Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays

Abstract

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Screening for Anticancer Activity of Leaf Ethanolic Extract of Alpinia elegans (“tagbak”) on Human Cancer Cell Lines

“…As of writing, only one other study has looked into the potential anti-cancer activity of A. elegans, which has been used as medicinal plant for hemoptysis and urticaria (Quisumbing, 1978) with evidence of anti-microbial properties (Oliveros and Bruce, 1991), and as natural insect pest maanagement in rice fields (Calumpang et al, 2013). Volatile plant-derived products from essential oil of A. elegans rhizomes have been shown to have cytotoxic activity in Caco-2 colon cancer cells (Houdkova et al, 2020). This study, the first done in A. elegans leaves, showed anti-cancer activity against A549 lung, HCT116 colorectal and HEPG2 hepatocellular carcinoma cells, although at high concentrations of crude leaf ethanolic extract.…”

Thymol Disrupts Cell Homeostasis and Inhibits the Growth of Staphylococcus aureus

“…All plates were placed in a humidified incubator at 37°C for 24/48 hours. Centrifugal 96-well plate and lower ambient temperature are helpful in eliminating steam influence on test results before test [ 43 ].…”

Characterization of Bioactivities and Biosynthesis of Angucycline/Angucyclinone Derivatives Derived from Gephyromycinifex aptenodytis gen. nov., sp. nov.

“…The main cultures were incubated with continuous shaking at 200 rpm on an orbital shaker (Model 5000I, VWR, Radnor, PA, USA), cultured at 28 • C for 10 days as previously described [11] for the pre-culture and sampled daily. For the enhancing experiments, glass beads (100 g/L, with diameters ranging from 100 to 2000 µm, Merck, Darmstadt, Germany) were added to the culture media to test the potential effects on the productivity of the active metabolites [37].…”

“…The antimicrobial activity was determined against two Gram-positive bacterial strains, namely, Staphylococcus aureus ATCC 12600 and Bacillus subtilis ATCC 6051, and one yeast Candida albicans ATCC 10231 using standard microplate assays [37,38]. For cytotoxic activity assay, three human-derived cancerous cell lines, namely, human leukemic cell line HL-60, human hepatocellular carcinoma cell line Bel-7402 and human lung cancer cell line A549, were used according to a standard MTT method [39] and performed in triplicate in a microplate reader.…”