In Vitro Growth-Inhibitory Effect of Cambodian Essential Oils Against Pneumonia Causing Bacteria in Liquid and Vapour Phase and Their Toxicity to Lung ﬁbroblasts

South African Journal of Botany 118 (2018) 85–97

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South African Journal of Botany

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In vitro growth-inhibitory effect of Cambodian essential oils against pneumonia causing bacteria in liquid and vapour phase and their toxicity to lung ﬁbroblasts

M. Houdkova a,K.Urbanovab, I. Doskocil c, J. Rondevaldova a,P.Novyd,S.Nguone,R.Chrunf,L.Kokoskaa,⁎ a Department of Crop Sciences and Agroforestry, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Praha 6 – Suchdol, Czech Republic b Department of Sustainable Technologies, Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Praha 6 – Suchdol, Czech Republic c Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Praha 6 – Suchdol, Czech Republic d Department of Quality of Agricultural Products, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamycka 129, 165 21 Praha 6 – Suchdol, Czech Republic e Department of Food Processing, Faculty of Agriculture and Food Processing, University of Battambang, National Rd 5, 02352-Sangkat Praek Preah Sdach, Battambang City, Cambodia f Department of Food Biotechnology, Faculty of Agro-Industry, Royal University of Agriculture, P.O. Box 2696, Khan Dangkor, Phnom Penh, Cambodia article info abstract

Article history: Essential oils hydrodistilled from seven Cambodian plant species (Alpinia oxymitra, Boesenbergia rotunda, Received 6 February 2018 Cinnamomum cambodianum, Citrus lucida, Limnophila aromatica, Rhodamnia dumetorum,andSindora siamensis) Received in revised form 26 April 2018 were tested for their in vitro growth-inhibitory effect against pneumonia causing bacteria (Haemophilus Accepted 6 June 2018 influenzae, Streptococcus pneumoniae, Staphylococcus aureus) using the broth microdilution volatilisation method. Available online xxxx Additionally, a modified thiazolyl blue tetrazolium bromide assay was performed for evaluation of their cytotoxic fi Edited by S Van Vuuren activity to human lung cells. All essential oils exposed some antibacterial ef cacy; however, only A. oxymitra rhizome oil was active against all bacteria tested. A. oxymitra pericarp oil was found as the most effective Keywords: antibacterial agent against H. influenzae in liquid and solid medium with the respective lowest minimum inhib- Antibacterial activity itory concentrations of 64 and 32 μg/mL. Due to its high value for 80% inhibitory concentration of proliferation Broth microdilution volatilisation (N512 μg/mL), this essential oil may be considered as safe to human lung cell lines. Using dual-column/dual- Cytotoxicity detector system GC/MS analysis, β-pinene was identified as the main constituent of A. oxymitra leaves, pericarp GC/MS analysis and rhizome oils, while volatile oil from A. oxymitra seeds consisted predominantly of shyobunol. The major Plant volatiles constituents of B. rotunda, C. lucida, L. aromatica, R. dumetorum,andS. siamensis oils were ocimene, decyl acetate, Vapour limonene, caryophyllene epoxide, and β-bourbonene, respectively. 1,8-cineole was the major compound of C. cambodianum bark and leaf essential oils. Based on these results, A. oxymitra pericarp oil can be considered as an effective antibacterial agent with application potential for the development of inhalation therapy against respiratory infections. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction antibiotic therapy can considerably reduce fatal cases of pneumonia (Sazawal and Black, 2003), nevertheless many low-income countries Pneumonia belongs to the leading causes of morbidity and mortality, have limited access to health services and synthetic drugs as well, especially in low-income countries. The majority of severe episodes oc- whereas less than 40% of children are treated with antibiotics in curs in children under ﬁve years, the elderly and immuno-compromised Cambodia (WHO, 2017). individuals (Nguyen et al., 2017). In Cambodia, about 9100 children die The plant essential oils are of great potential for the development of from pneumonia every year (Ginsburg et al., 2014) and according to the novel antimicrobial preparations. They have been widely used for their World Health Organisation (WHO) (WHO, 2017) only 64.2% of children diverse biological effects since the Middle Ages (Bakkali et al., 2008). with pneumonia symptoms are taken to an appropriate healthcare pro- Since the presence of volatile compounds is characteristic for some vider. This acute respiratory infection of lung parenchyma is caused plant taxa, chemotaxonomic research is a frequent approach to their by bacterial pathogens such as Haemophilus inﬂuenzae, Streptococcus exploration. Due to the volatility of essential oils, they are suitable for in- pneumoniae,andStaphylococcus aureus (Mandell, 2015). A timely halation therapy, which is an effective way for the healing of respiratory ailments such as pneumonia. Their vapours can act directly on the site of ⁎ Corresponding author. infection in the respiratory system and simultaneously restrict systemic E-mail address: [email protected] (L. Kokoska). exposure, degradation of active components in the gastrointestinal tract

https://doi.org/10.1016/j.sajb.2018.06.005 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved. 86 M. Houdkova et al. / South African Journal of Botany 118 (2018) 85–97 andassociatedtoxicity(Kuzmov and Minko, 2015). In addition, essen- R. dumetorum,andS. siamensis were collected from various districts of tial oils contain a broad spectrum of chemically diverse substances Cambodia (Cardamom Mountains, Elephant Mountains, Chant Saen with antimicrobial effect: thus it is more difficult for bacterial pathogens Commune in Oudong District) from wild populations of at least three to develop resistance to these multi component mixtures than to single- independent plants. B. rotunda, C. lucida,andL. aromatica were pur- ingredient conventional antibiotics (Yang et al., 2015). During the last chased in local markets (Psar Thmei, Chbar Ampov, and Cham Kar few years, several inhalation devices and suitable delivery systems for Dong in Phnom Penh). Identification of species was performed in the essential oils in the treatment of respiratory infections (e.g. pocket field by ethnobotany expert Prof Ladislav Kokoska, currently head of inhaler, aromatherapy patch, decongestant on a foraminous carrier, the Laboratory of Ethnobotany and Ethnopharmacology of the Faculty and encapsulated essential oils) have been developed and patented of Tropical AgriSciences, Czech University of Life Sciences Prague. (Horvath and Acs, 2015). Voucher specimens were deposited in the herbarium of the Department In Cambodia, after several decades of human destruction and the of Botany and Plant Physiology of the Faculty of Agrobiology, Food and collapse of all social welfare systems during the Pol Pot regime, medic- Natural Resources of the Czech University of Life Sciences Prague inal plants are considered as a very important factor for health security, (Czech Republic). A detailed description of collected plant samples is and traditional Khmer herbal medicine remains the oldest and the summarised in Table 1. most accessible source of primary health care (Bith-Melander and Efird, 2008). Cambodia also possesses rich natural resources and unique 2.2. Preparation of essential oils original ecosystems e.g. the Cardamom Mountains, which contain a number of endemic plant taxa belonging to essential oil-bearing fami- Essential oils were obtained by hydrodistillation of dried plant mate- lies such as Zingiberaceae, Lauraceae and Myrtaceae (Chassagne et al., rial (except C. lucida fruit peel which was obtained by the grating 2016). Nevertheless, scientific validation and identification of many of fresh fruits using a stainless steel grater) in 1 L of distilled water for Cambodian medicinal plants, as well as assessment of their anti- 3 h using a Clevenger-type apparatus (Merci, Brno, CZ) according to infective properties, active substances content and safety, are desirable. the procedures described in the European pharmacopoeia (2013).The Recently, several in vitro studies have investigated biological activity and essential oils were stored in sealed glass vials at 4 °C. The data on yields revealed some antibacterial potential of essential oils derived from dif- (v/w, based on the dry plant weight) of obtained essential oils are ferent parts of Cambodian plant species that are easily available in tradi- shown in Table 1. tional markets and in wild nature (Norajit et al., 2007; Phanthong et al., 2013). However, no experiments determining their antibacterial poten- 2.3. Bacterial strains and culture media tial in vapour phase against pathogens causing pneumonia had been carried out until now. The following standard strains of the American Type Culture In this article, we report a detailed examination of in vitro growth- Collection (ATCC) were used: Haemophilus influenzae ATCC 49247, inhibitory effect of essential oils from seven Cambodian medicinal and Staphylococcus aureus ATCC 29213, and Streptococcus pneumoniae ATCC edible plant species against pneumonia causing bacteria in liquid and 49619. The cultivation and assay media (broth/agar) were Mueller- vapour phase by using a new broth microdilution volatilisation method Hinton (MH) complemented by yeast extract and Haemophilus Tested recently developed by our team (Houdkova et al., 2017). This is the Medium (H. influenzae), MH (S. aureus), and Brain Heart Infusion first practical application of this novel method in the field of essential (S. pneumoniae). The pH of broths was equilibrated to a final value of oils. Additionally, the cytotoxicity and chemical composition of tested 7.6 using Trizma base (Sigma-Aldrich, Prague, CZ). All microbial strains essential oils were analysed with the aim of assessing the relationship and cultivation media were purchased from Oxoid (Basingstoke, UK). between their antimicrobial potential, chemistry and safety for treat- Stock cultures of bacterial strains were cultivated in appropriate me- ment of pneumonia. dium at 37 °C for 24 h prior to the testing and then the turbidity of the bacterial suspension was adjusted to 0.50 McFarland standard using 2. Materials and methods Densi-La-Meter II (Lachema, Brno, CZ) to get the final concentration of 107 CFU/mL. The susceptibilities of H. influenzae, S. aureus,and 2.1. Plant material S. pneumoniae to ampicillin (84.5%, CAS 69-52-3), oxacillin (86.3%, CAS 7240-38-2) and amoxicillin (90%, CAS 26787-78-0), respectively, pur- Based on chemotaxonomic criteria, seven local plant species (Alpinia chased from Sigma-Aldrich (Prague, CZ), were checked as positive anti- oxymitra K. Schum., Boesenbergia rotunda (L.) Mansf., Cinnamomum biotic controls (CLSI, 2015). cambodianum Lecomte, Citrus lucida (Scheff.) Mabb., Limnophila aromatica (Lam.) Merr., Rhodamnia dumetorum (DC.) Merr. & L.M. Perry, Sindora 2.4. Antimicrobial assay siamensis Miq.) were selected as phytochemically less explored repre- sentatives of taxa containing essential oils. The plant material was col- The antibacterial potential of plant essential oils in liquid and va- lected between July and September 2016. A. oxymitra, C. cambodianum, pour phase was determined using a broth microdilution volatilisation

Table 1 Plant species selected for antibacterial and cytotoxicity testing.

Scientiﬁc name Family Collection number Area of collection Part used Weight of Essential oil Essential oil sample (g) yield % (v/w) colour

Alpinia oxymitra K. Schum. Zingiberaceae 02463KBFR6 Mt Aoral Leaves 49.00 0.35 Colourless Pericarp 12.30 0.32 Pale yellow Rhizomes 125.40 0.04 Colourless Seeds 21.90 4.65 Colourless Boesenbergia rotunda (L.) Mansf. Zingiberaceae – Chbar Ampov Rhizomes 109.90 0.34 Colourless Cinnamomum cambodianum Lecomte Lauraceae 02455KBFR7 Mt Aoral Bark 44.50 0.45 Pale yellow Leaves 41.70 0.38 Pale yellow Citrus lucida (Scheff.) Mabb. Rutaceae 02476KBFRA Cham Kar Dong Fruit peel 74.50 0.43 Pale yellow Limnophila aromatica (Lam.) Merr. Plantaginaceae 02469KBFRC Psar Thmei Aerial part 13.80 1.20 Slightly yellow Rhodamnia dumetorum (DC.) Merr. & L.M.Perry Myrtaceae 02458KBFRA Oudong Leaves 164.80 0.18 Bright yellow Sindora siamensis Miq. Leguminosae 02481KBFR6 Angkor Chey Fruit husk 44.00 0.45 Pale yellow M. Houdkova et al. / South African Journal of Botany 118 (2018) 85–97 87 method (Houdkova et al., 2017). The experiments were performed in μg/mL. The levels of cytotoxic effect were classified according to the standard Nunclon 96-well microtiter plates (well volume = 400 μL), Special Programme for Research and Training in Tropical Diseases covered by tight-fitting lids with flanges designed to reduce evapora- (WHO – Tropical Diseases) as cytotoxic (IC50 b 2.00 μg/mL), moderately tion (Thermo Scientific, Roskilde, DK). Initially, 30 μL of agar was pipet- cytotoxic (IC50 2.00–89.00 μg/mL), and non-toxic (IC50 N 90.00 μg/mL). ted into every flange on the lid except the outermost flanges and Furthermore, 80% inhibitory concentration of proliferation (IC80) was inoculated with 5 μL of bacterial suspension after agar solidification. calculated as equivalent to MIC endpoint usually defined as 80% bacte- In the second part of this method, each sample of volatile oils was rial growth inhibition (Kokjohn et al., 2003) and therapeutic indices dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich, Prague, CZ) (TI), defined as the ratio of IC50 or IC80 and MIC values, were determined at maximum concentration of 1%, and diluted in an appropriate broth with the aim of comparing the amount of effective antibacterial agents medium. Seven two-fold serially diluted concentrations of samples with the quantity causing toxicity (Trevor et al., 2015). starting from 1024.00 μg/mL were prepared for all essential oils. The final volume in each well was 100 μL. The plates were then inoculated 2.7. Gas chromatography–mass spectrometry analysis (GC/MS) with bacterial suspension using a 96-pin multi-blot replicator (National Institute of Public Health, Prague, CZ). The wells containing in- For determination of the main components of essential oils tested, oculated and non-inoculated broth were prepared as growth and purity GC/MS analysis was carried out using the dual-column/dual-detector controls simultaneously. The outermost wells were left empty to pre- gas chromatograph system Agilent GC-7890B equipped with autosampler vent edge effect. Finally, clamps (Lux Tool, Prague, CZ) were used for Agilent 7693, two columns, a fused-silica HP-5MS column (30 m × fastening the plate and lid together, with the handmade wooden pads 0.25 mm, film thickness 0.25 μm, Agilent 19091s-433) and a DB-17MS (size 8.5 × 13 × 2 mm) for better fixing and the microtiter plates were column (30 m × 0.25 mm, film thickness 0.25 μm, Agilent 122–473), incubated at 37 °C for 24 h. The minimum inhibitory concentrations and a flame ionisation detector (FID) coupled with single quadrupole (MICs) were evaluated by visual assessment of bacterial growth after mass selective detector Agilent MSD-5977B (Agilent Technologies, colouring of a metabolically active bacterial colony with thiazolyl blue Santa Clara, CA, USA). The operational parameters were: helium as car- tetrazolium bromide dye (MTT) in a concentration of 600.00 μg/mL rier gas at 1 mL/min, injector temperature 250 and 200 °C for HP-5MS (Sigma-Aldrich, Prague, CZ) when the interface of colour change from and DB-17MS, respectively. The oven temperature was raised from 50 yellow to purple (relative to that of colours in control wells) was re- to 300 °C for HP-5MS and from 50 to 280 °C for DB-17MS. Samples of es- corded in broth and agar. The MIC values were determined as the lowest sential oils diluted in n-hexane for GC/MS (Merck KGaA, Darmstadt, DE) concentrations that inhibited bacterial growth compared with the at concentration 1 μg/mL and 1 μL of solution was injected in splitless compound-free control and expressed in μg/mL (in the case of vapour mode. The mass detector was set to following conditions: ionisation phase also in μg/cm3, where 256.00, 128.00, 64.00, 32.00, 16.00, 8.00, energy 70 eV, ion source temperature 200 °C, scan time 1 s, mass 4.00, and 2.00 μg/cm3 are real values for 1024.00, 512.00, 256.00, range 30–600 m/z. 128.00, 64.00, 32.00, 16.00, and 8.00 μg/mL, respectively). The DMSO The identification of constituents was based on comparison of assayed as the negative control at concentration of 1% did not inhibit their retention indices (RI) and retention times (RT) with the National any of the strains tested either in broth or agar media. All experiments Institute of Standards and Technology Library ver. 2.0.f (NIST, USA), as were carried out in triplicate in three independent experiments and re- well as authentic standards (Sigma-Aldrich, Prague, CZ) and literature sults were expressed as median/modal MICs values. (Adams, 2007).TheRIwerecalculatedforcompoundsseparatedbythe HP-5MS column using the retention times of n-alkanes series ranging 2.5. Cell culture from C9 to C29 (Sigma-Aldrich, Prague, CZ). For each essential oil analysed, the final number of compounds was calculated as the sum of Primarily, lung fibroblast cells MRC-5 obtained from ATCC components simultaneously identified using both columns and the re- (Manassas, VA, USA), were propagated in Eagle's Minimum Essential maining constituents identified by individual columns only. The relative Medium (EMEM) supplemented with 10% foetal bovine serum (FBS), percentage contents of essential oil components were determined by FID 2mMglutamine,10.00μL/mL non-essential amino acids, and 1% and indicated for both columns. penicillin–streptomycin solution (10,000.00 units/mL of penicillin and 10.00 mg/mL of streptomycin), all these components purchased from 3. Results Sigma-Aldrich (Prague, CZ). The cells were preincubated in 96-well microtiter plates at a density of 2.5 × 103 cellsperwellfor24hat37°Cina In this study, eleven essential oils derived from different parts of humidified incubator in an atmosphere of 5% CO2 in air. seven Cambodian medicinal and edible plant species were obtained in yields ranging from 0.04 to 1.20% (v/w). All essential oils exhibited a cer- 2.6. Cytotoxicity assay tain degree of antibacterial activity in liquid and vapour phase against at least one bacteria associated with respiratory system infections. The modified method for toxicity assessment of volatile agents The results of their in vitro growth-inhibitory effect against three bacte- (Houdkova et al., 2017) based on metabolization of MTT to blue rial strains using the broth microdilution volatilisation method are formazan by mitochondrial dehydrogenases in living lung cells previ- summarised in Table 2. The MTT assay performed with the lung fibro- ously described by Mosmann (1983) was used. At first, twelve two- blast cells showed the potential safety of certain essential oils. The results fold serially diluted concentrations ranging from 512.00 to 0.25 μg/mL of cytotoxicity assay are listed in Table 3. Based on GC/MS analysis, it was of essential oils dissolved in DMSO were prepared. The microtiter found that monoterpenoids and sesquiterpenoids were the leading plates were covered by EVA capmats™ at 37 °C in a humidified atmo- compounds present in the essential oils tested. The complete chemical sphere of 5% CO2 in air and cultivated for 72 h. Thereafter, MTT reagent composition is provided in Table 4. (1.00 mg/mL) in EMEM solution was added to each well and plates were incubated for an additional 2 h under the same conditions. The 3.1. Antibacterial activity media were removed and the intracellular formazan product was dissolved in 100 μL of DMSO. The solvent used did not affect the viability All essential oils tested in this study exposed some antibacterial of the lung cells at the concentrations tested. The absorbance was mea- efficacy; however, only the essential oil from A. oxymitra rhizomes sured at 555 nm and the percentage of viability was calculated when was active against all bacteria tested. In general, the effectiveness of es- compared to an untreated control. The results of the cytotoxicity effect sential oils varied substantially ranging from 64.00 to 1024.00 μg/mL in were expressed as half maximal inhibitory concentration (IC50)in broth and from 32.00 to 1024.00 μg/mL on agar. 88 M. Houdkova et al. / South African Journal of Botany 118 (2018) 85–97

Table 2 Antibacterial activity of essential oils and antibiotics in liquid and vapour phase against pneumonia causing bacteria.

Bacteria/grown medium/MIC

Haemophilus inﬂuenzae Staphylococcus aureus Streptococcus pneumoniae ATCC 49247 ATCC 29213 ATCC 49619

Broth Agar Broth Agar Broth Agar

Essential oils Part used (μg/mL) (μg/mL) (μg/cm3)(μg/mL) (μg/mL) (μg/cm3)(μg/mL) (μg/mL) (μg/cm3)

Alpinia oxymitra Leaves 512.00 512.00 128.00 1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Pericarp 64.00 32.00 8.00 512.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Rhizomes 128.00 64.00 16.00 256.00 N1024.00 N256.00 128.00 N1024.00 N256.00 Seeds 512.00 256.00 64.00 1024.00 1024.00 256.00 N1024.00 N1024.00 N256.00 Boesenbergia rotunda Rhizomes N1024.00 1024.00 256.00 N1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Cinnamomum cambodianum Bark N1024.00 1024.00 256.00 N1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Leaves 1024.00 512.00 128.00 N1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Citrus lucida Fruit peel 512.00 256.00 64.00 N1024.00 N1024.00 N256.00 N1024.00 1024.00 256.00 Limnophila aromatica Aerial part N1024.00 1024.00 256.00 N1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Rhodamnia dumetorum Leaves 1024.00 1024.00 256.00 N1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00 Sindora siamensis Fruit husk 256.00 256.00 64.00 1024.00 N1024.00 N256.00 N1024.00 N1024.00 N256.00

Positive antibiotic control Amoxicillin ––––––0.25 ND ND Ampicillin 0.5 0.25 0.0625 –––––– Oxacillin –––0.25 ND ND –––

MIC: minimum inhibitory concentration; ND: not determined; −:nottested.

In liquid phase, the lowest MIC value was observed for A. oxymitra C. cambodianum bark, and L. aromatica oils were effective against pericarp (64.00 μg/mL) against H. influenzae,followedbyA. oxymitra rhi- H. influenzae in vapour phase, whereas no activity has been observed zomes with MIC 128.00 μg/mL against H. influenzae and S. pneumoniae. in broth. Similarly, S. pneumoniae was susceptible to C. lucida oilinva- Moderate antibacterial activity was produced by A. oxymitra leaves pour phase only. Opposite results were found in the case of volatile and seeds, C. lucida,andS. siamensis with MICs ranging from 256.00 to oils from A. oxymitra leaves, pericarp, rhizomes, and S. siamensis,which 512.00 μg/mL. C. cambodianum leaves and R. dumetorum possessed inhibited growth of S. aureus and S. pneumoniae in broth medium with- only weak inhibitory effect (1024 μg/mL). No antibacterial activity in out effect in vapour phase. broth medium was determined for B. rotunda, C. cambodianum bark, and L. aromatica. 3.2. Cytotoxicity As well as in broth, A. oxymitra pericarp was the most effective antibacterial agent against H. influenzae in gaseous phase with MIC Values of IC50 and IC80 for lung fibroblasts varied substantially 32.00 μg/mL. In addition, A. oxymitra rhizomes effectively inhibited in ranges 1.98–225.35 μg/mL and 10.93–395.55 μg/mL, respectively. growth of H. influenzae on agar medium at concentration 64.00 μg/mL. In the case of A. oxymitra pericarp oil, IC80 value was not detected Other essential oils, A. oxymitra leaves and seeds, C. cambodianum leaves, (IC80 N 512.00 μg/mL). The comparison of MIC and IC80 values suggests C. lucida, and S. siamensis possessed moderate antibacterial activity in A. oxymitra pericarp oil as an effective antimicrobial agent that is non- vapour phase with MICs ranging from 256.00 to 512.00 μg/mL. A low toxic to lung fibroblasts. inhibitory activity was observed for B. rotunda, C. cambodianum bark, A. oxymitra seeds, C. lucida,andC. cambodianum bark and leaves

L. aromatica and R. dumetorum against H. influenzae (1024.00 μg/mL). were found to be non-toxic to lung cells with respective IC50 values All essential oils affected growth of H. influenzae on agar, whereas 225.35, 182.52, 133.94, and 112.16 μg/mL. Moderately toxic were essen- S. aureus and S. pneumoniae were inhibited only by A. oxymitra seeds tial oils of other parts of A. oxymitra, B. rotunda, L. aromatica, and and C. lucida oils, respectively. S. siamensis (IC50 value ranging from 5.98 to 29.96 μg/mL). R. dumetorum In Table 2, it can be seen that some essential oils showed significant appeared to be the most cytotoxic with IC50 value 1.98 μg/mL. differences in liquid and vapour phase MICs. Their two times lower Similarly to IC50, in the case of IC80 the lowest cytotoxicity was values on agar than in broth were determined for essential oils observed for essential oils of A. oxymitra pericarp, C. cambodianum bark, from A. oxymitra pericarp, rhizomes and seeds, C. cambodianum leaves, A. oxymitra seeds, C. lucida, C. cambodianum leaves, and B. rotunda with and C. lucida fruit peel against H. influenzae. In addition, B. rotunda, IC80 values N512.00, 395.90, 391.55, 380.30, 364.34, and 106.34 μg/mL, respectively. Moderate toxicity was recorded for L. aromatica,

Table 3 A. oxymitra leaves and rhizomes, S. siamensis, and R. dumetorum (IC80 Cytotoxicity of essential oils in the lung ﬁbroblast cells. value range 10.93–50.78 μg/mL).

Essential oils Part used IC ±SD(μg/mL) IC ±SD(μg/mL) 50 80 3.3. GC/MS analysis Alpinia oxymitra Leaves 7.05 ± 1.32 41.54 ± 11.69 Pericarp 29.96 ± 0.00 N 512.00 In the essential oils of A. oxymitra leaves, pericarp, rhizomes, and Rhizomes 7.06 ± 0.87 16.17 ± 4.55 ﬁ Seeds 225.35 ± 37.42 391.55 ± 13.4 seeds, a total of 56, 77, 52, and 62 compounds were identi ed Boesenbergia rotunda Rhizomes 22.09 ± 6.87 106.34 ± 13.43 using both HP-5MS/DB-17MS columns, representing 93.69/92.58, Cinnamomum cambodianum Bark 133.94 ± 12.92 395.90 ± 14.51 88.05/90.96, 91.68/91.13, and 95.00/93.58% of their total contents, Leaves 112.16 ± 58.97 364.34 ± 27.44 respectively. The analyses showed that the major constituents of Citrus lucida Fruit peel 182.52 ± 1.99 380.30 ± 1.43 Limnophila aromatica Aerial part 16.62 ± 5.63 50.78 ± 2.54 A. oxymitra leaves, pericarp, and rhizomes oils were monoterpenoids Rhodamnia dumetorum Leaves 1.98 ± 1.17 10.93 ± 0.29 and sesquiterpenoids in respective total contents 76.16/75.90, 55.39/ Sindora siamensis Fruit husk 5.98 ± 2.65 15.99 ± 5.69 58.55, 84.55/88.41, and 17.34/16.60, 27.48/29.03, 2.52/2.56%, whereas in A. oxymitra seeds essential oil, sesquiterpenoids were the most IC50: half-maximal inhibitory concentration of proliferation in μg/ml; IC80: 80% inhibitory concentration of proliferation in μg/mL; SD: standard deviation. dominant components (88.51/85.07%). In A. oxymitra leaves, β-pinene M. Houdkova et al. / South African Journal of Botany 118 (2018) 85–97 89

(58.40/59.06%) was the main compound followed by caryophyllene ep- 4. Discussion oxide (6.07/5.61%), α-pinene (4.79/5.01%), caryophyllene (2.98/3.03%), and myrtenol (2.66/2.52%). Furthermore, a relatively high amount of From a chemotaxonomic point of view, Lauraceae, Myrtaceae, sabinene (2.59/3.41%) was detected. Similarly, the essential oil from Rutaceae, and Zingiberaceae are typical families containing essential A. oxymitra pericarp consisted chieflyofβ-pinene (29.75/31.31%), oils with antimicrobial properties (Joshi et al., 2010; Victorio, 2011; caryophyllene epoxide (20.18/19.70%), myrtenol (5.48/5.18%), trans- Raut and Karuppayil, 2014). In correspondence with this, the essential pinocarveol (4.71/3.59%), and pinocarvone (3.65/4.35%). Then perillol oils from A. oxymitra and B. rotunda (Zingiberaceae), C. cambodianum was one of the main compounds found only by DB-17MS column (Lauraceae), C. lucida (Rutaceae), L. aromatica (Plantaginaceae), (5.03%). In A. oxymitra rhizomes oil, β-pinene (50.23/53.22%) prevailed R. dumetorum (Myrtaceae), and S. siamensis (Leguminosae) were ob- as well, followed by o-cymene (8.79/19.99%), α-pinene (5.10/5.58%), tained using hydrodistillation, whereas the oils from A. oxymitra leaves, and myrtenol (3.31/1.22%). 1,8-cineole was detected present also pericarp and seeds, C. lucida fruit peel, R. dumetorum leaves, and in a significant amount by HP-5MS column only (5.17%). In contrast to S. siamensis fruit husk were isolated for the first time. essential oils derived from other parts of A. oxymitra,sesquiterpenoids As far as antibacterial activity of plant species tested in this study is shyobunol (21.65/4.15%), germacrene D (16.45/16.28%), cubebol considered, B. rotunda essential oil is the only one previously tested for (16.21/16.28%), 6-epi-shyobunol (9.78/12.44%), and germacrene-D- its antimicrobial effect. It exhibited growth-inhibitory activity with MIC 4-ol (8.43/7.54%) were the main components of seed volatile oil. More- value 12,500.00 μg/mL against S. aureus (Norajit et al., 2007). In the case over, the presence of aromadendrene (4.73%) was found by DB-17MS of L. aromatica, our results can be supported by studies of Visutthi column. (2016) who described growth-inhibitory action of its methanolic/ The analysis of C. cambodianum revealed the same number of com- ethanolic extracts against various strains of S. aureus (MIC values rang- ponents 76 for both bark and leaf oils, representing 95.07/91.85 ing 2500.00–2600.00 μg/mL). Similarly, as in our previous research and 90.70/92.77% of total volatiles content, respectively. The oils (Houdkova et al., 2017), different MIC values of essential oils were re- mainly contained monoterpenoids (80.54/70.11 and 59.85/62.88%) corded in broth and agar medium, which could be caused by the diverse followed by sesquiterpenoids (14.44/21.19, 30.10/29.07%). 1,8-cineole volatility of samples tested (Kusumoto and Shibutani, 2015), as well as was the major compound in both bark and leaf oils (31.80/33.46, by varying levels of bacterial sensitivity in liquid and solid medium 30.54/33.09%). Other components of C. cambodianum bark oil were (Jiang et al., 2013). According to our best knowledge, this is the first re- α-terpineol (9.52/7.95%), α-pinene (8.79/5.89%), borneol (5.71/4.97%), port on antibacterial activity of A. oxymitra, C. cambodianum, C. lucida, terpinen-4-ol (5.39/2.47%), and β-spathulenol (5.32% on DB-17MS L. aromatica, R. dumetorum,andS. siamensis essential oils. Moreover, column). The essential oil of C. cambodianum leaves was also rich in the growth-inhibitory effects of essential oils from all seven species espathulenol (7.58/8.18%), α-terpineol (6.54/6.44%), β-spathulenol were assessed for the first time in vapour phase in this study. (5.84/3.60%), and linalool (4.66/4.16%). Although there is a lack of data on antibacterial activity of the above- In B. rotunda and L. aromatica oils, 36 and 47 compounds were iden- mentioned essential oils, several studies reporting the antibacterial tified that represent 98.62/98.20 and 97.52/95.72% of their total respec- potential of their main components have recently been published. tive contents. The essential oil of B. rotunda was composed especially For example, a significant growth-inhibitory effect of α-andβ-pinenes, of monoterpenoids that were present in the amount of 96.65/96.02%. constituents of R. dumetorum and A. oxymitra leaves, pericarp, and rhi- The most abundant component was ocimene (27.99/27.62%), followed zomes, has previously been reported against methicillin-resistant by geraniol (24.41/24.04%), d-camphor (18.56/19/12%), 1,8-cineole S. aureus with respective MIC values 4.15 and 6.25 μg/mL (da Silva (5.50/5.58%), and camphene (5.44/5.47%). Similarly, monoterpenoids et al., 2012). Ocimene, geraniol, and camphor, dominant monoterpenoids was the predominant class of compounds in L. aromatica oil with total of B. rotunda oil, also exhibited growth-inhibitory effect against various contents 85.30/81.56%. Sesquiterpenoids were also represented in sig- bacterial pathogens (Chen and Viljoen, 2010; Iscan, 2017). Moreover, nificant amounts (10.60/13.00%) with the main component being limo- the antibacterial potential of geraniol and camphor vapours was deter- nene (48.31/48.08%), followed by 3-p-menthen-7-al (20.08/20.26), mined as well (Inouye et al., 2001). A certain degree of anti-S. aureus myrtanol acetate (7.73/2.97%), α-humulene (2.59/5.68%), and perillyl effect has previously been attributed to 1,8-cineole and terpinen-4-ol, acetate (2.82/2.83%). which were detected as the major constituents of both C. cambodianum When the essential oils of C. lucida, R. dumetorum, and S. siamensis bark and leaf oils (Van Vuuren and Viljoen, 2007; Iscan, 2017). According were analysed, 49, 70, and 72 compounds were identified, representing to the previous studies (Mahboubi and Feifabadi, 2009; Dahham et al., 98.31/96.75, 89.98/92.15, and 91.37/90.48% of their total contents, re- 2015), decanal and β-caryophyllene, abundant compounds of essential spectively. The essential oil of R. dumetorum was rich in sesquiterpenoids oil from C. lucida, produced antibacterial effect against clinical strains (51.12/6.25%), and monoterpenoids (36.84/85.42%) with the major of S. aureus and other bacterial pathogens with respective MIC values compounds being caryophyllene epoxide (33.29/4.51%), α-pinene 62.50–125.00 μg/mL and 0.61–2.86 μg/mL. Antibacterial properties were (26.09/73.53%), humulene-1,2-epoxide (2.48/0.39), and caryophyllene also described for caryophyllene epoxide (Yang et al., 1999) and limo- (2.41/0.57%). Moreover based on the analysis with DB-17MS column, nene (Van Vuuren and Viljoen, 2007; Iscan, 2017), which are present in limonene (1.42/1.73%), trans-verbenol (1.18/1.64%), and α-pinene L. aromatica and R. dumetorum oils respectively. Therefore, it is possible epoxide (0.51/1.21) were present in a relatively high amount. Similarly, to suppose that the above-mentioned constituents can significantly S. siamensis oil consisted mainly of sesquiterpenoids (75.71/73.66%) contribute to the antibacterial effects of essential oils analysed in this and monoterpenoids (15.38/16.48%) with the main constituents being study when they lead to the disintegration of pathogen cell walls and β-bourbonene (27.49/28.54%), caryophyllene epoxide (14.52/12.37%), membranes, deformation of cells and reduction in nucleus cytoplasm α-pinene (6.69/7.35%), espathulenol (4.48/3.74%), and β-pinene (Nguyen et al., 2018). (4.40/4.73%). On DB-17MS column, a significant amount of trans-α- Several studies have previously evaluated the cytotoxic effect of ex- bergamotene (3.24/4.20%) was detected. In contrast to others oils tracts and their compounds isolated from B. rotunda and L. aromatica analysed in this study, the major constituents of C. lucida oil were esters (Isa et al., 2012) against carcinogenic lung cell lines; however, the data (59.06/37.51%), predominated by decyl acetate (49.44/27.55%) and on inhalation toxicity of the essential oils tested in this study and their dodecenyl acetate (9.26/8.89%), followed by carbonylic compounds safety for healthy lung tissues are missing. Nevertheless, a number of (15.61/34.85%) and sesquiterpenoids (14.77/14.00%). Aliphatic alcohols studies concerning the toxic properties of constituents present in essen- (7.70/7.81%) were also present in significant amounts with the main tial oils from our samples have previously been published. Nielsen et al. components being decanal (13.60/12.42%), caryophyllene epoxide (2005) investigated the acute inhalation effect of α-pinene vapours on (5.81/6.36%), and decan-1-ol (5.34/5.35%). the respiratory system in mice, where no animal died at the maximum 90 Table 4 Chemical composition of essential oils tested.

Component RI Plant species/column/relative contents [%] Identiﬁcationc

Publisheda Observedb Aol Aop Aor Aos Br Ccb Ccl Cl La Rd Ss

HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17

Hydrocarbons Nonane 900 f –e ––––––––––––––0.03 –– –– –– GC–MS, Std 1,5,5-Trimethyl-6-methylene- d 1340 –– –– –– –– –– –– –– –– 0.04 –––––GC–MS, RI cyclohexene Tetradeca-1,13-diene d 1347 –– 0.18 –––––––––––––––––––GC–MS, RI Cyclohexadecane 1881 1897 –– –– –– –– –– –– –– –– –– 0.18 –––GC–MS, RI Heneicosane 2100 2186 –– –– 1.49 –––––––––––––––––GC–MS, RI, Std Tricosane 2300 2300 –– 0.67 0.72 –– –– –– –– –– –– –– –– –– GC–MS, RI, Std Pentacosane 2500 2500 –– 0.69 0.62 –– –– –– –– –– –– –– –– –– GC–MS, RI, Std Group sum [%] 0.00 0.00 1.54 1.34 1.49 0.00 0.00 0.00 0.00 0.00 0.09 0.55 0.75 0.82 0.00 0.03 0.04 0.00 0.18 0.00 0.00 0.00

Carbonylic compounds 2-Hexanone 788 f –– –– –0.05 –– –– –– –– –– –– –– –– GC–MS .Hukv ta./SuhArcnJunlo oay18(08 85 (2018) 118 Botany of Journal African South / al. et Houdkova M. Octanal 998 1004 –– 0.03 – – – 0.28 0.43 –– –– –– 0.02 –––––––GC–MS, RI Nonanal 1100 1104 –– 0.09 0.09 – –––––––––0.38 0.38 –– –– –– GC–MS, RI 3-Methylacetophenone 1182 f –– –– – 0.06 –– –– –– –– –– –– –– –– GC–MS Decanal 1201 1207 –– –– – – 0.01 –––––––13.60 12.42 –– –– –– GC–MS, RI Benzylacetone 1218 1249 –– –– – –––0.07 0.05 –– –– –– –– –– –– GC–MS, RI trans-2-Decenal 1255 1264 –– –– – – 0.02 ––––––––0.09 –– –– –– GC–MS, RI Undecanal 1306 1309 –– –– – –––––––0.09 – 0.94 0.83 –– –– –– GC–MS, RI Dodecanal 1408 1410 –– –– – –––––0.09 0.55 0.66 0.82 – 20.98 –– –– –– GC–MS, RI Tridecanal 1510 1504 –– –– – –––––––––0.42 –––––––GC–MS, RI Flavesone 1547 1552 –– –– – –––––––––––––0.08 –––GC–MS, RI Tetradecanal 1612 1619 –– –– – –––––––––0.25 0.15 –– –– –– GC–MS, RI Leptospermone 1630 1628 –– –– – –––––––––––––0.72 –––GC–MS, RI Group sum [%] 0.00 0.00 0.12 0.09 0.00 0.11 0.31 0.43 0.07 0.05 0.00 0.00 0.00 0.00 15.61 34.85 0.00 0.00 0.80 0.00 0.00 0.00

Aliphatic alcohols Hexan-1-ol 870 g –– –– –– –– –– –– –– 0.10 0.10 –– –– –– GC–MS 1-Octen-3-ol 978 978 –– –– –– –– –– –– –– –– 0.20 0.23 –– –– GC–MS, RI, Std Octan-1-ol 1068 1070 –– –– –– –– –– –– –– 0.19 0.27 –– –– –– GC–MS, RI Nonan-1-ol 1169 1173 –– –– –– –– –– –– –– 0.39 0.43 –– –– –– GC–MS, RI Decan-1-ol 1269 1277 –– –– –– –– –– –– –– 5.34 5.35 –– –– –– GC–MS, RI Undecan-1-ol 1370 1379 –– –– –– –– –– –– –– 0.53 0.53 –– –– –– GC–MS, RI Dodecan-1-ol 1470 1477 –– –– –– –– –– –– –– 1.15 1.13 –– –– –– GC–MS, RI Hexadecan-1-ol 1875 1885 –– 0.48 0.45 –– –– –– –– –– –– –– –– –– GC–MS, RI

d – Octadecen-1-ol 2065 –– –– –– –– 0.30 0.24 –– –– –– –– –– –– GC–MS, RI 97 Group sum [%] 0.00 0.00 0.48 0.45 0.00 0.00 0.00 0.00 0.30 0.24 0.00 0.00 0.00 0.00 7.70 7.81 0.20 0.23 0.00 0.00 0.00 0.00

Fatty acids Decanoic acid d 1392 –– –– –– –– –– –– –– 0.23 0.32 –– –– –– GC–MS, RI Dodecanoic acid 1568 1577 –– –– –– –– –– –– –– 0.50 0.97 –– –– –– GC–MS, RI Hexadecanoic acid 1984 1969 –– –– –– –– –– –– –– 0.11 0.64 –– –– –– GC–MS, RI Group sum [%] 0.84 1.93 0.00 0.00 0.00 0.00 0.00 0.00

Esters Ethyl hexanoate d 1000 –– –– –– –– –– –– –– 0.04 0.17 –– –– –– GC–MS, RI Ethyl octanoate 1193 1198 –– –– –– –– –– –– –– 0.28 0.17 –– –– –– GC–MS, RI Octyl acetate d 1212 –– –– –– 0.02 ––––––––0.73 –– –– –– GC–MS, RI Benzyl isobutyrate d 1301 –– –– –– –– –– –– –– –– –– 0.24 –––GC–MS, RI Benzyl 2-methylbutyrate d 1391 –– –– –– –– –– –– –– –– –– 0.61 0.09 –– GC–MS, RI trans-Methyl cinnamate d 1391 –– –– –– –– 1.47 1.73 –– –– –– –– –– –– GC–MS, RI Decyl acetate 1408 1424 –– –– –– –– –– –– –– 49.44 27.55 –– –– –– GC–MS, RI Dodecenyl acetate d 1617 –– –– –– –– –– –– –– 9.26 8.89 –– –– –– GC–MS, RI Tetradecenyl acetate d 1812 –– –– –– –– –– –– –– 0.04 –––––––GC–MS, RI Dibutyl phthalate 1970 1982 –– –– –– –– –– –– –– –– –– 0.12 –––GC–MS, RI Methyl tetradecanoate d f –– –0.25 –– –– –– –– –– –– –– –– –– GC–MS Group sum [%] 0.00 0.00 0.00 0.25 0.00 0.00 0.02 0.00 1.47 1.73 0.00 0.00 0.00 0.00 59.06 37.51 0.00 0.00 0.97 0.09 0.00 0.00 Monoterpenoids Tricyclene 926 925 –– –– ––––0.23 0.23 0.08 –––––––––––GC–MS, RI α-Thujene 930 929 0.15 0.17 0.03 0.04 0.22 0.22 –– –– 0.56 0.19 0.35 0.40 –– 0.19 0.05 0.08 0.18 0.04 – GC–MS, RI α-Pinene 939 937 4.79 5.01 2.43 2.64 5.10 5.58 0.38 0.51 0.87 0.89 8.79 5.89 3.11 3.36 0.01 – 1.70 1.70 26.09 73.53 6.69 7.35 GC–MS, RI, Std α-Fenchene 952 f –– –– –0.04 –– –– –– –– –– –– –– –– GC–MS Rosefuran df –– –– –– –– –0.05 –– –– –– –– –– –– GC–MS Cosmene df –– –– –– –– –0.10 –– –– –– –– –0.11 –– GC–MS Camphene 954 952 0.07 0.06 0.17 0.10 0.22 0.18 –– 5.44 5.47 4.64 0.75 0.73 0.66 –– 0.04 – 0.04 0.08 0.18 0.22 GC–MS, RI, Std Thuja-2,4(10)-diene 957 960 –– –– –0.02 –– –– –– –– –– –– 0.13 0.45 – 0.25 GC–MS, RI Sabinene 975 978 2.59 3.41 0.53 0.77 – 1.13 –– –– 0.78 0.85 1.56 1.57 –– 0.06 0.09 –– 0.14 0.21 GC–MS, RI β-Pinene 979 985 58.40 59.06 29.75 31.31 50.23 53.22 –– 0.17 – 4.39 2.33 2.22 2.91 0.06 0.07 0.22 – 0.44 0.89 4.40 4.73 GC–MS, RI, Std β-Myrcene 990 992 0.30 – 0.03 – 0.29 – 2.41 3.20 1.11 1.50 1.31 0.36 0.42 –––0.17 0.36 –– 0.14 – GC–MS, RI, Std 2,3-Dehydro-1,8-cineole 991 994 –– –– –– –– –– –– 0.16 0.03 –– –– –– –– GC–MS, RI Pseudolimonene 1004 1007 –– 0.02 ––0.04 –– –– –– –– –– –– –– –– GC–MS, RI α-Phellandrene 1002 1008 –– –– –– 0.14 0.17 –– 0.11 0.61 – 0.19 –– –– –– –– GC–MS, RI, Std p-Mentha-1,5,8-triene 1135 1008 –– –– –– –– –– –– –– –– –– –0.10 –– GC–MS, RI δ-2-Carene 1002 f –– –– –– –– –– –0.28 –– –– –– –– –– GC–MS o-Cymenene df –– –0.03 –– –– –– –0.08 –– –– –– –– –– GC–MS α-Terpinene 1017 1020 –– –– –– –– –– 0.86 0.38 0.42 0.50 –– –– –– –– GC–MS, RI, Std p-Cymene 1024 1027 –– 1.17 1.51 –– 0.04 0.07 –– 1.77 – 1.91 – 0.04 0.04 –– 0.47 0.41 0.42 0.64 GC–MS, RI –– –– ––––––––––––– – o-Cymene 1026 1028 0.59 0.78 8.79 14.99 0.09 GC MS, RI 85 (2018) 118 Botany of Journal African South / al. et Houdkova M. Limonene 1029 1032 0.94 0.95 0.74 0.84 1.60 1.92 – 0.42 1.61 2.02 – 1.14 – 1.04 –– 48.31 48.08 1.42 1.73 0.55 0.71 GC–MS, RI β-Phellandrene 1029 f – 0.08 –– –0.04 – 2.88 –– –0.35 – 0.56 –– –– –– –– GC–MS β-Terpinene 1071 1034 –– –– –– 2.48 ––––2.72 –– –– –– –– –– GC–MS, RI 1,8-Cineole 1031 1035 0.12 – 0.21 – 5.17 –––5.50 5.58 31.80 33.46 30.54 33.09 –– –– –– –– GC–MS, RI, Std trans-β-Ocimene 1050 1039 –– –– –– –– 3.34 2.92 –– –– –– –– –0.12 –– GC–MS, RI Ocimene 1050 1049 –– –– –– 0.37 0.51 27.99 27.62 –– 0.43 0.50 –– 0.18 0.21 –– –– GC–MS, RI γ-Terpinene 1059 f –– –– –– –– –0.07 1.14 0.57 0.82 1.00 –– –– –– –– GC–MS, Std trans-Sabinene hydrate 1070 1070 0.18 0.14 – 0.04 –– –– –– –– 0.29 0.23 –– –– –– –– GC–MS, RI cis-Sabinene hydrate 1070 1071 –– 0.05 –––––––0.13 0.13 –– –– –– –– 0.06 – GC–MS, RI cis-Linalool oxide (furanoid) 1072 1075 –– –– –– –– –– –– –– –– –– 0.03 –––GC–MS, RI f L-Menthol 1172 –– –– –– –– –– –– –– –– –– –– –0.05 GC–MS, Std Camphenilone 1082 1089 –– 0.03 –––––––––––––––––––GC–MS, RI Terpinolene 1088 1092 –– –– –– –– 0.19 0.20 0.58 0.50 0.31 0.37 –– 0.02 –––––GC–MS, RI, Std Linalool 1096 1102 – 0.14 0.16 0.12 –– 0.17 0.22 1.55 1.48 0.27 2.34 4.66 4.16 0.17 0.11 0.28 0.29 0.54 0.89 –– GC–MS, RI, Std 4-Thujanol d 1102 0.28 –––––––––––––––––––––GC–MS, RI α-Pinene epoxide 1099 1103 – 0.05 –– 0.13 –––0.15 0.17 –– –– –– –0.07 0.51 1.21 0.31 0.15 GC–MS, RI Hotrienol 1101 1107 –– –– –– –– –– –– 0.17 0.17 –– –– –– –– GC–MS, RI cis-Verbenol 1141 1111 –– –– –– –– –– –– –– –– –– 1.44 1.09 – 0.12 GC–MS, RI Fenchol d 1119 –– 0.23 – 0.21 0.22 –– –– 0.14 0.14 –– –– –– –– –– GC–MS, RI trans-p-Menth-2,8-dien-1-ol d 1125 –– –– –– –– –– –– –– –– 0.05 –––––GC–MS, RI cis-2-Menthenol 1121 1126 0.05 – 0.25 – 0.11 – 0.01 –––0.17 0.09 0.21 0.16 –– –– –– –– GC–MS, RI α-Campholenal 1126 1131 –– 0.29 0.25 0.15 0.18 –– –– –– –0.05 –– –– 0.49 0.39 0.09 0.08 GC–MS, RI f Allo-ocimene 1132 –– –– –– –– –– –– –– –– –– –0.19 –– GC–MS – 97 Terpinen-1-ol 1133 f –– –– –– –– –– –0.11 –– –– –– –– –– GC–MS trans-2-Menthenol 1140 f –– –– –– –– –– 0.11 0.05 – 0.24 –– –– –– –– GC–MS Neo-allo-ocimene 1144 1132 –– –– –– –– 0.64 0.66 –– –– –– –– –– –– GC–MS, RI Limonen-1,2-epoxide d 1138 –– –0.02 –– –– –– –– –– –– 1.02 1.01 –– –– GC–MS, RI trans-Limonene epoxide 1142 1142 –– –– –– –– –– –– –– –– 0.46 0.48 –– –– GC–MS, RI trans-Pinocarveol 1139 1145 2.04 1.74 4.71 3.59 2.03 1.49 –– –– –0.17 – 0.29 –– –– –– –– GC–MS, RI Sabinol 1142 1145 –– –– –– –– –– –– –– –– –– –– 0.57 0.70 GC–MS, RI Myroxide 1145 1147 –– –– –– –– 0.12 0.13 –– –– –– –– –– –– GC–MS, RI trans-Verbenol 1144 1149 0.06 – 0.19 –––––––––––––––1.18 1.64 0.36 – GC–MS, RI Camphene hydrate 1149 1155 –– 0.07 – 0.05 0.05 –– 0.57 0.67 0.18 0.09 –– –– –– –– –– GC–MS, RI Camphor 1146 1156 –– –– –– –– 18.56 19.12 1.71 0.84 –– –– –– –– –– GC–MS, RI, Std β-Pinene epoxide 1159 f –– –– –0.18 –– –– –– –– –– –– –– –– GC–MS β-Terpineol 1159 f –– –0.08 –– –– –– –– –– –– –– –– –– GC–MS trans-β-Terpineol 1163 1168 –– –– –– 0.01 –––––––––––––––GC–MS, RI Pinocarvone 1164 1169 0.95 0.96 3.65 4.35 2.31 2.40 –– –– –– –0.14 –– –– 0.24 0.24 0.25 0.18 GC–MS, RI Borneol 1169 1172 0.08 – 0.31 – 0.26 –––0.41 – 5.71 4.97 1.41 –––0.18 0.21 0.06 –––GC–MS, RI, Std f L-Menthol 1172 –– –– –– –– –– –– –– –– –– –– –0.05 GC–MS, Std Terpinen-4-ol 1177 1183 0.45 0.55 0.67 0.83 0.88 1.06 0.02 – 0.31 – 5.39 2.47 3.04 4.36 –– 0.04 – 0.32 0.14 0.08 0.05 GC–MS, RI p-Cymene-8-ol 1182 1190 –– 0.38 – 0.35 0.19 –– –– –– –– –– –– 0.56 0.12 –– GC–MS, RI 91 (continued on next page) 92 Table 4 (continued)

Component RI Plant species/column/relative contents [%] Identiﬁcationc

Publisheda Observedb Aol Aop Aor Aos Br Ccb Ccl Cl La Rd Ss

HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17

Isopinocarveol d 1192 0.14 –––––––––––––––––––––GC–MS, RI Cinerone 1183 f –– –– –– –– –0.07 –– –– –– –– –– –– GC–MS Cryptone 1185 1193 –– –– –– 0.07 0.10 –– –– –– –– –– –– –– GC–MS, RI Methyl salicylate 1191 f –– –– –– –– –– –– –– –– –0.06 –– –– GC–MS Myrtenal 1195 f –– –– –1.61 –– –– –0.09 – 0.15 –– –– –0.22 – 0.33 GC–MS α-Terpineol 1188 1196 0.31 0.28 1.24 1.09 1.13 1.19 –– 0.59 0.57 9.52 7.95 6.54 6.44 – 0.19 0.04 0.12 0.68 0.66 0.14 0.14 GC–MS, RI cis-Dihydrocarvone df –– –0.35 –– –– –– –– –– –– –– –– –– GC–MS, Std Myrtenol 1195 1203 2.66 2.52 5.48 5.18 3.31 1.22 –– –– –0.02 0.27 0.04 –– –– 0.36 0.29 0.62 0.28 GC–MS, RI cis-Piperitol 1196 1213 –– –– –– –– –– 0.05 –––––––––––GC–MS, RI Cosmen-2-ol d 1210 –– –– –– –– 0.22 0.12 –– –– –– –– –– –– GC–MS, RI trans-Carveol 1216 1214 –– 0.12 0.09 –– –– –– –– –– –– –– –– –– GC–MS, RI 3-p-Menthen-7-al d 1215 –– –– –– –– –– –– –– –– 20.08 20.26 –– –– GC–MS, RI Verbenone 1205 1217 –– 0.15 0.13 – 0.04 –– –0.08 –– –– –– –– 0.59 0.62 0.22 0.23 GC–MS, RI cis-Carveol 1229 1224 –– –0.03 –– –– –– –– –– –– 0.12 – 0.25 0.12 0.06 0.06 GC–MS, RI .Hukv ta./SuhArcnJunlo oay18(08 85 (2018) 118 Botany of Journal African South / al. et Houdkova M. 2-Hydroxycineole 1219 1229 –– 0.09 – 0.06 0.14 –– –– –– –– –– –– –– –– GC–MS, RI Citronellol 1225 1230 –– –– –– –– –– –– –– –– –– –– 0.06 – GC–MS, RI, Std Nerol 1229 1257 –– –– –– 0,01 – 0,15 0,07 –– 0.14 0.10 –– –– –– –– GC–MS, RI Isobornyl formate 1239 1235 –– 0.04 –––––––––––––––––––GC–MS, RI Neral 1238 1245 –– –– –– –– 0.44 0.42 –– –– –– –– –– –– GC–MS, RI Cuminaldehyde 1241 1247 –– 0.15 ––0.03 –– –– –– –– –– –– –– –– GC–MS, RI Carvacrol methyl ether 1244 1248 –– –– 0.11 –––––––––––––––––GC–MS, RI Carvone 1243 1250 –– 0.12 – 0.04 –––––––––––0.11 0.09 0.07 –––GC–MS, RI, Std Piperitone 1252 1261 –– –– –– 0.01 –––––––––––––––GC–MS, RI Geraniol 1252 1265 –– –– –– –– 24.41 24.04 –– –– –– –– –– –– GC–MS, RI, Std Citral 1240 1274 –– –– –– 0.02 –––––––––––––––GC–MS, RI, Std Geranial 1267 1277 –– –– –– –– 1.99 1.77 –– –– –– –– –– –– GC–MS, RI p-Menth-2-en-1,4-diol 1269 1277 –– 0.06 0.05 –– –– –– –– –– –– –– –– –– GC–MS, RI trans-Shisool d 1277 –– –– –– –– –– –– –– –– –0.53 –– –– GC–MS, RI Perillal 1271 1283 –– –– –– –– –– –– –– –– 2.18 2.81 –– –– GC–MS, RI Bornyl acetate d 1292 0.04 ––––0.04 0.02 –––0.26 0.19 0.14 0.12 –– –– –– –– GC–MS, RI, Std p-Menth-1-en-9-ol d 1293 –– –– –– –– –– –– –– –– 2.08 2.17 –– –– GC–MS, RI Safrole 1287 1294 –– –– –– –– –– –– –– –– –– 0.03 –––GC–MS, RI p-Cymen-7-ol 1290 f –– –0.08 –– –– –– –– –– –– –– –– –– GC–MS Perillol df –– –5.03 –– –– –– –– –– –– –– –– –– GC–MS Methyl myrtenate 1294 1303 0.33 –––––––––––––––––––––GC–MS, RI trans-Pinocarvyl acetate 1298 1304 –– –– –– –– –– –– –– –– –– 0.52 –––GC–MS, RI –– –– –– –– –– ––––––––––– –

Carvacrol 1299 1304 0.05 GC MS, RI, Std – δ-Terpinyl acetate 1317 1323 –– –– 0.03 –––––––––––––––––GC–MS, RI 97 p-Mentha-1,4-dien-7-ol d 1328 0.35 – 1.71 – 0.94 –––––––––––––––––GC–MS, RI Myrtenyl acetate d 1331 0.20 –––––––––––––––––0.03 –––GC–MS, RI Terpinyl acetate 1349 1355 –– –– 0.53 0.99 –– –– –– –– –– –– –– –– GC–MS, RI Neryl acetate 1361 1366 –– –– –– –– –– –– –– –– 0.04 –––––GC–MS, RI Sobrerol d 1387 –– 0.16 – 0.09 –––––––––––––0.14 –––GC–MS, RI Myrtanol acetate 1381 1424 –– –– –– –– –– –– –– –– 7.73 2.97 –– –– GC–MS, RI Methyleugenol 1403 1406 –– –– –– –– –– 0.04 –––––––––––GC–MS, RI α-Ionone 1430 1435 0.09 –––––––––––––––––––––GC–MS, RI Geranyl acetone 1455 1456 –– –– –– –– –– –– –– –– –– 0.13 –––GC–MS, RI (1S,2S,4S)-Trihydroxy- d 1487 –– –– 0.21 –––––––––––––––––GC–MS, RI p-menthane Group sum [%] 76.16 75.90 55.39 58.55 84.55 88.41 6.16 8.08 96.65 96.02 80.54 70.11 59.85 62.88 0.28 0.53 85.30 81.56 36.84 85.42 15.38 16.48

Sesquiterpenoids δ-EIemene 1338 1344 –– –– –– –– –– 0.14 0.05 0.07 –––––0.07 –––GC–MS, RI Elemene isomer 0064 1345 –– –– –– 0.05 –––––0.80 0.53 –– –– –– –– GC–MS, RI α-Cubebene 1388 1357 –– –– –– 0.03 –––0.20 –––––––––0.33 – GC–MS, RI α-Longipinene 1352 1363 –– –– –– 0.08 –––––––––––––––GC–MS, RI Cyclosativene 1371 f –– –– –– –– –– –0.16 0.06 –––––––––GC–MS α-Copaene 1374 1385 0.14 0.21 –– –– 1.15 1.67 –– 0.88 0.67 0.48 0.50 –– 0.19 – 0.21 – 2.38 3.52 GC–MS, RI α-Ylangene 1375 1381 –– –– –– –0.15 –– –– –– –– –– –– 0.06 – GC–MS, RI Isoledene 1376 f –– –– –– –– –– 0.15 0.41 0.10 0.13 –– –– –– –– GC–MS β-Bourbonene 1388 1395 0.06 0.06 0.24 –––––––––––0.06 –––––27.49 28.54 GC–MS, RI β-Elemene 1390 1399 0.36 0.71 –– –– 0.66 0.63 –– 0.26 0.18 0.13 0.06 –– –– 0.45 –––GC–MS, RI α-Bourbonene d 1407 –– –– –– –– –– –– –– –– –– –– 0.41 0.24 GC–MS, RI β-Maaliene d 1420 –– –– –– –– –– 0.13 0.07 –– –– –– –– –– GC–MS, RI α-Gurjunene 1409 1421 –– –– –– 0.03 0.04 –– –– 0.15 0.06 –– –– –– –– GC–MS, RI α-Cedrene 1411 1422 –– –– –– –– –– 0.13 ––0.64 –– –– –– 0.65 0.33 GC–MS, RI cis-α-Bergamotene 1412 1423 –– –– –– –– –– –– 0.45 –––––––0.30 – GC–MS, RI β-Funebrene 1414 f –– –– –– –– –– –– –– –– –– –– –0.51 GC–MS β-Caryophyllene 1419 1431 2.98 3.03 – 0.02 –– 1.72 2.60 –– –0.82 1.35 1.31 4.96 3.73 1.54 1.47 2.41 0.57 0.72 0.72 GC–MS, RI, Std Selina-5,11-diene d f –– –– –– –– –– –0.11 0.01 0.15 –– –– –– –– GC–MS β-Cedrene 1420 f –– –– –– –– –– –– –– –– –– –– 0.09 – GC–MS β-Ylangene 1420 f –– –– –– –1.96 –– –– –– –– –– –– 2.09 1.32 GC–MS α-Guaiene 1439 f –– –– –– –0.10 –– –– –– –– –– –– –– GC–MS β-Cubebene 1388 1440 – 0.05 0.05 ––––0.31 –– –– –– –– –– –– –– GC–MS, RI β-Copaene 1432 1441 0.03 –––––0.50 1.41 –– –– –– –– –– –– 1.97 1.84 GC–MS, RI Perillyl acetate d 1443 –– –– –– –– –– –– –– –– 2.82 2.83 –– –– GC–MS, RI trans-α-Bergamotene 1434 1444 –– –– –– –– –0.10 –– –– 0.37 –––––3.24 4.20 GC–MS, RI Cadina-3,5-diene 1458 f –– –– –– –0.12 –– –– –– –– –– –– –– GC–MS Aromadendrene 1441 1451 –– –0.22 –– –4.73 –– 0.83 1.05 0.61 1.06 –– –– –0.08 –– GC–MS, RI 85 (2018) 118 Botany of Journal African South / al. et Houdkova M. Isogermacrene D d 1457 –– –– –– 0.20 –––––––––––––1.94 1.80 GC–MS, RI α-Himachalene 1451 1463 –– –– –– 0.14 –––––––––––0.83 – 0.58 0.55 GC–MS, RI Sesquisabinene 1459 1465 –– –– –– –– –– –– –– 0.12 0.35 –– –– 2.63 2.29 GC–MS, RI trans-β-Farnesene 1456 1461 –– –– –– –– –– –– –– –– 1.11 0.90 –– –– GC–MS, RI α-Humulene 1454 1466 0.67 0.68 –– –– 1.00 1.86 –– –– –– 0.55 0.43 2.59 5.68 0.45 0.07 0.40 – GC–MS, RI, Std Alloaromadendrene 1460 1473 0.36 0.36 –– 0.03 – 3.47 –––––––––––––––GC–MS, RI Dehydro-Aromadendrene 1462 1463 –– –– –– –– –– –– 0.05 0.44 –– –– –– –– GC–MS, RI Eudesma-1,4(15),11-triene d 1471 –– –– –– –– –– –– 0.15 0.23 –– –– –– –– GC–MS, RI 9-epi-(E)-Caryophyllene 1466 1473 –– 0.19 –––––––––––––––––––GC–MS, RI γ-Gurjunene 1477 1483 –– –– –– –– –– 0.12 0.13 0.14 0.15 –– –– 0.09 – 0.36 0.65 GC–MS, RI γ-Muurolene 1479 1486 0.21 0.19 0.13 0.07 –– –0.42 –– 0.73 0.27 –– –– 0.03 – 0.34 – 0.61 0.71 GC–MS, RI α-Curcumene 1480 1489 –– –– –– –– –– 0.14 – 0.22 – 0.01 –––––0.58 0.85 GC–MS, RI α-Amorphene 1484 f –– –– –– –– –– –– –0.13 –– –– 0.74 0.10 –– GC–MS Isocaryophyllene d f –– –– –– –– –– –– –– –– –– –– –0.08 GC–MS β-Selinene 1490 1498 0.32 0.51 –– –– –– –– –– –– –– –– –– GC–MS, RI Bicyclosesquiphellandrene 1490 f –– –– –– –0.06 –– –– –– –– –– –– –– GC–MS β-Guaiene 1490 f –– –– –– –– –– –0.01 –– –– –– –– –0.39 GC–MS trans-β-Bergamotene d 1495 –– –– –– –– –– –– –– 0.36 –––––0.35 – GC–MS, RI Eremophilene d 1499 –– –– –– –– –– –– –– –– 0.03 – 0.15 – 0.18 – GC–MS, RI Germacrene D 1485 1501 –– –– –– 16.45 16.28 –– –– –– –– –– –– 0.27 – GC–MS, RI Bicyclogermacrene 1494 f –– –– –– –– –– 1.20 1.03 3.62 2.15 –– –– –– –– GC–MS Epicubebol 1494 1505 –– –– –– 0.67 1.65 –– –– –– –– –– –– 0.31 0.69 GC–MS, RI –

γ-Amorphene 1495 f –– –– –– –0.56 –– –– –– –– –– –– –– GC–MS 97 Cubebol 1515 1505 0.14 0.41 –– –– 16.21 20.21 –– –– –– –– –– –– 0.48 0.60 GC–MS, RI Viridiﬂorene 1496 1507 –– –– –– –– 1.20 1.03 – 1.29 –– –– –– –– GC–MS, RI Aciphyllene 1501 1507 –– –– –– –– –– –– –– –– 0,02 – 0.81 –––GC–MS, RI Epishyobunone d 1505 –– –– –– 0.09 –––––––––––––––GC–MS, RI α-Muurolene 1499 1509 0.15 0.16 0.11 0.18 –– 0.71 1.49 –– 0.38 0.15 –– –– –– –– 0.10 – GC–MS, RI Cuparene 1504 f –– –– –– –– –– –– –0.28 –– –– –– –– GC–MS α-Farnesene 1505 1511 –– –– –– 0.45 0.49 0.08 ––– ––0.07 0.10 –– –– GC–MS, RI, Std β-Bisabolene 1505 f –– –– –– –– –– –– –0.08 1.12 1.70 –– –– 0.17 0.29 GC–MS cis-γ-Bisabolene 1507 1516 –– –– –– –– –– –– 0.18 –––––––––GC–MS, RI β-Curcumene 1515 1518 –– –– –– –– –– –– –– –– –– –– 0.02 – GC–MS, RI Sesquicineole 1516 1522 –– –– –– –– –– –– –– –– –– –– 0.22 0.36 GC–MS, RI γ-Cadinene 1513 1525 –– 0.15 0.31 –– –– –– 0.36 –––––0.05 – 0.15 –––GC–MS, RI δ-Cadinene 1523 1525 0.42 –––––––––––0.30 0.87 –– 0.11 –––0.34 – GC–MS, RI β-Sesquiphellandrene 1522 1532 –– –– –– –– –– –– –– –– –– –– –– GC–MS, RI β-Cadinene 1472 1534 –– –– –– –– –– 2.27 0.72 –– –– –– –– –– GC–MS, RI cis-Calamenene 1529 1534 0.30 0.25 –– –– –– –– –0.39 – 0.06 –– –– 0.23 – 0.07 0.15 GC–MS, RI Cadina-1,4-diene 1534 1543 –– –– –– –– –– 0.14 –––––––––––GC–MS, RI 6-epi-Shyobunol d 1548 –– –– –– 9.78 12.44 –– –– –– –– –– –– –– GC–MS, RI

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Table 4 (continued)

Component RI Plant species/column/relative contents [%] Identiﬁcationc

Publisheda Observedb Aol Aop Aor Aos Br Ccb Ccl Cl La Rd Ss

HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17 HP-5 DB-17

trans-α-Bisabolene 1549 1550 –– –– –– –– –– –– –– 0.50 0.44 –– –– –– GC–MS, RI α-Cadinene 1538 1552 –– –– –– 0.05 1.02 –– –– –– –– –– –– –– GC–MS, RI α-Calacorene 1545 1555 0.04 –––––––––0.29 0.09 – 0.03 –– –– –– –– GC–MS, RI Cadala-1(10),3,8-triene d 1555 –– –– –– –– –– –– –– –– –– 0.10 –––GC–MS, RI Elemol 1549 1560 –– –– 0.05 – 0.30 –––––––––––––––GC–MS, RI

cis-Sesquisabinene hydrate 1544 1563 –– –– –– –– –– –– –– –– –– –– 0.79 0.91 GC–MS, RI 85 (2018) 118 Botany of Journal African South / al. et Houdkova M. β-Vetivenene 1555 f –– –– –– –– –– –– –0.03 –– –– –– –– GC–MS β-Spathulenol 1578 1567 –– 0.92 0.80 –– –– –– 0.51 5.32 5.84 3.60 –– –– 0.47 0.38 –– GC–MS, RI trans-Nerolidol 1563 1569 0.14 0.18 –– –– 0.10 – 0.05 0.06 – 0.23 0.40 0.30 –– 0.30 0.25 –– –– GC–MS, RI Palustrol 1568 f –– –– –– –– –– –– –0.32 –– –– –– –– GC–MS 1,5-Epoxysalvial-4(14)-ene d 1575 –– –– –– –– –– –– –– –– –– –– 1.75 1.83 GC–MS, RI Norbourbonone 1571 1576 –– –– –– –– –– –– –– –– –– –– 0.32 – GC–MS, RI Globulol 1590 1582 –– –– –– 0.63 –––0.22 0.25 0.53 1.51 –– –– 0.06 –––GC–MS, RI Espathulenol d 1594 –– –– –– –– –– –– 7.58 8.18 –– –– –– 4.48 3.74 GC–MS, RI Germacrene-D-4-ol 1575 1596 –– –– –– 8.43 7.54 –– –– –– –– –– –– –0.18 GC–MS, RI Viridiﬂorol 1592 1599 –– –– –– –– –– 1.57 1.33 –– –– –– –– –– GC–MS, RI Caryophyllene epoxide 1583 1600 6.07 5.61 20.18 19.70 1.17 1.20 –– –– –– –– 5.81 6.36 0.13 0.68 33.29 4.51 14.52 12.37 GC–MS, RI Isoaromadendrene epoxide 1579 1603 –– –– –– –– –– –– –– –– 0.52 0.17 0.53 – 0.25 1.01 GC–MS, RI Gleenol 1587 f –– –– –– –– –– –0.07 –– –– –– –– –– GC–MS Cubeban-11-ol 1595 1607 –– –– –– –– –– 0.47 –––––––––––GC–MS, RI Salvialenone 1594 1609 –– –– –– –– –– –– –– –– –– –– 0.23 0.50 GC–MS, RI Guaiol 1600 1610 –– –– –– –– –– –– 1.13 –––––––––GC–MS, RI Rosifoliol 1600 1615 –– –– –– –– –– 0.38 2.19 1.28 1.68 –– –– –– –– GC–MS, RI Epiglobulol 1588 1619 0.10 –––––––––0.26 0.27 0.33 0.29 –– –– –– –– GC–MS, RI trans-Longipinocarveol d 1620 –– –– –– –– –– –– –– –– –– –– –– GC–MS, RI Humulene-1,2-epoxide 1608 1625 0.80 0.74 1.93 1.85 – 0.16 –– –– –– –– 0.59 0.59 0.97 0.92 2.48 0.39 2.81 1.52 GC–MS, RI γ-Eudesmol 1632 f –– –– –– –– –– –– –0.24 –– –– –– –– GC–MS α-epi-Cadinol 1640 1642 –– –– –– –0.27 –– –0.22 –– –– –– –– 0.08 – GC–MS, RI

–– –– –– –– –– –– –– –– –– ––– – –

Epicubenol 1613 1646 1.35 GC MS, RI 97 Longifolenaldehyde 1631 1646 0.14 –––––––––––1.31 –––––––0.05 – GC–MS, RI Cedrelanol d 1653 –– –0.18 –– –– –– –– –0.41 –– 0.12 –––––GC–MS, RI 10,10-Dimethyl-2,6- d 1651 –– –– –– –– –– –– –– 0.04 0.06 –– –– –– GC–MS, RI dimethylenebicyclo[7.2.0] undecan-5-ol 11,11-Dimethyl-4,8- d 1654 –– –0.49 –– –– –– –– –– 0.18 0.23 –– 1.45 0.30 0.17 – GC–MS, RI dimethylenebicyclo[7.2.0] undecan-3-ol α-epi-Muurolol 1642 1655 1.23 1.13 0.55 0.43 0.07 – 0.95 0.57 –– 0.54 0.24 –– –– –– –– –– GC–MS, RI α-Muurolol 1646 1659 0.34 0.49 0.16 0.36 –– –– –– –– –– –– –– –– –– GC–MS, RI Cubenol 1646 f –– –– –– –0.52 –– 0.89 0.14 –– –– –– –– –– GC–MS β-Eudesmol 1650 f –– –– –– –– –– –0.75 –– –– –– –– –– GC–MS Pogostole 1653 f –– –0.56 –– –– –– –– –– –– –– –– –– GC–MS Isospathulenol 1666 1659 –– –– –– –– –– 0.28 1.45 2.05 2.05 –– –– 0.41 –––GC–MS, RI δ-Cadinol 1645 1661 –– –– –– 0.54 –––––––––––––––GC–MS, RI α-Cadinol 1654 1668 1.86 1.83 0.94 1.11 0.23 0.09 1.11 1.45 –– 0.48 0.56 –– –– –– –– –– GC–MS, RI cis-10-Hydroxycalamenene 1661 1671 0.15 –––––––––––––––––––––GC–MS, RI Intermedeol 1666 1675 –– –– 0.24 0.26 –– –– –– –– 0.03 –––––––GC–MS, RI Neointermedeol 1656 1680 –– –– –– –– –– –– –– –– –– 1.20 –––GC–MS, RI trans-10-Hydroxycalamenene 1669 1681 0.07 –––––––––––––––––0.16 –––GC–MS, RI Cadalene 1676 1688 –– –– –– –– –– 0.26 0.50 –– –– –– –– –– GC–MS, RI Bulnesol 1671 f –– –– –– –0.37 –– –– –– –– –– –– –– GC–MS Mustakone 1677 1693 –– –– –– –– –– 0.23 –––––––––––GC–MS, RI α-Bisabolol 1685 1694 –– –– –– –– –– –– 0.12 – 0.03 –––––––GC–MS, RI, Std Aristol-1(10)-en-9-ol d 1699 –– –– –– –– –– –– –– –– –– 0.02 –––GC–MS, RI (1R,7S)-Germacra-4(15),5, d 1700 0.08 ––1.00 –– –– –– –– 0.66 0.20 –– –– 1.94 – 0.81 0.97 GC–MS, RI 10(14)-trien-1β-ol Shyobunol 1689 1724 –– –– –– 21.65 4.15 –– –– –– –– –– –– –– GC–MS, RI Eudesma-4,11-dien-2-ol 1690 f –– –– –– –– –– –– –0.07 –– –– –– –– GC–MS Aristolone 1763 1776 –– –– –– –– –– –– –– –– –– 0.27 –––GC–MS, RI Isolongifolol 1723 1742 –– 0.37 –––––––––––––––––––GC–MS, RI Oplopanone 1740 1753 0.18 –––––––––––––––––––––GC–MS, RI β-Costol 1767 f –– –– –– –– –– –– –– –– –– –0.15 –– GC–MS 15-Hydroxy-α-muurolene 1780 1758 –– –– –– 1.36 –––––––––––––––GC–MS, RI cis-Lanceol 1761 1785 –– 0.55 –––––––––––––––––––GC–MS, RI Ambrial d 1824 –– 1.01 1.56 0.73 0.85 –– –– –– –– –– –– –– –– GC–MS, RI Perhydrofarnesyl acetone d 1856 –– –– –– –– –– –– –– –– –– 0.34 0.09 –– GC–MS, RI Corymbolone d 1898 –– –– –– –– –– –– –– –– –– –– 0.11 – GC–MS, RI Farnesyl acetone d 1939 –– –– –– –– –– –– –– –– –– 0.12 –––GC–MS, RI Kessanyl acetate d f –– –0.19 –– –– –– –– –– –– –– –– –– GC–MS α-Vetivol d f –– –– –– –– –– –– –0.04 –– –– –– –– GC–MS 85 (2018) 118 Botany of Journal African South / al. et Houdkova M. Group sum [%] 17.34 16.60 27.48 29.03 2.52 2.56 88.51 85.07 0.13 0.16 14.44 21.19 30.10 29.07 14.77 14.00 10.60 13.00 51.12 6.25 75.71 73.66

Higher isoprenoids Geranyl linalool 2027 2039 –– –– –– –– –– –– –– –– 0.06 –––––GC–MS, RI Phytol 2114 2119 –– –– –– –– –– –– –– 0.05 –––––––GC–MS, RI Group sum [%] 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.00 0.06 0.00 0.00 0.00 0.00 0.00

Others 1-Vinyl-5,5-dimethyl[2.1.1] d 922 –– –– –– –– –– –– –– –– –– –– 0.28 0.34 GC–MS, RI bicyclohexane 3-Hydroperoxyhexane d 944 –– 0.05 ––––––––––––0.09 –– –– –– GC–MS, RI 1-(4-Methyl-3-cyclohexen- d 1152 –– –– –– –– –– –– –– –– 0.29 0.18 –– –– GC–MS, RI 1-yl)ethanol 4-Isopropenylcyclohexanone d 1161 –– –– –– –– –– –– –– –– 1.03 0.75 –– –– GC–MS, RI 2-Methylene-6,6- d 1161 0.19 0.08 0.38 0.10 – 0.05 –– –– –– –– –– –– –– –– GC–MS, RI dimethylbicyclo[3.2.0] heptan-3-ol cis-3-Hexenyl-α-methylbutyrate d 1234 –– –– –– –– –– –– –– –– –– 0.07 –––GC–MS, RI Cinerone d f –– –– –– –– –– –– –– –– –– –– –– GC–MS 5-Isopropenyl-2- d 1315 –– 2.38 – 1.50 –––––––––––––––––GC–MS, RI methylenecyclohexyl hydroperoxide Ledane d 1344 –– 0.23 –––––––––––––––––––GC–MS, RI – 6,6-dimethyl-bicyclo[3.1.1] df –– –0.38 –– –– –– –– –– –– –– –– –– GC–MS 97 hept-2-ene-2-carboxylic acid 3a,9-Dimethyldodecahydro- df –– –0.77 –– –– –– –– –– –– –– –– –– GC–MS 3H-cyclohepta[d]inden-3-one Labda-8(17),12-diene- d 2414 –– –– 1.62 –––––––––––––––––GC–MS, RI 15,16-dial Group sum [%] 0.19 0.08 3.04 1.25 3.12 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.09 1.32 0.93 0.07 0.48 0.28 0.34 Total identified 93.69 92.58 88.05 90.96 91.68 91.13 95.00 93.58 98.62 98.20 95.07 91.85 90.70 92.77 98.31 96.75 97.52 95.72 89.98 92.15 91.37 90.48 a) Data taken from Adams (2007) and from NIST (2017). b) Retention indices calculated from retention times on a HP-5MS column and based on C9–C29 alkanes. c) Identification method: GC–MS = Mass spectrum was identical to that of National Institute of Standards and Technology Library (ver. 2.0.f), RI = the retention index was consistent with that of the literature database, Std = constituent identity confirmed by co-injection of authentic standards. d) Literature data not available. e) Compound not detected in the sample. f) Retention indices were not calculated for compounds identified only by HP-17MS column. g) Retention indices were not calculated, as the retention times were outside the area of standard alkanes (C9–C29). . Aol — Alpinia oxymitra leaves, Aop — Alpinia oxymitra pericarp, Aor — Alpinia oxymitra rhizome, Aos — Alpinia oxymitra seeds, Br — Boesenbergia rotunda,Ccb— Cinnamomum cambodianum bark, Ccl — Cinnamomum cambodianum leaves, Cl — Citrus lucida,La— Limnophila aromatica,Rd.— Rhodamnia dumetorum,Ss— Sindora siamensis. 95 96 M. Houdkova et al. / South African Journal of Botany 118 (2018) 85–97 exposure of concentration 5213.00 ppm. Another in vivo test of acute with different polarities, non-polar HP-5MS and more polar DB-17MS. toxicity in rodents showed that 1,8-cineole was well tolerated In recent studies, it has been well described that the use of a simulta- up to a dose of 1500.00 mg/kg (Caldas et al., 2016) and an LD50 neous dual-column/dual-detector system increases the resolution of of α-terpineol was determined to be 2900.00 mg/kg (Bhatia et al., the analysis leading to the improved quantitation and identification of 2008) both being administered by oral route. Few reports have de- essential oil components (Marriott et al., 2001; Haggarty and Burgess, termined in vitro cytotoxic levels for volatiles to various cell cultures 2017). Based on these principles, four, eighteen, thirteen, and fourteen where α-pinene and β-caryophyllene (detected e.g. in A. oxymitra, additional compounds have been identified using the second column C. cambodianum, R. dumetorum,andS. siamensis oils) exhibited a good DB-17MS in oils of A. oxymitra leaves, pericarp, rhizomes, and seeds, re- safety profile for human lymphocytes (Amaral et al., 2016). In contrast, spectively. Similarly, five additional constituents were found in oil of B. caryophyllene epoxide, a dominant compound of R. dumetorum oil, rotunda, nineteen and twenty-two in oils of C. cambodianum bark and was responsible for the strong toxicity of a number of essential oils leaves, eight in C. lucida, three in oils of L. aromatica, seven in R. previously reviewed by Judzentiene et al. (2010). Although, borneol dumetorum, and eight components in oil of S. siamensis. Some compounds (found in C. cambodianum bark oil) was evaluated as toxic to human fi- detected by one column appeared as two peaks on the second column, broblasts (IC50 = 1.50 μg/mL) (Slamenova et al., 2009), C. cambodianum e.g. it was observed in the case of C. lucida essential oil, when the main bark essential oil was evaluated as non-toxic in our study. constituent decyl acetate, (49.44%) found on the HP-5MS column, was When comparing the bacterial growth-inhibitory and cytotoxic ef- separated into two different substances dodecyl acetate (27.55%) and fects of samples in this study, A. oxymitra pericarp oil has been deter- dodecanal (20.98%) on the DB-17MS column. It was explained by Liu mined as the most effective antimicrobial agent against H. influenzae and Hu (2007) that compounds, which have similar retention times on with the MIC value 32.00 μg/mL. According to the results of toxico- one column, usually have quite different retention times on the column logical evaluation, this oil is safe to lung cell lines at the IC80 level with different polarity. (N512.00 μg/mL) and a calculated TI value (N16.00) suggests that A. oxymitra pericarp oil could be considered as a potential antibacterial 5. Conclusion agent for inhalation treatment of respiratory infections (Becker, 2007).

However, TI calculated from IC50 value (29.96 μg/mL) indicated certain In this study, we reported the in vitro growth-inhibitory potential toxicological risk to the human lung cells (TI = 0.94). Therefore, more of eleven essential oils obtained by hydrodistillation from seven detailed toxicological assessments (especially in vivo) will be necessary Cambodian plant species (A. oxymitra, B. rotunda, C. cambodianum, for determination of its safety profile. C. lucida, L. aromatica, R. dumetorum,andS. siamensis) against pneumo- The chemical composition of essential oils from various parts of nia causing bacteria in liquid and vapour phase and their effect on the B. rotunda, L. aromatica, C. cambodianum, and C. lucida has previously proliferation of lung fibroblasts. All samples exhibited a certain degree been described; however, literature about chemical analysis of of antibacterial activity against at least one bacteria associated with A. oxymitra, C. cambodianum bark, C. lucida fruit peel, R. dumetorum, respiratory system infections, whereas cytotoxicity assay showed the and S. siamensis oils is not available. When comparing analytical data relative safety of some essential oils tested. As a result of this research, from this study with previously published works on B. rotunda rhizome A. oxymitra pericarp oil was found to be safe to human lung cell lines oil, its chemical composition corresponds to results of Phanthong et al. and at the same time, effective against H. influenzae in liquid and solid (2013),whodetectedocimene,geraniol,camphor,1,8-cineole,andcam- medium. Additionally, we analysed the chemical composition of volatile phene as its main constituents. The general prevalence of monoterpene oils by GC/MS using two capillary columns of different polarity. compounds in L. aromatica essential oils resembles the chemistry Monoterpenoids and sesquiterpenoids were the predominant classes of those previously isolated from plants collected in Bangladesh and identified in all essential oils analysed except C. lucida oil, where Vietnam (Bhuiyan et al., 2010; Dai et al., 2015). Nevertheless, there the major constituents were esters. This analysis also identified the were both quantitative and qualitative variations with respect to their presence of specific antimicrobial constituents such as β-caryophyllene, major components. While we found limonene (N48.00%) as the most caryophyllene epoxide, 1,8-cineole, decanal, α-pinene, β-pinene, and abundant compound, its amount described in literature was equal to terpinen-4-ol in the most effective essential oils, which disturb cell mem- or lower than 20.00%. On the other hand methyl benzoate, pulegone, brane structures of pathogens and thus contribute to their mode of anti- camphor, ocimene, and terpinolene, the main constituents of previous bacterial action. Our results suggest a potential of certain Cambodian analysis, were absent or detected in noticeably lower amounts in essential oils (e.g. A. oxymitra pericarp oil) for application in the inhala- our sample. Although linalool (33.10%) and terpinen-4-ol (12.30%) tion therapy against respiratory infections; however, further research fo- were previously identified as the main constituents of leaf oil from cused on in vivo evaluation will be necessary to be carried out in order to C. cambodianum growninVietnam(Son et al., 2014), we detected these verify its potential practical use. components in seven and four times lower quantities, respectively. The chemical composition of previously analysed essential oil from C. lucida Acknowledgments leaves (Supudompol, 2009) has a similar pattern of constituents to fruit peel investigated in this study, except for leaf oil's major compo- This research was financially supported by the Czech University of nent β-caryophyllene (26.60%) which was detected in a lower amount Life Sciences Prague (projects IGA 20175020 and CIGA 20175001), (b5.00%) in our sample. Decyl acetate, a predominated compound of Erasmus Mundus project ALFABET (No. 552071), and Foundation fruit peel oil, was identified in leaves at a four times lower amount. Chem- “Nadani Josefa, Marie a Zdenky Hlavkovych”. We thank Simean Sok ical profiles of 17 various Rhodamnia species have previously been exam- and Mister Khan for their assistance in collecting plant materials. The ined (Brophy et al., 1997), whereas R. argentea, R. australis,andR. whiteana authors are grateful to Michjal Ousej for providing language help. oils obtained by steam distillation were chemically similar to our sample of R. dumetorum. Nevertheless, none of these species contained as high a References quantity of caryophyllene epoxide as R. dumetorum did. The differences fi in qualitative and quantitative compositions of our, and previously Adams, R.P., 2007. Identi cation of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. 4th ed. Allured Publ. Corp, Carol Stream, Illinois, USA 978-19- analysed, essential oils can be caused by genetic and environmental fac- 326-3321-4. tors including geographical origin, as well as by using various methods Amaral, R.G., Baldissera, M.D., Grando, T.H., Couto, J.C.M., Posser, C.P., Ramos, A.P., Sagrillo, for their isolation (Kokoska et al., 2008; Rahimmalek et al., 2013). M.R., Vaucher, R.A., da Silva, A.S., Becker, A.P., Monteiro, S.G., 2016. Combination of α β fi the essential oil constituents -pinene and -caryophyllene as a potentiator of With the aim of achieving a higher quality identi cation of detected trypanocidal action on Trypanosoma evansi. Journal of Applied Biomedicine 14, constituents, the volatile oils analyses were conducted on two columns 265–272. M. Houdkova et al. / South African Journal of Botany 118 (2018) 85–97 97

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