected from a private property near Dutton, north of the Barossa Valley Region, South Australia (GPS Coordinates: 34.3297 S, 139.1616 E) in September 2013. The plant was typical of the narrow-leaved, reddish pink flowering form, which is the most common form of this species, growing naturally in the area and over a wide range of inland Australia. A voucher specimen (AD 271534) was deposited at the State Herbarium of South Australia, Adelaide and species

Structural determination

The structures of all compounds (14) were identified by analysing their mass and 1D/2D NMR spectra, and putative structures were then compared with published data. Compounds 13 were identified as flavanones while compound 4 was identified as a serrulatane diterpene.

All compounds (14) could be identified as previously reported compounds. On the basis of the mass and NMR data and comparison with literature data, we arrived at the assignments of pinobanksin (1) previously isolated from E.

Discussion

Eremophila alternifolia has been recorded as being a highly valued medicinal species by a number of Australian Aboriginal groups (Barr et al., 1993, Richmond and Ghisalberti, 1994). It has been used both internally and externally for a variety of symptoms indicative of bacterial infections (Barr et al., 1993, Richmond, 1993). Using a bioassay-guided approach we have identified three compounds with antibacterial activity against staphylococci from this species. The active compounds belong to two

Concluding remarks

Previous findings suggested that the antibacterial activity of the investigated Eremophila species was due to compounds from two different classes, o-naphthoquinones and serrulatanes (Forster et al., 1986, Ndi et al., 2007a, Ndi et al., 2007b). Interestingly, Eremophila alternifolia differs substantially from the (relatively few) other Eremophila species investigated thus far, in that active flavanones contribute to the antibacterial activity of E. alternifolia. In this study, we have

Acknowledgements

The authors would like to acknowledge the support of the Australian Government's Cooperative Research Centres Program (CRC WMI). We thank Dr Bradley Simpson for assistance with mass spectrometry. High resolution mass spectrometry was performed at Flinders Analytical, Flinders University, South Australia.

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