From Petri Dish to Patient: Bioavailability Estimation and Mechanism of Action for Antimicrobial and Immunomodulatory Natural Products.

The new era of multidrug resistance of pathogens against frontline antibiotics has compromised the immense therapeutic gains of the 'golden age,' stimulating a resurgence in antimicrobial research focused on antimicrobial and immunomodulatory components of botanical, fungal or microbial origin. While much valuable information has been amassed on the potency of crude extracts and, indeed, purified compounds there are too many reports that uncritically extrapolate observed in vitro activity to presumed ingestive and/or topical therapeutic value, particularly in the discipline of ethnopharmacology. Thus, natural product researchers would benefit from a basic pharmacokinetic and pharmacodynamic understanding. Furthermore, therapeutic success of complex mixtures or single components derived therefrom is not always proportionate to their MIC values, since immunomodulation can be the dominant mechanism of action. Researchers often fail to acknowledge this, particularly when 'null' activity is observed. In this review we introduce the most up to date theories of oral and topical bioavailability including the metabolic processes affecting xenobiotic biotransformation before and after drugs reach the site of their action in the body. We briefly examine the common methodologies employed in antimicrobial, immunomodulatory and pharmacokinetic research. Importantly, we emphasize the contribution of synergies and/or antagonisms in complex mixtures as they affect absorptive processes in the body and sometimes potentiate activity. Strictly in the context of natural product research, it is important to acknowledge the potential for chemotypic variation within important medicinal plants. Furthermore, polar head space and rotatable bonds give a priori indications of the likelihood of bioavailability of active metabolites. Considering this and other relatively simple chemical insights, we hope to provide the basis for a more rigorous scientific assessment, enabling researchers to predict the likelihood that observed in vitro anti-infective activity will translate to in vivo outcomes in a therapeutic context. We give worked examples of tentative pharmacokinetic assessment of some well-known medicinal plants.

Isolation and characterisation of (-)-genifuranal: the principal antimicrobial component in traditional smoking applications of Eremophila longifolia (Scrophulariaceae) by Australian aboriginal peoples.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Eremophila longifolia is considered by some Australian Aboriginal tribal groups to be among the most significant of the medicinal plants in contemporary and traditional use. Usage modalities traditionally involved lipophilic extraction into animal fats and most importantly, ceremonial or medicinal smoking applications, involving the fumigation of mothers and infants following childbirth or boys following circumcision. An attempt was made to replicate the smoking modalities used by Australian Aboriginal people in the laboratory to identify bioactive compounds. MATERIALS AND METHODS: Two methods were used to produce smoke extracts; smoke was channelled through a condenser then bubbled into solvent, or bubbled directly into H2O then partitioned into chloroform followed by butanol. Extracts were used, firstly for antimicrobial screening using micro-titre plate broth dilution to produce minimum inhibitory concentrations (MIC), and secondly for chemical analysis. Structure elucidation of an abundant compound isolated from the smoke extract was performed using 2D-NMR and derivatisation. RESULTS: Significant antimicrobial activity (<1.0 mg/ml) was produced using the smoke extracts against the Gram-positive species Staphylococcus aureus, Bacillus subtilis and the yeast Candida albicans. A major component of the smoke with strong antimicrobial activity (0.13-0.5 mg/ml) was isolated which we have named (-)-genifuranal. Structure elucidation using 2D-NMR and derivatisation demonstrated genifuranal to be 5,6-dihydro-4H-cyclopenta[c]furan-4-ylacetaldehyde. Genifuranal is not observed in the leaves before heating, but is produced in the smoking or heating process and is thought to derive from hydrolysis and rearrangement of geniposidic acid or a related glycoside. Only geographically specific specimens of Eremophila longifolia produced (-)-genifuranal, which strongly supports previous hypothesised geographical variation in traditional usage, reflective of phytochemical variation. CONCLUSION: It would appear that genifuranal is the medicinal principal involved in traditional use of Eremophila longifolia when smoking modalities are used. Topical treatments traditionally produced by lipophilic extraction into animal fats are not likely to have had genifuranal present, as the mechanism for its formation requires heat.

Cytogeography of essential oil chemotypes of Eremophila longifolia F. Muell (Scrophulariaceae).

Previous studies have demonstrated that the widely distributed desert plant Eremophila longifolia has at least six geographically defined essential oil chemotypes. The focus of the present study is to extend and enhance information concerning known chemotypes and to investigate the involvement of cell nuclei ploidy in this variation. Forty field collected specimens of E. longifolia were taken from most of the mainland states of Australia then subjected to hydrodistillation to produce essential oils, which were then chemically characterised. Ploidy was determined using relative fluorescence of cell nuclei stained with propidium iodide, measured in a flow cytometer. Using principal component analysis (PCA), at least three essential oil chemotypes, in addition to the six already described, were identified in the present study. Previously described high yielding essential oil chemotypes were also characterised in terms of diploidy. For the first time diploid populations were identified in New South Wales, correlating with high yielding isomenthone/menthone and karahanaenone chemotypes. Furthermore, the separate diploid population previously described from Western Australia was demonstrated to be the safrole/methyl eugenol type, which is restricted to a small geographic range in far north-west Western Australia (Murchison District). All other chemotypes were shown to be tetraploid, including apparently randomly emerging individuals, representative of chemotypes producing low yields of isomenthone/menthone and karahanaenone similar in composition to the high yielding diploid types.

Medicinal compounds, chemically and biologically characterised from extracts of Australian Callitris endlicheri and C.glaucophylla (Cupressaceae): used traditionally in Aboriginal and colonial pharmacopoeia.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Callitrisendlicheri and C.glaucophylla were highly valued by Australian Aboriginal people for use in medicinal applications. Pine needles were prepared using modalities of either smoking or topical preparations, requiring either aqueous or lipophilic extraction into animal fat. Extracts treated various ailments consistent with pathogenic infection, or other topical or tracheal ailments not clearly elucidated in ethnopharmacological records. AIM OF THE STUDY: Here we aim firstly to investigate antimicrobial activities of both smoke, essential oil and solvent extracts and secondly to chemically characterise significant volatile compounds potentially related to medicinal or antimicrobial activities. MATERIALS AND METHODS: Essential oils were produced using traditional hydrodistillation of pine needles collected from Callitrisendlicheri and C.glaucophylla. From the same material, solvent extracts were produced separately, using acetone and methanol, and then smoke extracts were produced with separate methods described herein, using fresh needles. All extracts were screened for antimicrobial activity against a range of bacterial organisms and sporicidal activity against pathogenic fungi (Trichophytonmentagrophytes, T.interdigitalis and T.rubrum). RESULTS: Essential oils produced only modest antibacterial activity and the Callitris endlicheri essential oil had moderate antifungal activity. Smoke extracts demonstrated considerable broad spectrum antimicrobial activity, but solvent extracts demonstrated more selective activity against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and the yeast Candida albicans. Chemical character of essential oils was consistent with previous studies; however, solvent and smoke extracts from fresh needles produced high concentrations of potentially medicinal abietane diterpenes, specifically pisiferal, pisiferol and ferruginol; well known from Japanese species with demonstrated bioactivity. CONCLUSION: The occurrence of these diterpenes and other phenolics, in conjunction with significant antimicrobial activities from the various extracts, is in alignment with the use of Australian Callitris species in Aboriginal medicinal practice.

Chemical and biological characterisation of solvent extracts and essential oils from leaves and fruit of two Australian species of Pittosporum (Pittosporaceae) used in aboriginal medicinal practice.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Although no known medicinal use for Pittosporum undulatum Vent. (Pittosporaceae) has been recorded, anecdotal evidence suggests that Australian Aboriginal people used Pittosporum angustifolium Lodd., G. Lodd. & W. Lodd. topically for eczema, pruritis or to induce lactation in mothers following child-birth and internally for coughs, colds or cramps. AIMS OF THE STUDY: Essential oil composition and bioactivity as well as differential solvent extract antimicrobial activity from Pittosporum angustifolium are investigated here first, to partially describe the composition of volatiles released in traditional applications of Pittosporum angustifolium for colds or as a lactagogue, and second to investigate antibacterial activity related to topical applications. Essential oils were also investigated from Pittosporum undulatum Vent., first to enhance essential oil data produced in previous studies, and second as a comparison to Pittosporum angustifolium. MATERIALS AND METHODS: Essential oils were hydrodistilled from fruit and leaves of both species using a modified approach to lessen the negative (frothing) effect of saponins. This was achieved by floating pumice or pearlite obsidian over the mixture to crush the suds formed while boiling. Essential oil extracts were analysed using GC-MS, quantified using GC-FID then screened for antimicrobial activity using a micro-titre plate broth dilution assay (MIC). Using dichloromethane, methanol, hexane and H(2)O as solvents, extracts were produced from leaves and fruit of Pittosporum angustifolium and screened for antimicrobial activity and qualitative phytochemical character. RESULTS: Although the essential oil from leaves and fruit of Pittosporum undulatum demonstrated some component variation, the essential oil from fruits of Pittosporum angustifolium had major constituents that strongly varied according to the geographical location of collection, suggesting the existence of at least two chemotypes; one with high abundance of acetic acid decyl ester. This chemotype had high antimicrobial activity whilst the other chemotype had only moderate antimicrobial activity against the three microbial species investigated here. This result may support the occurrence of geographical specificity with regard to ethnopharmacological use. Antimicrobial activity screening of the solvent extracts from Pittosporum angustifolium revealed the leaves to be superior to fruit, with water being the most suitable extraction solvent. CONCLUSION: To the best of our knowledge, this study constitutes the first time essential oils, and solvent extracts from the fruits of Pittosporum angustifolium, have been examined employing comprehensive chemical and biological analysis. The essential oil composition presented in this paper, includes components with structural similarity as chemosemiotic compounds involved in mother-infant identification, which may have significance with regard to traditional applications as a lactagogue.

Chemical and biological characterization of novel essential oils from Eremophila bignoniiflora (F. Muell) (Myoporaceae): a traditional Aboriginal Australian bush medicine.

Essential oils were extracted by hydrodistillation from the traditional Australian medicinal plant Eremophila bignoniiflora, characterized chemically and then screened for bioactivity. Characterization and quantification were completed using gas chromatography-mass spectrometry (GC-MS) and GC-flame ionization detection, respectively. Antimicrobial capacity was assessed using disc diffusion and micro-titre plate broth dilution and further characterized using thin layer chromatography followed by bioautography to assign activity to separated individual active components. Antifungal capacity was investigated using micro-titre plate broth dilution against pathogenic Trichophyton species. Free radical scavenging ability was assessed using the diphenylpicrylhydradyl reaction in methanol. The predominant components of the essential oil were fenchyl-acetate and bornyl-acetate. However, bioautography indicated antimicrobial ability to be largely linked to the less abundant, more polar constituents. Oils displayed only modest antifungal ability against pathogenic Trichophyton species associated with dermatophytosis, but moderate to high antimicrobial activity, particularly against the yeast Candida albicans and the bacteria Staphylococcus epidermidis. Essential oils exhibited relatively low free radical scavenging ability. Speculation over the role of essential oils in the traditional medicinal applications of E. bignoniiflora follows, exploring correlations between traditional use and investigated bioactivities.

Associations of HSP90 client proteins in human breast cancer.

BACKGROUND: HSP90 has been studied intensively as a therapeutic target, however little is known regarding specific interactions of the large number of HSP90 client proteins. Therefore, this study investigated HSP90 client proteins sensitive to the HSP90 inhibitor geldanamycin in tumour and healthy breast tissue. MATERIALS AND METHODS: Co-immunoprecipitation and SDS-PAGE were used to investigate protein interactions. Western blotting and LC-MS were used to infer protein identities. RESULTS: HSP90 client proteins were observed in 7 out of 11 breast cancer patients. Further experiments inferred HSP40, -56/FKBP52, -60, -70, -105 and lumican to associate with HSP90 and to belong to this group of geldanamycin-sensitive proteins. In one patient, a cancer-specific group of proteins was identified. In all experiments geldanamycin resistance was observed. CONCLUSION: HSP90 differentially associated with client proteins and this was patient dependent. Geldanamycin resistance and lack of HSP90 client protein expression may limit clinical applications of HSP90 inhibitors.

Identification of antibacterial constituents from the indigenous Australian medicinal plant Eremophila duttonii F. Muell. (Myoporaceae).

This paper reports on the isolation and identification of antibacterial constituents from the indigenous Australian medicinal plant Eremophila duttonii F. Muell. (Myoporaceae). Preparations derived from this plant are used by indigenous populations in the topical treatment of minor wounds, otitis and ocular complaints, and as a gargle for sore throat. Several authors have reported extracts of this plant to effect rapid bacteriolysis and inhibit growth of a wide range of Gram-positive micro-organisms. In other studies involving screening of native medicinal plants for antibacterial activity, extracts of Eremophila duttonii have been reported to consistently exhibit the highest potency amongst all species included. From a hexane extract, we identified two diterpenes of the serrulatane class, the principal constituents responsible for antibacterial activity and present as major constituents of the resinous leaf cuticle: serrulat-14-en-7,8,20-triol (1) and serrulat-14-en-3,7,8,20-tetraol (2). In addition, a hydroxylated furanosesquiterpene with mild antibacterial activity which appeared to be a novel compound was isolated from the extract and tentatively identified as 4-hydroxy-4-methyl-1-(2,3,4,5-tetrahydro-5-methyl[2,3'-bifuran]-5-yl) pentan-2-one. Minimum inhibitory concentrations for each of the compounds against three Gram-positive bacteria: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228) and Streptococcus pneumoniae (ARL 10582), were determined using a micro-titre plate broth dilution assay.

Enhancement of glutathione-dependent haemin degradation by ascorbic acid.

In the current work, we investigated the effect of ascorbic acid on GSH-mediated haemin degradation. GSH-mediated haemin degradation in the presence of ascorbic acid in phosphate-buffered saline and in erythrocyte ghosts was determined by recording absorbance at 365 and 399nm, respectively. Generation of intracellular H(2)O(2) was measured indirectly in terms of the inactivation of endogenous catalase in erythrocytes in the presence of 3-amino-1,2,4-triazole. Although ascorbic acid itself did not induce haemin degradation, it enhanced GSH-mediated haemin degradation. Experiments with catalase showed that H(2)O(2) was essential in this process. The oxidation of ascorbic acid in the presence of haemin was stimulated by GSH, suggesting that ascorbic acid can alter the mechanism of H(2)O(2) generation observed with GSH and haemin alone. These results suggest that enhancement of GSH-mediated haemin degradation by ascorbic acid may be due to an increase in the production of H(2)O(2) generated by GSH and haemin in the absence of ascorbic acid.

Chlorodinitrobenzene-mediated damage in the human erythrocyte membrane leads to haemolysis.

1-chloro-2,4-dinitrobenzene (CDNB), an intracellular glutathione-depleting agent, has been shown to have an adverse effect on erythrocyte membrane integrity. In the current study, we have demonstrated that CDNB caused haemolysis of human red blood cells (RBC) at higher concentrations (>or= 5 mM). The haemolysis induced by CDNB was preceded by the leakage of K(+) from the cells suggesting the colloid-osmotic nature of this lysis. The inclusion of molecules of increasing size in the extracellular media inhibited both the rate and extent of haemolysis thus supporting the proposal of CDNB-induced pore formation. The size of membrane lesions increased with an increase in the concentration of CDNB. SDS-PAGE demonstrated that CDNB causes the polymerisation and/or fragmentation of membrane proteins. Although CDNB has been shown to cause a drastic reduction in membrane thiols, our data suggest that the CDNB-induced formation of membrane disulfide bonds as a prima facie cause of permeability enhancement is unlikely.