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Journal of Essential Oil Research

ISSN: 1041-2905 (Print) 2163-8152 (Online) Journal homepage: http://www.tandfonline.com/loi/tjeo20

Chemical composition and antimicrobial activity ofEucalyptus radiata leaf essential oil, sampled overa 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, MaxleeneSandasi & Alvaro M. Viljoen (2016): Chemical composition and antimicrobial activity ofEucalyptus 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, 2016http://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

aDepartment of Pharmacy and Pharmacology, faculty of health sciences, university of the Witwatersrand, Johannesberg, south africa; bDepartment of Pharmaceutical sciences, tshwane university of technology, Pretoria, south africa; csaMrc herbal Drugs research unit, Department of Pharmaceutical sciences, tshwane university of technology, Pretoria, south africa

ABSTRACTThis 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

Eucalyptus radiata (syn. Eucalyptus australiana) is commonly referred to as the narrow-leaved peppermint, forth river pep-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

KEYWORDSseasonal variation; south africa; chemometric analysis; major compound; Mic

ARTICLE HISTORYreceived 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

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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

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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

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4 G. D. MAhuMAnE ET AL.

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 major compound determined from the mean ± 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-DA model was constructed on Pareto scaled data using two (1 + 1; predictive + orthogonal) components for both tar-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 leaves occupy the positive predictive component (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

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JournAL oF ESSEnTIAL oIL rESEArCh 5

Tabl

e 1.

 che

mic

al c

ompo

sitio

n of

E. r

adia

ta le

aves

ess

entia

l oil

for t

he p

erio

d Ja

nuar

y 20

14 to

Dec

embe

r 201

4.

not

es: a M

ajor

com

poun

ds; t

r (tr

ace

amou

nts <

0.1

).

Sum

mer

Autu

mn

Win

ter

Sprin

gSu

mm

erM

ean

± st

anda

rd

devi

atio

n (S

D)

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

oct

nov

Dec

rrI

Com

poun

dYo

ung

Ma-

ture

Youn

gM

a-tu

reYo

ung

Ma-

ture

Youn

gM

a-tu

reYo

ung

Ma-

ture

Youn

gM

a-tu

reYo

ung

Ma-

ture

Ma-

ture

Youn

gM

a-tu

reYo

ung

Ma-

ture

Youn

gM

a-tu

reYo

ung

Ma-

ture

Esse

ntia

l oil

yiel

d (%

; w

/w)

2.64

3.67

1.81

0.90

0.28

0.43

0.61

2.83

0.14

0.36

0.22

1.69

0.14

1.55

1.03

1.03

1.64

1.66

2.44

1.35

2.65

3.00

4.31

1.6

± 1

.2

1016

α-Pi

nene

2.3

2.2

1.2

2.0

3.6

5.1

2.5

2.2

2.3

2.6

0.4

3.1

2.1

2.6

2.6

1.2

1.3

1.5

2.5

3.8

2.5

2.3

1.2

2.3

± 1

.010

19α-

thuj

ene

0.2

0.2

0.1

0.2

0.1

0.3

0.2

0.2

0.2

0.1

tr0.

2tr

0.2

0.3

0.1

0.1

0.2

tr0.

20.

20.

10.

10.

2 ±

0.1

1104

β-Pi

nene

0.8

0.7

0.5

0.6

0.6

1.2

0.7

0.6

0.8

0.7

0.3

0.8

0.7

0.7

0.9

0.5

0.6

0.6

0.7

1.0

0.8

1.1

tr0.

7 ±

0.2

1117

sabi

nene

1.4

1.0

1.1

0.8

0.7

1.1

0.8

0.7

0.8

0.9

0.5

0.9

1.4

0.7

0.7

0.7

0.9

0.7

0.7

0.6

1.2

0.7

0.5

0.8

± 0

.311

59M

yrce

ne2.

01.

71.

41.

92.

23.

31.

61.

32.

01.

70.

82.

01.

71.

51.

91.

01.

11.

21.

53.

11.

33.

3tr

1.8

± 0

.711

74α-

terp

inen

e0.

10.

20.

20.

20.

30.

30.

20.

20.

30.

20.

10.

30.

20.

30.

30.

20.

10.

2tr

0.4

0.3

0.7

0.1

0.2

± 0

.111

94Li

mon

enea

6.3

6.5

4.6

4.4

5.5

12.8

6.4

6.3

8.3

6.5

3.6

7.4

6.1

6.4

6.5

4.2

4.6

5.1

5.9

9.4

6.7

13.0

3.7

6.5

± 2.

412

021.

8-Ci

neol

ea66

.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

1242

γ-te

rpin

ene

0.2

0.4

0.3

0.4

0.5

0.7

0.4

0.5

0.7

0.5

0.3

0.6

0.3

0.5

5.2

0.3

0.2

0.4

0.5

0.7

0.5

trtr

0.7

± 1

.012

50(E

)-β-

oci

men

e0.

40.

30.

30.

11.

30.

60.

30.

30.

40.

30.

40.

50.

30.

3tr

0.2

0.3

0.5

tr0.

70.

31.

30.

20.

4 ±

0.3

1270

p-cy

men

e0.

60.

50.

10.

60.

20.

80.

20.

30.

30.

30.

10.

30.

40.

30.

30.

30.

10.

3tr

0.2

0.1

0.1

0.1

0.3

± 0

.212

81te

rpin

olen

e0.

10.

10.

10.

10.

20.

20.

10.

10.

20.

1tr

0.1

0.1

0.1

tr0.

1tr

0.2

trtr

0.1

0.3

0.1

0.1

± 0

.113

82Z-

3-h

ex-e

n-1-

oltr

trtr

trtr

tr0.

10.

1tr

0.1

trtr

trtr

trtr

trtr

tr0.

10.

4tr

tr0.

1 ±

0.1

1541

lina

lool

0.5

0.4

0.7

0.3

0.3

0.3

0.5

0.5

0.5

0.5

0.6

0.2

0.3

0.3

0.4

0.2

0.5

0.4

tr0.

40.

3tr

0.2

0.4

± 0

.115

63tr

ans-

p-m

enth

-2-e

n-1-

ol

0.2

0.2

0.1

0.6

0.1

0.1

0.1

0.1

0.1

0.1

0.3

tr0.

10.

1tr

0.2

0.2

0.2

trtr

0.1

0.2

tr0.

2 ±

0.1

1602

terp

inen

e-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

1674

γ-te

rpin

eol

0.2

0.2

0.3

0.3

0.3

0.3

0.2

0.2

0.3

0.2

0.4

0.1

0.2

0.1

0.1

0.2

0.2

0.2

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6 G. D. MAhuMAnE ET AL.

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 the S-plot were identified and the corresponding compounds assigned to these retention/mass pairs (Table 2). Both the S-plot and Table 2 suggest that high levels of limonene and α-terpineol are consistent with younger leaves while α-pinene and 1,8-cineole are abundant in mature leaves. Using the tar-geted approach, the sample distribution shows minimal sep-aration between young and mature leaves and some overlap between the two classes as observed in the score plot (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 similarity in the biomarkers identified using the two different approaches, however, the targeted approached yielded less variables compared to the untargeted approach. The targeted 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

mature leaves are predominantly on the negative end. 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.

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JournAL oF ESSEnTIAL oIL rESEArCh 7

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 IDYoung leaves 17.66 92.9999 limonene

35.42 92.9999; 121.0000; 135.9999 α-terpineolMature 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 IDYoung leaves 35.38 α-terpineolMature 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).

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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 and terpineol) and summer seasons (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).

Table 4. list of biomarker compounds identified using the s-plot in the untargeted approach.

Season r.t (min) Mass Compound IDWinter 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 limonene18.33 107.9999; 111.0000;

138.9999; 153.99991,8-cineole

32.54 92.9999 terpinene-4-ol36.08 90.9998 α-terpineol

summer 9.81 90.9997; 93.0000 α-thujene17.94 90.9999; 92.9999 limonene18.01 107.0000; 135.9999 1,8-cineole19.56 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).

Table 5. list of biomarker compounds identified using the s-plot in the targeted approach.

Season r.t (min) Compound IDWinter 17.87 1,8-cineole

36.51 α-Elemenesummer 19.41 γ-terpinene

35.38 α-terpineol39.36 Geraniol

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JournAL oF ESSEnTIAL oIL rESEArCh 9

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

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Pathogens associated with dental infections

S. mutans L. acidophilus

0.000.501.001.502.002.503.003.504.004.505.00

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Pathogens associated with gastrointestinal/food-related infectionsE. coli B. cereus S. typhi S. sonnei L. monocytogenes

0.000.501.001.502.002.503.003.504.004.505.00

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Pathogens associated with respiratory infectionsC. neoformans M. catarrhalis K. pneumoniae S. agalactiae S. pneumoniae S. pyogenes

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Pathogens associated with wound/skin infectionsC. albicans E. faecalis P. aeruginosa MRSA S. aureus

(a)

(b)

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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.

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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 compounds1,8-cineole 2.00 2.00 2.00 2.00 2.00α-terpineol 0.88 0.75 0.75 1.00 1.00s-(-)-limonene 0.38 0.25 0.38 0.50 0.75r-(+)-limonene 0.38 0.25 0.25 0.50 0.63

1:1 Combinations1,8-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

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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,

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12 G. D. MAhuMAnE ET AL.

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; alaboratory distillation acquired essential oils; bcommercially 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 ControlB. 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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evaluate the toxicity profile of the essential oil to minimize risk of harm in a therapeutic context. Previously, it was noted that synergistic interactions occurred between an 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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