Journal of Experimental and Applied Animal Sciences

Volume 2, Number 2, pp. 211-222, 2017

Print ISSN 2314-5684 | Online ISSN 2314-5692
ORIGINAL ARTICLE

Antiasthma activity of Eucalyptus grandis essential oil and its

main constituent: Vasorelaxant effect on aortic smooth muscle
isolated from nomotensive rats
Oluwagbemiga S. Soyingbe1,2*, Tshepiso Jan Makhafola1, Bongiwe P. Mahlobo2,
Hussein M. Salahdeen3, Oladipupo A. Lawal1,4, Andy R. Opoku2

1
Department of Life and Consumer Sciences, College of Agriculture and Environmental Scienc-
es, University of South Africa, Private Bag X06, Florida 1710, South Africa; 2Department of Bi-
ochemistry and Microbiology, University of Zululand, Private Bag X1001, KwaDlangezwa 3886,
South Africa; 3Department of Physiology, College of Medicine, Lagos State University, PMB
21266Ikeja, Lagos, Nigeria; 4Department of Chemistry, Lagos State University, PMB 0001
LASU Post Office, Ojo, Lagos, Nigeria
DOI: https://doi.org/10.20454/jeaas.2017.1287

ABSTRACT
Asthma is a chronic inflammatory disorder of the airways. It is characterized by an in-
flammation of the airways causing dysfunction. Asthma is associated with widespread airflow
obstruction and increase in airway responsiveness to a variety of stimuli. An asthma attack is ac-
companied by wheezing, shortness of breath, chest tightness and coughing. The anti-asthmatic
and anti-cough activities of the essential oils and 1,8-cineole on rats were assessed. These activi-
ties were induced and challenged with histamine and acetylcholine using an ultrasonic nebulizer
for asthma and exposure to ammonia for coughs. Anti-inflammatory activities of the essential oils
and 1,8-cineole were determined using the cotton pellet granuloma test. Muscle contraction stu-
dies where carried out using the vascular reactivity on aortic smooth muscle. The oils exhibited
concentration dependent anti-asthma and anti-cough activities. Significantly, 1,8-cineole isolated
and purified from the essential oil showed a concentration dependent anti-inflammatory, anti-
cough and anti-asthma activity and The essential oils showed vasorelaxant activity. The ability of
the essential oils and 1,8-cineole to directly dilate smooth muscle and relieve the stress of bron-
chial contraction and obstruction of the airways passage validates the use of Eucalyptus grandis
as an anti-asthmatic and anti-cough agent. It is concluded that the essential oils have the potential
to be used as an anti-asthma and anti-cough therapy. This study also justifies its use by traditional
healers in the treatment of asthma and coughs in Zulu folklore medicine.
KEYWORDS: 1,8-cineole, Vasorelaxant, inflammation, Eucalyptus grandis, Cough, Asthma

211
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222
INTRODUCTION
Asthma is a common physiological
disorder encountered in clinical medicine. It
is a serious chronic inflammatory disorder that
affects the airways of both adults and children
(Masoli et al., 2004). It is generally character-
ized by inflammation of the airways, which
causes airways dysfunction (Patel et al.,
2009). There are various other symptoms of
asthma characterized by and associated with
airflow obstruction, which causes an associat-
ed increase in airway responsiveness to a vari-
ety of stimuli. An asthma attack is accompa-
nied by wheezing, shortness of breath, chest
tightness and coughing (Bousquet et al.,
2008). Asthma is currently a worldwide prob-
lem, with around 300 million people suffering
from it around the globe. About 250 thousand
annual world deaths are reported to be caused
by asthma (Archana et al., 2008). Asthma cas-
es are increasing at a rate of 50% every decade
and according to the WHO (2005), by the year
2020, Asthma will become the third leading
cause of death.
Inhaled bronchodilators and anti-
inflammatory drugs are available and are quite
effective in the management of asthma but,
they require long-term use and are associated
with various side effects (Bryan et al., 2000;
Angsten, 2000). To minimize and possibly
prevent these side effects alternative and com-
plementary medicine is being sought.
Eucalyptus grandis belongs to the
Myrtaceae family; it is native to Australia but
massive planting programs have been carried
out in the Republic of South Africa, Zambia
and Zimbabwe. Many species of the genus
Eucalyptus are cultivated Worldwide
(Juergens et al., 1998). Eucalyptus species are
used in folk medicine for a variety of medical
conditions in different part of World (Gomes
212
Carneiro et al., 1998). For instance, hot water
extracts of dried leaves of Eucalyptus citriodo-
ra (EC) are used in Brazil as anti- inflammato-
ry and analgesic remedies for the symptoms of
respiratory infection (Silva et al 2003). Euca-
lyptus globulus from Indian exhibits various
properties like antibacterial, antiseptic and an-
ticancer (Chalchat et al., 1995).
Eucalyptus grandis is a medicinal
plant indicated by Zulu traditional healers for
the treatment of respiratory tract infections,
bronchial infections, asthma and coughs; it is
administered via steam inhalation and also as
a concoction which is taken for the treatment
of asthma. The essential oil, obtained by hy-
dro-distillation of the leaves of the plant ex-
hibited antimicrobial activity against clinical
isolates and respiratory tract infectious bacte-
ria (Soyingbe et al., 2013; 2015). The main
constituent of the oil was identified as 1,8-
cineole (Soyingbe et al., 2013;2015). Essen-
tial oil isolated from eucalyptus species are al-
so widely used in pharmacological industries
(Gomes Carneiro et al., 1998). The aromatic
constituents of this oil are commercially avail-
able for the treatment of common cold and
other symptoms of respiratory infections
(Trigg 1996; Cockcroft et al., 1998). Ethnobo-
tanical survey of the local muthi market in
Empangeni, KZN also suggest the use of this
plant for the treatment of asthma and cold.
Since the biological and pharmacolog-
ical effects of any plant extract or isolated
compound may be affected by several factors
like time and period of collection, geograph-
ical origin and climatic conditions which in-
fluence the concentration of the active constit-
uents (Arogba, 1999) this study was undertak-
en to evaluate the anti-asthma and anti-cough
activity of the essential oil of Eucalyptus
grandis (cultivated in South Africa-Zululand)
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222
and 1,8-cineole isolated from it. The effect of
the oil and 1,8-cineole on the isometric con-
traction of isolated rat aortic rings was also
investigated. It is hoped that such investiga-
tion will help to verify the rationale behind the
plant’s use as a cure for these aforementioned
respiratory illnesses by the Zulu traditional
healers.
MATERIALS AND METHODS
Plant material & extraction of essential oil
The details of the collection and the
identification of the plant and the hydro-
distillation of the essential oil from the leaves
have already been reported (Soyingbe et al.,
2015). The oil was dried over anhydrous sodi-
um sulfate, dissolved in methanol and then
stored at -4°C until required.
Isolation of 1,8-cineole
The isolation and purification of com-
ponents from the essential oils of the fresh and
dry leaves of Eucalyptus grandis, were per-
formed using a column chromatography
packed with chromatographic silica gel
(0.063-0.200mM). Firstly 80 g of LC 60A 40-
63 silica gel was slurred with 120mL n-hexane
and poured into a 25mm I.D.×300mm glass
column, after which 3.2 grams of essential oil
was dissolved in 5 mL n-hexane. The mixture
was run through the silica gel column and
washed with n-hexane and ethanol to give two
fractions. Hexane was used to elute (20
ml/min) the terpene hydrocarbon from the
LC60A 40-63 silica gel, thereafter; two oxy-
genated compounds were easily washed off
the column with ethanol (20ml/min). These
fractions were then analyzed using thin layer
chromatography for purity. After this, a GC-
MS analysis was done on the ethanol fraction.
Animal care
Ethical clearance (UZREC 171110-
030 PGD 2013/26) was obtained from the Re-
213
search Animal Ethic Committee of the Uni-
versity of Zululand. Animals were collected
from the animal house in the Department of
Biochemistry, University of Zululand. Exper-
imental research was carried out following the
guideline for care and supervision of experi-
mental animals. The animals were housed in
standard cages and maintained at room tem-
perature with 12:12-h light: dark cycle. All
rats had free access to drinking water and
standard rat feed in the experimental environ-
ment for one week before the experiments
were carried out.
Anti-asthmatic activity
The animal experiments were carried
out using Sprague-Dawley rats. The rats were
divided into 5 groups: group 1 was the control
group that received only the carrier solvent
(10%Tween-20); group 2 was the positive
control, and were treated with Aminophylline
(125mg/kg body weight); groups 3, 4 and 5
received 50mg/kg, 100mg/kg and 250mg/kg
in body weight, respectively of the extract dis-
solved in 10%Tween-20. All the rats were fed
with a standard diet (Doghouse, SA) and wa-
ter ad libitum. The rats were administered
with their respective drugs through the oral
cannula for 3 consecutive days. One hour after
the last administration the rats were exposed
to irritant agents (histamine and acetylcholine
mixture 1:1) for 30seconds using an ultrasonic
nebulizer via whole body exposure (Kumar et
al., 2010). The latent periods of asthma were
recorded.
Anti-cough activity
The experimental design was similar to
that reported for the anti-asthma. Dextrome-
thorphan (5mg/kg of body weight) was used
as the positive control. Coughing was induced
by exposing rats to ammonia for 5 secs, and
the latent period of coughing (seconds from
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222
the expose to the first cough) and the number
of coughs during a 2-minute period recorded.
Anti-inflammatory activities
The proliferation phase of inflamma-
tion was investigated by the cotton pellet
granuloma model (Meier et al., 1950). The es-
sential oil at 50 and 150mg/kg, 1,8-cinoele at
10 mg/kg, and indomethacin as the control
drug at 10 mg/kg body weight were dissolved
in 10% Tween-20 and were given to the rats
orally. After 30min, the animals were anesthe-
tized. Sterile cotton pellets of 20mg each were
implanted at an interscapular depth under the
skin under sterile conditions. The essential oils
and control drug were administered daily for a
period of seven days. The rats were sacrificed
after anesthesia on the eighth day and the pel-
lets surrounded by granuloma tissues were
dissected out, weighed, dried for 24h at 40°C
and weighed again. The increment between
dry and wet pellet weights were taken as a
measure of granuloma formation and com-
pared with those of the control.
Vasorelaxant activity
Healthy, young adult, male and female
Wistar albino rats weighing 250-300g were
used. All the animals used were fasted for
18h, but still allowed access to water before
the commencement of the experiments. The
rats were sacrificed by cervical dislocation.
The thoracic aorta was quickly removed, freed
of connective tissue and placed in a Petri-dish
containing physiological salt solution (PSS).
The aortic lumen was gently flushed with PSS
and sectioned into 2mM ring segments. Each
aortic ring was suspended in a 60ml jacketed
tissue bath containing PSS with the following
composition (mmol/L): NaCl, 118.0; KCl, 4.7;
KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 15.0;
214
CaCl2, 1.6 and glucose, 11.5 (Ebeigbe and
Aloamaka, 1987; Obiefuna et al., 1991; Ojeik-
ere et al., 2003; Salahdeen and Murtala, 2012).
The temperature of the bath was maintained at
37°C and the solution bubbled with a 95%
O2:5% CO2 gas mixture (pH 7.35-7.40). An
initial tension of 2g was applied to all arterial
rings. An equilibration period of 60-90min
was allowed before the start of experiments,
and during this time it was stimulated thrice
with 10-6M noradrenaline for 5min at 30min
intervals. At the end of the equilibration peri-
od, the vessel was subjected to the following
procedures. Cumulative doses of noradrena-
line (NA) were added to the PSS to obtain
concentrations of 10-9 to 10-5M and the con-
centration–response curves (CRCs) were de-
termined. Following incubation with essential
oil the extract CRCs were determined for NA.
Statistical analysis
Statistical analyses of the differences
between mean values obtained were calculated
using Graphpad Prism 6. Data were subjected
to one way analysis of variance (ANOVA). P
values ≤0.05 were regarded as significant and
P values ≤0.01 as very significant.
RESULTS
Anti-asthma and anti-cough activity
The results presented in Figure 1 show
the anti-asthma activities of the essential oils,
the isolated 1,8-cineole and the control drug
aminophylline. The latent period in develop-
ing wheezing after being exposed to the irri-
tants was longer in rats which were adminis-
tered the control drug aminophylline while the
groups which were administered with the es-
sential oils showed a concentration dependent
activity in delaying wheezing associated with
asthma after exposure to the irritants.
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222

300 40
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t im e in s e c s

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c o n c e n t r a t io n m g /k g
C o n c e n t r a t io n m g /k g
Figure 1: Anti-asthma activity of the essential Figure 2: Anti-cough activity of the essential oils,
oils, 1,8-cineole and the control aminophylline 1,8-cineole and the control dextromethorphan (The
(The time in delaying the wheezing associated time in delaying the cough associated with asth-
with asthma after rats were exposed to the irritant ma).
agents, histamine and acetylcholine is presented).
50

The anti-cough activities of the essen-

40
tial oils, the isolated 1,8-cineole and the con-
N o of cough

trol drug dextromethorphan are presented in 30

Figures 2 and 3. The experimental groups

20
showed a concentration dependent activity * *
though they were not as effective as that of the 10

control drug dextromethorphan.

0
g

g
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The anti-cough activity of the essential

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oils showed a concentration dependent activity

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for both the number of coughs recorded in a 2

ry

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minutes period after exposure to ammonia and

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the delay in coughing after exposure. The de-

E

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lay in the time it took to develop a cough was C o n c e n t r a t io n m g /k g

longer at the highest concentration of the es-
sential oils for both the fresh and dry leaves Figure 3: The activity of the essential oils, 1,8-
and also for 1,8-cineole, as compared to the cineole and the control dextromethorphan on the
control group with no drugs. The number of number of coughs for 2minutes after 5seconds of
coughs counted for 2 minutes was also seen to exposure to ammonia.
decrease as the concentration increased.

215
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222

0 .4 Essential oils from fresh leaves

** **
In h ib it io n r a te

0 .3

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Essential oils from dry leaves

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1,8-cineole
E

Figure 4: Anti-inflammatory activity of the essen-

tial oils and the isolated 1,8-cineole on the cotton
pellet-induced granuloma.

The anti-inflammatory and vasorelaxant ac-

tivity NE
Cotton pellet-induced granuloma is
broadly used to evaluate the transudative and
proliferative components of chronic inflam-
mation (Winter et. al., 1957). The weight of
the wet cotton pellets correlates with transude
material, whereas the weight of dry pellet cor-
relates with the amount of granulomatous tis- NE Concentration (LogM)
sue. The results obtained from the anti-
inflammatory activity of the essential oils and Figure 5: Typical tracing showing concentration
the isolated 1,8-cineole on the cotton pellet- response curves of essential oils of E. grandis, 1,8-
cineoleand NE.
induced granuloma are presented in Figure 4.
It is apparent that the extracts exhibit a con-
The curves were evidence of the concentration
centration dependent anti-inflammatory activi-
dependent vasorelaxant activity of the oils.
ty.
Figure 6 shows the concentration dependent
The effects of the essential oils and the vasorelaxant activity of the essential oils and
isolated 1,8-cineole on aortic smooth muscle 1,8-cineole at different concentrations on the
are presented in Figures 5 – 9. Figure 5 shows aortic ring isolated from the rats. Figures 7 - 9
the concentration response curves for the es- shows the individual vasorelaxant activities of
sential oils of E. grandis and 1,8-cineole com- the essential oils and 1,8-cineole on the aortic
pared with noradrenaline.

216
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222

rings isolated from rat muscles which had pre- been reported to contain 60% of monoterpe-
viously been stimulated by noradrenaline. noids and citronellal (Silva et al., 2003), Euca-
lyptus tereticornis (ET) and Eucalyptus globu-
The essential oils and 1,8-cineole iso- lus (EG) contain 60- 90% of eucalyptol (Dam-
lated showed a concentration dependent vaso- janović-Vratnica et al., 2011) while Eucalyp-
relaxant activity on the aortic ring from rats. tus grandis contain eucalyptol 48-60% (Soy-
0 .6
ingbe et al., 2013a). Study indicates that cit-
ronellal is effective against bacterial and fun-
m a x im u m c o n t r a c t io n

gal infections (Pattnaik et al 1996); eucalyptol

0 .4
has been reported to inhibit the production of
tumor necrosis factor-α, interleukin-1β, leuko-
0 .2
triene B4, and thromboxane B2 in inflamma-
tory cells (Juetgens et al., 1998).

0 .0
le
s
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e
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in
le
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ry
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1
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fr

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E

N E c o n c e n t r a t io n ( L o g M )

Figure 6: Concentration response for noradrena-

line in aortic rings incubated with and without es-
sential oils and 1,8-cineole (0.5mg/ml). n= 6.

DISCUSSION AND CONCLUSION

The essential oil from E. grandis ex- Figure 7: Concentration response curve for nora-
hibited a complex spectrum Figure 10 and 11 drenaline in aortic rings incubated with and with-
out the essential oil from the fresh leaves (0.5
of compounds which were assigned by their
mg/ml). n=6
characteristic UV and mass spectrometric da-
ta. Two compounds, 1,8-cineole and terpinen- It is apparent that the essential oils
4-ol (Fig. 12), were isolated from the essential from the leaves of E. grandis and the 1,8-
oil. The GC-MS spectra showed them to be cineole (isolated from the oil) exhibit activity
pure compounds (Fig 13 and 14). in delaying asthma in rats exposed to the irri-
tant agents, histamine and acetylcholine (Fig.
The aromatic constituents of the oils of
1). The essential oil of lavender has been re-
Eucalyptus sp. are mainly eucalyptol (1,8-
ported to have the ability to assist with re-
cineole) and citronellal which are monoterpe-
sistance to asthma attack; this is triggered by
noids (Trigg, 1996; Cockeroft et al., 1998).
the strengthening of the immune system. Lav-
Previous phytochemical analysis has shown
ender oil also assists in treating other respira-
that the concentration of the monoterpenoids
tory problems such as bronchitis, sinus con-
varies among the Eucalyptus species with po-
gestion, laryngitis, tonsillitis and drug-
tential variations in their medicinal properties.
For example, Eucalyptus citrodora (EC) have

217
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222
resistant infections and fungal Infections (Ste-
venson, 2013).
Figure 8: Concentration response curve for nora-
drenaline in aortic rings incubated with and with-
out the essential oil from the dry leaves (0.5
mg/ml). n=6.
Figure 9: Concentration response curve for nora-
drenaline in aortic rings incubated with and with-
out the isolated 1,8-cineole (0.5 mg/ml). n=6.
Study on cardiovascular system indi-
cates that eucalyptol induces hypotension and
bradycardia in both anesthetized and con-
scious nomotensive rats (Lahlou et al., 2002).
The direct effects of the eucalyptol isolated
from Eucalyptus grandis on vascular smooth
218
muscles using isolated rat aorta show the con-
tractile response to noraderenalin which was
suppressed by the test samples (evident by the
shifting of concentration response curve to the
contractile agent to the right thereby depress-
ing the maximum response to each agonist). It
is well known that contraction of smooth mus-
cle preparations including aortic rings is de-
pendent upon an increase in the cytoplasmic
free Ca2+, which activates the contractile ele-
ments (Karaki and Weiss, 1988). The increase
in intracellular Ca2+ occurs either via influx
through voltage-dependant Ca2+ channels
(VDCs) or the release of Ca2+ from intracellu-
lar stores in the sarcoplasmic reticulum.
Figure 10: GC-MS Spectra for the essential oil of
the fresh leaves of Eucalyptus grandis.
Periodic depolarization and repolarization
regulates the spontaneous contraction of the
rings and at the height of depolarization, the
action potential appears as a rapid influx of
Ca2+ via VDCs (Brading, 1981). The inhibito-
 Soyingbe et al., 2017 Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222

ry effect of the essential oil on pre-contraction known cure for asthma, emphasis is being laid
with NE suggests that the inhibitory effect of on finding drugs to effectively manage the
this oil may be due to interference either with disease (Downs et al., 2001). Many of Chinese
the Ca2+ release or with the Ca2+ influx medi- formulations have been employed in the
ated through VDCs, in addition to other treatment of asthma.
mechanisms.

Our results are similar to other studies

of other compounds from essential oils, such
aseugenol and isoeugenol, which reportedly
produced vasodilatation (Nishijima et al.,
1998; Nishijima et al., 1999) and depress car-
diac contractions acting via calcium channels
(Damiani et al., 2002). The compound also af-
fected both time to peak tension and relaxation
time being slightly reduced, this is in agree-
ment with a calcium channel blocker action of
the compound (Mattiazzi and Garay, 1988). 1,8-cineole terpinen-4-ol
Figure 12: Structure of isolated compounds.
Abundance

TIC: BENGRR.D\data.ms
5800000 10.919

5600000

5400000

5200000

5000000

4800000

4600000

4400000

4200000

4000000

3800000

3600000

3400000

3200000

3000000

2800000

2600000

2400000

2200000

2000000

1800000

1600000 10.837

1400000

1200000
8.082
1000000
25.767
800000
10.704
600000

400000 25.920
19.972
111
.7
2170015.218
.6
200000 15.4
.592
6
99
7.888.6
.319
16 12.90215.411
2
77
2 .3
.0
1 3
81
.003 22.392252
17 6.1
2
24
.36 1
7
20
.84
7.1
.4 9
0336
17 74
37.62840.664 47.045

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
Time-->

Figure 11: GC-MS Spectra for the essential oil of

Figure 11: GC-MS Spectra for the essential oil of the dry leaves of Eucalyptus grandis.

the dry leaves of Eucalyptus grandis.

Current research on asthma is aimed at Figure 13: GC-MS Spectra of the isolated 1,8-
finding safer, better drugs to combat the dev- cineole.
astating effects of asthma and relieve the ef-
fects of asthma attacks. Since there is no

219
 Soyingbe et al., 2017
Figure 14: GC-MS Spectra of the isolated ter-
pinen-4-ol.
Several active components
phenethyl alcohol were investigated in vitro
and indicated an anti-asthmatic effect on the
contraction of isolated tracheal smooth mus-
cles in guinea pigs. This prevented histamine-
induced bronchoconstriction, thereby corrobo-
rating the traditional use of this formulation as
an anti-asthmatic agent (Chi et al., 2009).
Since the essential oils and 1,8-cineole from E
grandis have direct vasodilatory effect on
smooth muscle around the bronchial which
contracts during an asthma attack thereby ob-
structing the airways passage, coupled to the
anti-inflammatory activity of the oil and ter-
penoid oxide 1.8–cineole, which is also abun-
dantly present in the essential oils from Euca-
lyptus grandis, it is not unreasonable to con-
clude that the dilatory effect on smooth mus-
cle and the anti-inflammatory activity contrib-
ute to the antiasthma properties of the oils.
Journal of Experimental and Applied Animal Sciences. 2, 2: 211-222
The present results support the ethno-
medical use of Eucalyptus grandis as an anti-
asthmatic and anti-cough agent by traditional
healers. Additional experiments related to the
effects of this oil on contractile apparatus such
as the alteration of calcium sensitivity or regu-
latory proteins are needed.
ETHICS APPROVAL
Ethical clearance (UZREC 171110-
030 PGD 2013/26) was obtained from the Re-
search Animal Ethic Committee of the Uni-
versity of Zululand.
AUTHORS’ CONTRIBUTIONS
The biological assays analyses and
writing the draft manuscript were done by
SOS and BPM. The Vasorelaxant activity was
carried out by HMS and OAL. TJM has criti-
cally reviewed the manuscript and participated
in the study design and choice of assay meth-
ods. ARO conceived the idea, reviewed the
of draft and final manuscripts and interpretation
of results. All authors read and approved of
the final manuscript for submission
ACKNOWLEDGEMENT
We acknowledge the financial contri-
bution of University of Zululand Research
committee. The authors are grateful to the
University of Zululand research committee for
financial support.
CONFLICT OF INTERESTS
The authors declare that they have no
conflict of interests.
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