Eucalyptus radiata JEOR 2016

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Journal of Essential Oil Research
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Chemical composition and antimicrobial activity of
Eucalyptus radiata leaf essential oil, sampled over
a year
Gillian D. Mahumane, Sandy F. van Vuuren, Guy Kamatou, Maxleene Sandasi
& Alvaro M. Viljoen
To cite this article: Gillian D. Mahumane, Sandy F. van Vuuren, Guy Kamatou, Maxleene
Sandasi & Alvaro M. Viljoen (2016): Chemical composition and antimicrobial activity of
Eucalyptus radiata leaf essential oil, sampled over a year, Journal of Essential Oil Research,
DOI: 10.1080/10412905.2016.1175386
To link to this article: http://dx.doi.org/10.1080/10412905.2016.1175386
Published online: 02 May 2016.
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Journal of Essential Oil Research, 2016
http://dx.doi.org/10.1080/10412905.2016.1175386
Chemical composition and antimicrobial activity of Eucalyptus radiata leaf
essential oil, sampled over a year
Gillian D. Mahumanea
, Sandy F. van Vuurena
, Guy Kamatoub
, Maxleene Sandasib
and Alvaro M. Viljoenb,c
a
Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, Johannesberg, South Africa;
b
Department of Pharmaceutical Sciences, Tshwane University of Technology, Pretoria, South Africa; c
SAMRC Herbal Drugs Research Unit,
Department of Pharmaceutical Sciences, Tshwane University of Technology, Pretoria, South Africa
ABSTRACT
This study investigated the seasonal variation of the chemical composition and antimicrobial
activity of Eucalyptus radiata leaf essential oil. Young and mature Eucalyptus radiata leaf material
was collected monthly (January 2014 to December 2014), hydrodistilled and analyzed using GC-
MS. Essential oil yields ranged from 0.14% to 4.31% (w/w). The major compounds were 1,8-cineole
(65.7% ± 9.5), α-terpineol (12.8% ± 4.4) and limonene (6.5% ± 2.4). Chemometric tools were used
to determine seasonal variations, which showed slight variance in E. radiata chemistry between
seasons. The minimum inhibitory concentration (MIC) assay showed that the highest activity was
noted against the Streptococci (0.19–2.00 mg/mL) and Lactobacillus acidophilus (0.19–1.75 mg/mL).
The activity of the E. radiata leaf essential oil is dependent on the unique ratio of its compounds.
The E. radiata leaf essential oil showed good oil yields, a relatively consistent chemical profile and
noteworthy antimicrobial activity that rivals other commercial Eucalypt counterparts.
Introduction
Eucalyptusradiata(syn.Eucalyptusaustraliana)iscommonly
referredtoasthenarrow-leavedpeppermint,forthriverpep-
permint, grey peppermint or black peppermint tree (1–5).
Eucalyptus radiata belongs to the Myrtaceae family, which
is composed of numerous essential oil-bearing species, of
which the Eucalyptus species are well known.
The antimicrobial activity and chemical composition
of an essential oil is not static, but subject to variation,
influenced by factors such as harvest time, geographical
origin, leaf age, soil type, temperature and environmental
growth conditions (6–9). Consciousness of these factors
and how these parameters may influence the essential oil
yield, composition and bioactivity is important for com-
mercial development. Influence of these aforementioned
factors has previously been observed for several eucalypts
such as Eucalyptus saligna (variation in composition due
to leaf age), E. camaldulensis and E. globulus (variation
in composition due to season) (7, 10, 11). Although the
essential oil composition of E. radiata has been previously
reported (8, 12–15), information on seasonal variation
and leaf age is lacking. In light of this, the chemical com-
position of E. radiata leaf essential oil was investigated
in order to determine the chemotype grown in Tzaneen
(Limpopo province, South Africa). Furthermore, in view
of commercial interest, the effect of seasonal variation on
yield and chemical composition in samples obtained over
a 12-month period was determined in both young and
mature leaves.
Eucalyptus species are used for the treatment of a wide
range of infectious conditions with E. radiata being no
exception (16, 17). It is one of the most commonly used
of the Eucalyptus essential oils, and often preferred by
aroma therapists due to its pleasant fragrance (14, 18).
Eucalyptus radiata is used in the form of compresses,
poultices, massages, steam inhalations or applied slightly
diluted or concentrated for ear infections (19). Based on
reported therapeutic uses, independently or in combina-
tion with other essential oils, E. radiata essential oil is used
as a remedy for acne, wounds, cystitis, kidney infections,
respiratory conditions, vaginitis and dental conditions
(18–22). Its use for respiratory conditions is the most
extensive of all anti-infective properties reported (2, 20).
Although the antimicrobial activity of E. radiata has been
reported using the diffusion method (8, 23), limitations of
this method warrant further investigation using quantified
KEYWORDS
Seasonal variation; South
Africa; chemometric analysis;
major compound; MIC
ARTICLE HISTORY
Received 27 January 2016
Accepted 3 April 2016
© 2016 Informa UK Limited, trading as Taylor & Francis Group
CONTACT Sandy F. van Vuuren sandy.vanvuuren@wits.ac.za
Downloadedby[TheLibrary,UniversityofWitwatersrand]at03:0903May2016

2 G. D. Mahumane et al.
essential oil was then weighed and stored in tightly sealed
amber bottles at ± 4°C until further analysis. Oil yields
were determined on the basis of determining the weight
of fresh plant material without taking into account mois-
ture content. The oil of Eucalyptus camaldulensis was also
obtained using the hydrodistillation method. Eucalyptus
globulus, E. dives, E. smithii, E. radiata (hereafter referred
to as E. radiata comm due to the acquisition from a com-
mercial source) and E. citriodora oil samples were all com-
mercially acquired from Pranarôm (Belgium).
Chemical composition analysis
The essential oils were analyzed by gas chromatography
(Agilent 6890N GC) coupled to mass spectrometry with
a flame ionization detector (5973 MS) (26). A volume
of 1 μL was injected using a split ratio (200:1) with an
auto-sampler at 24.79 psi and an inlet temperature of
250°C. The GC system equipped with a HP-Innowax
polyethylene glycol column 60 m × 250 μm i.d. × 0.25
μm film thickness was used. The oven temperature was
set at 60°C for the first 10 minutes, rising to 220°C at
a rate of 4°C/min and held for 10 minutes and then
rising to 240°C at a rate of 1°C/min. Helium was used
as a carrier gas at a constant flow of 1.2 mL/minute.
The spectrum was obtained on electron impact at 70
eV, scanning from 35 m/z to 550 m/z. The peak areas of
all GC constituents were individually expressed as per-
centages of the total of all the peak areas as determined
by flame ionization detection (FID, 250°C). n-Alkanes
were used as reference points in the calculation of rela-
tive retention indices (RRI). Identification of chemical
components were made by comparing the mass spectra
from the total ion chromatogram, retention indices and
library searches using NIST® and Mass Finder® Flavour®
libraries.
Untargeted and targeted GC-MS analysis
The GC-MS chromatograms were analyzed using both
targeted and untargeted approaches, independently. In
the untargeted analysis, full scan GC-MS chromatograms
were analyzed using MarkerLynxTM
software version 4.1
(Waters, Manchester, United Kingdom) where peak selec-
tion and alignment were performed. To achieve this, a
method was set up specifying parameters that would iden-
tify the minimum and maximum peak thresholds, identify
peak shifts and eliminate noise that would interfere with
peak alignment. Peak alignment was performed with ion
fragments originating from chromatographic peaks across
the whole chromatogram for all samples. The resulting
amplitude data were further analyzed by multivariate anal-
ysis algorithms in SIMCA-P+13.0 (Umetrics AB, Malmo,
methodology. Limited data is available on the micro-
dilution minimum inhibitory concentration (MIC) assay
when investigating E. radiata (14, 15, 24). In this study the
antimicrobial activity of the E. radiata leaf essential oil is
screened against micro-organisms selected based on the
anti-infective claims in order to establish a rationale for
its use. Due to the growing interest in the use of essential
oils in the food and pharmaceutical industries, antimi-
crobial activity of E. radiata oil was considered for both
young and mature leaf samples and compared to essential
oils from other commercially available Eucalyptus spe-
cies. Furthermore, a comprehensive investigation of the
annual composition and role of the major compounds
independently and in selected combinations is provided in
order to determine if the major compounds play a role in
the antimicrobial activity. Correlation between the essen-
tial oil chemistry and antimicrobial activity is provided
using chemometric analysis.
Materials and methods
Plant material and distillation of essential oil
Fresh leaves were collected monthly (at ± 30-day inter-
vals) from a cultivated site in Magoebaskloof, north of
Polokwane, Limpopo Province, South Africa for a period
of one calendar year (January 2014 to December 2014). In
an effort to reduce the number of variables (i.e. different
growth conditions/soil type), E. radiata leaves were col-
lected within the same study area, from selected trees in
the study site. Young and mature leaves were distinguished
by phenotypical differences. This was achieved with the
assistance of the resident farmer Mr. Bruce Stumbles. The
weather conditions varied, characterized by high rainfall
(35% average chance of precipitation) and high temper-
atures (average daily ± 27°C high and ± 17°C daily low)
in summer and spring; and lower temperatures (average
daily ± 19°C high and ± 7°C daily low) and low rain-
fall (4% average chance of precipitation) in autumn and
winter (25). Previous studies have reported variation in
essential oil composition between young and mature leaf
oils of another Eucalyptus species, Eucalyptus saligna (7).
Therefore, the monthly plant samples comprised of both
young and mature leaves to determine if variation exists
in the current study. Voucher specimens were recorded
in the medicinal and aromatic plant register kept at the
Department of Pharmacy and Pharmacology, University
of the Witwatersrand. The essential oil was obtained via
hydrodistillation (26). A known quantity (130–1100g)
of weighed fresh leaf material was subjected to hydro-
distillation using a Clevenger-type apparatus, within 32
hours of harvesting in order to prevent loss of any volatile
compounds. The leaves were distilled for three hours. The

Journal of Essential Oil Research 3
Sweden). In the targeted approach, the peak areas of all
constituents were individually expressed as percentages
of total peak areas as determined by GC-FID through
manual integration. Identification of the constituents
was based on retention times, retention indices, authentic
standards and spectral library data from Mass Finder®
and
NIST®
. The collated chromatographic data were captured
in Microsoft Excel®
and exported into SIMCA-P+13.0 for
further analysis.
Multivariate analysis
The aligned data from MarkerLynxTM
, as well as targeted
GC-MS data, were analyzed in SIMCA-P+13.0 to observe
variance and clustering patterns. Principal component
analysis (PCA) an unsupervised linear algorithm that
converts data to a new coordinate system and investigates
systematic variance within the data was performed as the
initial step. The models generated were evaluated by con-
sidering the scores scatter plot, which provides informa-
tion on the spatial distribution of observations. Following
PCA, orthogonal projections to latent structures discri-
minant analysis (OPLS-DA) was applied to investigate
variation that is related to the maturity (mature versus
young). This was achieved by assigning a class identifier
(Class 1 = mature; Class 2 = young) that was modeled as a
Y-variable. To assess seasonal variation, the samples were
classified according to seasons where class 1 was assigned
to summer months (September, October, November,
December, January, February and March) and class 2 to
winter months (April, May, June, July and August). The
OPLS-DA models enabled separation of systematic vari-
ation (orthogonal) to the variation of interest (predictive)
as observed in the score plot. An S-plot was used to iden-
tify marker constituents responsible for the separation of
the different classes.
Antimicrobial activity
The antimicrobial activity was evaluated against selected
pathogens related to the claimed therapeutic applica-
tion of the essential oil. These included the pathogens
related to skin infections; Gram-positive Staphylococcus
aureus ATCC 25923, methicillin-resistant S. aureus
ATCC 33592, Enterococcus faecalis ATCC 29212; Gram-
negative Pseudomonas aeruginosa ATCC 27853, and the
yeast Candida albicans ATCC 10231. Pathogens associated
with gastro-intestinal disorders; Gram-positive Bacillus
cereus ATCC 11778, Listeria monocytogenes ATCC
19111, and Gram-negative Escherichia coli ATCC 25922,
Salmonella typhimurium ATCC 14028, and Shigella son-
nei ATCC 9290, were included with pathogens associated
with respiratory conditions (Gram-positive Streptococcus
pneumoniae ATCC 49619, Streptococcus agalactiae ATCC
55618, Streptococcus pyogenes NHLS 8668), Gram-
negative Klebsiella pneumoniae ATCC 13883, Moraxella
catarrhalis ATCC 23246 and the yeast Cryptococcus
neoformans ATCC 14116. Pathogens associated with
dental conditions (Gram-positive Lactobacillus acidophi-
lus ATCC 314, Streptococcus mutans ATCC 10919) were
also included. All reference cultures were provided by the
Department of Pharmacy and Pharmacology, University
of the Witwatersrand, South Africa. A waiver for the use
of micro-organisms was granted by the University of
the Witwatersrand Human Research Ethics Committee
(Reference W-CJ-140627-1).
Cultures used in this study were grown in Tryptone
Soya broth (TSB, Sigma-Aldrich), with the exception of
the Streptococci and L. acidophilus which were grown in
Mueller Hinton broth (MHB, Oxoid) enriched with 5%
sheep blood. The broth microdilution method was used to
determine the minimum inhibitory concentration (MIC)
in order to evaluate the antimicrobial efficacy (27). A 100
μL of sterile broth (TSB or MHB) was transferred into
each well of a 96-well micro-titre plate. Stock solutions
of 100 μL of the essential oil samples, prepared to a con-
centration of 32 mg/mL in acetone were transferred into
the first row of the 96-well micro-titre plate and the serial
doubling dilution technique was employed. Ciprofloxacin
(Sigma-Aldrich) at a 0.01 mg/mL stock concentration was
used as a positive control for bacteria, with the exceptions
of S. mutans, L. acidophilus, S. pyogenes, S. pneumoniae
and S. agalactiae, where penicillin (Sigma-Aldrich) was
used. Amphotericin B (Sigma-Aldrich) at a 0.1 mg/mL
stock concentration was used when testing the yeasts.
Negative controls (acetone-water mixture) were included
to assess the antimicrobial effect of the solvent, and a cul-
ture control of sterile broth was included in order to eval-
uate the ability of the media to support microbial growth.
Thereafter, 100 μL of a standardized culture suspension
(approximately 1 × 106
colony forming units (CFU)/mL)
prepared as a 0.5 McFarland standard was added to each
of the wells. Each plate was subsequently covered with
sterile adhesive micro-titre plate sealing tape (NUNC™)
in order to prevent evaporation of volatile essential oil
components during incubation. Broth prior to use was
checked for turbidity to assess sterility. An inoculum of
the standardized culture was streaked on an appropriate
agar plate for single colonies to check for purity of the
culture. Incubation conditions for aerobic pathogens were
37°C for 24 hours and 37°C for 48 hours for bacterial
and yeast cultures respectively. Streptococci and L. aci-
dophilus species were grown under anaerobic conditions
using the candle jar method. After incubation, 40 μL of
a 0.04% w/v solution of p-Iodonitrotetrazolium chloride
indicator (INT) (Sigma-Aldrich) was added to each well

antimicrobial studies were compared to the results of the
whole essential oil in order to determine the role of these
compounds in the observed antimicrobial activity of this
oil. The ΣFIC was calculated according to the following
equations;
*where (a) is the MIC of one component in the combina-
tion and (b) is the MIC of the other component. The sum
of the FIC, is thus calculated as:
Results and discussion
The essential oil yield ranged from 0.14% to 4.31% (w/w)
for both young and mature leaf samples throughout
the sampling period (Table 1). The highest yields were
obtained during peak summer (December and January)
for both young (2.64–3.00%) and mature (3.67–4.31%)
leaf samples. In general, mature leaves produced higher
essential oil yields in comparison to the younger leaves.
Eucalyptus radiata is regarded as a high essential oil yield-
ing species and the expected yield is estimated between
2.50% and 3.50% (1, 12, 30, 31). However, yields outside
the expected range, as high as 9.00% have been reported
(13). In this study, seasons producing high rainfall and
high temperatures (summer) resulted in higher yields
in comparison to low rainfall, low temperate seasons
(autumn and winter). This correlation is in corroboration
with those reported for other Eucalyptus species (10). The
significance of leaf age was pronounced during autumn
and winter, with young leaves producing on average, two
times less oil in comparison to mature leaves.
A total of twenty-six compounds were identified, which
accounted for 93.5–99.5% of the total oil composition. The
majorcompounddeterminedfromthemean±SD(standard
deviation) of the monthly samples throughout the sampling
period was 1,8-cineole (65.7% ± 9.5). Other compounds
present in appreciable amounts were α-terpineol (12.8% ±
4.4) and limonene (6.5% ± 2.4) (Table 1 and Figure 1). An
OPLS-DAmodelwasconstructedonParetoscaleddatausing
two(1+1;predictive+orthogonal)componentsforbothtar-
geted and untargeted data. Figure 2a is the score plot for the
untargeted data showing subtle differences between young
and mature E. radiata leaves. The plot shows that young
leavesoccupythepositivepredictivecomponent(Pp1)while
FIC (i) =
MIC of (a*) combined with (b*)
MIC of (a) independently
FIC (ii) =
MIC of (b) combined with (a)
MIC of (b) independently
ΣFIC = FIC(i)
+ FIC(ii)
of the micro-titre plate and allowed to develop until a
color change (with reference to the culture control) was
observed. Results were read after 3 hours for all bacte-
rial cultures grown in TSB and after 24 hours for yeast
strains and cultures grown in MHB. The MIC was read
as the lowest concentration at which no visible growth
(no color change observed from the plate) was observed
after the addition of an indicator. The antimicrobial assays
were performed in duplicate (to check for accuracy and
re-tested where variance was observed) and undertaken
on consecutive days.
Interactive efficacy
The antimicrobial activities of the major compounds
identified in the essential oils were assessed singularly
and in combination using the MIC method previously
described against the pathogens that were most suscep-
tible to the E. radiata leaf essential oil. Combination
studies were undertaken to establish if any synergistic
interactions were apparent between major compounds.
The compounds 1,8-cineole at 98.0% purity (Lot
1054365), (+)-α-terpineol at 97.0% purity (Lot 427741/1)
and S-(-)-limonene at 99.0% purity (Lot 054076) were
obtained from Fluka. R-(+)-Limonene at 97.0% purity
(Lot 301Tl-101) was obtained from Sigma-Aldrich. These
compounds were prepared at starting concentrations of
32 mg/mL. The sum of the fractional inhibitory concen-
tration (ΣFIC) was used to determine the interaction
using 1:1 combinations of the compounds. Instead of
100 μL of sample added in to the first row of each well, a
1:1 ratio (50 μL of compound A and 50 μL of compound
B) was introduced into the first row of the micro-titre
plate. The sum of the fractional inhibitory concentration
(ΣFIC) was calculated and classified as either synergistic
(ΣFIC ≤0.50), additive (> 0.50 ΣFIC ≤ 1.00), indifferent
(> 1.00 ΣFIC ≤ 4.00) or antagonistic (ΣFIC >4.00) (26).
The FIC method is based on the principle that each test
agent is responsible for half of the antimicrobial activ-
ity of the combination mixture (28). The limitation with
FIC calculations is that: (a) the two compounds in com-
bination may not have the same dose response and (b)
plants do not accumulate compounds in 1:1 ratios (28,
29). To account for this, further combination studies were
additionally conducted on the major compounds at the
relative ratios (mean annual compositional ratio, Table
1) in which they naturally appeared in the E. radiata leaf
essential oil. For evaluation at the relative ratios the com-
pound mixtures comprised: 1,8-cineole (84 μL): α-terpi-
neol (16 μL), 1,8-cineole (95 μL): S-(-)-limonene (5 μL),
1,8-cineole (95 μL): (R)-(+)-limonene (5 μL), α-terpineol
(77 μL): S(-)-limonene (23 μL), α-terpineol (77 μL): (R)-
(+)-limonene (23 μL). Independent and combination

Table1. ChemicalcompositionofE.radiataleavesessentialoilfortheperiodJanuary2014toDecember2014.
Notes:a
Majorcompounds;tr(traceamounts<0.1).
SummerAutumnWinterSpringSummer
Mean±
standard
deviation
(SD)
JanFebMarAprMayJunJulAugSepOctNovDec
RRICompoundYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
ture
Ma-
tureYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
tureYoung
Ma-
ture
Essentialoil
yield(%;
w/w)
2.643.671.810.900.280.430.612.830.140.360.221.690.141.551.031.031.641.662.441.352.653.004.311.6±1.2
1016α-Pinene2.32.21.22.03.65.12.52.22.32.60.43.12.12.62.61.21.31.52.53.82.52.31.22.3±1.0
1019α-Thujene0.20.20.10.20.10.30.20.20.20.1tr0.2tr0.20.30.10.10.2tr0.20.20.10.10.2±0.1
1104β-Pinene0.80.70.50.60.61.20.70.60.80.70.30.80.70.70.90.50.60.60.71.00.81.1tr0.7±0.2
1117Sabinene1.41.01.10.80.71.10.80.70.80.90.50.91.40.70.70.70.90.70.70.61.20.70.50.8±0.3
1159Myrcene2.01.71.41.92.23.31.61.32.01.70.82.01.71.51.91.01.11.21.53.11.33.3tr1.8±0.7
1174α-terpinene0.10.20.20.20.30.30.20.20.30.20.10.30.20.30.30.20.10.2Tr0.40.30.70.10.2±0.1
1194Limonenea
6.36.54.64.45.512.86.46.38.36.53.67.46.16.46.54.24.65.15.99.46.713.03.76.5±2.4
12021.8-Cineolea
66.968.668.066.063.841.666.666.356.267.252.473.071.475.166.077.373.669.272.053.169.347.779.065.7±9.5
1242γ-Terpinene0.20.40.30.40.50.70.40.50.70.50.30.60.30.55.20.30.20.40.50.70.5trtr0.7±1.0
1250(E)-β-Ocimene0.40.30.30.11.30.60.30.30.40.30.40.50.30.3tr0.20.30.5Tr0.70.31.30.20.4±0.3
1270p-Cymene0.60.50.10.60.20.80.20.30.30.30.10.30.40.30.30.30.10.3Tr0.20.10.10.10.3±0.2
1281Terpinolene0.10.10.10.10.20.20.10.10.20.1tr0.10.10.1tr0.1tr0.2Trtr0.10.30.10.1±0.1
1382Z-3-Hex-en-
1-ol
trtrtrtrtrTr0.10.1tr0.1trTrtrTrtrtrtrtrTr0.10.4trtr0.1±0.1
1541Linalool0.50.40.70.30.30.30.50.50.50.50.60.20.30.30.40.20.50.4Tr0.40.3tr0.20.4±0.1
1563Trans-p-
menth-2-en-
1-ol
0.20.20.10.60.10.10.10.10.10.10.3Tr0.10.1tr0.20.20.2Trtr0.10.2tr0.2±0.1
1602Terpinene-4-ol1.11.20.91.70.51.31.41.72.01.41.90.20.91.11.31.11.01.31.71.50.12.40.71.2±0.6
1674γ-Terpineol0.20.20.30.30.30.30.20.20.30.20.40.10.20.10.10.20.20.2Tr0.3tr0.30.20.2±0.1
1689Neral0.10.20.20.50.10.10.20.20.30.20.4Tr0.10.1tr0.10.20.2Tr0.30.2tr0.20.2±0.1
1701α-Terpineola
12.611.015.013.66.713.712.914.418.812.327.47.010.77.610.49.511.913.110.517.011.416.410.112.8±4.4
1740Geranial0.50.30.40.70.20.20.20.30.40.20.6Tr0.20.10.30.10.40.4Trtr0.2tr0.10.3±0.2
1743γ-Elemene0.20.40.30.54.64.10.50.60.90.50.40.20.20.2tr0.10.10.2Tr0.30.30.30.10.7±1.2
1822Geraniol1.81.02.70.20.73.61.71.82.11.46.30.91.50.91.51.11.461.02.51.32.91.21.8±1.3
2141Spathulenoltr0.1Tr0.10.10.70.10.10.20.10.1TrtrTrtrtrtrtrTrtrtr0.1tr0.2±0.2
2181γ-Eudesmol0.10.20.10.30.40.70.10.10.20.20.20.1tr0.10.10.1tr0.1Tr0.20.1trtr0.2±0.2
2235α-Eudesmol0.10.30.10.40.41.00.20.20.20.20.20.10.10.1tr0.1tr0.1Trtr0.1trtr0.2±0.2
2245β-Eudesmol0.10.40.10.50.41.20.20.10.20.30.30.10.10.1tr0.1tr0.1Trtr0.10.3tr0.3±0.3
Totalarea
percentage
(%)
98.898.398.897.093.895.398.499.498.798.898.098.199.199.598.899.098.898.097.095.897.993.597.8

the low modeled variance of 28% (Pp1 = 0.28) related to
this distinguishing feature. To further investigate the chem-
ical features responsible for these observed differences, an
S-plot was constructed and analyzed (Figure 2b). Variables
of high correlation and covariance, on the extreme ends of
theS-plotwereidentifiedandthecorrespondingcompounds
assigned to these retention/mass pairs (Table 2). Both the
S-plot and Table 2 suggest that high levels of limonene and
α-terpineolareconsistentwithyoungerleaveswhileα-pinene
and1,8-cineoleareabundantinmatureleaves.Usingthetar-
getedapproach,thesampledistributionshowsminimalsep-
aration between young and mature leaves and some overlap
betweenthetwoclassesasobservedinthescoreplot(Figure
3a). Statistically, only 21% (Pp1) of the modeled variance
was attributed to leaf maturity, which is lower than in the
untargeted approach (28%). Biomarker identification using
the S-plot displayed only two variables attributed to this
observation (Figure 3b; Table 3). Interesting to note was the
similarityinthebiomarkersidentifiedusingthetwodifferent
approaches, however, the targeted approached yielded less
variablescomparedtotheuntargetedapproach.Thetargeted
approach identified α-terpineol as a marker in young leaves
while 1,8-cineole was also identified for mature leaves.
Seasonal variation was assessed using a two (1+1;
predictive + orthogonal) component model based on
Pareto scaled data for targeted and untargeted approaches.
Using the untargeted approach, a clear seasonal separa-
tion of the samples based on summer and winter was
observed along the predictive component (Figure 4a).
A 14% modeled variance (Pp1 = 0.14) was recorded for
matureleavesarepredominantlyonthenegativeend.Partial
overlap is observed among the samples which could explain
Figure 2. An OPLS-DA score plot showing distribution of young
and mature E. radiata leaves based on untargeted GC-MS analysis
(A), an S-plot displays variables of high correlation and covariance
responsible for separation of young ( top right) and mature
( bottom left) plants (B).
Figure 1. Total ion chromatogram of a South African sample of Eucalyptus radiata leaf essential oil with chemical structures of major
compounds 1,8-cineole, α-terpineol and limonene.

winter season. In addition to limonene and 1,8-cineole,
α-thujone and γ-terpinene are among the list of com-
pounds that dominate during the summer season but
occur at lower levels during winter. Using the targeted
approach, 14% variation (Pp1 = 0.14) was also modeled
for seasonal variation, however, the clustering pattern
in the score plot was not as clear as observed using the
untargeted approach (Figure 5a). Again, fewer variables
were identified from the S-plot as biomarkers responsible
for this variation (Figure 5b). Table 5 lists the biomarkers
showing again that 1,8-cineole is correlated with winter
months while γ-terpinene is associated with the summer
months corroborating the untargeted results. A few addi-
tional compounds were also identified using the targeted
approach.
The chemical composition of the leaf oil of E.
radiata obtained through different studies has
this distinguishing feature which suggests variance in E.
radiata chemistry between seasons. Other variation in
the data set not related to the seasons was observed along
the orthogonal component (Po1 = 33%), which accounts
for higher variability in the sample set. To investigate the
variables related to the seasonal variation observed, the
extreme ends of the S-plot were assessed for biomarker
retention mass pairs and the corresponding compounds
identified (Figure 4b; Table 4). Table 4 shows α-pinene,
sabinene, limonene, 1,8-cineole, terpinene-4-ol and ter-
pineol as dominant compounds in the plants during the
Table 2. List of biomarker compounds identified using the S-plot in the untargeted approach.
Leaf age R.t (min) Mass Compound ID
Young leaves 17.66 92.9999 Limonene
35.42 92.9999; 121.0000; 135.9999 α-Terpineol
Mature leaves 9.56 93.000 α-Pinene
18.33 80.9999; 84.000; 92.9999; 107.9999; 111.0000; 138.9999; 153.9999 1.8-Cineole
Figure 3. An OPLS-DA score plot showing distribution of young
and mature E.radiata leaves based on targeted GC-MS analysis(A),
an S-plot displays variables of high correlation and covariance
responsible for separation of young ( bottom left) and mature
( top right) plants (B).
Table 3. List of biomarker compounds identified using the S-plot
in the targeted approach.
Leaf age R.t (min) Compound ID
Young leaves 35.38 α-Terpineol
Mature leaves 17.87 1.8-cineole
Figure 4. An OPLS-DA score plot showing distribution of summer
and winter E. radiata leaves based on untargeted GC-MS analysis
(A), an S-plot displays variables of high correlation and covariance
responsible for separation of summer ( top right) and winter
( bottom left) plants (B).

The antimicrobial activity of the E. radiata leaf
essential oil samples against the eighteen test patho-
gens is summarized in Figure 6 (a, b, c and d). A review
proposed that for essential oils, an MIC value of 2.00
mg/mL or lower should be considered noteworthy (33).
Therefore, noteworthy activity was observed through-
out the sampling period from monthly samples of
both young and mature leaf oils (Figure 6a, b, c and
d) for 11 of the 18 test pathogens. The most suscep-
tible micro-organisms were the Streptococci and L.
acidophilus, particularly S. mutans with an MIC range
between 0.25–1.00 mg/mL and L. acidophilus with
an MIC of 0.19–1.75 mg/mL (Figure 6a). Among the
gastrointestinal-related pathogens, L. monocytogenes
and B. cereus were the most susceptible with MIC
been reported. 1,8-Cineole (72.5%), α-terpineol
(11.6%) and limonene (4.5%) were also reported as
the major compounds of an oil sample from India
(12). Furthermore, 1,8-cineole (80.8%), α-terpineol
(6.4%) and limonene (3.7%) were also reported as the
major compounds of an oil sample from Zambia (13).
1,8-Cineole (69.5%), α-pinene (11.9%) and trans-
pinocarveol (4.8%) were reported as the major com-
pounds from a Tunisian oil sample (8). 1,8-Cineole
(82.7%), α-terpineol (7.0%) and α-pinene (3.7%)
were reported as the major compounds of a German
sample (14). Limonene (68.51%), α-terpineol (8.60%)
and α-terpinyl acetate (6.07%) were reported as
the major compounds of the E. radiata oil sample
from Portugal (15). The South African harvested
young and mature E. radiata leaf essential oil sam-
ples contained similar major compounds reported
by the majority of these previous studies (Table 1).
Variations in the compound ratios were observed
which may be influenced by the differences in geo-
graphical locality and growth conditions of the E.
radiata samples.
Changes in chemical composition due to leaf age
were noted at different levels of maturity. Higher levels
of limonene and α-terpineol were consistent with young
leaves, while higher levels of α-pinene and 1,8-cineole
were consistent with mature leaves. A similar difference
in leaf oil composition due to leaf age has been previously
noted (7).
The use of untargeted approaches in the analysis of
multivariate data provides a comprehensive and rapid
analysis of data. The biomarkers varied between the winter
(α-pinene, sabinene, limonene, 1,8-cineole, terpinene-4-ol
andterpineol)andsummerseasons(limonene,1,8-cineole,
α-thujone and γ-terpinene) (Table 4). These differences in
chemical composition due to seasonal variation highlight
the significant role of seasonal variation on Eucalyptus
leaf essential oil composition, as noted in previous studies
(11, 32).
in the untargeted approach.
Season R.t (min) Mass Compound ID
Winter 9.56 90.9997; 93.0000 α-Pinene
13.74 84.0000; 80.9999; 92.9999;
95.9999
Sabinene
17.37 106.9999; 121.0000 Limonene
18.33 107.9999; 111.0000;
138.9999; 153.9999
1,8-Cineole
32.54 92.9999 Terpinene-4-ol
36.08 90.9998 α-Terpineol
Summer 9.81 90.9997; 93.0000 α-Thujene
17.94 90.9999; 92.9999 Limonene
18.01 107.0000; 135.9999 1,8-Cineole
19.56 93.0000 γ-Terpinene
Figure 5. An OPLS-DA score plot showing distribution of summer
and winter E.radiata leaves based on targeted GC-MS analysis (A),
an S-plot displays variables of high correlation and covariance
responsible for separation of summer ( bottom left) and winter
( top right) plants (B).
in the targeted approach.
Season R.t (min) Compound ID
Winter 17.87 1,8-Cineole
36.51 α-Elemene
Summer 19.41 γ-Terpinene
35.38 α-Terpineol
39.36 Geraniol

the non-resistant S. aureus strain with an MIC range
of (1.00–3.00 mg/mL) (Figure 6d).
Previous antimicrobial investigations on E. radiata
included measures of vapor activity (34) and diffusion
assays (8, 23). The lipophilic and volatile nature of essen-
tial oils may not allow for easy diffusion through the agar
and may lead to loss of a portion of the essential oil dur-
ing the pre-diffusion stage in agar diffusion assays. Also,
vapor composition may not reflect the composition of
the whole essential oil, thus making these earlier results
values between 0.25–1.00 mg/mL and 0.25–2.00 mg/
mL, respectively (Figure 6b). Among the respiratory-
related pathogens, S. agalactiae (0.19–1.00 mg/mL) and
S. pneumoniae (0.19–1.00 mg/mL) were the most sus-
ceptible (Figure 6c). Pseudomonas aeruginosa showed
the highest sensitivity with an MIC range of 0.50–1.50
mg/mL among the wound/skin-related pathogens
(Figure 6d). Interestingly, similar activity was observed
against the methicillin-resistant Staphylococcus strain
(MRSA) with an MIC range of 0.50–3.00 mg/mL and
0.00
0.50
1.00
1.50
2.00
2.50
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
MIC(mg/mL
Pathogens associated with dental infections
S. mutans L. acidophilus
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Mature
Young
Mature
Young
Mature
Young
Mature
Young
MIC(mg/mL)
Pathogens associated with gastrointestinal/food-related infections
E. coli B. cereus S. typhi S. sonnei L. monocytogenes
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Mature
Young
Mature
Young
Mature
Young
Mature
Young
MIC(mg/mL)
Pathogens associated with respiratory infections
C. neoformans M. catarrhalis K. pneumoniae S. agalactiae S. pneumoniae S. pyogenes
0.00
1.00
2.00
3.00
4.00
5.00
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Young
Mature
Mature
Young
Mature
Young
Mature
Young
Mature
Young
MIC(mg/mL)
Pathogens associated with wound/skin infections
C. albicans E. faecalis P. aeruginosa MRSA S. aureus
(a)
(b)
(c)
(d)
Figure 6. Antimicrobial activity (mean MIC expressed in mg/mL) of monthly young and mature E. radiata leaf essential oil samples
across one year sampling period, against micro-organisms associated with dental (a), gastrointestinal (b), respiratory (c) and wound (d)
infections.

In contrast variation in activity was observed during the
winter months, whereby S. mutans was more susceptible
than L. acidophilus to the essential oil (Figure 6a). These
differences in activity can be attributed to the differences
in dominant compounds between the two seasons.
Listeria monocytogenes contamination is problematic
in the food industry, often resulting in compromised
food quality and safety (39). Among the gastrointestinal/
food-related pathogens, L. monocytogenes was the most
susceptible (0.25–1.00 mg/mL (Figure 6b). Eucalyptus leaf
extracts have been approved as food additives (40), there-
fore the noteworthy antimicrobial activity of the E. radiata
leaf essential oil shows potential for use as a preservative.
Unlike the dental pathogens (S. mutans and L. acido-
philus), no significant variation in antimicrobial activ-
ity against the gastrointestinal/food-related pathogens
was observed between the summer and winter months
(Figure 6b). Gastrointestinal/food-related pathogens were
less sensitive to the differences in dominant essential oil
compounds between the two seasons in comparison to
dental pathogens.
Eucalyptus oil is predominantly used in the treatment
of respiratory disorders (7, 41). Eucalyptus radiata is no
exception and the oil has been termed the ‘the oil of res-
piration’ (14, 20). The noteworthy antimicrobial activity
of the E. radiata oil against these respiratory pathogens
not only shows that there is some in vitro rationale
behind its use for respiratory disorders, but also high-
lights the potential for application in the management
of respiratory conditions associated with S. agalactiae
(0.19–1.00 mg/mL) and S. pneumoniae (0.19–1.00 mg/
preliminary (23, 35). The MIC method is the preferred
method for antimicrobial evaluation of plant studies and
essential oils (26, 36). Therefore, only studies reporting
broth microdilution (MIC) assay results were considered
for comparison. To the best of our knowledge, only three
other studies have reported the antimicrobial efficacy of
E. radiata leaf essential oil using the quantitative MIC
method (14, 15, 24), but not to the comprehensive nature
as reported herein.
The properties exhibited by an essential oil are deter-
mined by its unique qualitative and quantitative chemical
composition, which has been shown to vary according to
seasonal variation and leaf age for the E. radiata species
(Tables 1–3, Figures 1–3). Furthermore, the antimicrobial
activities of essential oils have been linked to monoterpe-
nes (8, 17). Eucalyptus radiata leaf essential oil comprises
of various monoterpenes (Table 1).
Noteworthy activity (0.19–1.75 mg/mL) against dental
pathogens (S. mutans and L. acidophilus) is aligned with
previous findings on cariogenic and periodontopathic
micro-organisms which previously reported E. radiata
oil to have anti-adhesion activity against S. mutans (24).
The monoterpenes linalool and α-terpineol possess strong
antibacterial activity against periodontopathic and car-
iogenic micro-organisms (37, 38). α-Terpineol was one
of the dominant compounds during winter and summer
(Tables 4–5), which could explain the noteworthy antimi-
crobial activity of the E. radiata leaves essential oil against
dental pathogens across the sampling period. Dental
pathogens, S. mutans and L. acidophilus showed similar
susceptibility to the essential oil in the summer months.
Table 6. Mean MIC (mg/mL) for the major compounds independently and in combination with ΣFIC (in brackets), determined for 1:1
combinations and combinations at various ratios (relative to essential oil composition in Table 1).
Note: Values in bold demonstrate synergistic activity.
Compound
Pathogens
L. acidophilus S. pyogenes S. mutans S. pneumoniae S. agalactiae
Independent compounds
1,8-Cineole 2.00 2.00 2.00 2.00 2.00
α-Terpineol 0.88 0.75 0.75 1.00 1.00
S-(-)-Limonene 0.38 0.25 0.38 0.50 0.75
R-(+)-Limonene 0.38 0.25 0.25 0.50 0.63
1:1 Combinations
1,8-Cineole:α-Terpineole 1.00 (0.82) 1.00 (0.92) 1.00 (0.92) 1.50 (1.13) 1.50 (1.13)
1,8-Cineole: S-(-)-Limonene 0.50 (0.79) 0.50 (1.13) 0.25 (0.40) 0.25 (0.31) 0.50 (0.46)
1,8-Cineole:R-(+)-Limonene 0.50 (0.79) 0.50 (1.13) 0.25 (0.56) 0.25 (0.31) 0.50 (0.53)
α-Terpineole: S-(-)-Limonene 0.25 (0.48) 0.25 (0.67) 0.25 (0.50) 0.25 (0.38) 0.38 (0.44)
α-Terpineole:R-(+)-Limonene 0.25 (0.48) 0.25 (0.67) 0.25 (0.67) 0.25 (0.38) 0.25 (0.33)
S-(-)-Limonene: R-(+)-Limonene 0.25 (0.67) 0.25 (1.00) 0.50 (1.67) 0.25 (0.50) 0.25 (0.37)
Various ratios (relative to essential oil composition in Table 1)
1,8-Cineole:α-Terpineol 2.00 (1.64) 1.00 (0.92) 1.00 (1.83) 1.00 (0.75) 2.00 (1.50)
1,8-Cineole: S-(-)-Limonene 1.00 (2.25) 1.00 (2.25) 1.50 (2.35) 1.00 (1.25) 2.00 (1.83)
1,8-Cineole:R-(+)-Limonene 1.00 (1.57) 2.00 (4.50) 1.00 (2.25) 1.00 (1.25) 2.00 (2.09)
α-Terpineole:S-(-)-Limonene 0.50 (0.94) 0.50 (1.33) 0.25 (0.50) 0.25 (0.38) 1.00 (1.17)
α-Terpineole:R-(+)-Limonene 0.50 (0.94) 0.50 (1.33) 0.25 (0.67) 0.25 (0.38) 1.00 (1.29)
Control (Penicillin) 0.31 x 10−3
0.31 x 10−3
0.16 x 10−3
1.25 x 10−3
0.31 x 10−3

additive or indifferent effects (28). All the 1:1 combina-
tions demonstrated reduced MIC values for at least one
of the paired compounds. From the 1:1 combinations, the
α-terpineol: S-(-)-limonene combination resulted in the
highest number of synergistic interactions with synergy
observed against L. acidophilus, S. mutans, S. pneumoniae,
S. agalactiae and additive effects noted against S. pyogenes
(Table 6).
The relative ratio combinations produced ΣFIC val-
ues ranging from 0.38 to 4.50 (Table 6). Less synergy was
observed at these various ratios in comparison to com-
binations at 1:1 ratios. The general pattern identified was
that when limonene is in lower quantities the antimi-
crobial activity of the combination decreases. Although,
1,8-cineole represents the highest proportion of the E.
radiata essential oil composition, these results indicate
that the major compound (in the highest proportion) is
not necessarily the most potent (Table 6). Instead, the
results show that in general, limonene (both (+) and (-)
isomers tested) is the more active compound from the
three major compounds tested (Table 6). In contrast to
previous reports (29), this study found that both enanti-
omers of limonene displayed similar antimicrobial activity
against the selected test pathogens.
The antimicrobial activity of the compounds at 1:1
ratios was lower than the activity of at least one of the
compounds independently. These results indicate that
interactions exist between these major compounds
found within the E. radiata leaf essential oil sample, and
these interactions have the ability to alter (enhance or
reduce) the antimicrobial activity of the combination.
Furthermore, combinations containing limonene as one
of the compounds generally resulted in enhanced anti-
microbial activity (synergistic and additive outcomes).
Plants do not accumulate compounds in 1:1 ratios (Table
1). Thus, the major compounds were further combined
at the relative ratios (mean annual compositional ratio,
Table 1) in which they naturally appeared within the
whole E. radiata oil. Table 6 shows that the ratio at which
various compounds occur within the essential may be a
determinant factor to whether antimicrobial activity is
enhanced or not.
It is important to keep in mind that, E. radiata leaf
essential oil contains a variety of other compounds with
antibacterial activity such as; myrcene, linalool, β-pinene,
α-pinene, terpinolene to name a few (17, 43). Further
research into the antimicrobial properties of these minor
compounds independently and in combination with the
major compounds is recommended to gain a more holis-
tic understanding of their role in the activity of this E.
radiata essential oil. Essential oils have been reported
to exhibit higher antimicrobial activity than their major
compounds (44). For this study, the combination of the
mL). Similar to gastro-intestinal/food-related pathogens,
no significant variation in antimicrobial activity against
the respiratory pathogens was observed between the sum-
mer and winter months (Figure 6c). The differences in
dominant compounds between the two seasons did not
affect activity. This observation may be attributed to the
presence of α-terpineol, limonene and 1,8-cineole. These
three compounds were the dominant compounds in both
winter and summer months. Furthermore, α-terpineol,
limonene and 1,8-cineole displayed noteworthy antibac-
terial activity against the respiratory-related pathogens (S.
agalactiae, S. pneumoniae and S. pyogenes) (Table 6) when
tested independently.
Traditionally, topical ointments containing Eucalyptus
oil were used in Aboriginal medicines for the healing of
wounds and fungal infections (7, 40, 42). Among the many
reported uses for E. radiata oil includes the treatment of
acne, vaginitis, and wound healing (18, 21). The note-
worthy antimicrobial activity displayed against pathogens
associated with wound/skin infections shows that there is
some in vitro rationale behind its used for wound infec-
tions (Figure 6d). Previously, poor-to-moderate activity
against MRSA (≥ 4 mg/mL) was noted (14). However, in
this study, noteworthy activity, as low as 0.50 mg/mL was
noted against the MRSA strain. This noteworthy activ-
ity was particularly observed in the summer months of
November and December, by both young and mature
leaf oil samples. It is interesting to note that during these
months, significant variation in the ratio of major com-
pounds was observed between young and mature leaf
samples (Table 1).
In an effort to better understand the relationship
between chemical composition and antimicrobial activ-
ity, the antimicrobial properties of the major compounds
were evaluated independently and in combination.
Antimicrobial activities of the major compounds were
evaluated independently and in combination (1:1 com-
bination and at the relative ratios they naturally occur in
the essential oil as reported in Table 1) in order to establish
interactions in relation to the antimicrobial activity of the
E. radiata leaf essential oil. These were evaluated against
micro-organisms showing the most promising antimicro-
bial activity.
The antimicrobial results (MIC values) of the major
compounds are shown in Table 6. Independently, the
major compounds exhibited varied noteworthy activities
against all five test pathogens. 1,8-Cineole had MIC values
of 2.00 mg/mL against all pathogens tested. α-Terpineol
displayed MIC values of 0.75–1.00 mg/mL and S-(-)-
limonene and R-(+)-limonene had MIC values of between
0.25 mg/mL and 0.75 mg/mL.
All 1:1 combinations produced ΣFIC values ranging
from 0.31 to 1.67 (Table 6), corresponding to synergistic,

Conclusion
This study is the first detailed (annual) report on the
yield, chemical composition and antimicrobial activity of
the essential oils from young and mature South African
harvested E. radiata leaves. The yield and chemical com-
position of essential oils obtained from both young and
mature E. radiata leaves are largely influenced by seasonal
variation, where high yields and higher cineole content
can be obtained under conditions of high rainfall and high
temperatures. Both young and mature E. radiata leaf oil
possess noteworthy antimicrobial activity against a broad
spectrum of pathogens (Gram-positive, Gram-negative
and yeast) and showed the highest potential for use against
the dental pathogens, S. mutans and L. acidophilus. The
E. radiata oil sample can be used as a substitute for other
Eucalyptus species based on the similarity of antimicro-
bial activity against the test pathogens. The correlation
between the chemical composition and the antimicrobial
activity is related to the presence of the major compounds.
Limonene had the highest antimicrobial activity and the
strongest influence on the strength of the antimicrobial
activity of the combinations. Depending on the ratio of
the compounds, synergistic interactions may be observed.
In summary, the South African E. radiata leaf essen-
tial oil showed good oil yields, a relatively consistent
chemical profile and noteworthy antimicrobial activity.
The combination of these properties makes E. radiata oil
appealing as a worthwhile source of essential oil, with
potential for use as a commercial antimicrobial. In con-
tribution to the body of knowledge of its real world use,
this study provides an in vitro antimicrobial rationale
behind the broad anti-infective traditional uses of the
essential oil. Follow-up studies should be conducted to
major compounds showed MIC values similar to that of
the whole essential oil. Furthermore, from the results it is
evident that limonene (both enantiomers) has the most
contributory effect on the strength of the antimicrobial
activity.
In order for E. radiata to be considered as an additional
medicinal Eucalyptus essential oil for anti-infective use,
scientific data showing similar efficacy to commercial
Eucalyptus oils is needed. The antimicrobial efficacy of E.
radiata essential oil was evaluated in comparison to com-
mercially acquired and other popular and commercially
relevant Eucalyptus species such as E. globulus, E. camald-
ulensis, E. citriodora, E. dives and E. smithii (Table 7). The
essential oil samples from different Eucalyptus species pos-
sessed predominantly noteworthy antimicrobial activity
against all the micro-organisms (Table 7). All the essential
oils appeared to be more active against Streptococci and
L. acidophilus. This is in agreement with the findings for
the E. radiata leaf oil samples.
The Eucalyptus genus is known to have efficacy
against dental pathogens, hence the incorporation into
products like Colgate® Herbal® toothpaste (Colgate-
Palmolive Company, Gauteng, South Africa; toothpaste
containing Eucalyptus globulus leaf oil as an ingredient)
and Aquafresh® Herbal toothpaste (GlaxoSmithKline,
Gauteng, South Africa; toothpaste containing E. globu-
lus as an ingredient). The results of this study indicate
that the E. radiata essential oil test sample possesses
similar antimicrobial activity to all the Eucalyptus
essential oils (Table 7). Even though E. globulus is the
most documented and most commonly used species
(14, 18), equal credibility should be given to the E.
radiata essential oil based on how well it compares to
the other popular species.
Table 7. Antimicrobial activity (Mean (n= ≥ 2) MIC values in mg/mL of different Eucalyptus leaf essential oils.
Notes: Noteworthy activity is in bold; a
Laboratory distillation acquired essential oils; b
Commercially acquired essential oils; Ciprofloxacin was used as the control for
bacteria excluding Streptococci and L. acidophilus where penicillin was used as the control; Amphotericin B was used as the control for the yeast.
Pathogens
Eucalyptus species
radiataa
radiata commb
globulusb
camaldulensisa
citriodorab
smithiib
divesb
Control
B. cereus 0.50 1.50 0.25 0.25 1.00 2.00 1.00 0.039e−3
C. albicans 1.00 1.00 1.00 0.50 1.00 1.00 1.00 3.125e−3
C. neoformans 1.00 1.00 1.00 0.50 1.00 1.00 1.00 6.250e−3
E. faecalis 2.00 3.00 1.50 2.00 2.00 2.00 2.00 0.625e−3
E. coli 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.625e−3
K. pneumoniae 2.00 2.00 2.00 2.00 2.00 2.00 2.00 0.039e−3
L. acidophilus 0.50 1.00 1.00 0.38 0.75 1.00 1.00 0.310e−3
L. monocytogenes 0.75 1.00 0.50 0.50 0.50 1.00 1.00 0.625e−3
S. aureus 2.00 2.00 2.00 0.50 1.00 2.00 2.00 0.625e−3
Methicillin-resistant S. aureus 2.00 1.00 0.75 0.50 1.00 2.00 1.00 1.250e−3
M. catarrhalis 4.00 4.00 4.00 4.00 2.00 2.00 2.00 0.313e−3
P. aeruginosa 1.00 1.00 1.00 1.00 1.00 2.00 1.00 0.313e−3
S. typhimurium 2.00 2.00 4.00 2.00 3.00 2.00 2.00 0.039e−3
S. sonnei 3.00 1.50 3.00 2.00 1.00 2.00 1.50 0.625e−3
S. agalactiae 0.25 0.50 0.25 0.25 0.75 0.25 0.25 0.310e−3
S. mutans 0.50 0.50 0.25 0.25 0.50 0.50 0.25 0.160e−3
S. pneumoniae 0.25 1.00 2.00 1.00 1.00 2.00 1.00 1.250e−3
S. pyogenes 0.50 1.00 0.50 0.50 1.00 0.50 0.50 0.310e−3

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E. radiata essential oil sample and conventional antibi-
otics (15). However, the composition of the essential oil
differed in comparison to our sample. Therefore, further
combination studies should be conducted to evaluate
the potential of the South African E. radiata essential oil
to potentiate the antimicrobial activity of conventional
antibiotics/other essential oils as it is commonly used in
blends.
Acknowledgments
The authors would like to thank the National Research Foun-
dation (NRF) and the Faculty Research Committee (FRC)
(Faculty of Health Sciences, University of the Witwatersrand)
for financial assistance towards this research. Thanks to the
University of the Witwatersrand and Tshwane University of
Technology for the infrastructural support and for the resourc-
es provided for this research. Mr. Bruce Stumbles is acknowl-
edged for the continual and timely supply of plant material
used in this study.
Disclosure statement
The authors report no conflicts of interest.
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