Chemical Composition of Natural Hydrolates and Their Antimicrobial Activity on Arcobacter-Like Cells in Comparison with Other Microorganisms

Abstract

Hydrolates obtained via the hydrodistillation and steam distillation of Lavandulaangustifolia Mill., Syzygiumaromaticum L., Foeniculumvulgare Mill., and Laurusnobilis L. were analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography coupled to mass spectrometry (GC-MS). Additionally, the hydrolates were evaluated for antimicrobial activity (disk-diffusion and microdilution method), influence on biofilm formation (Christensen method) and cytotoxicity of concentrated hydrolates against human cell lines (A549) by xCELLigence system. Using chemical analysis, 48, 9, 13 and 33 different components were detected in lavender, clove, fennel and laurel hydrolates, respectively. Lavender hydrolate contained the largest proportion of 1,8-cineol, linalool furanoxide, and linalool. The main components of laurel hydrolate were 1,8-cineol, 4-terpineol and α-terpineol. Fenchone and estragole were the most abundant in fennel hydrolate, and eugenol and eugenyl acetate in clove hydrolate. Concentrated hydrolates showed significant antimicrobial activity. Clove hydrolate was among the most antimicrobially active agents, most preferably against C. albicans, with an inhibition zone up to 23.5 mm. Moreover, concentrated hydrolates did not show any cytotoxic effect again8 st human A549 cells. In the presence of the non-concentrated hydrolates, significantly reduced biofilm formation was observed; however, with concentrated clove hydrolate, there was an increase in biofilm formation, e.g., of A. thereius, A. lanthieri, and A. butzleri. Research shows new findings about hydrolates that may be important in natural medicine or for preservation purposes.

Keywords: Arcobacter-like bacteria; Foeniculum vulgare; Laurus nobilis; Lavandula angustifolia; Syzygium aromaticum; antimicrobial activity; biofilm formation; distillation; gas chromatography; hydrolates.

Figure 1

Biofilm formation in the presence of non-concentrated hydrolates (a) and concentrated hydrolates (b). Bars represent standard deviation, n = 3. H_HD—hydrolate obtained by hydrodistillation; H_SD—hydrolate obtained by steam distillation; H_{HD_SPE}—hydrolate obtained by hydrodistillation and 50× concentrated using solid phase extraction (SPE); H_{SD_SPE}—hydrolate obtained by steam distillation and 50× concentrated using SPE. Red lines—biofilm formation of strains in water (a), biofilm formation of strains in extraction solvent (b); values above/below the red lines—increased/reduced biofilm formation due to hydrolate presence.

Figure 2

Dynamic monitoring of cytotoxic response to different concentrations of the hydrolates concentrated by SPE. A549 cells were treated with selected concentrations of (a) lavender, (b) fennel, (c) bay leaves and (d) clove concentrated hydrolates. Cells treated with 0.1%, 0.4% and 0.8% ethanol were used as vehicle control and 5% DMSO treated cells were as positive control. Arrow showing time-point of treatment. Cell index values over 72 consecutive treatment hours were normalized to the time point of treatment.