Eremophila Longifolia Research Articles

Antibacterial and Antibiofilm Activity of Endophytic Alternaria sp. Isolated from Eremophila longifolia.

The threat to public health resulting from the emergence of antimicrobial resistance (AMR) is ever rising. One of the major bacterial pathogens at the forefront of this problem is methicillin-resistant Staphylococcus aureus, or MRSA, for which there is a great need to find alternative treatments. One of the most promising alternatives is endophytic fungi, which were shown to produce a vast array of bioactive compounds, including many novel antibacterial compounds. In this study, two endophytic Alternaria sp., EL 24 and EL 35, were identified from the leaves of Eremophila longifolia. Ethyl acetate (EtOAc) extracts of their culture filtrates were found to inhibit both methicillin-sensitive S. aureus ATCC 25923 and MRSA strains M173525 and M180920. The activity of each extract was shown to be greatly affected by the growth medium, with considerable reductions in minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) observed when tested in tryptic soy broth with glucose (TSBG) compared with Mueller-Hinton broth (MHB). Both extracts displayed significant (p ≤ 0.05) antibiofilm activity against all three S. aureus strains, the greatest of which was that of EL 35, which reduced biofilm formation by M180920 by 72%, while that of EL 24 resulted in a 57% reduction against ATCC 25923. Both extracts also disrupted established biofilms, of which the most effective was EL 35, which reduced the M180920 biofilm by 64%, while EL 24 also performed best against M180920, reducing biofilm by 54%. Gas chromatography-mass spectrometry (GC-MS) analysis of the EL 24 EtOAc extract revealed five known compounds. This study highlights the promise of endophytic fungi from Australian plants as a potential source of substances effective against important bacterial pathogens. Further understanding of the responsible compounds and their mechanisms could lead to the development of treatments effective against MRSA, as well as novel biofilm-resistant biomedical materials, contributing towards reducing the burden of AMR.

Serrulatane diterpenoids with unusual side chain modifications from root bark of Eremophila longifolia

The plant genus Eremophila is endemic to Australia and widespread in arid regions. Root bark extract of Eremophila longifolia (R.Br.) F.Muell. (Scrophulariaceae) was investigated by LC-PDA-HRMS, and dereplication suggested the presence of a series of diterpenoids. Using a combination of preparative- and analytical-scale HPLC separation as well as extensive 1D and 2D NMR analysis, the structures of 12 hitherto unreported serrulatane diterpenoids, eremolongine A-L, were established. These structures included serrulatanes with unusual side chain modifications to form hitherto unseen skeletons with, e.g., cyclopentane, oxepane, and bicyclic hexahydro-1H-cyclopenta[c]furan moieties. Serrulatane diterpenoids in Eremophila have recently been shown to originate from a common biosynthetic precursor with conserved stereochemical configuration, and this was used for tentative assignment of the relative and absolute configuration of the isolated compounds. Triple high-resolution α-glucosidase/α-amylase/PTP1B inhibition profiling demonstrated that several of the eremolongines had weak inhibitory activity towards targets important for management of type 2 diabetes.

The Diversity of Volatile Compounds in Australia's Semi-Desert Genus Eremophila (Scrophulariaceae).

Australia’s endemic desert shrubs are commonly aromatic, with chemically diverse terpenes and phenylpropanoids in their headspace profiles. Species from the genus Eremophila (Scrophulariaceae ex. Myoporaceae) are the most common, with 215 recognised taxa and many more that have not yet been described, widely spread across the arid parts of the Australian continent. Over the years, our research team has collected multiple specimens as part of a survey to investigate the chemical diversity of the genus and create leads for further scientific enquiry. In the current study, the diversity of volatile compounds is studied using hydrodistilled essential oils and leaf solvent extracts from 30 taxa. Several rare terpenes and iridoids were detected in chemical profiles widely across the genus, and three previously undescribed sesquiterpenes were isolated and are assigned by 2D NMR—E-11(12)-dehydroisodendrolasin, Z-11-hydroxyisodendrolasin and 10-hydroxydihydro-α-humulene acetate. Multiple sampling from Eremophila longifolia, Eremophila arbuscular, Eremophila latrobei, Eremophila deserti, Eremophila sturtii, Eremophila oppositifolia and Eremophila alternifolia coneys that species in Eremophila are highly chemovariable. However, taxa are generally grouped according to the expression of (1) furanosesquiterpenes, (2) iridoids or oxides, (3) mixtures of 1 and 2, (4) phenylpropanoids, (5) non-furanoid terpenes, (6) mixtures of 4 and 5, and less commonly (7) mixtures of 1 and 5. Furthermore, GC–MS analysis of solvent-extracted leaves taken from cultivated specimens conveys that many heavier ‘volatiles’ with lower vapour pressure are not detected in hydrodistilled essential oils and have therefore been neglected in past chemical studies. Hence, our data reiterate that chemical studies of the genus Eremophila will continue to describe new metabolites and that taxon determination has limited predictive value for the chemical composition.

Metabolites of endophytic fungi from Australian native plants as potential anticancer agents.

Interest in endophytes as natural sources for new medicines was inspired by the discovery of paclitaxel-producing endophytic fungi. This study investigated the anti-cancer activity of extracts of endophytes isolated from two Australian plants, Eremophila longifolia (EL) and Eremophila maculata (EM). Endophytes were isolated from surface-sterilised leaf tissue, grown as pure cultures and identified by sequencing of Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. To determine cytotoxicity, two leukaemic (MOLT-4, T-cell leukaemia; PreB-697, Pre-B leukaemia), a lung cancer cell line (A549) and a normal human fibroblast cell line were treated with endophyte extracts to assess cytotoxicity in relation to alternariol monomethyl ether (AME) and alternariol (AOH). Endophyte extracts that showed cell cytotoxicity were analysed by UV-HPLC to determine the metabolites. Pure AME and AOH, three extracts form Alternaria sp. (EM-6, EM-7 and EM-9) and one from Preussia minima (EL-14) were cytotoxic to the cancer cell lines. All cytotoxic endophytes contained AME and AOH, the most cytotoxic endophyte EM-6 also contained two unique peaks. These data indicate that these four endophyte extracts may have anti-cancer properties due to the presence of AME and AOH; however, the unique compounds found in the EM-6 extract may be exclusively cytotoxic and warrant further investigation.

Antibacterial Nerol Cinnamates from the Australian Plant Eremophila longifolia.

Two new antimicrobial agents, neryl ferulate (1) and neryl p-coumarate (2), were identified using bioassay-guided isolation from the leaves of Eremophila longifolia, which is a medicinal plant used by some Australian Aboriginal communities. Although gradual autoxidation of the nerol subunit hindered the initial attempts to purify and characterize 1 and 2, it was found that the autoxidation could be stopped through storage under argon at -20 °C. Biological evaluation showed that neryl ferulate (1) had moderate activity against various Gram-positive bacteria, while neryl p-coumarate (2) was active only against Enterococcus faecium.

Cytogeography of essential oil chemotypes of Eremophila longifolia F. Muell (Scrophulariaceae)

Previous studies have demonstrated that the widely distributed desert plant Eremophila longifolia has at least six geographically defined essential oil chemotypes. The focus of the present study is to extend and enhance information concerning known chemotypes and to investigate the involvement of cell nuclei ploidy in this variation. Forty field collected specimens of E. longifolia were taken from most of the mainland states of Australia then subjected to hydrodistillation to produce essential oils, which were then chemically characterised. Ploidy was determined using relative fluorescence of cell nuclei stained with propidium iodide, measured in a flow cytometer. Using principal component analysis (PCA), at least three essential oil chemotypes, in addition to the six already described, were identified in the present study. Previously described high yielding essential oil chemotypes were also characterised in terms of diploidy. For the first time diploid populations were identified in New South Wales, correlating with high yielding isomenthone/menthone and karahanaenone chemotypes. Furthermore, the separate diploid population previously described from Western Australia was demonstrated to be the safrole/methyl eugenol type, which is restricted to a small geographic range in far north-west Western Australia (Murchison District). All other chemotypes were shown to be tetraploid, including apparently randomly emerging individuals, representative of chemotypes producing low yields of isomenthone/menthone and karahanaenone similar in composition to the high yielding diploid types.

Isolation and characterisation of (−)-genifuranal: The principal antimicrobial component in traditional smoking applications of Eremophila longifolia (Scrophulariaceae) by Australian aboriginal peoples

Ethnopharmacological significanceEremophila longifolia is considered by some Australian Aboriginal tribal groups to be among the most significant of the medicinal plants in contemporary and traditional use. Usage modalities traditionally involved lipophilic extraction into animal fats and most importantly, ceremonial or medicinal smoking applications, involving the fumigation of mothers and infants following childbirth or boys following circumcision. An attempt was made to replicate the smoking modalities used by Australian Aboriginal people in the laboratory to identify bioactive compounds. Materials and methodsTwo methods were used to produce smoke extracts; smoke was channelled through a condenser then bubbled into solvent, or bubbled directly into H2O then partitioned into chloroform followed by butanol. Extracts were used, firstly for antimicrobial screening using micro-titre plate broth dilution to produce minimum inhibitory concentrations (MIC), and secondly for chemical analysis. Structure elucidation of an abundant compound isolated from the smoke extract was performed using 2D-NMR and derivatisation. ResultsSignificant antimicrobial activity (<1.0mg/ml) was produced using the smoke extracts against the Gram-positive species Staphylococcus aureus, Bacillus subtilis and the yeast Candida albicans. A major component of the smoke with strong antimicrobial activity (0.13–0.5mg/ml) was isolated which we have named (−)-genifuranal. Structure elucidation using 2D-NMR and derivatisation demonstrated genifuranal to be 5,6-dihydro-4H-cyclopenta[c]furan-4-ylacetaldehyde. Genifuranal is not observed in the leaves before heating, but is produced in the smoking or heating process and is thought to derive from hydrolysis and rearrangement of geniposidic acid or a related glycoside. Only geographically specific specimens of Eremophila longifolia produced (−)-genifuranal, which strongly supports previous hypothesised geographical variation in traditional usage, reflective of phytochemical variation. ConclusionIt would appear that genifuranal is the medicinal principal involved in traditional use of Eremophila longifolia when smoking modalities are used. Topical treatments traditionally produced by lipophilic extraction into animal fats are not likely to have had genifuranal present, as the mechanism for its formation requires heat.

Dietary characteristics of Emus (Dromaius novaehollandiae) in semi-arid New South Wales, Australia, and dispersal and germination of ingested seeds

The Emu (Dromaius novaehollandiae) eats a wide range of seeds and other plant material but little is known of the seeds eaten and dispersed in arid Australia. This study aimed to determine the species of seed and fruits eaten by Emus through the year and investigate the effect of the passage of seeds through the Emu gut on seed germination. Emu scats were opportunistically collected in all seasons of 2005 at Nanya Research Station, western New South Wales. Seeds of 29 plant species were retrieved, identified and counted from 221 scats. Seeds of perennial species, such as Belah (Casuarina pauper), Ruby Saltbush (Enchylaena tomentosa), Common Emu-bush (Eremophila glabra) and Nitre-bush (Nitraria billardierei), predominated. Seed composition of scats was more diverse in summer than in winter, autumn and spring. Seeds of Common Emu-bush, Berrigan (Eremophila longifolia) and Sweet Quandong (Santalum acuminatum) showed no germination after passage through the gut. Ruby Saltbush and Nitre-bush showed very low germination rates. The Emu is a generalist feeder, capable of ingesting and dispersing large numbers of plant species. Through high seed consumption and long gut-retention time, Emus could potentially play a significant role in long-distance dispersal of seeds and, through such dispersal, in the vegetation dynamics of arid Australia.

A possible role of partially pyrolysed essential oils in Australian Aboriginal traditional ceremonial and medicinal smoking applications of Eremophila longifolia (R. Br.) F. Muell (Scrophulariaceae)

Ethnopharmacological significanceEremophila longifolia is one of the most respected of the traditional medicines used by Australian Aboriginal people. Customary use involves smoldering the leaves over hot embers of a fire to produce an acrid smoke, believed to have therapeutic effects broadly consistent with antimicrobial, antifungal and anti-inflammatory capacity. Aim of the studyThe current study aims to examine the contribution of partially pyrolysed and non-pyrolysed essential oils in traditional usage of Eremophila longifolia. Materials and methodsNon-pyrolysed and partially pyrolysed essential oils were produced by hydrodistillation and part-wet/part-dry distillation, respectively. All samples were tested for antimicrobial activity by broth dilution. Some of these samples were further treated to an incrementally stepped temperature profile in a novel procedure employing a commercial thermocycler in an attempt to mimic the effect of temperature gradients produced during smoking ceremonies. Components from the pyrodistilled oils were compared with the non-pyrodistilled oils, using GC–MS, GC-FID and HPLC-PAD. The 2,2-diphenyl-1-picrylhydrazyl method, was used to compare free radical scavenging ability. ResultsPartially pyrolysed oils had approximately three or more times greater antimicrobial activity, enhanced in cultures warmed incrementally to 60°C and held for 30s and further enhanced if held for 2min. Partially pyrolysed oils showed a radical scavenging capacity 30–700 times greater than the corresponding non-pyrolysed oils. HPLC-PAD revealed the presence of additional constituents not present in the fresh essential oil. ConclusionThese results, by showing enhanced antimicrobial and antioxidant activities, provide the first known Western scientific justification for the smoking ceremonies involving leaves of Eremophila longifolia. During customary use, both partially pyrolysed as well as non-pyrolysed essential oils may contribute significantly to the overall intended medicinal effect.

Extracts of Eremophila longifolia inhibit the cariogenic activities of streptococcus mutans and streptococcus sobrinus

Plants belonging to the Eremophila genus are an integral part of the traditional medicine of indigenous Australian populations. Extracts of a number of Eremophilia species have demonstrated the presence of bioactivity, including inhibitory effects against Gram positive bacteria. The aim of the present study was to determine the activity of solvent and aqueous extracts of Eremophilia longifoliastem and leaves against Streptococcus mutans and Streptococcus sobrinus. A stem ethanol extract (SEE) demonstrated growth inhibition of the two cariogenic bacteria with a minimum inhibitory concentration (MIC) of 0.5% (w/v). Time-kill experiments indicated bactericidal activity by SEE within a 2 h period. Furthermore, this study assessed the anticariogenic activity of SEE in terms of its effect on glycolytic pH drop, viability of cells within an artificial biofilm and cell attachment to a membrane. In all experiments, SEE demonstrated significant anticariogenic activity compared with controls (p<0.05, Students t-test). SEE also compared favourably with commercial oral healthcare products. Preliminary phytochemical investigations suggested that the active components within SEE were phenolic compounds but unlikely to be flavonoids. Our study advocates SEE as a worthy candidate for further research into alternative chemotherapeutic approaches to dental caries. Key words: Eremophila longifolia, dental caries, biofilm, antibacterial.

Characterization and bioactivity of essential oils from novel chemotypes of Eremophila longifolia (F. Muell) (Myoporaceae): a highly valued traditional Australian medicine

Leaf specimens of Eremophila longifolia were collected from western New South Wales, Australia. Essential oils were extracted using hydro-distillation. Characterizations using GC-MS and GC-FID were consistent with those reported in previous work, but demonstrated greater chemo-variability. Antimicrobial activity from disc diffusions and broth dilutions indicated that oils dominated by hydrocarbon monoterpenes and monoterpenols had moderate antimicrobial activity for both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and S. epidermidis. Oils dominated by ketones demonstrated less activity but showed substantially greater yields in wet-weight leaves. Bioautography revealed the most active constituents to be related to the most abundant – karahanaenone and menthone – but active constituents also included d-limonene, borneol, sabinene, terpinolene and α-terpineol. Antifungal capacity for Trichophyton interdigitalis, rubrum and mentagrophytes was determined using an agar transplant method. Results indicated very high activity from oils rich in borneol and also for oils that were distilled from leaves accidentally burnt or partially pyrolysed during hydro-distillation. This serendipitous observation may have relevance given the traditional manner of use in smoking ceremonies. Free radical scavenging was determined using the DPPH method in methanol and antioxidant capacity with the FRAP assay. Results indicated that oils dominated by monoterpenols had moderate ability, but became very high when the oils were partially pyrolysed. Copyright © 2011 John Wiley & Sons, Ltd.

Intraspecific variation in essential oil composition of Eremophila longifolia F. Muell. (Myoporaceae): Evidence for three chemotypes

Volatile oils of Eremophila longifolia F. Muell. (Myoporaceae) leaves were obtained by hydrodistillation and analysed using GC–MS. A total of 33 compounds were identified in the oils examined and a high degree of intraspecific variability in chemical composition between specimens occurring in separate geographic localities was found. Multivariate statistical analysis of chemical composition of volatile oils enabled classification of three chemotypes in this species.

The Australian desert shrub Eremophila longifolia (R. Br.) F. Muell. (Schrophulariaceae), as a potential source of karahanaenone and other terpenoids

Eremophila longifolia (R. Br.) F. Muell. (Schrophulariaceae) is a shrub endemic to the arid and semi-arid regions of Australia. The plant is used in contemporaneous indigenous traditional medicine and has been cited extensively in medical ethnobotanical literature [1]. This work forms part of an ongoing project investigating variation in essential oil yield and composition from this species. Here we report on the discovery of two novel essential oil chemovarieties, one rich in isomenthone (5-methyl-2-propan-2-ylcyclohexan-1-one), and the other yielding large quantities of the monocyclic heptene ketone karahanaenone (2,2,5,-trimethyl-4-cyclohepten-1-one), Yield and composition was investigated for hydrodistilled leaf essential oils of specimens occurring in semi-arid areas in the state of NSW. Essential oil composition was investigated using a combination of GC/GC-MS. The identity of karahanaenone was established by NMR spectroscopy. Isomenthone type specimens produced essential oils (3.12% –5.65% w/w yield) containing 61.1% –86.7% isomenthone. Karahanaenone type specimens produced essential oils (0.46% –1.90% w/w yield) containing 81.0% –82.2% karahanaenone. This relatively rare natural product and its derivatives are of considerable value to the flavour and fragrance industry and several methods of synthesis from a variety of precursors have been developed [2–5]. Given the potential commercial value of these essential oils as a source of naturally occurring karahanaenone and other compounds, we believe further investigation is warranted into the geographic distribution of these varieties, along with assessment of commercial viability as an agricultural crop for oil production in Australia.

Anti-Mycobacterial Activity of Extracts Derived from Australian Medicinal Plants

Extracts of seventeen traditional Australian medicinal plants used to treat infections and respiratory conditions were tested for anti-mycobacterial activity against the fast-growing strains, Mycobacteriumfortuitum andM. smegmatis. Four extracts, the aerial parts of Pterocaulon sphacelatum (Asteraceae), the bark and leaves of Acacia ligulata (Mimosaceae), the leaves and stems of Eremophila alternifolia (Myoporaceae) and the leaves of Eremophila longifolia, showed activity against M. smegmatis only, while the two Eremophila extracts were also active against M. fortuitum. The minimum inhibitory concentrations ranged from 20-66 mg mL -1. The identification of the anti-mycobacterial compounds from these extracts may yield new and effective agents to combat diseases caused by Mycobacterium species.

Long-term changes in vegetation, gradual and episodic, on the TGB Osborn Vegetation Reserve, Koonamore, South Australia (1926 - 2002)

The TGB Osborn Vegetation Reserve at Koonamore, South Australia, is a 390-ha exclosure in semi-arid chenopod shrubland. The area was heavily overgrazed in 1925 when it was fenced to exclude sheep. Permanent quadrats and photopoints have been maintained to the present. Feral rabbits were sometimes numerous until the mid-1970s but have since been controlled. The records represent 50 years without sheep grazing, followed by 26 years without either sheep or rabbits. Dramatic seedling establishment events have occurred since 1978 for the following species: Acacia aneura Benth., Myoporum platycarpum R.Br., Senna artemesioides subsp. coriacea Randell, S. artemesioides subsp. petiolaris Randell, Acacia burkittii Benth., Dodonaea attenuata A.Cunn., Eremophila longifolia (R.Br.) F.Muell., E. sturtii R.Br. and Maireana pyramidata (Benth.) Paul G.Wilson. However, the chenopod shrubs Atriplex vesicaria Benth. and A. stipitata Benth. increased earlier and did not respond in the same way to episodic rainfall events or rabbit control. Numbers of Alectryon oleifolius (Desf.) S.T.Reynolds and Casuarina pauper F.Muell. ex L.A.S.Johnson have remained almost unchanged, whereas Maireana sedifolia (F.Muell.) Paul G.Wilson and M. astrotricha (L.Johnson) Paul G.Wilson have shown a very gradual increase over time. The data show evidence for both episodic and gradual change among different species.

Inhibition of platelet aggregation and 5-HT release by extracts of Australian plants used traditionally as headache treatments

To identify potential migraine therapeutics, extracts of eighteen plants were screened to detect plant constituents affecting ADP induced platelet aggregation and [ 14C]5-hydroxytryptamine (5-HT) release. Extracts of the seven plants exhibiting significant inhibition of platelet function were reanalysed in the presence of polyvinyl pyrrolidone (PVP) to remove polyphenolic tannins that precipitate proteins. Two of these extracts no longer exhibited inhibition of platelet activity after removal of tannins. However, extracts of Crataegus monogyna, Ipomoea pes-caprae, Eremophila freelingii, Eremophila longifolia, and Asteromyrtus symphyocarpa still potently inhibited ADP induced human platelet [ 14C]5-HT release in vitro, with levels ranging from 62 to 95% inhibition. I. pes-caprae, and C. monogyna also caused significant inhibition of ADP induced platelet aggregation. All of these plants have been previously used as traditional headache treatments, except for C. monogyna which is used primarily for protective effects on the cardiovascular system. Further studies elucidating the compounds that are responsible for these anti-platelet effects are needed to determine their exact mechanism of action.

The effects of grazing on topfeed in central Australia

A system of recording grazing effects on edible trees and shrub is described. Results of observations using this method are recorded. Acacia aneura and Atalaya hemiglauca are the topfeed species most grazed in Central Australia, but several less common species, Ventilago viminalis, Eremophila longifolia and Santalum lanceolatum are more palatable. A pat soft leaf is preferred by graying cattle, but spines and viscid or oily leaves are deterrents.