Properties of the bovine viral diarrhoea virus replicase in extracts of infected MDBK cells

An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially doublestranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5–10mM Mg2+ and although it was also active in 1–2mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.

Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca2+ channels

Structurally novel compounds able to block voltage-gated Ca2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca2+ concentration ([Ca2+]i) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K+-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K+-depolarised Ca2+ influx, with IC50 values calculated to be 234, 548 and 209 μg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.

Identification and simultaneous analysis of harmane, harmol, isovitexin and vitexin in Passiflora incarnata extracts with a novel HLPC method

A high performance liquid chromatographic method for the simultaneous analysis of two flavonoids (iso-vitexin and vitexin), and three indole alkaloids (harmane, harmine, and harmol) was developed. This method was then utilised to quantitate levels of these five constituents in methanolic extracts of Australian Passiflora incarnata. HPLC analysis was performed using a Waters™ Novapak C18 (150 × 4 mm, 4 μm) column, with a gradient solvent system of methanol-water-acetic acid. Detection was achieved by PDA UV (254 nm) and fluorescence (excitation 254 nm, emission 414 nm), utilising the external standard method to obtain quantification.

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

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

Simultaneous determination of aldrin, dieldrin, endrin, heptachlor, and p,p'-DDT in medicinal plant extracts using a novel high performance liquid chromatography method

A high performance liquid chromatographic method for the simultaneous determination of five organochlorine pesticides (aldrin, p,p’-DDT, dieldrin, endrin, and heptachlor) was developed. The method was used to determine the levels of these pesticides in medicinal plant samples. Analysis was carried out using a Merck LiChrospher 100 RP C18 (5 μm) column with a gradient solvent system of acetonitrile-water and PDA UV detection (224 nm). Quantification was carried out by the external standard method. The limit of detection for the utilized method was below the local legal limits (ANZFA) for similar plant materials for all 5 pesticides excepting endrin. Medicinal plant extracts were further analyzed by conventional GC-ECD and GC-NPD means using SPE and GPC cleanup as required.

Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer

RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract. © CSIRO 2008.

Anti-staphylococcal activity of C-methyl flavanones from propolis of Australian stingless bees (Tetragonula carbonaria) and fruit resins of Corymbia torelliana (Myrtaceae)

Propolis of Australian stingless bees (Tetragonula carbonaria, Meliponini) originating from Corymbia torelliana (Myrtaceae) fruit resins was tested for its antimicrobial activities as well as its flavonoid contents. This study aimed at the isolation, structural elucidation and antibacterial testing of flavanones of C. torelliana fruit resins that are incorporated into stingless bee propolis. Flavanones of this study were elucidated by spectroscopic and spectrometric methods including UV, 1D and 2D NMR, EI-MS, ESI-MS and HR-MS. The results indicated known C-methylated flavanones namely, 1 (2S)-cryptostrobin, its regioisomer 2 (2S)- stroboponin, 3 (2S)- cryptostrobin 7-methyl ether, and 6 (2S)- desmethoxymatteucinol, and known flavanones 4 (2S)- pinostrobin and 5 (2S)- pinocembrin as markers for C. torelliana fruit resins and one propolis type. Ethanolic preparations of propolis were shown to be active against Staphylococcus aureus (ATCC 25923) and to a lesser extent against Pseudomonas aeruginosa (ATCC 27853). C. torelliana flavanones inhibited the growth of S. aureus therefore contributing to the antibacterial effects observed for Australian stingless bee propolis extracts.

The effects of salivary gland extracts from Boophilus microplus ticks on mitogen-stimulated bovine lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.

An innovative microplate assay to facilitate the detection of antimicrobial activity in plant extracts.

A microplate assay was modified for the detection of antimicrobial activity in plant extracts. The aim was to develop an in vitro assay that could rapidly screen plant extracts to provide quantitative data on inhibition of microbial growth. A spectrophotometric assay using a microplate with serial dilutions of the plant extract and the bacteria was developed. Two bacteria, Staphylococcus aureus and Escherichia coli, were used for this study. Essential oils, oregano (Origanum vulgare) and lemon myrtle (Backhousia citriodora), and three active components carvacrol, thymol and citral were evaluated. The reproducibility of the assay was high, with correlation coefficients (r aureus and E. coli between 0.9321 and 0.9816. Similarly, r and 0.9814. This assay could also be used to measure antimicrobial activity in plant extracts which vary in pH and color.

Collation of health literature for tropical and exotic fruits and extracts

This report collates data on the nutrient and phytochemical content of tropical exotic fruits, the evidence for health effects from consumption of these fruit and the use of extracts from edible and non-edible parts of these plants. The knowledge of Australian fruit compared with that grown overseas is presented together with opportunities for future work by Australian researchers. Opportunities for developing commercial extracts for use as food or nutraceutical uses are also presented.

Mango fruit peel and flesh extracts affect adipogenesis in 3T3-L1 cells

Obesity is associated with many chronic disease states, such as diabetes mellitus, coronary disease and certain cancers, including those of the breast and colon. There is a growing body of evidence that links phytochemicals with the inhibition of adipogenesis and protection against obesity. Mangoes (Mangifera indica L.) are tropical fruits that are rich in a diverse array of bioactive phytochemicals. In this study, methanol extracts of peel and flesh from three archetypal mango cultivars; Irwin, Nam Doc Mai and Kensington Pride, were assessed for their effects on a 3T3-L1 pre-adipocyte cell line model of adipogenesis. High content imaging was used to assess: lipid droplets per cell, lipid droplet area per cell, lipid droplet integrated intensity, nuclei count and nuclear area per cell. Mango flesh extracts from the three cultivars did not inhibit adipogenesis; peel extracts from both Irwin and Nam Doc Mai, however, did so with the Nam Doc Mai extract most potent at inhibiting adipogenesis. Peel extract from Kensington Pride promoted adipogenesis. The inhibition of adipogenesis by Irwin (100 mu g mL(-1)) and Nam Doc Mai peel extracts (50 and 100 mu g mL(-1)) was associated with an increase in the average nuclear area per cell; similar effects were seen with resveratrol, suggesting that these extracts may act through pathways similar to resveratrol. These results suggest that differences in the phytochemical composition between mango cultivars may influence their effectiveness in inhibiting adipogenesis, and points to mango fruit peel as a potential source of nutraceuticals.

Polyphenolic contents and the effects of methanol extracts from mango varieties on breast cancer cells

Bioactivities of peel and flesh extracts of 3 genetically diverse mango (Mangifera indica L.) varieties were studied. Nam Doc Mai peel extracts, containing the largest amounts of polyphenols, were associated with an effect on MCF-7 viable cell numbers with an IC50 (dose required for 50% inhibition of cell viability) of 56 μg/mL and significantly (p<0.01) induced cell death in MDA-MB-231 cells, compared with other varieties. Hydrophilic fractions of Nam Doc Mai peel extracts had the highest bioactivity values against both MCF-7 and MDA-MB-231 cells. Soluble polyphenols were present in the largest amounts in most hydrophilic fractions. The Nam Doc Mai mango variety contains high levels of fruit peel bioactivity, which appears to be related to the nature of the polyphenol composition.

Bioactive Properties of Mushroom (Agaricus bisporus) Stipe Extracts

The antibacterial activity and total phenolic (TP) content of Agaricus bisporus stipes were assessed using solvent and water extracts to determine its bioactivity. Extraction methods included accelerated solvent extraction (ASE) and hot water followed by membrane concentration. Water extract from ASE had the highest TP of 1.08 gallic acid equivalents (GAE)/g dry weight (DW) followed by ethanol at 0.61 mg GAE/g DW and 0.11 mg GAE/g DW for acetone. Acetone extracts inhibited Escherichia coli and Staphylococcus aureus at less than 50%; ethanol inhibited E. coli at 61.9% and S. aureus at 56.6%; and ASE water inhibited E. coli at 78.6% and S. aureus at 65.4%. The TP content of membrane concentrated extract of mushroom was 17 mg GAE in 100 mL. Membrane concentrated water extracts had a higher percentage inhibition on S. aureus than E. coli. Overall, the results were promising for further application of mushroom stipe extracts as a functional food additive. Practical Applications Mushrooms are known for their health benefits and have been identified as a good source of nutrients. The highly perishable nature of mushrooms warrants further processing and preservation to minimize losses along the supply chain. This study explores the possibility of adding value to mushroom stipes, a by-product of the fresh mushroom industry. The extracts assessed indicate the antibacterial activity and phenolic content, and the potential of using these extracts as functional ingredients in the food industry. This study provides valuable information to the scientific community and to the industries developing novel ingredients to meet the market demand for natural food additives.