Pharmaceutical and preclinical evaluation of Advax adjuvant as a dose-sparing strategy for ant venom immunotherapy.

A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.

Receiver Operating Characteristic curve analysis determines association of individual potato foliage volatiles with onion thrips preference, cultivar and plant age.

Tomato spotted wilt virus (TSWV) causes sporadic but serious disease in Australian potato crops. TSWV is naturally spread to potato by thrips of which Thrips tabaci is the most important. Prior studies indicated possible non-preference of potato cultivars to T. tabaci. Select potato cultivars were assessed for non-preference to T. tabaci in paired and group choice trials. Cultivars 'Bismark', 'Tasman' and 'King Edward' were less preferred than 'Atlantic', 'Russet Burbank' and 'Shepody'. Green leaf volatiles were sampled using solid-phase microextraction from the headspace of potato cultivars of two ages that differed in T. tabaci preference. Analysis of headspace volatile data using Receiver Operating Characteristic curves identified individual volatiles associated with T. tabaci preference and non-preference, young and old plants and individual cultivars. These data could be used to inform breeding programs for selection of T. tabaci resistance to assist with TSWV management, and biological testing of novel thrips management compounds.

GC-MS method validation and levels of methyl eugenol in a diverse range of tea tree (Melaleuca alternifolia) oils.

Tea tree oil distilled from Melaleuca alternifolia has widespread use in the cosmetic industry as an antimicrobial as well as for other functions in topical products. Concerns were first raised by the European Commission's Scientific Committee on Consumer Products in 2004 about the level of the potentially carcinogenic phenylpropanoid compound methyl eugenol in tea tree oil. Limits on oil content in different types of cosmetic products were set based on a reported upper level of 0.9% methyl eugenol in the oil. A previous publication indicated that these levels were based on oil from a Melaleuca species not used in the commercial production of oil. Even the highest recorded levels in Melaleuca alternifolia, the overwhelmingly most common species used, were ∼15 times less than this, meaning that more oil could be safely used in the products. The current study, including details on methodology and reproducibility, extends that work across a suite of 57 plantation-sourced oils from a range of geographical locations and production years, as well as many Australian and international commercial oils. Lower levels of methyl eugenol in oils of known provenance were confirmed, with a recorded range of 160-552 ppm and a mean of 337 ppm. Analysis of variance showed methyl eugenol levels in Australian plantation oils to be correlated to the geographical region but not to the year of production. Average methyl eugenol levels in commercial oils were significantly lower, and these samples were divided into an authentic group and a group that were suspected of being adulterated based on an independent test. Authentic commercial oils had similar levels of methyl eugenol to Australian provenance material, whilst the oils classed as suspect had significantly lower levels.

Determination of Enantiomeric Distribution of Terpenes for Quality Assessment of Australian Tea Tree Oil.

A number of papers have appeared in recent years proposing the use of enantiomeric ratios of key monoterpenes in Australian tea tree oil (TTO) for detection of adulterated oils. There are however a range of reported values, even from exactly the same suite of authentic oils, and we address here probable reasons for these differences and stress the importance of establishing reference ratios within each laboratory based on oils of known provenance. Any biological variation in the ratio for the key terpene terpinen-4-ol has been demonstrated to be effectively unmeasurable, because the standard deviation on multiple measurements of the same oil is of the same order as that of multiple authentic oils.

Hypolipidaemic effect of crude extract from Carpobrotus rossii (pigface) in healthy rats.

Carpobrotus rossii (CR) was used by the Aboriginal population and early European settlers both as a food and therapeutic agent. Based on the presence of flavonoids in CR and results from our previous in vitro investigations, this study aimed to determine whether consumption of CR crude leaf extract: (a) affected lipoprotein profile, resting glucose, systolic blood pressure and vascular function, and (b) produced toxic effects (haematological measures, organ weight) in healthy rats. Male Hooded-Wistar rats (~230 g) were supplemented for 4 weeks with CR extract in their drinking water (35 mg/kg body weight daily). CR extract produced a significant decrease (18%, p=0.033) in atherogenic lipoproteins (but not high density lipoprotein). CR supplemented animals showed no signs of haematological toxicity and body and organ weight, daily fluid and food consumption and in vitro vascular responsiveness were similar for both groups. CR also increased (40%, p=0.049) the renal concentration of 3-hydroxy-3-methylglutaric acid (HMG), consistent with HMG-containing CR flavonoids being bioavailable, and therefore possessing the potential to interfere with cholesterol synthesis pathways. CR extract appears to be safe to ingest and may reduce cardiovascular risk.

Metabolomics reveals increased isoleukotoxin diol (12,13-DHOME) in human plasma after acute Intralipid infusion.

Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipid and saline into 10 healthy men in a double-blind, placebo-controlled experiment and used GC/MS, LC/MS, and NMR to profile the small-molecule composition of their plasma before and after infusion. Multivariate statistical analysis of the 40 resulting plasma samples revealed that after Intralipid infusion, a less-well-characterized pathway of linoleic acid metabolism had resulted in the appearance of (9Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DHOME, P < 10(-3)), a leukotoxin that has powerful physiological effects and is known to inhibit the neutrophil respiratory burst. Intralipid infusion caused increased plasma 12,13-DHOME. Given that 12,13-DHOME is known to directly affect neutrophil function, we conclude that untargeted metabolomics may have revealed a hitherto-unknown mechanism of intralipid-induced immunosuppression.

Glycosidic conjugates of C13 norisoprenoids, monoterpenoids, and cucurbates in Boronia megastigma (Nees).

Analysis of a methanolic extract of marc from Boronia megastigma (Nees) using LC-MS (APCI, nominal mass) provided strong evidence for the presence of both glycosides and malonyl glycosides of methyl cucurbates, C13 norisoprenoids including megastigmanes, and monoterpene alcohols. Subsequent fractionation of an extract from the marc using XAD-2 and LH 20 chromatography followed by LC-UV/MS-SPE-NMR and accurate mass LC-MS resulted in the isolation and identification of (1R,4R,5R)-3,3,5-trimethyl-4-[(1E)-3-oxobut-1-en-1-yl]cyclohexyl ß-D-glucopyranoside (3-hydroxy-5,6-dihydro-ß-ionone-ß-D-glucopyranoside); 3,7-dimethylocta-1,5-diene-3,7-diol-3-O-ß-D-glucopyranoside; and a methyl {(1R)-3-(ß-D-glucopyranosyloxy)-2-[(2Z)-pent-2-en-1-yl]cyclopentyl}acetate stereoisomer (a methyl cucurbate-ß-D-glucopyranoside); and provided evidence for 3,7-dimethylocta-1,5-diene-3,7-diol-3-O-(6'-O-malonyl)-ß-D-glucopyranoside in boronia flowers.

Auxin biosynthesis in pea: characterization of the tryptamine pathway.

One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.

A novel compound from celery seed with a bactericidal effect against Helicobacter pylori.

OBJECTIVES: The aim was to purify and characterise an antimicrobial component from celery (Apium graveolens) seeds, which have been used for centuries as a herbal medicine with reported antibacterial effects. METHODS: A crude alcoholic extract of celery seeds was fractionated by organic solvent extractions, column chromatography and HPLC. Fractions were assayed for antimicrobial activity against the gastric pathogen Helicobacter pylori and other bacteria. The purified antibacterial component was characterised via MS and NMR. Preliminary investigation of its mechanism of action included morphological studies, incorporation of macromolecular precursors, membrane integrity and two-dimensional protein electrophoresis. KEY FINDINGS: The purified component, termed 'compound with anti-Helicobacter activity' (CAH), had potent bactericidal effects against H. pylori; the minimum inhibitory concentration and minimum bactericidal concentration were 3.15 microg/ml and 6.25-12.5 microg/ml, respectively. CAH (M(r) = 384.23; empirical formula C(24)H(32)O(4)) had specific inhibitory effects on H. pylori and was not active against Campylobacter jejuni or Escherichia coli. MS and NMR data were consistent with a dimeric phthalide structure. The results appeared to rule out mechanisms that operated solely by loss of membrane integrity or inhibition of protein or nucleic acid synthesis. CONCLUSIONS: CAH may be suitable for further investigation as a potent agent for treating H. pylori infections.

Changes in some carotenoids and apocarotenoids during flower development in Boronia megastigma (Nees).

The carotenoid profile of an acetone extract from the flowers and leaves of Boronia megastigma (Nees) was examined. A comparison was made of the major carotenoids found in boronia flowers and leaves. The C-40 carotenoids beta-carotene, zeaxanthin, lutein, and neoxanthin were positively identified in boronia flowers using known standards, UV-vis spectra, and mass spectrometry. Two other carotenoids were tentatively assigned as the palmitic acid ester of 3-hydroxy-10'-apocaroten-10'-oic acid and 9,15,9'-tri-cis-zeta-carotene. Additionally, changes in the levels of C-40 carotenoids, C-27 apocarotenoids, and beta-ionone during flower development were measured. Significant increases in beta-carotene and apocarotenoids that could be derived from cleavage in the 9,10-position, including beta-ionone and various C-27 apocarotenoids, were observed at the time of flower opening. An increase in lutein, which is derived through an alternative biosynthetic pathway, was not observed during flower opening, thus indicating the possibility that the beta-carotene pathway was activated during flower opening in boronia. The understanding of these processes may assist in optimizing harvest and postharvest processes useful to the boronia extraction industry.

How do soil nutrients affect within-plant patterns of herbivory in seedlings of Eucalyptus nitens?

This study assessed how the palatability of leaves of different age classes (young, intermediate and older) of Eucalyptus nitens seedlings varied with plant nutrient status, based on captive feeding trials with two mammalian herbivores, red-bellied pademelons (Thylogale billardierii), and common brushtail possums (Trichosurus vulpecula). Seedlings were grown under three nutrient treatments (low, medium and high), and we determined how palatability was related to chemical and physical characteristics of the leaves. Pademelons ate more older leaves than young and intermediate leaves for all treatments. This pattern was best explained by sideroxylonals (formylated phloroglucinol compounds known to deter herbivory by other marsupials), and/or essential oil compounds that were present in lower concentrations in older leaves. In the low-nutrient treatment, possums also ate more of the older leaves. However, in the medium- and high-nutrient treatments, possums ate more intermediate leaves than older leaves and showed a behavioural preference for young leaves (consuming younger leaves first) over intermediate and older leaves, in spite of high levels of sideroxylonals and essential oils. The young leaves did, however, have the highest nitrogen concentration of all the leaf age classes. Thus, either sideroxylonals and essential oils provided little or no deterrent to possums, or the deterrent was outweighed by other factors such as high nitrogen. This study indicates that mammalian herbivores show different levels of relative use and damage to leaf age classes at varying levels of plant nutrient status and, therefore, their impact on plant fitness may vary with environment.

Spatial scale of the patchiness of plant poisons: a critical influence on foraging efficiency.

Generalist mammalian browsers and folivores feed on a range of chemically different plant species, which may assist them in diluting toxins and diversifying nutrient consumption. The frequency and order in which their diets are mixed are important determinants of intake. As a result, the degree of plant heterogeneity in an environment, and the spatial scale at which this occurs, should directly influence herbivore foraging decisions. We tested whether altering the Spatial scale of plants, and thus plant secondary metabolites (PSMs), affected foraging efficiency of a generalist folivore, the common brushtail possum (Trichosurus vulpecula). First, we demonstrated that possums were able to consume more from a mixed diet of two chemically different species, Eucalyptus globulus and E. tenuiramis, than when either of these species was offered alone. We then tested whether altering the spatial scale between E. globulus and E. tenuiramis, as small- or large-scale plant heterogeneity "patches," affected possum foraging behavior and, ultimately, their foraging efficiency. Possums increased their foraging efficiency when the spatial scale of plant heterogeneity was small rather than large. We argue that the ability to regularly switch diets, when plant spatial distribution is at a small scale, reduces the negative effects of PSM ingestion. We predict that the heterogeneity of plant patches, in relation to PSM distribution, and the scale at which this occurs across a landscape, are critical factors that influence foraging efficiency and, ultimately, fitness of mammalian herbivores. This research provides a fundamental link between plant chemistry, foraging, and habitat heterogeneity.

Inheritance of resistance to mammalian herbivores and of plant defensive chemistry in an Eucalyptus species.

Hybridization in plants provides an opportunity to investigate the patterns of inheritance of hybrid resistance to herbivores, and of the plant mechanisms conferring this resistance such as plant secondary metabolites. We investigated how inter-race differences in resistance of Eucalyptus globulus to a generalist mammalian herbivore, Trichosurus vulpecula, are inherited in their Fl hybrids. We assessed browsing damage of 3-year-old trees in a common environment field trial on four hybrid types of known progeny. The progeny were artificial intra-race crosses and reciprocal inter-race F1 hybrids of two geographically distinct populations (races) of E. globulus north-eastern Tasmania and south-eastern Tasmania. Populations of trees from north-eastern Tasmania are relatively susceptible to browsing by T. vulpecula, while populations from south-eastern Tasmania are more resistant. We assessed the preferences of these trees in a series of paired feeding trials with captive animals to test the field trial results and also investigated the patterns of inheritance of plant secondary metabolites. Our results demonstrated that the phenotypic expression of resistance of the inter-race Fl hybrids supported the additive pattern of inheritance, as these hybrids were intermediate in resistance compared to the pure parental hybrids. The expression of plant secondary metabolites in the Fl hybrids varied among major groups of individual compounds. The most common pattern supported was dominance towards one of the parental types. Together, condensed tannins and essential oils appeared to explain the observed patterns of resistance among the four hybrid types. While both chemical groups were inherited in a dominant manner in the inter-race Fl hybrids, the direction of dominance was opposite. Their combined concentration, however, was inherited in an additive manner, consistent with the phenotypic differences in browsing.

Novel detection of formylated phloroglucinol compounds (FPCs) in the wound wood of Eucalyptus globulus and E. nitens.

This study characterized the chemical responses of Eucalyptus globulus and Eucalyptus nitens to artificial inoculation with a basidiomycete decay fungus. Nine-year-old trees responded to mechanical wounding or inoculation with the decay fungus by producing new wound wood characterized by the presence of dark extractives 17 months after wounding. Analysis of crude wound wood extracts by HPLC coupled to negative ion electrospray mass spectrometry revealed the presence of a complex mixture of many unidentified formylated phlorglucinol compounds (FPCs), in addition to a diverse range of other polyphenolic compounds (hydrolyzable tannins, proanthocyanidins, flavanone glycoside, stilbene glycosides). Prior to this study, FPCs have only been reported from leaves and buds of Eucalyptus spp. Unequivocal evidence for the presence of macrocarpal A and B, and sideroxylonal A and B in the crude extracts was obtained, as well as evidence for a wide range of as yet unreported FPCs. Subsequent preliminary in vitro fungal and bacterial bioassays did not support an antimicrobial role for FPCs in host-pathogen interactions in eucalypts.

C-27 apocarotenoids in the flowers of Boronia megastigma (Nees).

Five C-27 apocarotenoids were detected in acetone extracts of the flowers of Boronia megastigma (Nees) using HPLC with UV-vis photodiode array and MS detection. Comparison of methylated and unmethylated extracts aided identification when considered with the UV-vis and MS data. The five apocarotenoids identified here were hydroxy-apo-10'-carotenoic acid (1), methyl hydroxy-apo-10'-carotenoate (2), apo-10'-carotenoic acid (3), apo-10'-carotenal (4), and methyl apo-10'-carotenoate (5). The data obtained was not sufficient to allow the specific isomeric forms to be unequivocally identified. The results further support speculation that the C-13 norisoprenoids found in boronia are derived from C-40 carotenoids. Possible parent molecules of beta-ionone, an important component of boronia extract, were identified. An understanding of C-13 norisoprenoid biosynthesis may assist in the selection and postharvest processing of boronia flowers for flavor and fragrance applications.