Prior to the advances in modern medicine that have occurred during the last century, traditional remedies were relied upon to treat a myriad of diseases, and this medicinal knowledge has been recorded for many civilisations globally. In contrast, the First Australians did not record their knowledge of Australian plants and instead relied on the oral transfer of this knowledge between generations. The general lack of written records has resulted in considerable loss of knowledge of First Australian traditional healing methods, or incorrect or incomplete documentation when recorded by European immigrants ^[1]. Considerable ethnobotanical, pharmacognostic, and pharmacological work is required to record and safeguard this knowledge for future generations.

As a result of its geographic isolation, Australian flora and fauna evolved separately from species in other regions, with minimal spread globally or minimal introduction of plants from other regions through most of the estimated 50,000 years that Australia has been populated by humans. This has resulted in one of the highest floral diversities and degrees of endemism globally. Furthermore, the size and wide array of Australian environmental conditions has resulted in plant species that have adapted to survive in these conditions. As plants produce secondary metabolites to assist in their survival, the diversity of unique plant species growing in harsh environments provides a largely untapped reservoir of natural products for drug discovery.

The First Australians have developed a detailed understanding of the medicinal potential of Australian flora through 50,000 years of trial and observation. Numerous plants were used therapeutically by individual tribal groups, and the ancestral knowledge has been transmitted orally from generation to generation. Unfortunately, few written accounts of the plant species used medicinally as well as of their preparation and application exist, with most records written from the perspective of European settlers. This has led to many dangerous or potentially fatal misrepresentations about the usage of plants by the First Australians. For example, Lassak and McCarthy ^[2] provide an account of the use of a native cycad fruit as a food. An early account by European settlers lists the fruit as a food without describing its preparation. Notably, the fruit contains substantial levels of toxic compounds. The First Australians placed the fruit in a stream of running water for several days before they considered it safe to consume. Reporting the fruit as a food without including details of its preparation resulted in several early settlers becoming seriously ill. An understanding of not only the species used by the First Australians, but also of their preparation and application methods is important for safe usage and also to highlight plant species worthy of further evaluation as drug leads.

Cold water decoctions were commonly used by the First Australians to prepare medicines ^[2]. As metal or clay containers were not readily available to the First Australians, it was difficult for them to boil water to prepare hot water infusions. Instead, most traditional medicines used by the First Australians were produced by crushing the plant material and soaking it in wooden bowls. The liquid was strained off, and the medicine could be drunk or used as a body wash. The plant material was also often pounded into a paste and mixed with animal fat to produce an ointment that was applied topically to the affected area. Alternatively, smoking ceremonies and smoke inhalation were also particularly widespread amongst First Australians, yet despite this, limited studies are yet to examine the effects of pyrolytic activity on the phytochemistry or therapeutic properties of Australian medicinal plants. One exception was a study that reported substantial phytochemical changes following the burning of Eremophila longifolia (R.Br.) F.Muell. leaves, as they would have been used in many traditional healing practices ^[3]. Instead, most laboratory-based studies have prepared and tested either essential oils or solvent extracts, and substantial further work is required to test First Australian medicines in the form similar to how they were traditionally used to validate their activity and to highlight new drug leads.

The taxonomy of the family Scrophulariaceae is complex and previously included approximately 5000 species from 275 genera. However, following reclassification, the family now consists of approximately 1830 species across 62 genera. The genus Eremophila (Tribe Myoporeae) is one of the largest genera of Scrophulariaceae, consisting of more than 260 species, all of which are endemic to Australia [4,5,6]^[4][5][6]. The genus is widespread in arid areas of mainland Australia (none occur naturally in Tasmania), particularly in Western Australia, South Australia, and the Northern Territory. Whilst individual species can range in size from prostrate shrubs (e.g., Eremohilia serpens Chinnock) to small trees (e.g., Eremophila bignoniflora (Benth.) F.Muell.), most species grow as compact, low-growing shrubs. Figure 1a shows the growth form of Eremophila maculata (Ker Gawl.) F.Muell., which is characteristic of many species of this genus. The size and shape of Eremophila spp. is also variable, although the leaves are generally relatively small (as shown for E. maculata in Figure 1b) and may be shiny or hairy. The flowers also share common features across the genus, generally having five petals per flower, which are joined at their base, forming a tube (Figure 1b). The flower color can vary widely between species, with red-, purple-, lilac mauve-, cream-, white-, or even green-coloured flowers being common across many species. Notably, many Eremophila spp. share common names, which include emu bush or poverty bush, which relate to the belief that emus eat the fruit of those species and to the arid/poor environment in which they grow. Alternatively, multiple Eremophila spp. are also known as fuchsia bush due to the superficial resemblance of their flowers to those of some Fuchsia spp.

The First Australians had substantial knowledge of Eremophila spp. and utilised multiple species medicinally. Indeed, an earlier study noted that of ~70 species that had been identified as being used therapeutically by First Australians from central Australia, approximately a third of those species belonged to either the Eremophila or Acacia genera ^[7]. Of these, Eremophila alternifolia R.Br. was highlighted because of its importance to the First Australians [6,8]^[6][8], and it is still considered to be one of the most important medicinal plants amongst central Australian Aboriginal populations.

2. Ethnopharmacology

The hostile environment and conditions of Australia led the First Australians to develop therapeutic methods using elements readily available to them in their environment. The local flora consists of a myriad of unique species, some of which have been exploited by the First Australians for their therapeutic properties. The genus Eremophila was widely used by the First Australian communities in all of the regions in which they grew to treat a wide variety of illnesses and medical complaints. Of the Eremophila spp., approximately twenty species were most frequently used as medicines ^[7]. Supplementary Table S1 records and summarises the ethnobotanical knowledge and traditional therapeutic uses of Eremophila spp. as traditional medicines, whilst Supplementary Table S2 summarises the studies undertaken to validate their therapeutic properties. The use of Eremophila spp. preparations to treat bacterial infections is particularly well reported. Eremophila alternifolia R.Br., Eremophila duttonii F.Muell., Eremophila freelingii F.Muell., Eremophila gilesii F.Muell., Eremophila latrobei F.Muell., Eremophila longifolia (R.Br.) F.Muell., and Eremophila sturtii R.Br. were traditionally used as antiseptic/antibacterial therapies to treat wounds and skin infections. Decoctions prepared from the leaves of E. alternifolia, E. duttoni, E. freelingi, E. gilesii, E. longifolia, and E. strutii were used topically as a hot bath by the First Australians to treat skin infections [1,2]^[1][2]. Similarly, decoctions prepared using young Eremophila bignoniiflora leaves were applied topically to treat bacterial skin diseases and wound infections. Alternatively, the leaves and twigs were wrapped around the head for the treatment of sinusitis and to relieve nasal congestion. The leaves of E. latrobei were prepared as a decoction and used as an antibacterial mouthwash/gargle to relieve sore throats. Decoctions of the leaves of E. longifolia were also applied as an eye wash and antiseptic for ophthalmic problems [1,2]^[1][2]. The First Australians also prepared a body scrub by mixing Eremophila dalyana leaf decoctions with animal fat. This was applied directly to the chest to treat chest pain [2,7]^[2][7]. Similarly, decoctions prepared from Eremophila duttonii, Eremophila elderi, or Eremophila latrobei leaves were applied to the affected area to alleviate the symptoms of rheumatism ^[2]. Rheumatism was also treated by the topical application of Eremophila gilesii decoctions to the affected area. In contrast, Eremophila maculata leaves are applied directly to the body as a poultice to treat rheumatism and chest pain. In addition to topical application, several Eremophila spp. preparations were consumed to treat bacterial diseases. This was particularly evident for the treatment of gastrointestinal bacterial infections. Decoctions of Eremophila sturtii and Eremophila freelingii leaves were consumed by several First Australian groups to fight gastrointestinal diseases and food poisoning [1,2]^[1][2]. Similarly, Eremophila goodwinii leaf decoctions were ingested to purge the digestive system and to improve gastrointestinal health. Interestingly, several Eremophila spp. were also noted for their laxative effects, which may in part be responsible for the use of Eremophila spp. decoctions to improve gastrointestinal health. Eremophila bignoniiflora decoctions were particularly noted for their laxative effects. Decoctions prepared from the fruit of this species were ingested to purge the gastrointestinal tract of microbial pathogens during severe gastrointestinal illness. Eremophila cuneifolia leaf decoctions wresearchers also consumed to treat headaches as well as to alleviate other aches and pains [1,2,6,10]^[1][2][6][9]. Eremophila spp. preparations were also used for the treatment of viral respiratory diseases. The use of Eremophila spp. decoctions by multiple First Australian groups to treat colds and influenza were particularly well reported [1,2,6,10]^[1][2][6][9]. Eremophila spp. decoctions were also useful in alleviating the symptoms of these diseases, including headaches. Whilst multiple species were used to treat viral respiratory diseases, the consumption of Eremophila alternifolia, Eremophila fraseri, Eremophila freelingii, and Eremophila longifolia leaf decoctions were most frequently reported for the treatment of colds. Additionally, Eremophila sturtii leaf infusions were used as hot baths and for steam inhalation for colds and influenza. Several Eremophila species were also used by the First Australians as a body wash for the treatment of scabies infestations (caused by the ecto-parasite Carcoptes scabiei L.). In particular, Eremophila dalyan, Eremophila latrobei, and Eremophila paisley decoctions and infusions were reported to be effective against this parasite ^[2]. Whilst reports are scarce about the use of Eremophila spp. preparations against other ecto-parasites, it is possible that they may also be useful against lice, fleas, bedbugs, and mites, although this remains to be verified.

3. Medicinal Properties and Therapeutic Effects

3.1. Antibacterial Activity

Multiple studies have screened Eremophila spp. extracts for medically relevant bioactivities. Of these, the antibacterial properties have been the most extensively reported. One study screened multiple species for antibacterial activity and reported an interesting trend [11]^[10]. That study noted a correlation between the relative levels of the antimicrobial compounds present in Eremophila spp. that produce a sticky, oily, or waxy resin layer on green leaves and branches. Therefore, the high-resin-producing Eremophila spp. should be prioritised as candidates for future studies evaluating the antimicrobial properties of Eremohilia spp. extracts and isolated compounds. That study reported that Eremophila lucida leaf extracts exhibited noteworthy antibacterial activity against several Gram-positive bacterial species, including several Staphylococcus and Streptococcus species. The authors subsequently used a bioactivity-driven separation approach to isolate three major constituents from the leaf resin. The resin was initially extracted with acetone, and the extract was then fractionated with hexane. The hexane fraction displayed good antibacterial activity against Staphylococcus aureus and was therefore used to isolate the sesquiterpenoid farnesal and the diterpenoid viscidane (5α-hydroxyviscida-3,14-dien-20-oic acid). Viscidane had particularly good antibacterial activity, with MIC values of 65 μg/mL recorded against several S. aureus strains. Another study by the same group examined the antimicrobial activity of E. alternifolia leaf extracts and the isolated fractions using broth microdilution methods, and MIC and MBC values were reported [12]^[11]. The authors of that study identified the flavanones pinobanksin, pinobanksin-3-acetate, and pinobanksin-3-cinnamate as well as the serrulatan diterpene 8-hydroxyserrulat-14-en-19-oic acid as particularly promising. Of these components, all except pinobanksin had substantial antibacterial activity. Notably, the serrulatan diterpenoid component was identified as being widespread amongst Eremophila spp. and may therefore contribute to the antibacterial properties of those species. However, the most potent antibacterial activity was measured for pinobanksin-3-cinnamate against several S. aureus strains (MIC values 10–20 µM), including methicillin-resistant and biofilm-forming strains. In contrast, all of the isolated E. alternifolia leaf compounds were completely ineffective against the Gram-negative bacterium Escherichia coli. Similarly, a recent study reported that Eremophila alternifolia leaf extracts have substantial antifungal and antibacterial activity against multiple pathogens [13]^[12]. That study also demonstrated that the extracts inhibited several important cellular pathways and disrupted membrane integrity in Gram-positive bacterial species. The same study determined that several isolated E. alternifolia compounds have significant antifungal activity against the fungi Cryptococcus gattii, and Cryptococcus neoformans as well as against the yeasts Candida albicans, Candida krusei, and Candida glabrata. Another study screened 39 plant species (including six Eremophila spp.) for antibacterial activity using an agar well-diffusion assay [14]^[13]. They reported that Eremophila duttonii had particularly good growth-inhibitory activity against Bacillus cereus, Enterobacter faecalis, Staphylococcus aureus, and S. pyogenes, with 10, 9, 12, and 14 mm inhibition zones measured, respectively. Notably, this was considered to be the best antibacterial activity measured for any of the plant extracts tested (on the basis of the measured inhibition zones). The authors also reported growth-inhibitory activity against B. cereus (albeit with substantially small inhibition zones). In contrast, all of the Eremophila spp. extracts (including E. duttonii) were completely ineffective against Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Salmonella typhimurium, indicating that the extract components inhibit bacterial growth via selective mechanisms. That study did not quantify the potency of the extracts by determining MIC values for bacterial growth inhibition. Therefore, is it not possible to fully evaluate the antibacterial activity of the extracts nor to compare the activity with other studies. Notably, the choice of a solid phase diffusion assay may not have provided the best indication of the antibacterial activities of those extracts. The movement of molecules within solid-phase agar gels is dependent on the physiochemical characteristics of the molecules in the tested extracts. In general, large or low-polarity molecules diffuse poorly with agar gels [15]^[14], providing small inhibition zones that do not necessarily correspond to activity in biological systems. Eremophila spp. extracts are rich in terpenoids, which have relatively low solubility in aqueous solutions. It is likely that the bacterial-growth-inhibitory activity was substantially underestimated in that study due to the choice of assay, and further studies using liquid dilution-based MIC assays are required to confirm and quantify the antibacterial activity of these extracts. A different study used both disc diffusion assays and fluorescein diacetate (FDA) bactericidal assays to evaluate the growth-inhibitory activity of some diterpenoids isolated from Eremophila sturtii leaf extracts [16]^[15]. Unfortunately, that study focussed on the isolated compounds and did not test the crude extract for inhibitory activity. Thus, it is not possible to validate the traditional use of E. sturtii preparations against the tested bacterial species. However, noteworthy activity was reported for 3,8-dihydroxyserrulatic acid and serrulatic acid against S. aureus. Serrulic acid was a particularly good inhibitor of that bacterium, with a minimum bactericidal activity (MBC) of 15 μg/mL. A noteworthy although substantially higher MBC (200 μg/mL) was also determined for 3,8-dihydroxyserrulatic acid. However, both compounds were ineffective against E. coli and P. aeruginosa and against the yeast Candida albicans. As plant extracts often contain multiple compounds that may potentiate each other’s activity, testing only the isolated compounds without also testing the crude extract in parallel may have resulted in interesting therapeutic properties being overlooked, and future studies should also test E. sturtii solvent extractions against the same bacterial pathogens. A recent study reported that similar compounds isolated from Eremophila glabra leaf extracts also had noteworthy antibacterial activity [17]^[16]. The authors of that study isolated and screened seven serrulatan diterpenes and three flavonoids for antibacterial activity. All of the isolated compounds were tested against Staphylococcus aureus (NCTC 10442) and Staphylococcus epidermidis (ATCC 14990) using an agar diffusion antimicrobial test. Two flavonoids (hispidulin and jacesidin) as well as three diterpenoids (18-acetoxy-8-hydroxyserrulat-14-in-19-oic acid, 8,18,20-triacetoxyserrulat-14-in-19-oic acid, and 20-acetoxy-8-hydroxyserrulat-14-in-19-oic acid) displayed good growth-inhibitory activity against both bacteria, with minimum inhibitory concentrations (MICs) ranging from 32 to 512 μg/mL. However, whilst agar diffusion assays are suitable for polar flavonoid compounds, the lower solubility of the diterpenoids in aqueous solutions may have resulted in the potency of those compounds being underestimated. The MIC values of these compounds should be re-evaluated in more appropriate assays. Despite this, the serrulatan diterpene 20-acetoxy-8-hydroxyserrulat-14-en-19-oic acid had the highest apparent growth-inhibitory activity against S. epidermidis (MIC = 32 μg/mL), although it displayed only low inhibitory activity against S. aureus. In contrast, the flavonoid compound jaceosidine displayed the highest growth-inhibitory activity against S. aureus (MIC = 128 μg/mL). Decoctions prepared from Eremophila serrulata leaves were also traditionally used to treat bacterial infections. In one study, E. serrulata leaves were extracted with diethyl ether, and then the extract was fractionated using RP-HPLC [18]^[17]. Several compounds with antibacterial activity were isolated and identified. The compounds 9-methyl-3-(4-methyl-3-pentenyl)-2,3-dihydronaphtho[1,8-bc] pyran-7,8-dione and 8,20-diacetoxyserrulat-14-en-19-oic acid displayed particularly good antimicrobial activity against Staphylococcus aureus (ATCC 29213), with MIC values ranging from 15.6 to 250 μg/mL. Both of these compounds also inhibited the growth of other Gram-positive bacteria, including Streptococcus pyogenes and Streptococcus pneumonia, although they were ineffective against all of the Gram-negative bacteria species tested. Another study by the same group screened two surrulatane diterpenoids isolated from Eremophila neglecta to determine the growth-inhibitory activity against an extended panel of human bacterial pathogens (including several MRSA strains) as well as multiple Gram-positive and Gram-negative species using broth dilution assays [19]^[18]. The serrulatan compounds 8,19- dihydroxyserrulat-14-ene and 8-hydroxyserrulat-14-en-19-oic acid were both effective inhibitors of all of the Gram-positive bacteria tested, with MIC values between 3 and 165 μM and MBC values between 6 and 330 μM. Notably, both compounds had noteworthy anti-mycobacterial activity against Mycobacterium fortuitum and Mycobacterium chelonae. In contrast, Moraxella catarrhalis was the only Gram-negative bacterium of the panel tested that was inhibited by the serrulatane diterpenoids with MICs and MBCs of 3–20 μM. The lack of activity against most of the Gram-negative bacteria may be related to their decreased passage through the cell wall of Gram-negative bacteria. It is likely that the relatively large molecular size and bicyclic conformation of serrulatans may inhibit their entry into the size-selective porin channels of the outer membrane of the Gram-negative cell wall, thereby preventing the compounds from reaching the inner membrane and cytoplasm of Gram-negative bacterial species [20]^[19]. Of further interest, a different study also reported that 8-hydroxyserrulat-14-en-19-oic acid isolated from E. neglecta leaves inhibits the formation of S. epidermidis and S. aureus biofilms and also has a dispersing effect on established biofilms [21]^[20]. Essential oils prepared by the hydro-distillation of Eremophila longifolia leaves were tested using both disc diffusion and broth dilution assays [22]^[21]. The authors detected antibacterial components using bioautography and reported an interesting trend. Essential oils rich in monoterpenes and hydrocarbon monoterpenols had moderate antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria, including S. aureus and S. epidermidis, whereas essential oils that contained ketone compounds as the dominant components displayed substantially lower antibacterial activity. Another study by the same group tested E. bignoniiflora leaf essential oil for antibacterial activity against a panel of bacteria [23]^[22]. The oil showed moderate to high activity against all of the Gram-positive organisms, yet only low growth-inhibitory activity against the Gram-negative bacteria. Bioautography of the Gram-positive bacteria indicated that the antimicrobial activity was related to the higher-polarity components of the essential oil, instead of to fenchyl, bornyl, and nerol acetate, which were the major constituents of the essential oils.

3.2. Antifungal and Antiviral Properties

Multiple Eremophila spp. also have antifungal properties. Several studies examined the antifungal properties of Eremophila spp. in parallel with their antibacterial activity. Some of those studies have already been summarised in the preceding section. Additionally, a recent study screened E. alternifolia leaf extracts and isolated compounds for antiviral activity and reported substantial activity [13]^[12]. The authors tested the extract and isolated compounds using disk diffusion and broth microdilution assays against ten clinically relevant yeast and mould species. The most potent activity was observed for the diterpene compound 8,19-dihydroxyserrulat-14-ene against Cryptococcus gattii and Cryptococcus neoformans, with MICs comparable to those of amphotericin B. This compound was similarly active against six Candida species (including C. albicans, C. krusei, and C. glabrata) and is therefore a promising drug target for the development of a novel therapy to treat infections of Cryptococcus spp. and other yeast species. The authors of that study reported that 8,19-dihydroxyserrulat-14-ene inhibits several biosynthetic pathways and compromises cell membrane integrity, thereby resulting in cell death. A further study reported that essential oils produced by hydro-distillation of Eremophila longifolia leaves had good inhibitory activity against the human dermal pathogens Trichophyton interdigitale, Trichophyton rubrum, and Trichophyton mentagrophytes [22]^[21]. The authors also evaluated the phytochemistry of the essential oils and reported that the antifungal activity was related to the borneol content of the oils. Interestingly, the authors also noted substantially greater antifungal activity when essential oils were prepared using leaves that were burned or partially pyrolysed during hydro-distillation. This is a particularly interesting finding and may provide some explanation as to why the First Australians frequently burned the leaves of this species in smoking ceremonies. The authors of that study reported that monoterpenol-dominated oils had moderate antifungal activity, although this activity increased significantly when the oils were partially pyrolysed. The E. longifolia essential oils were also effective against Candida albicans, possibly indicating that this species may be a good general fungicide. A similar study by the same group also tested an essential oil prepared from Eremophila bignoniiflora leaves using broth dilution assays against the same Trichophyton species, with moderate activity reported against all dermatophytes and against C. albicans [23]^[22]. Several Eremophila spp. also have antiviral bioactivities. Indeed, a study was conducted using Eremophila latrobei subsp. glabra extracts to examine their antiviral activity [24]^[23]. Notably, Eremophila latrobei subsp. glabra leaf extracts displayed antiviral activity against Ross River virus (RRV) at non-cytotoxic concentrations, indicating its potential for therapeutic use. The study was conducted on 40 plant species used traditionally by First Australians to treat viral diseases. However, that study did not determine the antiviral components, nor was the antiviral mechanism determined. Substantially more work is required to thoroughly evaluate the potential of this species to treat Ross River fever. Additionally, the extracts should also be evaluated for antiviral activity against other viral pathogens, particularly against other RNA viruses.