Chemical and Biological Investigations of Yaegl Medicinal Plants

<p dir="ltr">The Yaegl Country Aboriginal people of northern New South Wales, Australia, have used plants as medicines over many generations. This includes topical application of leaf preparations of <i>Alphitonia excelsa </i>to treat sores, wounds and skin infections, and use of water decoctions from <i>Smilax australis </i>leaves orally as a “blood cleanser” and for “diabetes”, and for antiseptic purposes. Building on previous investigations of the Indigenous Bioresources Research Group (IBRG) of Macquarie University, the aim of this research project was to investigate the phytochemistry and biological properties of these plants, in alignment with their customary uses by Yaegl Country people. Literature reviews were conducted on both the <i>Alphitonia </i>genus and <i>Smilax australis </i>and select other <i>Smilax </i>species to provide comprehensive accounts on their customary uses, phytochemistry and biological activities. Antibacterial, antioxidant and anti-inflammatory testing of <i>Alphitonia excelsa </i>and <i>Smilax australis </i>were conducted on extracts and partitions of these plants. This included the <i>n</i>-hexane, dichloromethane, ethyl acetate, methanol and water sequential extracts of <i>Alphitonia excelsa </i>leaves; the <i>n</i>-hexane, dichloromethane, ethyl acetate, and <i>n</i>-butanol sequential partitions of the 70% aqueous ethanol extracts of <i>Smilax australis</i>; and water extracts of leaves of both plants – at room temperature for <i>Alphitonia excelsa </i>and at room temperature and with boiling water for <i>Smilax australis</i>, to best mimic their customary uses. Both plants exhibited good antioxidant properties, with the ethyl acetate and direct water extracts of <i>Alphitonia excelsa </i>and all the partitions and water extracts of <i>Smilax australis </i>showing very strong antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. The ethyl acetate extract of <i>Alphitonia excelsa </i>and ethyl acetate partitions and room temperature direct water extract of <i>Smilax australis </i>also showed anti-inflammatory properties, significantly suppressing the secretion of the cytokine interleukin IL-1β. The direct water extract of <i>Smilax australis </i>additionally significantly suppressed the secretion of the cytokine Il-6. Limited antibacterial activity was observed for both plants, with <i>n</i>-hexane, dichloromethane, and ethyl acetate extracts of <i>Alphitonia excelsa </i>and the <i>n</i>-hexane, dichloromethane and ethyl acetate partitions and water extracts of <i>Smilax australis </i>showing low-moderate antibacterial activity against <i>Staphylococcus aureus </i>for the disc diffusion assay. For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, weak antibacterial activity was observed for both plants against <i>Staphylococcus aureus</i>. No antibacterial activity was observed for any of the extracts/partitions against <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa </i>or <i>Salmonella </i>ser. <i>typhimurium</i>. Phytochemical screening of the extracts and partitions for total phenolic, flavonoid and tannin contents identified that for all the extracts and partitions with strong antioxidant activity, there were significant levels of phenolic compounds present. The <i>n</i>-hexane and dichloromethane extracts and partitions of <i>Alphitonia excelsa </i>and <i>Smilax australis </i>were examined by gas chromatography-mass spectrometry (GC-MS) coupled with NIST and Wiley databases, and the ethyl acetate, methanol, <i>n</i>-butanol, water and direct water extracts were investigated by ultra-high-performance liquid chromatography coupled with electrospray ionisation-quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) and database searches. GC-MS analysis of the <i>Alphitonia excelsa n</i>-hexane and dichloromethane extracts, confirmed the presence of nonacosane, phytol, palmitic acid, lupeol and methyl linolenate in high abundances. These are medicinally significant compounds known for their anti-inflammatory, antioxidant and/or antimicrobial activities. None of these compounds have been previously reported in <i>Alphitonia excelsa</i>. For <i>Smilax australis, </i>GC-MS analyses of the <i>n</i>-hexane partitions confirmed the presence of ethyl linoleate, ethyl linolenate, ethyl palmitate, ethyl stearate, palmitic acid, phytol and β-sitosterol, with some variation between the leaves collected at different times across 2020-2022. For the dichloromethane partitions, eicosane, ethyl palmitate, lupeol, nonacosane, palmitic acid, phytol and β-sitosterol were identified, but there was considerable variability in their abundances across the different collections. The compounds identified are medicinally significant, having been reported for their anti-inflammatory, antioxidant and/or antimicrobial activities. None of these compounds have been previously reported in <i>Smilax australis</i>. For <i>Alphitonia excelsa</i>, the LC-MS analyses led to tentative identification of twenty-two metabolites classified into flavonoids, phenolic acids and triterpenoids; betulinic acid, kaempferol, protocatechuic acid, quercetin, rutin, and salicylic acid were confirmed with authentic standards. Eighteen metabolites were tentatively identified in <i>Smilax australis </i>and classified into flavonoids, and phenolic acids, with apigenin 7-<i>O</i>-neohesperidoside, catechin, chlorogenic acid, epicatechin, procyanidin B2, rutin and vitexin confirmed with authentic standards. Many of the compounds identified in both plants have been previously reported for their antioxidant, antibacterial and anti-inflammatory activities<i>. </i>Further bioassay guided isolation of the ethyl acetate extract of <i>Alphitonia excelsa </i>led to the isolation of two bioactive flavonoids, kaempferol and quercetin, which have been well documented for their antioxidant, antibacterial and anti-inflammatory activities<i>. </i>The findings of antioxidant, antimicrobial (albeit weak-moderate) and anti-inflammatory properties for extracts and solvent partitions from leaves of <i>Alphitonia excelsa </i>and <i>Smilax australis</i>, and the findings of some of these extracts and partitions being rich sources of antioxidant phenolics and other medicinally important compounds, aligns well with the customary uses of these two plants by Yaegl Country and other Aboriginal peoples and shows the significance of Aboriginal knowledge systems.</p>