In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes.

1. The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes. 2. Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway. 3. Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH. 4. Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by ß-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite. 5. This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.

Supplementation with Brazil nuts and green tea extract regulates targeted biomarkers related to colorectal cancer risk in humans.

Se and green tea have been shown in epidemiological, observational and preclinical studies to be inversely related to the risk of developing colorectal cancer (CRC). However, there are limited studies to evaluate their regulatory effects on genes/proteins that relate to CRC oncogenesis in human subjects, such as selenoproteins, WNT signalling pathway, inflammation and methylation. This study examined the effects of supplementation of Se using Brazil nuts and green tea extract (GTE) capsules, alone and in combination, on targeted biomarkers. In total, thirty-two volunteers (>50 years of age) with plasma Se≤1·36 µmol/l were randomised to one of three treatment groups: nine to Se (approximately 48 µg/d) as six Brazil nuts, eleven to four GTE capsules (800 mg (-)-epigallocatechin-3-gallate) and twelve to a combination of Brazil nuts and GTE. Blood and rectal biopsies were obtained before and after each intervention. Plasma Se levels, rectal selenoprotein P (SePP) and ß-catenin mRNA increased significantly in subjects consuming Brazil nuts alone or in combination, whereas rectal DNA methyltransferase (DNMT1) and NF-κB mRNA were reduced significantly in subjects consuming GTE alone or in combination. None of the interventions significantly affected rectal acetylated histone H3 or Ki-67 expression at the protein level or plasma C-reactive protein. Effects of the combination of Brazil nuts and GTE did not differ from what would be expected from either agent alone. In conclusion, supplementation of Brazil nuts and/or GTE regulates targeted biomarkers related to CRC oncogenesis, specifically genes associated with selenoproteins (SePP), WNT signalling (ß-catenin), inflammation (NF-κB) and methylation (DNMT1). Their combination does not appear to provide additional effects compared with either agent alone.

Combination chemotherapy with cyclophosphamide, epirubicin and 5-fluorouracil causes trabecular bone loss, bone marrow cell depletion and marrow adiposity in female rats.

The introduction of anthracyclines to adjuvant chemotherapy has increased survival rates among breast cancer patients. Cyclophosphamide, epirubicin and 5-fluorouracil (CEF) combination therapy is now one of the preferred regimens for treating node-positive breast cancer due to better survival with less toxicity involved. Despite the increasing use of CEF, its potential in causing adverse skeletal effects remains unclear. Using a mature female rat model mimicking the clinical setting, this study examined the effects of CEF treatment on bone and bone marrow in long bones. Following six cycles of CEF treatment (weekly intravenous injections of cyclophosphamide at 10 mg/kg, epirubicin at 2.5 mg/kg and 5-flurouracil at 10 mg/kg), a significant reduction in trabecular bone volume was observed at the metaphysis, which was associated with a reduced serum level of bone formation marker alkaline phosphatase (ALP), increased trends of osteoclast density and osteoclast area at the metaphysis, as well as an increased size of osteoclasts being formed from the bone marrow cells ex vivo. Moreover, a severe reduction of bone marrow cellularity was observed following CEF treatment, which was accompanied by an increase in marrow adipose tissue volume. This increase in marrow adiposity was associated with an expansion in adipocyte size but not in marrow adipocyte density. Overall, this study indicates that six cycles of CEF chemotherapy may induce some bone loss and severe bone marrow damage. Mechanisms for CEF-induced bone/bone marrow pathologies and potential preventive strategies warrant further investigation.

Polyandric acid A, a clerodane diterpenoid from the Australian medicinal plant Dodonaea polyandra, attenuates pro-inflammatory cytokine secretion in vitro and in vivo.

Dodonaea polyandra is a medicinal plant used traditionally by the Kuuku I'yu (Northern Kaanju) indigenous people of Cape York Peninsula, Australia. The most potent of the diterpenoids previously identified from this plant, polyandric acid A (1), has been examined for inhibition of pro-inflammatory cytokine production and other inflammatory mediators using well-established acute and chronic mouse ear edema models and in vitro cellular models. Topical application of 1 significantly inhibited interleukin-1ß production in mouse ear tissue in an acute model. In a chronic skin inflammation model, a marked reduction in ear thickness, associated with significant reduction in myeloperoxidase accumulation, was observed. Treatment of primary neonatal human keratinocytes with 1 followed by activation with phorbol ester/ionomycin showed a significant reduction in IL-6 secretion. The present study provides evidence that the anti-inflammatory properties of 1 are due to inhibition of pro-inflammatory cytokines associated with skin inflammation and may be useful in applications for skin inflammatory conditions including psoriasis and dermatitis.

Learning from both sides: Experiences and opportunities in the investigation of Australian aboriginal medicinal plants.

With one of the oldest surviving cultures in the world, Australian Aboriginal people have developed immense knowledge about the diverse Australian flora. Western scientific investigation of some Australian Aboriginal medicinal plants has demonstrated interesting pharmacological activities and chemistry, however the majority of these species have not yet been extensively examined. We argue that research that is locally initiated and driven by Indigenous traditional owners in collaboration with Western scientists has significant potential to develop new plant-based products. Locally driven medicinal plants research in which traditional owners work as researchers in collaboration with University-based colleagues in the investigation of medicines rather than "stakeholders" or "informants" is one model that may be used in characterising plants with the potential to be developed into sustainable plant-based medicinal products with commercial value. Our team has taken this approach in research located both on traditional homelands and in the laboratory. Research being conducted by the University of South Australia and Chuulangun Aboriginal Corporation has led to patent filing for protection of intellectual property associated with novel compounds and extracts with the potential for development through cosmetic, complementary medicine and pharmaceutical routes. Ongoing research is examining the commercial developmental pathways and requirements for product development in these spaces. This review will address the opportunities that might exist for working in partnership with Australian Indigenous communities, some of the scientific knowledge which has been generated so far from our work together and the lessons learnt since the inception of the collaboration between the Chuulangun Aboriginal Corporation and scientists from the University of South Australia.

Flavonoids from the leaves and stems of Dodonaea polyandra: a Northern Kaanju medicinal plant.

Three prenylated flavonoids 5,7,4'-trihydroxy-3'(3-methylbut-2-enyl)-3-methoxy flavone, 5,7-dihydroxy-3'(3-methylbut-2-enyl)-3,4'-dimethoxy flavone and 5,7,4'-trihydroxy-3',5'(3-methylbuyt-2-enyl)-3-methoxy flavone together with three other known flavonoids were isolated from the medicinal plant Dodonaea polyandra. The plant is used in the traditional medicine system of Northern Kaanju people of Cape York Peninsula, Queensland, Australia. The extracts studied have previously been found to possess anti-inflammatory activity. Successive fractionation of leaf and stem extracts by column and high performance liquid chromatography led to the isolation of these compounds. Their structures were determined using a number of spectroscopic techniques including 1D and 2D NMR and high resolution mass spectroscopy. The structural elucidation is reported herein accompanied by full ¹H and ¹³C NMR spectroscopic data. Spectroscopic data of known compounds was in agreement with that previously reported in literature.

Consumers' views of pharmacogenetics--A qualitative study.

BACKGROUND: Adverse drug reactions are recognized as a significant public health issue. Pharmacogenetics (PGx) provides a potential means of preventing some adverse drug reactions by predicting the optimal medication dose for an individual; however, PGx is rarely used in clinical practice. Thus far, there have been few studies investigating consumers' perceptions of the barriers to the implementation of PGx in clinical practice. OBJECTIVES: This study explored the views of the general public regarding their current use of medications, and their experiences of side effects and opinions on PGx. METHODS: Members of the general public who suffered a chronic medical condition and/or had an immediate family member with a chronic medical condition were recruited to form 5 separate focus groups (n=35). Three separate age ranges were used in the focus groups. A questioning route was developed and used in focus groups to determine participants' experiences with medication use and opinions on PGx (referred to as "Personalized Medicine"). Focus group discussions were transcribed by 2 separate investigators, and qualitative analysis, based on the framework approach, was applied to the data. Data were independently coded to identify key themes then compared both within and between focus groups. RESULTS: A common theme was a desire to have a holistic approach to disease diagnosis and medication selection. A wide range of views were expressed by the focus group participants. Concerns were raised regarding the current level of side effects experienced with medications. Storage and privacy of genetic information, and the costs involved, were also seen as potential barriers to implementation of PGx. CONCLUSIONS: PGx testing was seen as a potential positive contribution, but only if other factors were considered during the prescribing process. As participants desired a high level of information and effective communication from their health-care professionals, PGx education of clinicians and pharmacists will be essential to satisfy consumers' requirements.

Atorvastatin-induced cell toxicity in yeast is linked to disruption of protein isoprenylation.

Statins, used to treat hypercholesterolemia, are one of the most frequently prescribed drug classes in the developed world. However, a significant proportion of users suffer symptoms of myotoxicity, and currently, the molecular mechanisms underlying myotoxicity remain ambiguous. In this study, Saccharomyces cerevisiae was exploited as a model system to gain further insight into the molecular mechanisms of atorvastatin toxicity. Atorvastatin-treated yeast cells display marked morphological deformities, have reduced cell viability and are highly vulnerable to perturbed mitochondrial function. Supplementation assays of atorvastatin-treated cells reveal that both loss of viability and mitochondrial dysfunction occur as a consequence of perturbation of the sterol synthesis pathway. This was further investigated by supplementing statin-treated cells with various metabolites of the sterol synthesis pathway that are believed to be essential for cell function. Ergosterol, coenzyme Q and a heme precursor were all ineffective in the prevention of statin-induced mitochondrial disruption and cell death. However, the addition of geranylgeranyl pyrophosphate and farnesyl pyrophosphate significantly restored cell viability, although these did not overcome petite induction. This highlights the pleiotropic nature of statin toxicity, but has established protein prenylation disruption as one of the principal mechanisms underlying statin-induced cell death in yeast.