Binding of the plant-derived toxin simplexin to bovine protein kinase C: insights from molecular dynamics.

Pimelea poisoning of cattle is toxicologically linked to the activation of bovine protein kinase C (PKC) by the plant-derived toxin simplexin. To understand the affinity of PKC for simplexin, we performed molecular dynamics (MD) studies of simplexin, simplexin analogues, and several other activators of PKC. Binding enthalpy calculations indicated that simplexin had the strongest affinity for PKCα-C1B among the activators studied. Key to simplexin's affinity is its ability to form more hydrogen bonds to PKC, compared to the other activators. The C-3 carbonyl group and C-20 hydroxyl group of simplexin were identified as especially important for stabilizing the PKC binding interaction. The hydrophobic alkyl chain of simplexin induces deep membrane embedding of the PKC-simplexin complex, enhancing the protein-ligand hydrogen bonding. Our findings align with previous experiments on structure-activity relationships (SAR) for simplexin analogues, and provide insights that may guide the development of interventions or treatments for Pimelea poisoning.

Analysis of Pyrrolizidine Alkaloids in Stingless Bee Honey and Identification of a Botanical Source as Ageratum conyzoides.

Stingless bee honeys (SBHs) from Australian and Malaysian species were analysed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the presence of pyrrolizidine alkaloids (PAs) and the corresponding N-oxides (PANOs) due to the potential for such hepatotoxic alkaloids to contaminate honey as a result of bees foraging on plants containing these alkaloids. Low levels of alkaloids were found in these SBHs when assessed against certified PA standards in targeted analysis. However, certain isomers were identified using untargeted analysis in a subset of honeys of Heterotrigona itama which resulted in the identification of a PA weed species (Ageratum conyzoides) near the hives. The evaluation of this weed provided a PA profile matching that of the SBH of H. itama produced nearby, and included supinine, supinine N-oxide (or isomers) and acetylated derivatives. These PAs lacking a hydroxyl group at C7 are thought to be less hepatoxic. However, high levels were also observed in SBH (and in A. conyzoides) of a potentially more toxic diester PA corresponding to an echimidine isomer. Intermedine, the C7 hydroxy equivalent of supinine, was also observed. Species differences in nectar collection were evident as the same alkaloids were not identified in SBH of G. thoracica from the same location. This study highlights that not all PAs and PANOs are identified using available standards in targeted analyses and confirms the need for producers of all types of honey to be aware of nearby potential PA sources, particularly weeds.

Identification of Acid Hydrolysis Metabolites of the Pimelea Toxin Simplexin for Targeted UPLC-MS/MS Analysis.

Pimelea poisoning of cattle is a unique Australian toxic condition caused by the daphnane orthoester simplexin present in native Pimelea pasture plants. Rumen microorganisms have been proposed to metabolise simplexin by enzymatic reactions, likely at the orthoester and epoxide moieties of simplexin, but a metabolic pathway has not been confirmed. This study aimed to investigate this metabolic pathway through the analysis of putative simplexin metabolites. Purified simplexin was hydrolysed with aqueous hydrochloric acid and sulfuric acid to produce target metabolites for UPLC-MS/MS analysis of fermentation fluid samples, bacterial isolate samples, and other biological samples. UPLC-MS/MS analysis identified predicted hydrolysed products from both acid hydrolysis procedures with MS breakdown of these putative products sharing high-resolution accurate mass (HRAM) fragmentation ions with simplexin. However, targeted UPLC-MS/MS analysis of the biological samples failed to detect the H2SO4 degradation products, suggesting that the rumen microorganisms were unable to produce similar simplexin degradation products at detectable levels, or that metabolites, once formed, were further metabolised. Overall, in vitro acid hydrolysis was able to hydrolyse simplexin at the orthoester and epoxide functionalities, but targeted UPLC-MS/MS analysis of biological samples did not detect any of the identified simplexin hydrolysis products.

How is Trehalulose Formed by Australian Stingless Bees? - An Intermolecular Displacement of Nectar Sucrose.

Trehalulose, a rare sucrose isomer, is a dominant sugar in stingless bee honey, with traces of the trisaccharide erlose. Incubating sucrose solutions with macerated stingless bee parts (head, thorax, and abdomen) from Tetragonula carbonaria, we observed that sucrose isomerization occurs predominantly in the head incubations, with trehalulose constituting 76.2-80.0% of total detected sugar. By contrast, sucrose hydrolysis occurred in stingless bee abdomen incubations, with glucose and fructose observed as 48.6-51.7% and 48.3-49.7%, respectively, of total detected sugar. Incubating glucose/fructose (1:1) solutions with any bee part did not result in trehalulose formation. In addition, by tracing the 13C isotope-labeled monosaccharide moieties throughout the isomerization from sucrose to trehalulose and erlose, for the first time, the mechanism was established as an enzymatic double displacement reaction. Sucrose acts as a glucose donor giving a ß-d-glucosyl enzyme intermediate with fructose release as demonstrated by mixed isotope products. Glucosylation of fructose (inter- or intramolecularly) with isomerization forms trehalulose (favorable), while glucosylation of sucrose forms erlose (less favorable).

Unique physicochemical properties and rare reducing sugar trehalulose mandate new international regulation for stingless bee honey.

Stingless bee honey (SBH) of four stingless bee species (Heterotrigona itama, Geniotrigona thoracica, Tetragonula carbonaria, and Tetragonula hockingsi) from two geographic regions (Malaysia and Australia, n = 36) were studied for their physicochemical parameters, including total phenolic and multi-elemental contents. Sugar analysis confirmed the prominent presence of trehalulose in all samples. All SBH failed to meet the CODEX Standard for honey moisture, free acidity, and total fructose plus glucose levels. One-way ANOVA, principal component analysis (PCA) and hierarchical component analysis (HCA) confirm distinctive differences between Australian and Malaysian SBH with Australian SBH having significantly (P < 0.05) higher pH, total phenolic, total mineral, and electrical conductivity attributes. Toxic elements in all SBH were much lower than standard CODEX limit. The species G. thoracica produced honey with significantly (P < 0.05) higher trehalulose than both Australian bee species. This research will add value to SBH and hasten new international standards ensuring commercial viability, safety, quality, and marketability of SBH.

Feeding Sugars to Stingless Bees: Identifying the Origin of Trehalulose-Rich Honey Composition.

The beneficial disaccharide, trehalulose, is a feature of stingless bee honey, while not dominant in any other foods. By experimentally feeding sugar solutions to confined colonies of the Australian stingless bee Tetragonula carbonaria, the origin of trehalulose has now been established. Complete conversion of fed sucrose was observed, by analysis of the honey, forming trehalulose (64-72%) with lesser erlose (18-23%), fructose (9-12%), and minor glucose detected. Remarkably, feeding solutions of glucose/fructose (1:1) mixtures did not result in trehalulose/erlose formation. Hence, stingless bees with natural access to floral nectar high in sucrose will produce honey high in trehalulose, with its associated beneficial properties. Any temptation to artificially increase trehalulose content by feeding sucrose to stingless bees would produce "fake" honey lacking key natural phytochemicals available to the foraging bee. The sucrose-fed fake and natural honey were however readily distinguished via isotope ratio mass spectrometry δ13C values, to combat such potential indirect adulteration.

Extraction and determination of the Pimelea toxin simplexin in complex plant-polymer biocomposites using ultrahigh-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry.

In the present paper, we describe how a robust and fundamental methodology was developed for extraction and determination of a principal natural toxin compound, simplexin, from a series of bulk biocomposites. These complex matrices were fabricated by direct encapsulating either ground plant particles or an ethanolic crude extract of the Australian toxic pasture plant Pimelea trichostachya in the biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Proton nuclear magnetic resonance spectroscopy was initially employed to examine the chemical compositions of these complicated systems. Then, a more sensitive strategy was developed and validated by combining solid-phase extraction and ultrahigh-performance liquid chromatography hyphenated with a quadrupole Orbitrap mass spectrometer for the quantification of simplexin embedded in different biocomposites. Satisfactory linearity (R2 > 0.99) and recovery ranges (86.8-116%) with precision (relative standard deviations) of between 0.2 and 13% (n = 3) were achieved from seven biocomposites. The established protocol was further shown to be accurate and reliable in confirming the homogeneous distribution of the simplexin in different biocomposite formulations. A limited mass transfer of simplexin (< 3.5%) from one of the biocomposites into a simulated but sterilized in vitro rumen environment after a 10-day incubation was also revealed by utilizing the method. This quantitative analysis of targeted natural product within plant material-integrated polymeric platforms has potential application when controlled release is required in the bovine rumen and other biological systems. Graphical abstract.

Occurrence of environmental contaminants (pesticides, herbicides, PAHs) in Australian/Queensland Apis mellifera honey.

Honey is a popular agricultural product containing mostly sugars and water, but due to its nutritious components and natural production by honeybees (Apis mellifera) from floral nectar, it is marketed as a premium health food item. As environmental monitors, honeybees can potentially transfer environmental contaminants to honey. Whilst pesticides can have ubiquitous presence in agricultural and urban areas, polycyclic aromatic hydrocarbons (PAHs) can be more prevalent in higher density urban/industrial environments. Australian beehives are customarily located in rural areas/forests, but it is increasingly popular to keep hives in urban areas. This study assessed the levels of environmental contaminants in honeys (n = 212) from Queensland/Australian sources including rural, peri-urban and urban areas. Honey samples were analysed by LC-MS/MS and GC-MS/MS for 53 herbicides, 83 pesticides, 18 breakdown products (for certain pesticides/herbicides) and 33 PAHs and showed low/negligible pesticide, herbicide and PAHs contamination, consistent regardless of honey origins.

Toxin Degradation by Rumen Microorganisms: A Review.

Animal feeds may contain exogenous compounds that can induce toxicity when ruminants ingest them. These toxins are secondary metabolites originating from various sources including plants, bacteria, algae and fungi. Animal feed toxins are responsible for various animal poisonings which negatively impact the livestock industry. Poisoning is more frequently reported in newly exposed, naïve ruminants while 'experienced' ruminants are observed to better tolerate toxin-contaminated feed. Ruminants can possess detoxification ability through rumen microorganisms with the rumen microbiome able to adapt to utilise toxic secondary metabolites. The ability of rumen microorganisms to metabolise these toxins has been used as a basis for the development of preventative probiotics to confer resistance against the poisoning to naïve ruminants. In this review, detoxification of various toxins, which include plant toxins, cyanobacteria toxins and plant-associated fungal mycotoxins, by rumen microorganisms is discussed. The review will include clinical studies of the animal poisoning caused by these toxins, the toxin mechanism of action, toxin degradation by rumen microorganisms, reported and hypothesised detoxification mechanisms and identified toxin metabolites with their toxicity compared to their parent toxin. This review highlights the commercial potential of rumen inoculum derived probiotics as viable means of improving ruminant health and production.

Mineral and Trace Element Analysis of Australian/Queensland Apis mellifera Honey.

Honey is an extensively utilized sweetener containing sugars and water, together with small quantities of vitamins, minerals, fatty acids, amino acids and proteins. Naturally produced by honeybees (Apis mellifera) from floral nectar, honey is increasingly sold as a health food product due to its nutritious features. Certain honeys are retailed as premium, trendy products. Honeybees are regarded as environmental monitors, but few reports examine the impact of environment on Australian honey trace elements and minerals. In higher density urban and industrial environments, heavy metals can be common, while minerals and trace elements can have ubiquitous presence in both agricultural and urban areas. Honey hives are traditionally placed in rural and forested areas, but increasingly the trend is to keep hives in more urban areas. This study aimed to determine the levels of 26 minerals and trace elements and assess elemental differences between honeys from various regional Queensland and Australian sources. Honey samples (n = 212) were acquired from markets, shops and supermarkets in Queensland while urban honeys were purchased online. The honey samples were classified into four groups according to their regional sources: urban, rural, peri-urban and blend honey. Elemental analyses of honey were performed using ICP-MS and ICP-OES after microwave and hot block digestion. Considerable variations of essential trace elements (Co, Cu, Cr, Fe, Mn, Mo and Zn) and mineral levels (Ca, K, Mg, Na and P) were found in honeys surveyed. There were significant differences (p < 0.05) between urban and rural honey samples for B, Na, P, Mn, K, Ca and Cu. Significant differences (p < 0.05) were also found between blend and urban honey samples for K, Cu, P, Mn, Sr, Ni, B and Na. Peri-urban versus urban honeys showed significant differences in P, K and Mn. For rural and peri-urban honeys, the only significant difference (p < 0.05) was for Na. Toxic heavy metals were detected at relatively low levels in honey products. The study revealed that the Queensland/Australian honey studied is a good source of K and Zn and would constitute a good nutritional source of these elements.

A review on Pimelea poisoning of livestock.

Pimelea poisoning of cattle, historically known as St. George Disease or Marree Disease, is a prevailing issue in arid grazing regions of inland Australia. Ingestion of the toxic native Pimelea species that contain the secondary metabolite simplexin, a diterpene orthoester with potent protein kinase C activity, induces diarrhoea, characteristic oedema and potentially fatal right-sided heart failure in cattle. Outbreaks of toxic Pimelea in the grazing field depend on seasonal prevalence. However, all stages of the plant carry the toxin, from seeds, juvenile plants to dead plant material. Livestock generally avoid consuming green Pimelea plants and only consume toxic Pimelea when pasture is minimal or where Pimelea growing through grass tussocks results in inadvertent ingestion. Our knowledge base of Pimelea poisoning has greatly improved with past research, yet the health hazards for livestock grazing in Pimelea affected pastures remains a significant issue whilst the ongoing search to develop effective strategies to mitigate poisoning continues. The goal of this review is to collate historical and recent research giving an overview of the current understandings of Pimelea poisoning, the toxin, its toxic effects and progress made towards remedies to alleviate the effects of Pimelea intoxication.

Stingless bee honey, a novel source of trehalulose: a biologically active disaccharide with health benefits.

Stingless bee (Meliponini) honey has long been considered a high-value functional food, but the perceived therapeutic value has lacked attribution to specific bioactive components. Examination of honey from five different stingless bee species across Neotropical and Indo-Australian regions has enabled for the first time the identification of the unusual disaccharide trehalulose as a major component representing between 13 and 44 g per 100 g of each of these honeys. Trehalulose is an isomer of sucrose with an unusual α-(1 → 1) glucose-fructose glycosidic linkage and known acariogenic and low glycemic index properties. NMR and UPLC-MS/MS analysis unambiguously confirmed the identity of trehalulose isolated from stingless bee honeys sourced across three continents, from Tetragonula carbonaria and Tetragonula hockingsi species in Australia, from Geniotrigona thoracica and Heterotrigona itama in Malaysia and from Tetragonisca angustula in Brazil. The previously unrecognised abundance of trehalulose in stingless bee honeys is concrete evidence that supports some of the reported health attributes of this product. This is the first identification of trehalulose as a major component within a food commodity. This study allows the exploration of the expanded use of stingless bee honey in foods and identifies a bioactive marker for authentication of this honey in associated food standards.

Analysis of Pyrrolizidine Alkaloids in Queensland Honey: Using Low Temperature Chromatography to Resolve Stereoisomers and Identify Botanical Sources by UHPLC-MS/MS.

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples.

Pyrrolizidine Alkaloids of Blue Heliotrope (Heliotropium amplexicaule) and Their Presence in Australian Honey.

Blue heliotrope (Heliotropium amplexicaule) is an invasive environmental weed that is widely naturalized in eastern Australia and has been implicated as a source of pyrrolizidine alkaloid (PA) poisoning in livestock. Less well-documented is the potential of such carcinogenic alkaloids to contaminate honey from bees foraging on this plant species. In this study, the PA profile of H. amplexicaule plant material, determined by HRAM LC-MS/MS, revealed the presence of nine PAs and PA-N-oxides, including several PAs and PA-N-oxides of the indicine class, which have not previously been reported. The predominant alkaloid, indicine, represents 84% of the reduced PA content, with minor alkaloids identified as intermedine and the newly reported helioamplexine, constituting 7 and 9%, respectively. NMR analysis confirmed the identity of helioamplexine as a previously unreported indicine homologue. This is the first report of the isolation of intermedine, helioamplexine, and 3'-O-angelylindicine from H. amplexicaule. Also described is the identification of N-chloromethyl analogues of the major alkaloids as isolation-derived artifacts from reactions with dichloromethane. Analysis of regional-market honey samples revealed a number of honey samples with PA profiles analogous to that seen in H. amplexicaule, with measured PA contents of up to 2.0 µg of PAs per gram of honey. These results confirm the need for honey producers to be aware of H. amplexicaule as a potential PA source, most particularly in products where honey is sourced from a single location.

(4+3) Cycloaddition Reactions of N-Alkyl Oxidopyridinium Ions.

N-Methylation of methyl 5-hydroxynicotinate followed by reaction with a diene in the presence of triethylamine afforded (4+3) cycloadducts in good to excellent yields. High regioselectivity was observed with 1-substituted and 1,2-disubstituted butadienes. Density functional theory calculations indicate that the cycloaddition involves concerted addition of the diene onto the oxidopyridinium ion. The process provides rapid access to bicyclic nitrogenous structures resembling natural alkaloids.

Synthesis and evaluation of anticancer natural product analogues based on angelmarin: targeting the tolerance towards nutrient deprivation.

Inspired by nature: Angelmarin is an anticancer natural product with potent antiausterity activity, that is, selective cytotoxicity towards nutrient-deprived, resistant cancer cells. Through structure-activity relationship studies, three analogues were identified as lead compounds for the develpoment of molecular probes for the investigation of the mode of action and biological targets of the antiausterity compounds.

3,4'-Linked bis(piperidines) related to the haliclonacyclamine class of marine alkaloids: synthesis using crossed-aldol chemistry and preliminary biological evaluations.

Compounds 2-5, incorporating various elements of the 3,4'-bis(piperidine) core associated with the sponge-derived alkaloid haliclonacyclamine A (HA, 1), have been prepared through, inter alia, aldol-type reactions of N-substituted piperidin-4-ones and certain derivatives. Screening of these compounds in various assays, including an ecological one, reveals that compound 5 exhibits allelochemical properties similar to those associated with HA itself.

Selective manipulation of steroid hydroxyl groups with boronate esters: Efficient access to antigenic C-3 linked steroid-protein conjugates and steroid sulfate standards for drug detection.

The temporary protection of 17alpha-alkyl-5alpha-androstane-3beta,16beta,17beta triols as boronate esters is an efficient method for their regioselective functionalisation. This has been applied to the synthesis of protein-steroid conjugates 7-10 suitable for the development of immunoassays targeting classes of steroids banned from competition in Australian horse racing and other sports. The synthesis of steroids sulfate conjugates 42 and 44 for use as reference standards is also reported.

Enantioselective total synthesis of (-)-dactylolide.

[reaction: see text] The enantioselective total synthesis of (-)-dactylolide is reported. The absolute stereochemistry of the tetrahydropyran was established by catalytic asymmetric Jacobsen hetero-Diels-Alder reaction. The remote C19 stereocenter was introduced by a sequence of chelation-controlled Grignard addition and Ireland-Claisen rearrangement.

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.