Chemical and biological studies of medicinal plants used by the Yaegl Aboriginal community of Australia

"A thesis submitted as partial fulfilment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia, 2013".Bibliography: pages 193-210.Chapter 1. Introduction -- Chapter 2. Selection of plants and methods for screening and isolation of bioactive compounds -- Chapter 3. Chemical and biological studies on Lophostemon suaveolens -- Chapter 4. Chemical and biological studies on Alphitonia excelsa -- Chapter 5. Ethical engagement with community and capacity strengthening -- Chapter 6. Conclusion and future directions."This PhD study was based on the ethnomedicinal knowledge of the Yaegl Aboriginal community of northern NSW, Australia. It follows previous investigations of the Indigenous Bioresources Research Group (IBRG) on first-hand documentation of and preliminary screening of some medicinal plants used by the Yaegl community for treatment of wounds, sores and skin infections. The overall aim of this project was to isolate and indentify bioactive components from two medicinal plants of the Yaegl community. Two Indigenous Australian medicinal plants, Lophostemon suaveolens and Alphitonia excelsa were selected for detailed chemical and biological studies. This selection was on the basis of a literature review on the Yaegl medicinal plants documented by the IBRG for the treatment of wounds, sores and skin infections, preliminary screening results of previous IBRG researchers, and specific Yaegl community requests. Antimicrobial activity (disc diffusion, MTT microdilution and TLC bioautography assays), anti-inflammatory activity (NO, TNF-α, PGE2 and COX inhibitory assays) and antioxidant activity (ORAC assay) of these two plants were evaluated. Leaves of L. suaveolens were extracted sequentially with n-hexane, DCM, EtOAc and MeOH. Significant antimicrobial activity was observed with the n-hexane and DCM extracts with IC90 <50 μg/mL against antibiotic sensitive and resistant strains of Staphylococcus aureus (MRSA and MDRSA) and Streptococcus pyogenes in the MTT microdilution assay. Both extracts also showed significant NO inhibitory activity in RAW264 cells with IC50 43.9 μg/mL and 4.6 μg/mL, respectively, for the n-hexane and DCM extracts. These extracts (n-hexane and DCM) did not show TNF-α or PGE2 inhibitory activity. GC-MS analysis of the oily n-hexane extract identified 16 components including the well known bioactive compounds aromadendrene (15.47%), spathulenol (12.46%), globulol (4.47%), epiglobulol (2.69%), phytol (2.84%), β-caryophyllene (2.53%) and α-humulene (1.52%). These compounds were reported for having antimicrobial (aromadendrene, spathulenol, globulol, β-caryophyllene, α-humulene and phytol), anti-inflammatory (β-caryophyllene) and cytotoxic (β-caryophyllene, spathulenol and α-humulene) properties. Further fractionation of the DCM extract by normal plase silica gel column chromatography yielded five major bioactive fractions that showed good antimicrobial (IC90 <50-1000 μg/mL range against Streptococcus pyogenes and methicillin sensitive and resistant strains of Staphylococcus. aureus), NO inhibitory (IC50 <12 μg/mL), PGE2 inhibitory (<20 μg/mL) and modest antioxidant (1000-2700 μM TE/g) activities. Further bioassay guided studies on these active fractions using different chromatographic procedures led to the isolation of betulinic acid and eucalyptin from the n-hexane and DCM extracts. Eucalyptin has been previously reported to have antimicrobial properties and betulinic acid is a well known anti-inflammatory compound. This is the first report of bioassay studies and isolation of bioactive compounds from L. suaveolens. Leaves of A. excelsa were extracted sequentially with n-hexane, DCM, EtOAc and MeOH. They were also extracted with water to mimic some preparation practices of the Yaegl community. The EtOAc extract showed promising antimicrobial activity (IC90 <50 μg/mL against S. pyogenes and IC90 500-1000 μg/mL against antibiotic sensitive and resistant strains of S. aureus) in the MTT microdilution assay. The EtOAc extract also showed potent nitric oxide (NO) inhibition (IC50 10.7 μg/mL) and promising antioxidant (3.70x10³ μM TE/g) activities. The MeOH extract of A. excelsa displayed moderate levels of antimicrobial (IC90 1000 and 62.5 μg/mL against S. aureus and S. pyogenes, respectively, in MTT microdilution assay) and anti-inflammatory (IC50 30.5 μg/mL in NO inhibition assay) activities. The water extract showed good anti-inflammatory activity in the COX inhibitory assay and modest antioxidant activity in the ORAC assay. Further bioassay guided isolation of the EtOAc extract led to the isolation of two bioactive flavonoids, kaempferol and quercetin. Both isolated compounds showed antimicrobial activity (IC90 =<62.5 μg/mL against S. pyogenes, sensitive and resistant strains of S. aureus) in the MTT microdilution assay. This is the first report of isolation of these two compounds from A. excelsa. Nurturing and sustaining a strong relationship with the Yaegl Indigenous people and providing capacity strengthening opportunities for the community was an essential part of this research. This is aligned with the best ethical practices essential for working with Indigenous people on their knowledge systems. For this PhD study, this included participation in meetings and workshops with the Yaegl community members; presenting and providing feedback on the research work and inviting feedback by the community to guide the research; and organising and participating in an education program with the local youth to enhance educational outcomes, as requested by the Yaegl community.Mode of access: World Wide Web.1 online resource (xix, 235 pages) illustrations (some coloured), maps (coloured

Antimicrobial activity of customary medicinal plants of the Yaegl Aboriginal community of northern New South Wales, Australia : a preliminary study

BACKGROUND: This study is a collaboration between Macquarie University researchers and the Yaegl Aboriginal Community of northern NSW, Australia to investigate the antimicrobial potential of plants used in the topical treatment of wounds, sores and skin infections. Based on previously documented medicinal applications, aqueous and aqueous ethanolic extracts of Alocasia brisbanensis, Canavalia rosea, Corymbia intermedia, Hibbertia scandens, Ipomoea brasiliensis, Lophostemon suaveolens and Syncarpia glomulifera and the aqueous extracts of Smilax australis and Smilax glyciphylla were tested against common wound pathogens, including antibiotic resistant bacterial strains. METHODS: Plant material was prepared as aqueous extractions modelled on customary preparations and using 80% aqueous ethanol. Extracts were assayed against a selection of clinically relevant Gram positive (Streptococcus pyogenes and sensitive and resistant strains of Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium) bacteria and a fungus (Candida albicans) using disc diffusion and MTT microdilution methods. Viability of treated microorganisms was determined by subculturing from microdilution assays. RESULTS: The extracts of Corymbia intermedia, Lophostemon suaveolens and Syncarpia glomulifera had promising levels of antimicrobial activity (MIC 31-1,000 µg/mL) against both antibiotic sensitive and resistant Staphylococcus aureus as well as the fungus Candida albicans (clinical isolate). CONCLUSION: Aqueous and 80% aqueous ethanolic extracts of Lophostemon suaveolens, Corymbia intermedia and Syncarpia glomulifera exhibited promising levels of antimicrobial activity against a range of both antibiotic sensitive and resistant strains of microorganisms. This is the first report of antimicrobial activities for C. intermedia and L. suaveolens and the leaves of S. glomulifera. This study demonstrates the value of customary knowledge in the identification of new sources of antimicrobial treatments.7 page(s

Additional file 1. of Antimicrobial activity of customary medicinal plants of the Yaegl Aboriginal community of northern New South Wales, Australia: a preliminary study

Picture showing typical results of the disc diffusion assay

Bioactivity and chemical characterisation of Lophostemon suaveolens - an endemic Australian Aboriginal traditional medicinal plant

Lophostemon suaveolens is a relatively unexplored endemic medicinal plant of Australia. Extracts of fresh leaves of L. suaveolens obtained from sequential extraction with n-hexane and dichloromethane exhibited antibacterial activity in the disc diffusion and MTT microdilution assays against Streptococcus pyogenes and methicillin sensitive and resistant strains of Staphylococcus aureus (minimum bactericidal concentration < 63 μg/mL). The dichloromethane extract and chromatographic fractions therein inhibited nitric oxide in RAW264.7 murine macrophages (IC₅₀ 3.7–11.6 μg/mL) and also PGE₂ in 3T3 murine fibroblasts (IC₅₀ 2.8–19.7 μg/mL). The crude n-hexane, dichloromethane and water extracts of the leaves and chromatographic fractions from the dichloromethane extract also showed modest antioxidant activity in the ORAC assay. GC–MS analysis of the n-hexane fraction showed the presence of the antibacterial compounds aromadendrene, spathulenol, β-caryophyllene, α-humulene and α-pinene and the anti-inflammatory compounds β-caryophyllene and spathulenol. Fractionation of the dichloromethane extract led to the isolation of eucalyptin and the known anti-inflammatory compound betulinic acid.4 page(s

Phytochemical Analysis and Understanding the Antioxidant and Anticancer Properties of Methanol Extract from Litsea glutinosa: In Vitro and In Vivo Studies

International audienceLitsea glutinosa (L. glutinosa) is considered an evidence-based medicinal plant for the treatment of cancer, the leading cause of death worldwide. In our study, the in vitro antioxidant and in vivo anticancer properties of an essential ethno-medicinal plant, L. glutinosa, were examined using non-toxic doses and a phytochemical analysis was executed using gas-chromatography–mass-spectrometry. The in vitro antioxidant study of the L. glutinosa methanolic extract (LGBME) revealed a concentration-dependent antioxidant property. The bark extract showed promising antioxidant effects in the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. The strongest antioxidant activity was demonstrated at the maximum concentration (50 µg/mL). The IC50 values of the LGBME and BHT were 5.51 and 5.01 µg/mL, respectively. At the same concentration, the total antioxidant capacity of the LGBME was 0.161 µg/mL and the ferric reducing antioxidant power assay result of the LGBME was 1.783 µg/mL. In the cytotoxicity study, the LD50 of the LGBME and gallic acid were 24.93 µg/mL and 7.23 µg/mL, respectively. In the in vivo anticancer-activity studies, the LGBME, particularly at a dose of 150 mg/kg/bw, showed significant cell-growth inhibition, decreased tumor weight, increased mean survival rate, and upregulated the reduced hematological parameters in EAC (Ehrlich’s ascites carcinoma)-induced Swiss albino mice. The highest cell-growth inhibition, 85.76%, was observed with the dose of 150 mg/kg/bw. Furthermore, the upregulation of pro-apoptotic genes (p53, Bax) and the downregulation of anti-apoptotic Bcl-2 were observed. In conclusion, LGBME extract has several bioactive phytoconstituents, which confirms the antioxidant and anticancer properties of L. glutinosa