Plantago squarrosa Murray extracts inhibit the growth of some bacterial triggers of autoimmune diseases: GC-MS analysis of an inhibitory extract

Ankylosing spondylitis, multiple sclerosis, rheumatoid arthritis and rheumatic fever are autoimmune inflammatory diseases that may be triggered in genetically susceptible individuals by specific bacterial pathogens. Inhibiting the growth of these bacteria with high antioxidant plant extracts may inhibit the aetiology of these diseases, as well as inhibiting the later phase symptoms. P. squarrosa extracts were analysed for antioxidant activity using a DPPH free radical scavenging assay. Bacterial growth inhibitory activity was evaluated using disc diffusion assays and the activity was quantified by MIC determination. The extracts were screened for toxicity by A. franciscana nauplii assays. The most potent antibacterial extract (ethyl acetate) was analysed by GC-MS headspace profile analysis and compounds were identified with reference to a phytochemical database. All extracts displayed strong DPPH radical scavenging activity. The ethyl acetate extract was particularly potent (IC₅₀ 1.4 µg/mL), whilst the other extracts also had significant radical scavenging activity (IC₅₀ values between 11 and 22 µg/mL). Notably, the bacterial growth inhibitory activity of the extracts correlated with their DPPH radical scavenging activity. The ethyl acetate extract, which had the greatest DPPH scavenging activity, generally displayed the most potent bacterial growth inhibitory activity. This extract was particularly potent against P. mirabilis, P. vulgaris and A. baylyi (MIC values of 484, 575 and 880 µg/mL, respectively). It also inhibited P. aeruginosa and S. pyogenes growth, albeit with higher MICs (1600-3700 µg/mL). All other extract-bacteria combinations were either inactive or resulted in mid-low potency inhibition. All extracts were non-toxic in the A. franciscana bioassay (LC₅₀ substantially > 1000 µg/mL). In total, 89 unique mass signals were identified in the P. squarrosa ethyl acetate extract by non-biased GC-MS headspace analysis. A number of compounds which may contribute to the antibacterial activity of this extract have been highlighted.