David N Leach

The role of structure and molecular properties of terpenoids in determining their antimicrobial activity

The minimum inhibitory concentrations (MIC) of 60 terpenoids against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans have been determined. Hierarchical cluster analysis was used to group the compounds into five groups according to their activity patterns against the four microorganisms. K-Means cluster analysis was then used to confirm these groupings and to show the differences in the activity patterns of the groups. Ten molecular properties of the terpenoids, either calculated via molecular modelling or determined by direct measurement, were then used as variables in a forward stepwise discriminant analysis to identify which variables discriminated between groups. Low water solubility of Group IV compounds, mainly hydrocarbons and acetates, was found to be associated with their relative inactivity. The remaining groups, all containing oxygenated terpenoids, showed characteristic but distinct activity patterns towards the four test organisms. Hydrogen bonding parameters were found to be associated with antimicrobial activity in all cases. Activity against Gram-negative E. coli and P. aeruginosa was associated with a combination of a hydrogen bonding and size parameters. This was not found to be the case for the Gram-positive S. aureus or the yeast C. albicans. Copyright

Anti-inflammatory activity of Chinese medicinal vine plants

Anti-inflammatory activities of ethanol extracts from nine vine plants used in traditional Chinese medicine to treat inflammatory conditions were evaluated against a panel of key enzymes relating to inflammation. The enzymes included cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), phospholipase A(2) (PLA(2)), 5-lipoxygenase (5-LO) and 12-lipoxygenase (12-LO). The vine plants studied were: the stem of Spatholobus suberectus Dunn, the stem of Trachelospermum jasminoides Lem., the root from Tripterygium wilfordii Hook. f., the stem of Sinomenium acutum Rehder and Wilson, the stem of Piper kadsura (Choisy) Ohwi, the stem of Polygonum multiflorum Thunb., the root and stem from Tinospora sagittata Gagnep., the root of Tinospora sinensis (Lour.) Merrill, and the stem of Clematis chinensis Osbeck. All of the plant extracts showed inhibitory activities against at least one of the enzymes in various percentages depending upon the concentrations. The extract from S. suberectus was found to be active against all enzymes except COX-2. Its IC(50) values were 158, 54, 31 and 35 microg/ml in COX-1, PLA(2), 5-LO and 12-LO assays, respectively. T. jasminoides showed potent inhibitory activities against both COX-1 (IC(50) 35 microg/ml) and PLA(2) (IC(50) 33 microg/ml). The most potent COX-1, COX-2 and 5-LO inhibition was observed in the extract of T. wilfordii with the IC(50) values of 27, 125 and 22 microg/ml, respectively. The findings of this study may partly explain the use of these vine plants in traditional Chinese medicine for the treatment of inflammatory conditions.

A cross-cultural study: anti-inflammatory activity of Australian and Chinese plants.

In this study, in vitro inhibitory effects of 33 ethanol extracts obtained from 24 plant species (representing 11 different families) on cyclooxygenase-1 (COX-1) were evaluated. The plant materials selected for this study have been used in aboriginal medicine in Australia and traditional medicine in China for the treatment of various diseases that are considered as inflammation in nature, e.g. asthma, arthritis, rheumatism, fever, edema, infections, snakebite and related inflammatory diseases. All of the selected plants, with one exception, showed inhibitory activity against COX-1, which supports their traditional uses. The most potent COX-1 inhibition were observed from the extracts of Acacia ancistrocarpa leaves (IC(50)=23 microg/ml). Ficus racemosa bark, Clematis pickeringii stem, Acacia adsurgens leaves, Tinospora smilacina stem and Morinda citrifolia fruit powder exhibited inhibition of COX-1 with the IC(50) of 100, 141, 144, 158 and 163 microg/ml, respectively. Aspirin and indomethacin used as the reference COX-1 inhibitors in this study inhibited COX-1 with IC(50) of 241 and 1.2 microg/ml, respectively. The findings of this study may explain at least in part why these plants have been traditionally used for the treatment of inflammatory conditions in Australian aboriginal medicine and traditional Chinese medicine.

An agar dilution method for the determination of the minimum inhibitory concentration of essential oils

Abstract Methods for the measurement of the minimum inhibitory concentration (MIC) of antibacterial agents have been developed for water-soluble substances and require adaptation for use with water-insoluble essential oils. This paper reports a standardized agar dilution MIC method, using 0.5% v/v Tween 20 as a dispersant, which provides a reliable and reproducible technique. The method was tested using two Melaleuca alternifolia oil samples with two different levels of terpinen-4-ol (37% and 45% v/v). The MIC values of die tea tree oil samples against a wide selection of bacteria, moulds and yeast are reported.

Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae)

The composition and yield of oil in 615 trees representing the natural populations of Melaleuca alternifolia, or tea tree, was investigated. A sixth distinct oil chemotype was identified. Of the six chemotypes, one chemotype is dominated by terpinen-4-ol, one by 1,8-cineole, one by terpinolene and the remaining three chemotypes are all dominated by 1,8-cineole and differ in either terpinen-4-ol or terpinolene content. Whilst most chemotypes are present throughout the distribution range, a definite correspondence of oil types with geographic location was found. Terpinen-4-ol types predominate in and around the Bungawalbin basin in the Casino area of northern New South Wales (NSW), high 1,8-cineole types predominate toward the southern end of the distribution around Grafton and terpinolene types predominate in southern Queensland. Preliminary formulae have been developed to allow comparisons of oil data obtained by steam distillation with a static headspace gas chromatography method.

Antioxidant activity of 45 Chinese herbs and the relationship with their TCM characteristics

Here, 45 Chinese herbs that regulate blood circulation were analyzed for antioxidant activity using the oxygen radical absorbance capacity (ORAC) assay. A recent publication by Ou et al. identified a close relationship between in vitro antioxidant activity and classification of Chinese herbs as yin or yang. The 45 Chinese herbs in this study could be assigned the traditional characteristics of natures (cold, cool, hot and warm), flavors (pungent, sweet, sour, bitter and salty) and functions (arresting bleeding, promoting blood flow to relieve stasis, nourishing blood and clearing away heat from blood). These characteristics are generalized according to the theory of yin and yang. We identified a broad range, 40–1990 µmol Trolox Equivalent/g herbs, of antioxidant activity in water extracts. There was no significant correlation between ORAC values and natures or functions of the herbs. There was a significant relationship between flavors and ORAC values. Bitter and/or sour herbs had the highest ORAC values, pungent and/or sweet herbs the lowest. Other flavors had intermediate values. Flavors also correspond with the yin/yang relationship and our results are supportive of the earlier publication. We reported for the first time antioxidant properties of many Chinese herbs. High antioxidant herbs were identified as Spatholobus suberectus vine (1990 µmol TE/g), Sanguisorba officinalis root (1940 µmol TE/g), Agrimonia pilosa herb (1440 µmol TE/g), Artemisia anomala herb (1400 µmol TE/g), Salvia miltiorrhiza root (1320 µmol TE/g) and Nelembo nucifera leaf (1300 µmol TE/g). Antioxidant capacity appears to correlate with the flavors of herbs identified within the formal TCM classification system and may be a useful guide in describing their utility and biochemical mechanism of action.

In vitro Cytotoxicity of Australian Tea Tree Oil using Human Cell Lines

Abstract Cytotoxicity of Australian tea tree oil (oil of Melaleuca alternifolia) and its major oxygenated monoterpenes: terpinen-4-ol, 1,8-cineole and α-terpineol were investigated using the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay at two exposure times: 4 and 24 h on five different human cell lines. These cell lines included: Hep G2, a heptaocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line; and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. The overall rating for cytotoxicity of tea tree oil and its components was α-terpineol>tea tree oil>terpinen-4-ol> 1,8-cineole and with comparison with the controls used mercuric chloride>tea tree oil>aspirin. Antimicrobial activity (MICs) displayed a similar pattern where α-terpineol>terpinen-4-ol>tea tree oil> 1,8-cin...

The use of HPLC protein profiles in fish species identification

Abstract A method for the rapid identification of fish species by high performance liquid chromatographic (HPLC) analysis of water-soluble sarcoplasmic protein extracts is described. The method relies on the visual comparison of a star-symbol plot constructed from the samples HPLC profile with those obtained from the mean profile data of morphologically identified species. A library of plots is presented for 15 common edible marine fish species from eastern Australian waters, but any suitable data can be used for star construction. The data presented are applicable to the identification of samples from any season or location, whether they are raw, gamma-irradiated, or dried with infra-red radiation. The procedure involves a single 10 min extraction followed by a 60 min analysis, without the use of an internal standard.

Alkylamides from echinacea modulate induced immune responses in macrophages.

The ability of Echinacea and its components to alter the immune response was examined in vitro in a macrophage cell line under either basal or immunostimulated conditions. Potential immunostimulatory and inflammatory activity was determined using a nuclear transcription factor (NFκB) expression, tumour necrosis factor α (TNFα) and nitric oxide (NO) production as biomarkers. In the absence of alternate stimulation, the only significant effects seen were a decrease in NFκB expression by a 2-ene alkylamide ((2E)-N-isobutylundeca-2-ene-8,10-diynamide (1)) and a decrease in TNFα levels by cichoric acid and an Echinacea alkylamide fraction (EPL AA). When the cells were stimulated by lipopolysaccharide (LPS), inhibition of the increased NFκB expression levels was caused by cichoric acid, an Echinacea preparation (EPL), EPL AA and a 2,4-diene ((2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (2)). Increases in TNFα levels were inhibited by cichoric acid, EPL and EPL AA but enhanced by 1 in the presence of LPS, while only EPL AA was able to inhibit the stimulated increases in NO. When using phorbol myristate acetate to stimulate the cells, NFκB and NO levels were unaffected by Echinacea or its components while only cichoric acid and 2 inhibited TNFα levels. Although cichoric acid was found to have an effect, it is probably not an important contributor to the Echinacea modulation of the immune response in vivo, as it is not bioavailable. Echinacea appears to attenuate the response of macrophages to an immune stimulus and its combination of phytochemicals exhibits different pharmacological properties to one or more of the isolated major individual components.

Chemical composition and antioxidant and anticandidal activities of essential oils from different wild moroccan thymus species

Samples of the aerial parts of Thymus broussonetii, T. ciliatus, T. leptobotrys, T. maroccanus, T. pallidus, T. satureioides, and T. serpyllum collected from different natural regions in southern and south‐western Morocco were analyzed for their qualitative and quantitative essential oil profiles. In total, 46 compounds, representing more than 99% of the oils, were characterized. Monoterpenes, both hydrocarbons (12.9–58.0%) and oxygenated monoterpenes (38.8–81.1%), were the principal classes of compounds for most of the thyme species studied. Cluster analysis allowed the classification of the species into three main groups: a carvacrol group (Group I), comprising the species T. maroccanus and T. leptobotrys, a linalyl acetate and (E)‐nerolidol group (Group II), represented by T. serpyllum, and a thymol and/or carvacrol, γ‐terpinene, and p‐cymene group (Group III), composed of T. satureioides, T. broussonetii, T. ciliatus, and T. pallidus. The essential oils were screened for their antioxidant and anticandidal activities. The data showed that the oils obtained from T. leptobotrys and T. maroccanus (carvacrol group) possessed the highest antioxidant activities as assessed by the determination of the DPPH free radical‐scavenging ability and the ferric‐reducing potential. The anticandidal assays indicated that the highest activity was noticed for the essential oil isolated from T. leptobotrys.