Journal of Essential Oil Bearing Plants

ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: https://www.tandfonline.com/loi/teop20

Chemical Composition and Antifungal Activity of

Essential Oils Extracted from Leaves of Eucalyptus
Melliodora and Eucalyptus Anceps Grown in Rwanda

Daniel Umereweneza, Théoneste Muhizi, Théoneste Kamizikunze & Jean

Pierre Nkurunziza

To cite this article: Daniel Umereweneza, Théoneste Muhizi, Théoneste Kamizikunze & Jean
Pierre Nkurunziza (2019) Chemical Composition and Antifungal Activity of Essential Oils Extracted
from Leaves of Eucalyptus�Melliodora and Eucalyptus�Anceps Grown in Rwanda, Journal of
Essential Oil Bearing Plants, 22:1, 151-158, DOI: 10.1080/0972060X.2019.1585297

To link to this article: https://doi.org/10.1080/0972060X.2019.1585297

Published online: 19 Mar 2019.

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 TEOP 22 (1) 2019 pp 151 - 158 151
ISSN Print: 0972-060X
ISSN Online: 0976-5026

Chemical Composition and Antifungal Activity of Essential Oils Extracted from

Leaves of Eucalyptus Melliodora and Eucalyptus Anceps Grown in Rwanda

Daniel Umereweneza 1,2*, Théoneste Muhizi 1,

Théoneste Kamizikunze 1, Jean Pierre Nkurunziza 1

1
Department of Chemistry, College of Science and Technology,
University of Rwanda, Rwanda
2
Department of Chemistry-BMC, Uppsala University, Sweden
Received 22 June 2018; accepted in revised form 17 November 2018

Abstract: Essential oils were extracted from leaves of Eucalyptus melliodora and Eucalyptus anceps
by steam distillation and chemically analysed using GC/MS.The main components obtained in both Eucalyptus
species oils were, monoterpenes: 1,8-cineole (47.7 %, 33.49 %) and α-pinene (7.8 %, 13.69 %) as dominant
components, and sesquiterpenes: aromandrene (1 %, 18.01 %) and allo-aromandrene (1.5 %, 2.37 %), respectively.
Caryophyllene (5.1 %) and bicyclogermacrene (4.6 %) were found only in E. melliodora. The antifungal efficacy
of these essential oils was assessed against R. nigricans, A. flavus, A. niger, A. parasiticus, F. oxysporum and
P. digitatum.The obtained results indicated that essential oil from E. melliodora was the most effective against
all tested fungi with minimal inhibitory concentration (MIC) at 3.3 mg/mL for A. flavus. The anti-aflatoxin
production test was conducted with essential oil from E. melliodora and A. flavus and A. parasiticus, as
aflatoxins producers. The findings clearly showed that concentrations of 6 μL/mL and 7 μL/mL of the essential
oil completely inhibited production of aflatoxins by A. parasiticus and A. flavus, respectively. However, further
studies on this essential oil should be conducted to assure its standard use as antiaflatoxin agent.

Keywords: E. melliodora, E. anceps, essential oils, antifungal activity, mycotoxins, food

preservation.
Introduction
Eucalyptus is a diverse genus belonging to the
Myrtaceae family 1,2, this plant genus includes
about 900 species and subspecies. In 1788 through
the operculate nature of the flower which lacks
conspicuous petals and sepals, a French botanist,
Charles Louis L’Héritier de Brutelle has coined
this term of eucalyptus for this genus which
means ‘well covered’ 3. It is an evergreen tall
tree, native to Australia, successfully introduced
worldwide and now extensively cultivated in many
other countries including Rwanda.
A wide variety of secondary metabolites oc-
curring in large concentrations are found in Eu-
*Corresponding author (Daniel Umereweneza)
E-mail: < umereweneza@gmail.com, daniel.umereweneza@kemi.uu.se >
calyptus and its relatives 4. Different scientific
works reported antimicrobial properties of essen-
tial oils from the leaves of Eucalyptus 5,6, and
safeness of these aromatic compounds on human
health. Therefore, Eucalyptus essential oils have
been approved as food additives and preserva-
tives in Japan 7.
Food manufacturers include preservatives in
food products to prevent them from spoilage by
different microorganisms including molds and bac-
teria 8. In spite of the introduction of these chemi-
cals to increase shelf life of food, problems asso-
ciated to food contamination are still found in dif-
ferent countries.
© 2019, Har Krishan Bhalla & Sons
 Daniel Umereweneza et al., / TEOP 22 (1) 2019 151 - 158
During storage and transportation, a number of
fungal pathogens attack the food items leading to
their qualitative and quantitative loss 9. Therefore
foodstuffs should be stored and transported prop-
erly for future needs. However some Rhizopus,
Aspergillus, Penicillium and Fusarium species
are able to grow under particularly dry conditions
and attack stored foodstuffs. Not only do such
fungi damage the grains or render them unpalat-
able, but also they may excrete toxins that can
cause illness or even death to consumers 8.
Due to climatic factors including temperature,
relative humidity and moisture content of stored
foodstuffs, fungal population continue to grow on
food especially in tropical and sub-tropical regions
of the world and cause economic and social prob-
lems. Around a third of all foodstuffs produced
for world’s population are lost from field to con-
sumer, amounting to roughly 1.3 billion metric tons
per annum 10,11.
Though data on current situation on fungal food
spoilage are scarce in Rwanda, the border rejec-
tion that the country has suffered could be an alert
for the presence of aflatoxins in higher concen-
trations than regulatory levels in food commodi-
ties, which are used as food for daily life of
Rwandan population 12.
Synthetic fungicides such as sodium ortho-
phenylphenate, thiabendazole and imazalil are cur-
rently used as primary means to fight against fungi
and they have shown efficacy. However some of
them have indicated side effect to human health
and/or are creating environmental problems. In
addition, resistance cases of some of the target
microorganisms to these synthetic fungicides have
been observed 13.
Due to these remarked challenges and because
of the increased consumer numbers of demand
for both safer and natural compounds used to pre-
serve foods, natural fungicides have been inten-
sively investigated 14.
Plant extracts including essential oils (EO) may
provide an alternative to safe food preservative
agents.EO are known to be less likely associated
with the development of resistance by fungi, as
observed with synthetic fungicides, and are less
hazardous to the environment and human health
15
. In addition, reports describing the antifungal
activity of essential oils extracted from Eucalyp-
152
tus anceps and Eucalyptus melliodora grown
in Rwanda and their use in foodstuffs preserva-
tion are scanty. Therefore, this study aims to
analyse the essential oils composition of Eucalyp-
tus species viz. E. anceps and E. melliodora, to
determine their effect on the growth of food spoil-
age fungi: Rhizopus sp., Aspergillus sp., Peni-
cillium sp. and Fusarium sp. and to test their in-
hibitive capacity for aflatoxin production by A.
parasiticus and A. flavus.
Materials and methods
Plant material and essential oil extraction
Fresh and mature leaves of E. anceps and E.
melliodora were harvested in the month of Febru-
ary 2018, from the Ruhande arboretum, Huye dis-
trict in Southern Province of Rwanda (2°36’48.9"S
29°45’25.7"E). The Eucalyptus species were
authenticated by botanists of the University of
Rwanda. The essential oils were extracted from
fresh leaves by steam distillation using a vertical
steam distillation unit. A 2-l flask containing 200 g
of chopped and homogenized leaves material was
heated during 4 h and the vapor condensed and
separated throughout an oil/water separator. Crude
essential oil extract was obtained. Essential oil was
dried over anhydrous sodium sulphate and used
for further experiments prior to the determination
of the chemical composition and the assessment
of antifungal activity tests.
Determination of extraction yield
The yield in essential oils was calculated as fol-
lows:
Yield ( %) = (Weo/W1) x 100
Where Weo is the weight of dry essential oil and
W1 is the weight of fresh leaves before extrac-
tion.
Chemical analysis of essential oils
The main constituents were analyzed on Gas
Chromatography apparatus, Shimadzu QP2010
Plus Gas Chromatography, equipped with flame
ionization detector (FID). An HP-5MS (30 m x
0.25 mm x 0.25 μm) capillary column coated with
non-polar cross-linked fused silica. The oven tem-
perature was maintained at 40oC for 1min and
then increased progressively to 70oC at the rate
of 3oC/min. After 1 min the temperature was again
 Daniel Umereweneza et al., / TEOP 22 (1) 2019 151 - 158
increased with a rate of 15oC/min from 70oC for
1 min, and then increased to 220oC for 10 min
before sample injection. Helium was used as a
carrier gas at the flowrate of 1.2 mL/min. To en-
hance the sensitivity for minor constituents, 10 %
(v/v) solution of each essential oil in hexane were
prepared. While the major constituents were de-
termined using a 1 % (v/v) solution of essential oil
in hexane. To conduct chemical analysis, one mi-
cro liter of the solution was injected at 220oC and
the effluent obtained from GC column was di-
rectly introduced into mass spectrometer with m/
z 5-500 mass range. Scanning was done at an
interval of 0.5 sec with a scanning speed of 1000
amu/s and ionization voltage of 70 eV. Identifica-
tion of components was based on computer li-
brary software and by comparing the obtained
retention time indices (RI) with those from the
literature 16, 17. Quantification of different constitu-
ents, expressed in percentage, was done by peak
area normalization measurements.
Fungi strains and inoculum preparation
Fungi strains, Rhizopus nigricans, Aspergil-
lus flavus, Aspergillus niger, Aspergillus para-
siticus, Fusarium oxysporum and Penicilium
digitatum were cultured and isolated. These fungi
strains were grown on Potato Dextrose Broth
(PDB) medium at 30oC for 24 h in a shaking in-
cubator at 180 rpm. Fungi cells were separated
from the medium by centrifugation and suspended
in distilled and sterilized water for further use.
Determination of minimum inhibitory concen-
tration (MIC)
The minimum inhibitory concentration of essen-
tial oils against test fungi strains (Rhizopus
nigricans, Aspergillus flavus, Aspergillus
Niger, Fusarium oxysporum and Penicilium
digitatum) was determined by twofold dilution
method 18. Samples were dissolved in methanol
according to their known weights. The methanol
solutions were added to PDB to obtain different
final concentrations ranging from 0.27 to 15 μL/
mL. A 5 mm diameter fungal disc containing a
10-day old culture of each test strains was inocu-
lated to the Petri dishes treated with essential oil
solution of each eucalyptus species. The Petri
153
dishes were incubated at 27oC for 7 days; the
experiment was conducted in triplicate. The low-
est essential oil concentrations that prevented the
visible fungi growth were taken as minimum in-
hibitory concentration (MIC) 19.
Determination of minimum fungicidal concen-
tration (MFC)
The in vitro fungicidal activity (MFC) was de-
termined as described by Espinel-Ingroff et al.
20
. After 72 h of incubation, 20 μL from each well
content that showed no visible growth (growth
inhibition of over 98 %), from the last positive well
(growth similar to that for the growth control well),
and from the growth control (essential oil-free
medium) were subcultured onto PDB plates. The
plates were incubated at 27°C until growth was
seen in the growth control subculture. The mini-
mum fungicidal concentration was regarded as
the lowest extract concentration that did not yield
any fungal growth on the solid medium used.
Antiaflatoxin test
Antiaflatoxin test was carried out using desic-
cated Coconut Agar medium (DCA) as described
by Adjou et al. 21. DCA medium with different
concentrations varying from 1 to 7 μL/mL of the
essential oil were prepared using Tween-80 as
solvent. About 20 mL of the medium were poured
in each glass Petri-dish test containing DCA. Then
A. parasiticus or A. flavus was inoculated with
single spores and petri dishes were incubated at
30oC for 48 h. Experiments without essential oil
were used as control. The reverse side of each
plate, which consists of single large colony, was
observed under long wave (365 nm) UV for blue/
blue green fluorescence.
Statistical analysis
Antifungal activity of EOs from E. anceps and
E. melliodora was tested. Each experiment was
conducted in triplicate and mean values were pre-
sented. Data analysis was performed with statis-
tical package (SPSS version 16.0)
Results and discussion
Average yield of essential oil
E. anceps gave a slightly lower yield (0.80 %)
 Daniel Umereweneza et al., / TEOP 22 (1) 2019 151 - 158 154
than E. melliodora (0.92 %). These results are drene (4.8 %) was only present in E. melliodora.
close to those reported by Coppen 22 for the same Some sesquiterpenes such as aromandrene (1 %,
species but they are lower than those reported by 18.01 %) and allo-aromandrene (1.5 %, 2.37 %)
Zrira et al. 23. The observed differences could be were identified in both species whereas caryo-
due to different parameters which include plant phyllene (5.1 %) and bicyclogermacrene (4.6 %)
species, its origin and climate. were only found in E. melliodora. Some alcohols:
globulol (3.5 %, 6.27 %), viridiflorol (4.2 %, 1.07
Chemical composition of essential oils %) and spathulenol (4.6 %, 0.14 %), as well as γ-
The results of essential oil analysis are presented eudesmol (0.12 %) were also detected. The com-
in Table 1 and 2. It is important to mention that position and components concentrations of these
only compound whose concentration was equal essential oils follow the same pattern as essential
or higher than 0.1 % has been reported. The main oils analysed in Australia 24, 25 and in Morocco 23.
components in E. melliodoraand E. anceps oils
were, respectively, monoterpenes: 1,8-cineole Antifungal activity
(47.7 %, 33.49 %), α-pinene (7.8 %, 13.69 %), Results of minimum inhibitory concentration
limonene (0.9 %, 0.39 %), and p-cymene (1.8 %, (MIC) and minimum fungicidal concentration
0.51 %). It is important to note that α-phellan- (MFC) are presented in table 3. For the essential
Table 1. Chemical constituents of essential oil of E. anceps

No. RI Compound %

1 939 α-Pinene 13.69

2 952 Camphene 0.11
3 978 β-Pinene 0.12
4 1016 p-Cymene 0.51
5 1024 Limonene 0.39
6 1025 1 ,8-Cineole 33.49
7 1105 Isoamyl isovalerate 0.12
8 1125 Fenchol 0.25
9 1132 trans-Pinocarveol 11.78
10 1144 trans-p-Menth-2-en-1-ol 0.11
11 1164 Pinocarvone 3.32
12 1178 trans-p-Mentha-1(7),8-dien-2-ol 0.47
13 1189 α-Terpineol 0.34
14 1215 trans-p-Mentha-1,8-dien-6-ol 0.83
15 1407 β-Elemene 0.51
16 1449 Aromadendrene 18.01
17 1475 allo-Aromadendrene 3.37
18 1484 α-Selinene 0.47
19 1502 α-Bulnesene 0.79
20 1542 α-Calacorene 0.51
21 1578 Globulol 6.27
22 1585 Spathulenol 0.14
23 1588 Viridiflorol 1.16
24 1645 β-Eudesmol 0.12
Total 96.88

R.I: Retention index

 Daniel Umereweneza et al., / TEOP 22 (1) 2019 151 - 158 155
Table 2. Chemical constituents of essential oil of E. melliodora

No. RI Compound %

1 939 α-Pinene 7.8

2 1002 α-Phellandrene 4.8
3 1016 p-Cymene 1.8
4 1024 Limonene 0.9
5 1025 1,8-Cineole 47.7
6 1105 Isoamyl isovalerate 0.4
7 1125 Fenchol 0.2
8 1132 Trans-Pinocarveol 2.7
9 1164 Pinocarvone 0.5
10 1179 Terpinen-4-ol 0.7
11 1189 α-Terpineol 1.8
12 1362 α-Cubebene 0.1
13 1413 α-Gurjunene 0.5
14 1417 Caryophyllene 5.1
15 1449 Aromadendrene 1.0
16 1475 allo-Aromadendrene 1.5
17 1482 β-Bicycloelemene 0.1
18 1484 Viridiflorene 2.7
19 1490 Bicyclogermacrene 4.6
20 1578 Globulol 3.5
21 1585 Spathulenol 4.6
22 1588 Viridiflorol 4.2
Total 97.2

R.I: Retention index

Table 3. Minimum inhibitory concentration (MIC)
and minimum fungicidal concentration (MFC)

Fungal strain E. melliodora oil (mg/mL) E. anceps oil (mg/mL)

MIC MFC MIC MFC

R. nigricans 3.50±0.01 7.00±0.03 8.80±0.04 18.00±0.08

A. flavus, 3.30±0.09 6.60±0.05 6.90±0.06 13.90±0.09
A. niger 7.20±0.03 14.40±0.01 15.80±0.01 32.00±0.04
F. oxysporum 3.50±0.07 7.00±0.08 8.70±0.03 17.50±0.06
P. digitatum 8.10±0.10 16.00±0.04 18.00±0.09 38.00±0.11
oil extracted from E. melliodora, the MIC var- E. anceps essential oil was most effective against
ied from 3.5 to 8.10 mg/mL. A. flavus showed A. flavus (6.90 mg/ml) and least effective against
the lowest MIC, whereas P. digitatum had the P. digitatum (18 mg/ml). Analogous to E.
highest MIC. The MFC followed similar pattern melliodora, E. anceps essential oil showed higher
with higher concentrations varying from 8.80 to MFC values than MIC values but they followed
18.00 mg/mL. For the essential oil extracted from the same pattern.
E. anceps, MIC varies from 6.90 to 18 mg/mL. Fungicidal activity of Eucalyptus essential oils
 Daniel Umereweneza et al., / TEOP 22 (1) 2019 151 - 158 156
has been extensively investigated in literature. Due

μL/mL μL/mL μL/mL μL/mL μL/mL μL/mL μL/mL μL/mL

to the complexity and high variability among Eu-

-
-
-
-
-
-
-
-
calyptus essential oils, the antifungal efficacy of
essential oil is mainly either attributed to the over-
all synergistic effects of all the major and minor

+++
++
+
6

-
-
-
-
-
compounds or to the bioactivity of the major com-
pounds.

+++
+++
+++
+++
Mishra et al. 26 reported that Eucalyptus es-

-
-
-
-
sential oil compounds such as limonene, linalool,
γ-terpinene, p-cimene, α-pinene and α-terpineol

+++
+++
+++
+++
exhibit antimicrobial activity. Inouye et al. 27 sug-

A. flavus

+
4

-
-
-
gested that terpineol was the main contributor to
the bioactivity. Although our study was not able

+++
+++
+++
+++
+++
+
3

-
-
to identify the principal bioactive component of

Table 4. A. parasiticus and A. flavus antiaflatoxinogenic test

bright fluorescence (+++)

the analysed essential oils, the latter suggestion
agrees with the results of antifungal activity as-

+++
+++
+++
+++
+++
+
2

-
-
say of the analysed essential oils because E.
anceps oil which has less α-terpineol content (0.24

+++
+++
+++
+++
+++
%) than E. melliodora (2.8 %) showed lower

+
1

-
-
antifungal activity.
Vilela et al. 28 demonstrated that the bioactivity

+++
+++
+++
+++
+++
++
does not necessarily depend on the concentration 0

-
-
because E. globulus demonstrated very poor fun-

moderate fluorescence (++);

gicidal activity against A. flavus and A.
Day μL/mL μL/mL μL/mL μL/mL μL/mL μL/mL μL/mL μL/mL

parasiticus although 1,8-cineole was the major

7

-
-
-
-
-
-
-
-
component (80 %). Gilles et al. 5 also showed that
geraniol, which represented 3.4 % of the essen-
tial oil of E.staigeriana had antimicrobial activity
6

-
-
-
-
-
-
-
-
against S. aureus three to seven times higher than
that of 1,8-cineole (63 %). However, Van Vuuren

+++
and Viljoen 29 reported that limonene and 1,8-cin- ++
+
+
5

-
-
-
-

eole have synergistic bioactivity effects.

A. parasiticus

+++
+++
+++
++

weak fluorescence (+);

+
4

-
-
-

Antiaflatoxin activity
Antiaflatoxin activity test was conducted for E.
melliodora essential oil on A. flavus and A.
+++
+++
+++
+++
+++
+
3

-
-

Parasiticus. Results presented in table 4, showed

that E. melliodora essential oil has important afla-
+++
+++
+++
+++
+++

toxin inhibition potential. The antiaflatoxin efficacy

+
2

-
-

increased with essential oil concentration. At the

concentration of 6 μL/mL for A. parasiticus and
+++
+++
+++
+++
+++

No fluorescence (-);

7 μL/mL for A. flavus, the aflatoxin production

+
1

-
-

was completely inhibited. Therefore, the essen-

tial oils analysed are good candidates for food
+++
+++
+++
+++
+++
++
0

-
-

preservation against aflatoxin production. How-

ever, it is important to note that chemical compo-
sition of natural essential oils depends on differ-
1
2
3
4
5
6
7
8

ent factors including storage and environmental

 Daniel Umereweneza et al., / TEOP 22 (1) 2019 151 - 158
conditions. Hence, essential oils standardization
is recommended prior to large scale application in
food preservation industries 30.
Conclusion
E. melliodora and E. anceps essential oils were
extracted and chemically analysed. Antifungal
activity of the essential oils was also evaluated
using different fungal strains: R. nigricans, A.
flavus, A. niger, A. parasiticus, F. oxysporum
and P. digitatum. Essential oil extracted from E.
anceps leaves was most effective against A.
flavus (6.90 mg/mL) and least effective against
P. digitatum (18 mg/mL). The findings of the
present study also showed that, at the concentra-
tion of 6 μL/mL for A. parasiticus and 7 μL/mL
for A. flavus, aflatoxin production was completely
inhibited. Therefore, these essential oils can be
157
considered as potential food preservative against
food spoilage fungi. However, essential oils should
be qualitatively standardized before practical ex-
ploitation because their chemical composition may
be greatly affected by harvest period, and by cli-
matic, seasonal, and geographical conditions.
Disclosure statement
No potential conflict of interest was reported
by the authors.
Acknowledgements
Authors are grateful to the Department of
Chemistry of the University of Rwanda for hav-
ing provided laboratory facilities and the Interna-
tional Science Programme (ISP), Uppsala Uni-
versity for financial support through a PhD schol-
arship offered to one of the authors (DU).

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