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A detailed close phytochemical investigation of the fruits of Ternstroemia cherryi led to the isolation and identification of the minor metabolite, ternstroenol F, which possessed the usual barrigenol-like terpenoid backbone. The notable difference was that this minor metabolite had the 2(E)-4(Z)-6(E)-decatrienoic acid forming an ester bond at C-22 of the oleanane backbone. Ternstroenol F was evaluated for its inhibitory effects on NO inhibition, cell viability and TNF- α release in RAW 264.7 macrophages, displaying an IC50 values of 0.23, 0.81 and 1.84 µM respectively.

Flowering plants in tropical rainforests rely heavily on pollen vectors for successful reproduction. Research into pollination systems in tropical rainforests is dominated by canopy species, while subcanopy plant–pollinator interactions remain under‐represented. The microclimate beneath the rainforest canopy is characterized by low light levels and is markedly different from the canopy environment that receives more light energy. We studied the floral attractants and floral visitors of a dioecious, subcanopy tree, Fontainea picrosperma (Euphorbiaceae), in the Wet Tropics bioregion of northern Queensland, Australia. We found that wind pollination is rare and male and female flowers do not produce nectar. Female flowers are likely pollinated due to their perceptual similarity to pollen‐offering male flowers. Female flowers had the same scent profile as male flowers, and floral scent was an important floral attractant that acted to regulate pollinator behavior. The two most abundant scent compounds present in the floral bouquet were benzyl alcohol and 4‐oxoisophorone. These compounds are ubiquitous in nature and are known to attract a wide variety of insects. Both day‐time and night‐time pollinators contributed to successful pollen deposition on the stigma, and diurnal flower visitors were identified from several orders of insects including beetles, flies, predatory wasps, and thrips. Fontainea picrosperma is therefore likely to be pollinated by a diverse array of small insects. Synthesis. Our data indicate that F. picrosperma has a generalist, entomophilous pollination syndrome. The rainforest subcanopy is a distinctive environment characterized by low light levels, low or turbulent wind speeds, and relatively high humidity. Female flowers of F. picrosperma exhibit cost‐saving strategies by not producing nectar and mimicking the smell of reward‐offering male flowers. Insects opportunistically forage on or inhabit flowers, and pollination occurs from a pool of small insects with low energy requirements that are found beneath the rainforest canopy.

The kernels of the Australian blushwood tree (

The long-standing perception of Protein Kinase C (PKC) as a family of oncoproteins has increasingly been challenged by evidence that some PKC isoforms may act as tumor suppressors. To explore the hypothesis that activation, rather than inhibition, of these isoforms is critical for anticancer activity, we isolated and characterized a family of 16 novel phorboids closely-related to tigilanol tiglate (EBC-46), a PKC-activating epoxytigliane showing promising clinical safety and efficacy for intratumoral treatment of cancers. While alkyl branching features of the C12-ester influenced potency, the 6,7-epoxide structural motif and position was critical to PKC activation in vitro. A subset of the 6,7-epoxytiglianes were efficacious against established tumors in mice; which generally correlated with in vitro activation of PKC. Importantly, epoxytiglianes without evidence of PKC activation showed limited antitumor efficacy. Taken together, these findings provide a strong rationale to reassess the role of PKC isoforms in cancer, and suggest in some situations their activation can be a promising strategy for anticancer drug discovery.

Background Tigilanol tiglate (TT) is a novel small molecule for intratumoral treatment of nonmetastatic mast cell tumors (MCTs) in dogs. In a randomized controlled clinical study, 75% of dogs that received a single TT treatment achieved complete resolution of the MCT by 28 days, with no recurrence in 93% of dogs at 84 days. Critical to TT's efficacy was the area of the wound (tissue deficit) after slough of the necrotic tumor relative to pretreatment tumor volume. Objectives To analyze data collected during the previous study to (a) describe wounds after slough of treated MCTs and (b) identify determinants of wound area and speed of wound healing. Methods Wound presence, condition, and area were determined from clinical records of 117 dogs over 84 days after a single intratumoral TT treatment. Results Tumor slough occurred 3 to 14 days after treatment, exposing granulation tissue in the wound bed. Wound area after tumor slough in general was related to pretreatment tumor volume, with maximal recorded wound area fully evident in 89% of dogs by day 7. In dogs achieving complete tumor resolution, all wounds were left to heal by secondary intention. Bandaging and other wound management interventions only were required in 5 dogs. Time to healing (ie, full re-epithelialization of treatment site) depended on wound area and location on the body, with most wounds being fully healed between 28 and 42 days after treatment. Conclusions Wound area and healing after slough of TT-treated tumors follow a consistent clinical pattern for most dogs.

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree ( Fontainea picrosperma) , and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)–dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing–associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.

Background Tigilanol tiglate (TT) is a novel small molecule approved by the European Medicines Agency for intratumoral treatment of mast cell tumors (MCTs) in dogs. In a randomized controlled clinical efficacy and safety study in the United States, 85 of 116 dogs that received a single TT injection achieved complete response (CR) of the treated MCT by day 28. Objective To evaluate the durability of the TT treatment response achieved at day 28 in the U.S. study by assessing MCT recurrence at the treatment site 6 and 12 months after TT administration. Animals Eighty-five dogs previously treated with TT. Methods Dogs that achieved CR at day 28 were assessed retrospectively for the presence or absence of MCT at the treatment site using records from clinical visits and telephone interviews with owners. Dogs unavailable at an assessment time were considered lost-to-follow-up and data for their last assessment used in the final analysis. Results By 12 months after TT treatment, 64 dogs remained evaluable, with 21 unavailable. Of evaluable patients, 57 (89%) remained tumor free at the treatment site and 7 (11%) had developed recurrence. All recurrences occurred within the first 6 months, predominantly (5/7, 71%) within the first 12 weeks. Conclusions and clinical importance Tigilanol tiglate provided a durable long-term local response for the treatment of MCT in dogs.

The Australian rainforest plant, Croton insularis, has provided a further casbane diterpene family member in the form of EBC-342, which contains a novel tetrahydrofuran ring. A combination of DP4 and TD-DFT CD methods were required to confirm both structure and absolute stereochemistry.

The kernels of the Australian blushwood tree (Fontainea picrosperma) are the source of the veterinary anticancer drug tigilanol tiglate (2a, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by 1H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, 2a), EBC-47 (4a), EBC-59 (5a), EBC-83 (6a), and EBC-177 (7a) were identified. By chemoselective acylation of EBC-46 (2a) and EBC-177 (7a) the natural triesters 2b and 7b and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester 2b in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate (2a) under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in F. picrosperma, suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.

Canine high-grade mast cell tumours (HGMCT) are associated with a poor prognosis, are inherently more invasive, and have higher rates of local recurrence. The primary aim of this retrospective study was to assess the efficacy of intratumoural tigilanol tiglate (TT) as a local treatment option. Eighteen dogs with mast cell tumours (MCT) cytologically diagnosed by veterinary pathologists as either high-grade or suspected high-grade MCT were treated with TT. The TT dose was based on tumour volume (0.5 mg TT/cm3 tumour volume) and delivered intratumourally using a Luer lock syringe and a fanning technique to maximise distribution throughout the tumour mass. Efficacy was assessed on the presence/absence of a complete response (CR) to therapy at days 28 and 84 using response evaluation criteria in solid tumours (RECIST). For dogs not achieving a CR after 28 days, the protocol was repeated with a second intratumoural TT injection. Ten out of 18 dogs (56%) in this study achieved and maintained a CR to at least 84 days after their first or second treatment. Six patients were alive and available for evaluation at 2 years, three of those were recurrence free, and a further three patients were recurrence free following a second treatment cycle. Tigilanol tiglate shows efficacy for local treatment of HGMCT, with higher efficacy noted with a second injection if a CR was not achieved following the first treatment. In the event of treatment site recurrence (TSR), the tumour may be controlled with additional treatment cycles. Tigilanol tiglate provides an alternative local treatment approach to dogs with HGMCT that would either pose an unacceptable anaesthetic risk or the tumour location provides a challenge when attempting surgical excision.

Antibacterial-activity-guided fractionation of a dichloromethane extract from the fruit of Cordyline manners-suttoniae and subsequent structure-activity investigations resulted in the identification of 10 new (1-10) and one known (11) 5α-spirostane saponin. The structures of the new compounds were established by 1D and 2D NMR analyses. The absolute configurations of the isolated compounds were determined by X-ray diffraction analysis or chemical derivatizations. The most active compound, suttonigenin F (6), inhibited the Gram-positive bacteria Staphylococcus aureus with MIC75 values that were comparable to those of the antibiotic chloramphenicol. Structure-activity relationships were also obtained from the assessment of antibacterial and cytotoxic activities of the isolated saponins.

Three new metabolites, 1-(4′′-Methoxyphenyl)-7-(3′,4′-di-hydroxyphenyl)-(E)-hept-2-ene (1), 1-(4′′-Methoxyphenyl)-7-(3′,4′-di-methoxyphenyl)-(E)-hept-2-ene (2), 1-(4′′-Methoxyphenyl)-7-(4′-methoxyphenyl)-(E)-hept-2-ene (3), and the known diaryl heptanoid (4) were identified following anti-inflammatory activity guided fractionation from the rhizomes of Pleuranthodium racemigerum (Zingiberaceae). These diaryl heptanoids (1 – 4) were evaluated for their inhibitory effects on NO production and downregulation of TNF-α in RAW 264.7 macrophages and N-11 microglial cell lines).

Investigation of the Australian rainforest plant Croton insularis revealed seven new cis-, two new trans-cyclopropane casbanes, and the first trans-cyclopropane seco-casbane. The relative configuration of the cyclopropane moiety for all compounds (EBC-182, 217, 218, 220, 343, 357, 358, 361, 365, 373; EBC=EcoBiotics Compound) was assigned using 13 C NMR data. Comparison of the experimental electronic circular dichroism (ECD) spectra with the theoretical curves, calculated by TD-DFT at the B3LYP/6-31+G**//B3LYP/6-31+G** level, in conjunction with NOE data afforded the absolute configuration. EBC-180, 181 and 220 displayed potent activity against cervical carcinoma (HeLa cells).

From a literature search, information has been compiled on the mycorrhizal status under field conditions of 20 or more species in each of 25 families. The percentage of species which are mycorrhizal ranged from 100% in seven families to 8% in Cruciferae, many families having additional species that are sometimes mycorrhizal. No family in the list was consistently non-mycorrhizal. Apart from the Ericaceae, the families were either predominantly ectomycorrhizal or predominantly VA mycorrhizal. However, almost all families had at least one example of each of these mycorrhizal types.

Background Cytochrome P450s (P450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. Results A comparative species leaf metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea P450s were investigated from leaf and root tissue. A total of 103 and 123 full-length P450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of P450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Representatives within the CYP71D and CYP726A were identified in Fontainea that are excellent candidates for diterpenoid synthesis, of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species and were significantly more highly expressed in root tissue compared to leaf tissue. Conclusion This study presents a comprehensive overview of the P450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.

Background: Cytochrome P450s (CYP450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. Results: A metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea CYP450s were investigated from leaf and root tissue. A total of 103 and 123 full-length CYP450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of CYP450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Fontainea CYP450 associated with diterpenoid biosynthesis were classified into 2 subfamilies (CYP71D and CYP726A), of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species, and significantly more highly expressed in root tissue compared to leaf tissue. Conclusion: This study presents a comprehensive overview of the CYP450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.

Two new phloroglucinols, acronyols A (1) and B (2) along with the four known (3–6) pholoroglucinols were identified following anti-inflammatory activity guided fractionation from the fruits of Acronychia crassipetala (family Rutaceae). The pholoroglucinols (1–6) were evaluated for their inhibitory effects on NO production and downregulation of TNF-α in RAW 264.7 macrophage cell lines.

Tigilanol tiglate is a novel small molecule approved as a veterinary pharmaceutical in Europe for intratumoural treatment of non-metastatic, non-resectable canine mast cell tumors. The drug has a "tumor agnostic" mode of action associated with induction of an acute inflammatory response at the treatment site, immune cell recruitment, and disruption of tumor vasculature. Consequently, tigilanol tiglate has potential in treating a range of tumor types in humans and companion animals. However, it is likely that species-specific dosing and concomitant medication protocols will be required, especially to manage the drug-induced acute inflammatory response at the treatment site. As an initial step in evaluating tigilanol tiglate for treating cutaneous tumors in horses, we developed an equine-specific protocol involving (a) a 30% reduction in intratumoural tigilanol tiglate dose rate compared to that used in dogs, and (b) a regime of concomitant medications to manage the drug-induced acute inflammatory response at the treatment site. Here we report a preliminary study in two horses using the protocol to treat (i) an aggressive fibroblastic sarcoid that had recurred following surgical excision and (ii) a fast-growing peri-ocular squamous cell carcinoma. Clinical response to tigilanol tiglate treatment in these cases was similar to that observed in canine and human patients. Localized inflammation and bruising developed rapidly at the treatment site with haemorrhagic necrosis of the tumor evident within 24 h. Slough of necrotic tumor mass occurred within 6-16 days followed by infill of the tissue defect and full re-epithelialisation of the treatment site with good functional outcome. Drug-induced inflammation and oedema at the treatment site were well controlled by the concomitant medications and largely resolved within 3 days, while the wound that formed following tumor slough healed uneventfully. Both patients displayed minor lethargy during the first 36 h after treatment and localized treatment-site discomfort was apparent over the first 3-5 days. There was no evidence of recurrence of the sarcoid at 93 days, or the squamous cell carcinoma at 189 days. The results from this study support continued development and evaluation of tigilanol tiglate as a potential future treatment option for cutaneous equine tumors.

Euphorbiaceae is a large and diverse family of herbs, shrubs and trees that includes a number of species of considerable economic importance as sources of food, medicines and raw materials. One member of this family, Fontainea picrosperma, is the source plant for the diterpene ester tigilanol tiglate, a natural product recently approved as a treatment for canine mast cell tumours. Here we report the development of reference transcriptomes from root and leaf tissues of F. picrosperma, which include core diterpene biosynthesis genes. A total of ~12 Gb of combined clean reads were generated for assembly into 167,566 contigs with a GC (guanine-cytosine) content of ~41%. Gene ontology showed that 2286 and 2504 transcripts were enriched in the cellular process and 2369 and 2529 transcripts were enriched in the metabolic process categories in leaf and root tissue, respectively. The reference transcriptome contains genes coding for core enzymes involved in common secondary metabolite biosynthetic pathways, including the diterpene biosynthesis pathway within the mevalonate (MVA) and 2-C-methyl-D-erythritol 4- phosphate (MEP) pathways. A phylogenetic analysis using these genes found that F. picrosperma clustered most closely to Jatropha curcas. We found a significantly higher concentration of tigilanol tiglate in F. picrosperma root tissue, which correlated with higher levels of gene expression for enzymes associated with the MVA (6 genes) and MEP (7 genes) pathways, and we hypothesise that the initial stages of tigilanol tiglate biosynthesis occur primarily in the roots of F. picrosperma. This study provides a resource for future gene-related biodiscovery investigations in F. picrosperma and diterpene biosynthesis, in particular for tigilanol tiglate and related macrocyclic diterpenes.

Structurally unique halimanes EBC-232 and EBC-323, isolated from the Australian rainforest plant Croton insularis, proved considerably difficult to elucidate. The two diastereomers, which consist an unusual oxo-6,7-spiro ring system fused to a dihydrofuran, were solved by unification and consultation of five in silico NMR elucidation and prediction methods [i.e., ACDLabs, olefin strain energy (OSE), DP4, DU8+ and TD DFT CD]. Structure elucidation challenges of this nature are prime test case examples for empowering future AI learning in structure elucidation.

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound re-epithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM–151 µM concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM–15.1 µM) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51–151 nM) and EBC-211 (1.51 nM–15.1 µM) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51–151 nM) with proliferation inhibitor, mitomycin C (1 μM), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 μM). By identifying how epoxy-tiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds.

Phytochemical investigation of the Australian rainforest plant leaves Waterhousia mulgraveana, yielded two rare oxidized xanthenes, mulgravanols A (1) and B (2) along with a new phloroglucinol, mulgravanol C (3). Mulgravanol A (1) is the first reported example of a complex xanthene flanked by a methine bridged phloroglucinol unit. All the compounds displayed moderate inhibitory effects on nitric oxide production and TNF-α release in RAW 264.7 macrophages (IC50) 42–55 μM. The structures of the new compounds were assigned based on a detailed spectroscopic interpretation.

Identification of potent natural products is a challenging task in which sophisticated separation processes including HPLC are employed. The bioactivity of HPLC fractions is determined with a bioassay, and the most potent compounds are progressed to structural elucidation. In pharmacology, the potency of a compound is expressed as the half-maximal effective concentration (EC

Anti-inflammatory activity guided isolation from Citrus garrawayi (Rutaceae) led to the discovery of five new garracoumarins A – E (1 - 5), and five known (6 – 10) isoprenylated furanocoumarins and the known sterol bourjotinolone A (11). Discussed herein is the anti-inflammatory structure activity relationship of the ten furanocoumarins and a detailed spectroscopic analysis leading to the structure elucidation of the new and known isoprenylated furanocoumarins.

Two novel (EBC-204 and EBC-205), together with three known, halimanes were isolated from the Australian rainforest plant Croton insularis. EBC-203 strongly inhibited K562 leukemia cell growth.

A detailed phytochemical investigation of the leaves of the Australian rainforest tree Eupomatia laurina, led to the discovery of five new neolignans, eupomatenes A – E and eight known compounds, eupomatenoid-2, trans-(2′S)-2-[1′-(4-methoxyphenyl)prop-2′-yl]anethol, chlorogenic acid, chlorogenic acid-methyl ester, tyrosol-1-O-β-xylopyranosyl-1(1 → 6)-O-β-glucopyranoside, leucoside, kaempferol-3-O-neohesperidoside, and pachypodol. The structures of all the compounds were determined by detailed spectroscopic analysis. All compounds were also evaluated for their anti-inflammatory properties by assessing their inhibitory effects on nitric oxide (NO) production and TNF- α release in RAW 264.7 macrophages. Whilst slight anti-inflammatory activity (in terms of inhibition of NO production) was observed with eupomatenes A – E, this was also associated with high levels of cell growth inhibition.

Cutaneous pigmented viral plaques is a disorder of epidermal growth caused by canine papillomavirus type 4 (CPV‐4). There is currently no standard of care for managing this condition and it has not been reported in the Hungarian Vizsla. This case series documents the clinical features of canine pigmented viral plaques in Hungarian Vizsla dogs and the treatment of a severe case using a novel topical agent tigilanol tiglate (EBC‐46). A 4‐year‐old spayed Hungarian Vizsla in Australia was presented for multiple cutaneous pigmented plaques extending from the ventral cervical region. Lesions were neither painful nor pruritic. The number and size of these sessile plaques increased over time, with the largest lesions eventually taking on an exophytic (wart‐like) appearance. These lesions did not affect the dog's wellbeing. Two much less severe cases in a 5‐year‐old Vizsla from the UK and a 7‐year‐old Vizsla from New Zealand were also diagnosed. Histology was consistent with papillomavirus‐induced pigmented plaques and CPV‐4 DNA sequences were amplified from paraffin‐embedded formalin‐fixed tissue using the polymerase chain reaction from the most severely affected patient. Topical imiquimod was ineffective although used for only a short time. Two topical applications of novel anti‐neoplastic diterpene ester tigilanol tiglate as a gel, 9 days apart, greatly reduced the size and number of lesions in a limited portion of skin treated, over the lateral hock. While CPV‐4 has been previously reported to cause pigmented plaques, most commonly on pug dogs, but sporadically on other breeds, this is the first report of this virus causing plaques in Hungarian Vizslas. The cases illustrate some of the difficulties in diagnosing papillomavirus‐induced disease in dogs, especially in its early stages. Topical tigilanol tiglate is a potentially useful topical therapy for this viral‐induced disorder of cell growth and represents a treatment deserving of further investigation.

Objective To evaluate the efficacy and safety of tigilanol tiglate (TT) for local intratumoral treatment of mast cell tumors (MCTs) in dogs. Methods A randomized controlled clinical study in 2 phases involving 123 dogs with cytologically diagnosed MCT. Phase 1 compared 81 TT‐treated dogs with 42 control dogs; phase 2 allowed TT treatment of control dogs and retreatment of dogs that failed to achieve tumor resolution after TT treatment in phase 1. Tigilanol tiglate (1 mg/mL) was injected intratumorally with dose based on tumor volume. Concomitant medications were used to minimize potential for MCT degranulation. Modified response evaluation criteria in solid tumors were used to evaluate treatment response at 28 and 84 days. Adverse events and quality of life were also assessed. Results A single TT treatment resulted in 75% complete response (CR) (95% confidence interval [CI] = 61‐86) by 28 days, with no recurrence in 93% (95% CI = 82‐97) of dogs by 84 days. Eight TT‐treated dogs that did not achieve CR in phase 1 achieved CR after retreatment, increasing the overall CR to 88% (95% CI = 77‐93). Control dogs had 5% CR (95% CI = 1‐17) at 28 days. Wound formation after tumor slough and wound size relative to tumor volume were strongly associated with efficacy. Adverse events typically were low grade, transient, and directly associated with TT's mode of action. Conclusions Tigilanol tiglate is efficacious and well tolerated, providing a new option for the local treatment of MCTs in dogs.

Investigation of the Australian rainforest plant Croton insularis led to isolation of the first casbane hydroperoxide diterpenes EBC-304 and EBC-320. Extensive DFT and electronic circular dichroism (ECD) calculations in combination with 2D NMR spectroscopy determined the absolute configurations. EBC-304 and EBC-320 both display significant cytotoxicity.

The seco-casbane framework, first isolated from Croton insularis, is surprisingly rare. Further evaluation of this Australian rainforest plant unearthed two additional members, EBC-328 and EBC-363. TD DFT predicted electronic circular dichroism (ECD) spectra were critical for remote absolute stereochemical determination. EBC-328 displayed cytotoxic activity towards leukemia cells (K562). Potential biosynthetic relationships are discussed. seco-Casbanes, which are considered very rare natural products, have only recently joined the ranks of other rarely occurring seco diterpenes, such as seco-lathyranes and seco-phorboids. Described herein are two further examples, EBC-328 and EBC-363 isolated from Croton insularis. TD DFT predicted ECD spectra were crucial for solving remote absolute stereochemistry.

Two new crotinsulidanes EBC-318 and EBC-339 were isolated from the Australian rain forest plant Croton insularis. Both compounds are characterised by a bicyclo[10.2.1]alkane skeleton, one of which contains a bridgehead double bond. Cancer cell line cytotoxicity and biosynthetic considerations are presented.

Chromatographic separation of the extracts of the Australian rainforest plant Ternstroemia cherryi led to the isolation of five undescribed barrigenol-like triterpenoids, ternstroenols A – E, from the fruits and three known ones from the leaves. Ternstroenols A – E represent a new form of structural diversity, being the first in its kind to incorporate a trans- 2, 4, 6- decatrienoyl moiety at C-22. The structures of the ternstroenols were assigned by detailed spectroscopic analysis, degradation and chemical derivatization. All compounds exhibited potent anti-inflammatory activity in LPS and IFN- γ activated RAW 264.7 macrophages, with IC50 values as low as 0.7 μM. Despite the remarkable potency, high levels of unwanted cell growth inhibition was also observed, which prompted their cytotoxic evaluation in U87/U251 human glioblastoma cell lines.

Identification of potent natural products is a challenging task in which sophisticated separation processes including HPLC are employed. The bioactivity of HPLC fractions is determined with a bioassay, and the most potent compounds are progressed to structural elucidation. In pharmacology, the potency of a compound is expressed as the half-maximal effective concentration (EC50), which refers to the concentration of a drug that induces a response halfway between the baseline and maximum. While expressing the potency of a compound by its EC50 value makes sense in a clinical context, it is counterintuitive in the context of bioactivity-guided purification, as the potency of a compound is inversely related to its EC50 value, and the most potent compound is the one with the lowest EC50. In natural products chemistry, it would be more logical if an increase in potency would be reflected by an increase of a parameter reflecting the potency. In this study, we introduce the term "effective dilution volume (EDV50)" as the reciprocal of the EC50 (1/EC50). We show how the EDV50 can be used to identify potent compounds in chromatographic separations, allowing to easily graph and identify anti-inflammatory compounds. We show two examples of this approach by overlaying an HPLC chromatogram with the EDV50 to point out the most potent compounds. We hope that the EDV50 will make the illustration of active fractions containing potent compounds in a chromatogram obvious for the reader and will become a useful graphic tool in the natural products literature in the future.

Gene flow via pollen movement affects genetic variation in plant populations and is an important consideration in plant domestication. Fontainea picrosperma is a subcanopy rainforest tree that is of commercial interest because it is the source of tigilanol tiglate, a natural product used for the treatment of solid tumors. We identify patterns of pollen-mediated gene flow within natural populations of F. picrosperma and estimate genetic parameters and genetic structure between adult and juvenile groups using microsatellite markers. Our results show pollination events occur over much shorter distances than reported for tropical canopy species. At least 63% of seeds are sired by male trees located within 30 m of the mother. On average, 27% of the local male population contributed to successful reproduction of F. picrosperma with most fathers siring a single seed, however, the contributions to reproduction were uneven. Larger male trees with more flowers had greater reproductive success than those with less flowers (P

Mast cell tumor (MCT) is the most common cutaneous neoplasm in dogs and wide surgical resection is the current first-line treatment. However, recurrence is common and often requires more specialist and expensive therapies. Tigilanol tiglate is a novel small molecule drug delivered by intratumoral injection that is currently under development to provide a new option for treating MCT. The aim of this study was to characterize a safe and effective dose of tigilanol tiglate for canine MCT and to gather preliminary data on the drug's pharmacokinetics. A multicenter, open-label, uncontrolled, non-randomized, dose de-escalation design was used. Eligibility was MCT stage I/IIa and a tumor size of 0.1-6.0 cm3. Dosing was based on tumor size (50% v/v tumor) and 3 drug concentrations (1.0, 0.5, 0.2 mg/mL) were evaluated. Twenty-seven dogs were treated in 3 dose de-escalation cohorts (10, 10, and 7 dogs, respectively). Efficacy at 21 days was defined using international accepted solid tumor response criteria (RECIST). Greatest efficacy (90% complete response) was observed at the highest drug concentration (1.0 mg/mL) and adverse events were generally low grade, mild and transient, and directly associated with the mode of action of the drug. Hematological and serum biochemistry were generally unremarkable with plasma concentration curves typical of a non-intravenous parenteral medication. Intratumoral treatment of MCT with tigilanol tiglate at a concentration of 1.0 mg/mL was highly efficacious and well-tolerated. These results support the drug's further development for the treatment of MCT and other solid tumors.