Local people in the Sudhnoti district of Pakistan share a rich practice of traditional medicine for the treatment of a variety of ailments. We selected nine plants from the Sudhnoti ethnopharmacological tradition used for the treatment of infectious and inflammatory disease. Our aim was to evaluate the in vitro anti-infective potential of extracts from these species against multidrug-resistant (MDR) ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Plant specimens were collected in the Sudhnoti district of Pakistan and vouchers deposited in Pakistan and the USA. Dried bulk specimens were ground into a fine powder and extracted by aqueous decoction and maceration in ethanol. Extracts were assessed for growth inhibitory activity against ESKAPE pathogens and biofilm and quorum sensing activity was assessed in Staphylococcus aureus. Cytotoxicity to human cells was assessed via a lactate dehydrogenase assay of treated human keratinocytes (HaCaTs). Four ethanolic extracts (Zanthoxylum armatum, Adiantum capillus-venaris, Artemisia absinthium, and Martynia annua) inhibited the growth of MDR strains of ESKAPE pathogens (IC50: 256 μg mL−1). All extracts, with the exception of Pyrus pashia and M. annua, exhibited significant quorum quenching in a reporter strain for S. aureus agr I. The ethanolic extract of Z. armatum fruits (Extract 1290) inhibited quorum sensing (IC50 32–256 μg mL−1) in S. aureus reporter strains for agr I-III. The quorum quenching activity of extract 1290 was validated by detection of δ-toxin in the bacterial supernatant, with concentrations of 64–256 μg mL−1 sufficient to yield a significant drop in δ-toxin production. None of the extracts inhibited S. aureus biofilm formation at sub-inhibitory concentrations for growth. All extracts were well tolerated by human keratinocytes (LD50 ≥ 256 μg mL−1). Chemical analysis of extract 1290 by liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) revealed the presence of 29 compounds, including eight with putative structural matches. In conclusion, five out of the nine selected anti-infective medicinal plants exhibited growth inhibitory activity against at least one MDR ESKAPE pathogen at concentrations not harmful to human keratinocytes. Furthermore, Z. armatum was identified as a source of quorum quenching natural products and further bioassay-guided fractionation of this species is merited.

Pseudomonas aeruginosa (P. aeruginosa), one of the dangerous multidrug resistance pathogens, orchestrates virulence factors production through quorum sensing (QS). Since the exploration of QS inhibitors, targeting virulence to circumvent bacterial pathogenesis without causing significant growth inhibition is a promising approach to treat P. aeruginosa infections. The present study has evaluated the anti-QS and anti-infective activity of epigallocatechin-3-gallate (EGCG), a bioactive ingredient of the traditional green tea, against P. aeruginosa. EGCG showed significant inhibitory effects on the development of biofilm, protease, elastase activity, swimming, and swarming motility, which was positively related to the production of C4-AHL. The expression of QS-related and QS-regulated virulence factors genes was also evaluated. Quantitative PCR analysis showed that EGCG significantly reduced the expression of las, rhl, and PQS genes and was highly correlated with the alterations of C4-AHL production. In-vivo experiments demonstrated that EGCG treatment reduced P. aeruginosa pathogenicity in Caenorhabditis elegans (C. elegans). EGCG increased the survival of C. elegans by 23.25%, 30.04%, and 36.35% in a dose-dependent manner. The findings of this study strongly suggest that EGCG could be a potential candidate for QS inhibition as an anti-virulence compound against bacterial infection.

Recently, an interest has surged in utilizing indigenous medicinal plants to treat infectious illnesses and extract bioactive substances, highlighting the need to analyze medicinal plants for phytochemicals and bioactivities. The present study was aimed to evaluate the impact of different solvent systems (aqueous, ethanol, and methanol) used for extraction on total phenolics, total flavonoids, antioxidant, and antibacterial activity of three medicinal plants of Azad Kashmir (Achillea millefolium, Bergenia ciliata, and Aloe vera). High phenolic content was found in methanol extracts of B. ciliata (27.48 ± 0.58 mg GAE/g dry weight), A. vera (25.61 ± 0.33 mg GAE/g dry weight), and A. millefolium (24.25 ± 0.67 mg GAE/g dry weight). High flavonoid content was obtained in the ethanol extract of A. millefolium (27.13 ± 0.64 mg QE/g dry weight), methanol extract of B. ciliata (17.44 ± 0.44 ± 0.44 mg QE/g dry weight), and the methanol extract of A. vera (14.68 ± 0.67 mg QE/g dry weight). Strong 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) was obtained with a methanol extract of B. ciliata (IC50 = 60.27 ± 0.20 µg/mL). With a zone of inhibition and a minimum inhibitory concentration ranging from 10.00 ± 0.66 to 24.67 ± 1.21 mm and 78 to 625 µg/mL, respectively, all of the studied plants demonstrated notable antibacterial activity against Staphylococcus aureus and Escherichia coli. A. vera showed greater antibacterial activity as compared to other plants under study while methanolic extract showed greater antibacterial activity than ethanolic and aqueous extract. The findings of this research support the use of these medicinal plants to treat a variety of diseases.