The aim of this study was to investigate the antimicrobial property of the compounds present in the lichen Usnea albopunctata. Ethyl acetate extract of the lichen was purified by column chromatography to yield a major compound which was characterised by spectroscopic methods as protocetraric acid. In this study, protocetraric acid recorded significant broad spectrum antimicrobial property against medically important human pathogenic microbes. The prominent antibacterial activity was recorded against Salmonella typhi (0.5 μg/mL). Significant antifungal activity was recorded against Trichophyton rubrum (1 μg/mL), which is significantly better that the standard antifungal agent. Protocetraric acid is reported here for the first time from U. albopunctata. Thus the results of this study suggest that protocetraric acid has significant antimicrobial activities and has a strong potential to be developed as an antimicrobial drug against pathogenic microbes.
Candida albicans is a widespread disease-causing yeast affecting humankind, which leads to urinary tract, cutaneous and various lethal systemic infections. As this infection rate steadily increases, it is becoming a significant public health problem. Recently, Caesalpinia bonduc has received much attention from researchers due to its diverse pharmacological properties, including antimicrobial effects. Accordingly, we first planned to explore the in-vitro anticandidal potential of three extracts obtained from C. bonduc seeds against four Candida species. Initially, the anticandidal activity of the seed extracts was checked by the microdilution technique. Out of three seed extracts tested, ethanolic extract of C. bonduc seed (EECS) recorded the best activity against C. albicans. Hence, we next aimed to find out the anticandidal mechanism of EECS in C. albicans. The liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis showed that the major compounds present in the EECS were tocopherols, fucosterol, linoleic acid, β-amyrin, β-sitosterol, campesterol, cassane furanoditerpene, Norcassane furanoditerpene and other diterpenes. To evaluate the cell death mechanism in C. albicans, a series of parameters related to apoptosis, viz., reactive oxygen species (ROS) production, membrane permeability, mitochondrial membrane potential, release of cytochrome c, DNA fragmentation, nuclear condensation, increased Ca2+ level in cytosolic and mitochondrial and activation of metacaspase, were analyzed. The results showed that EECS treatment resulted in the elevation of ROS, which leads to plasma membrane permeability in C. albicans. Annexin V staining further confirms the early stage of apoptosis through phosphatidylserine (PS) externalization. We further inspected the late apoptotic stage using DAPI and TUNEL staining assays. From the results, it can be concluded that EECS triggered mitochondrial dysfunction by releasing high levels of ROS, cytochrome c and Ca2+resulting in the activation of metacaspase mediated apoptosis, which is the central mechanism behind the cell death of C. albicans. Finally, a Galleria mellonella-C. albicans infection system was employed to assess the in-vivo potential of EECS. The outcomes displayed that the EECS considerably enhanced the recovery rate of G. mellonella larvae from infection after the treatment. Additionally, EECS also recorded low hemolytic activity. This study thus spotlights the anticandidal potential and mechanism of action of EECS against C. albicans and thus delivers a promising treatment approach to manage C. albicans infection in the future.
Candida species are currently developing resistance to prevailing commercially available drugs, which raises an instantaneous need to discover novel antifungals. To cope with this shocking situation, phytochemicals are the richest, safest, and most potent source of excellent antimicrobials with broad-spectrum activity. The aim of the current study is to explore the anticandidal potential of the various fractions purified from the hydroalcoholic extract of C. bonduc seed. Out of five fractions purified from the hydroalcoholic extract, fraction 3 (Fr. 3) recorded the best activity against C. albicans (8 μg/mL) and thus this species was chosen for further mechanism of action studies. The phytochemical examination reveals that Fr. 3 was found to contain steroids and triterpenoids. This was further supported by LC-QTOF-MS and GCMS analyses. Our findings show that Fr. 3 targets the ergosterol biosynthesis pathway in C. albicans by inhibiting the lanosterol 14-α demethylase enzyme and downregulating expression of its related gene ERG11. Molecular docking outcomes disclosed favorable structural dynamics of the compounds, implying that the compounds present in Fr. 3 would be able to successfully bind to the lanosterol 14-α demethylase, as evidenced by the docked compounds’ strong interaction with the target enzyme’s amino acid residues. Considering virulence factors, the Fr. 3 recorded significant antibiofilm activity as well as germ-tube reduction potential. Furthermore, Fr. 3 enhances the production of intracellular reactive oxygen species (ROS). This suggests that the antifungal activity of Fr. 3 was associated with membrane damage and the induction of ROS production, resulting in cell death. Fluorescence microscopic analysis of PI stained Candida further showed changes in the plasma membrane permeability, which causes severe loss of intracellular material and osmotic balance. This was demonstrated by the potassium ion leakage and release of genetic materials. Finally, the erythrocyte lysis assay confirmed the low cytotoxicity of Fr. 3. Both in silico and in vitro results suggest that Fr. 3 has the potential to propel forward novel antifungal drug discovery programmes.
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