Vapours of essential oils and their constituents as in vitro inhibitors of pneumonia causing microorganisms

Ing. Markéta Houdková

Doctoral thesis

Abstract:

With aim to develop effective proof-of-concept approach, which can be used in a development of new preparations for inhalation therapy, we designed a new microdilution volatilization method for in vitro fast screening and simple simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmatTM as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. First, antibacterial activity of six plant volatile compounds, namely carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone, was determined against pneumonia causing bacteria (Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae) using optimized novel method and the cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5 (Medical Research Council cell strain 5). Thereafter, essential oils (EOs) hydrodistilled from seven Cambodian (Alpinia oxymitra, Boesenbergia rotunda, Cinnamomum cambodianum, Citrus lucida, Limnophila aromatica, Rhodamnia dumetorum, and Sindora siamensis) and six Philippine (Alpinia brevilabris, Alpinia cumingii, Alpinia elegans, Callicarpa micrantha, Cinnamomum mercadoi, and Piper quinqueangulatum) plant species were tested for their in vitro growth-inhibitory effect against vH. influenzaev, S. aureus, and S. pneumoniae with aim to verify practical usability of novel broth microdilution volatilization method. Additionally, MTT assay was performed for evaluation of their cytotoxic activity to human lung cells. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128 µg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86–2.95 µg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71 µg/mL, however this compound produced only weak antibacterial potency with MICs 512–1024 µg/mL. In case of EOs, all samples exposed some antibacterial efficacy; however, only A. oxymitra rhizome EO was active against all bacteria tested. A. oxymitra pericarp EO was found as the most effective antibacterial agent against H. influenzae in liquid and solid medium with the respective lowest MIC of 64 and 32 µg/mL. Due to its high value for 80% inhibitory concentration of proliferation (> 512 µg/mL), this EO may be considered as safe to human lung cell lines. Other EOs showed moderate or weak antibacterial effect with MIC values ranging from 128 to 1,024 µg/mL and from 64 to 1,024 µg/mL in liquid and vapour phase, respectively. Using dual-column/dual-detector system for gas chromatography-mass spectrometry (GC-MS) analysis of Cambodian EOs, β-pinene was identified as the main constituent of A. oxymitra leaves, pericarp and rhizome EOs, while volatile oil from A. oxymitra seeds consisted predominantly of shyobunol. The major constituents of B. rotunda, C. lucida, L. aromatica, R. dumetorum, and S. siamensis EOs were ocimene, decyl acetate, limonene, caryophyllene epoxide, and β-bourbonene, respectively. Chemical analysis of EOs from Philippine plant species determined hedycaryol, β-pinene, caryophyllene epoxide, aristolochene, borneol, and linalool were identified as the major components of A. brevilabris, A. cumingii, A. elegans, C. micrantha, C. mercadoi, and P. quinqueangulatum leaves EOs, respectively. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents (EOs and their constituents) without need of special apparatus. Based on results of growth-inhibitory and lung toxicity effects, A. oxymitra pericarp EO can be considered as an

Abstract:

effective antibacterial agent with application potential for the development of inhalation therapy against respiratory infections. However, further research focused on in vivo evaluation of pharmacological effects and toxicological safety, will be necessary before its possible practical use.

Keywords

cytotoxicity, GC-MS analysis, plant volatile compounds, respiratory infections, antibacterial activity

Language used: English

Date on which the thesis was submitted / produced: 12. 9. 2018

Thesis defence

• Supervisor: prof. Ing. Ladislav Kokoška, Ph.D.
• Reader: Pavel Klouček, externi, Jiří Killer, externi

Czech University of Life Sciences Prague

Faculty of Tropical AgriSciences

Doctoral programme / field:
Agricultural Specialization / Tropical and Subtropical Agriculture