Journal of Visceral Surgery (2019) 156, 291—295

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ORIGINAL ARTICLE

In vitro evaluation of the protoscolicidal

effect of Eucalyptus globulus essential oil
on protoscolices of hydatid cyst compared
with hypertonic saline, povidone iodine and
silver nitrate
M. Moazeni a, S.V. Hosseini b, M.H. Al-Qanbar c,
A.M. Alavi a, H. Khazraei b,∗

a
Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
b
Colorectal Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
c
Kufa University, College of medicine, Kufa, Iraq

Available online 13 April 2019

KEYWORDS Summary
Echinococcus Objective: There are various protoscolicidal agents for inactivation of protoscoleces of hydatid
granulosus; cysts before and during surgical operation. The present study was aimed to evaluate the proto-
Eucalyptus globulus; scolicidal effect of two concentrations of Eucalyptus globulus on protoscoleces of Echinococcus
Povidone iodine; granulosus sensu lato under in vitro condition and to compare its efficacy with hypertonic saline,
Hypertonic Saline; povidone iodine and silver nitrate.
Silver nitrate Methods: Live protoscoleces obtained from the liver of naturally infected sheep were exposed
to 0.5% and 1% of Eucalyptus globulus essential oil, 5% hypertonic saline, 10% povidone iodine
and 0.5% silver nitrate for 1 and 3 minutes. Phosphate buffered saline was used as a negative
control. One percent eosin staining method was used to test the viability of protoscoleces in
different groups.
Results: While the mean percentage of dead protoscoleces was 6.08% in the control group, the
scolicidal power of 5% hypertonic saline was only 6.54% and 6.60% after 1 and 3 min respec-
tively. 0.5% E. globulus EO demonstrated 97.38% and 100% scolicidal activity after 1 and 3 min
respectively. The mean protoscolicidal power of 1% E. globulus EO, 10% povidone iodine and
0.5% silver nitrate was 100% after one minute.
Conclusions: According to the results of this study, E. globulus EO demonstrated high scolicidal
power in a short period of time. Hence, this herbal product could be considered as a potent
natural scolicidal agent that could be used before and during surgery of hydatid disease.
© 2019 Published by Elsevier Masson SAS.

∗ Corresponding author.
E-mail address: Khazraee@sums.ac.ir (H. Khazraei).

https://doi.org/10.1016/j.jviscsurg.2019.01.002
1878-7886/© 2019 Published by Elsevier Masson SAS.
292
Introduction
Human cystic echinococcos, a neglected disease that is
caused by the larval stage of Echinococcus granulosus, is
a progressive and chronic disease, affecting a large number
of people all around the world [1]. Hydatid cysts are formed
mostly in the liver and lungs and rarely in the other internal
organs such as kidneys, spleen, heart, central nervous sys-
tem and bones [2]. The most common surgical technique is
evacuation of the cyst contents and instillation of protoscol-
icidal agents into the cyst cavity to sterilize it and to prevent
the spread of infection and secondary cyst formation [3]. To
date, several protoscolicidal substances, like hydrogen per-
oxide, formalin, hypertonic saline, cetrimide, ethyl alcohol
and silver nitrate have been practiced throughout the world,
but many of them are together with side harmful effects [4].
For this reason recognition of new substances as robust pro-
toscolicidal agents with minimal side effects is essential.
Eucalyptus is a widely used tree that is cultivated in nearly
20 million hectares of agricultural lands of the world [5]. The
leaves essential oil of E. globulus with a number of biological
properties is a valuable source for traditional medicines [6].
This study was aimed to evaluate the protoscolicidal
effect of E. globulus essential oil in comparison with povi-
done iodine, hypertonic saline and silver nitrate.
Materials and methods
Preparation of scolicidal agents
Five percent hypertonic saline (Shahid Ghazi Co. Tabriz,
Iran), 10% povidone iodine (Tolid Daruo Co. Tehran, Iran)
and 0.5% silver nitrate (MERK, Germany) were prepared at
desired concentrations. Eucalyptus globulus EO [obtained
from the leaves of fresh plant] was purchased from Barij
essence pharmaceutical company (Kashan, Iran) and used at
concentrations of 0.5% and 1%. For preparation of 0.5% and
1% solution of E. globulus EO, 0.5 and 1 mL of the oil was
added to 99.5 and 99 mL of 1× PBS respectively. Few drops
of tween 80 (Sigma, Germany) were added to the solution
for better solubility.
Collection of protoscoleces
Sheep livers containing hydatid cysts were transferred to the
parasitology laboratory within an hour of slaughter. After
washing of infected livers, cyst surfaces were sterilized by
70% ethyl alcohol and the cyst contents were evacuated
completely and transferred into falcon tubes, where the
protoscoleces were precipitated and separated.
Viability test
Eosin staining assay was used to test the viability of the
protoscoleces. The scolices were exposed to the 0.1% stain
solution (1 g of eosin powder in 1000 mL distilled water)
for 15 minutes. Dead protoscoleces absorbed the dye and
got red color (Fig. 1), while the viable ones remained
colorless.
Protoscolicidal assay
At first 0.2 mL of pooled protoscoleces (containing about
1200 live protoscoleces) was poured into six test tubes
M. Moazeni et al.
(A-F) and exposed to 2 mL of 0.5% E. globulus EO (groups A),
1% E. globulus EO (groups B), 5% hypertonic saline (group
C), 10% povidone iodine (group D), 0.5% silver nitrate (group
E), as well as 1 × PBS + tween 80 (group F) as the control
group. The tubes’ contents were mixed gently and then
the tubes were incubated at room temperature for one and
three minutes. Subsequently, the supernatant of each tube
was cautiously discarded so that not to disturb the settled
protoscoleces. Then protoscoleces were washed three times
by adding and discarding 1 × PBS. In order to determine the
viability, 0.1% eosin solution was added to the treated pro-
toscoleces. After 15 min, protoscoleces were smeared on
manually scaled glass slides and covered with a cover glasses
(24 mm × 50 mm), and were observed under a light micro-
scope. Dead protoscoleces absorbed the dye and colored red
but alive ones remained colorless. The scolicidal activity of
different agents was determined by counting a minimum of
1000 protoscoleces. All agents were tested three times with
protoscoleces obtained from three different infected livers.
Statistical analysis
According to the results of the homogeneity test, the mean
protoscolicidal power of different agents were compared by
One-way analysis of variance (Anova) using the Tukey test.
The data are given as the mean ± SD. The statistical anal-
yses were performed with the SPSS statistics version 22.
P-values < 0.05 were considered significant.
Results
Protoscolicidal effects of different concentrations of the
essential oil of E. globulus, 5% hypertonic saline, 10% povi-
done iodine and 0.5% silver nitrate solutions comparing with
each other as well as with the control group are summarized
in Tables 1, 2 and 3 and Fig. 1. As shown in Table 1, while
the mean percentage of dead protoscoleces was 6.08% in the
control group, the mean protoscolicidal power of 1% E. glob-
ulus EO, povidone iodine and silver nitrate was 100% after
one minute. Accordingly, the potency of 0.5% E. globulus EO
and 5% hypertonic saline were 97.38%and 6.54% respectively.
Regarding to the data presented in Table 2, after three
min of exposure, 0.5% E. globulus EO showed 100% pro-
toscolicidal power, while in this time 5% hypertonic saline
exhibited only 6.60% protoscolicidal activity. The data pre-
sented in Tables 1 and 2 clearly indicated that 1% E. globulus
EO, silver nitrate and povidone iodine were powerful scoli-
cidal agents so that they killed all protoscoleces after one
minute.
Statistical analysis using the Anova test showed that
there was highly significant difference between the mean
protoscolicidal effects of 0.5% and 1% E. globulus EO as well
as silver nitrate and povidone iodine comparing with the
control group (P < 0.001). Conversely, 5% hypertonic saline
demonstrated so weak protoscolicidal potency that it could
be ignored (P = 1) (Table 3).
Discussion
The results of the present study revealed that, E .globulus
EO is a powerful scolicidal agent. According to WHO, ideal
scolicidal agents are those that are effective in low concen-
trations, stable in cyst fluid, able to kill the protoscoleces
in the cyst, non-toxic, easily available, inexpensive, easy to
In vitro protoscolicidal effect of Eucalyptus globulus essential oil 293

Figure 1. Protoscolicidal activity of 0.5% E. globulus EO, 1% E. globulus EO, 5% hypertonic saline, 10% povidone iodine (betadine) and
0.5% silver nitrate after 1 minute and following the eosin staining test.1: experiment 1, 2: experiment 2, 3: experiment 3.

Table 1 Scolicidal pover of Eucalyptus globulus essential oil (0.5% and 1%), 5% hypertonic saline, 10% povidone iodine,
and 0.5% silver nitrate after 1 min of exposure.
Scilicidal agent Experiments Protoscolices Dead protoscolices Mortality rate (%)
Eucalyptus globulus (0.5%) 1 1245 1185 92.21
2 1150 1138 98.96
3 1124 1104 98.20
Total 3519 3427 97.38
Eucalyptus globulus (1%) 1 1151 0 100
2 1123 0 100
3 1109 0 100
Total 3383 0 100
Hypertonic saline (5%) 1 1183 58 4.90
2 1090 65 5.96
3 1242 107 8.62
Total 3515 230 6.54
Povidone iodine 1 1114 0 100
2 1098 0 100
3 1157 0 100
Total 3369 0 100
Silver nitrate 1 1011 0 100
2 1124 0 100
3 1211 0 100
Total 3346 0 100
PBS and tween 80 (control) 1 1207 46 3.81
2 1140 68 5.96
3 1138 98 8.61
Total 3485 212 6.08

Table 2 Scolicidal pover of Eucalyptus globulus essential oil (0.5%) and 5% hypertonic saline after 3 min of exposure.
Scilicidal agent Experiments Protoscolices Dead protoscolices Mortality rate (%)
Eucalyptus globulus (0.5%) 1 1076 0 100
2 1118 0 100
3 1143 0 100
Total 3337 0 100
Hypertonic saline (5%) 1 1032 49 4.74
2 1059 66 6.24
3 1091 95 8.71
Total 3182 210 6.60
PBS and tween 80 (control) 1 1207 46 3.81
2 1140 68 5.96
3 1138 98 8.61
Total 3485 212 6.08
294 M. Moazeni et al.

Table 3 Scolicidal activity (mean ± SD) of different scolicidal agents after 1 min of exposure.
Scolicidal agent Scolicidal activity (mean ± SD) (%) P value
Eucalyptus globulus (0.5%) 96.45 ± 3.69 0.000
Eucalyptus globulus (1%) 100 ± 0 0.000
Hypertonic saline (5%) 6.49 ± 1.91 1.000
Povidone iodine (10%) 100 ± 0 0.000
Silver nitrate (0.5%) 100 ± 0 0.000
Control (dead protoscolices) (%) 6.12 ± 2.40 —
SD: standard deviation.

prepare and those that act in a short period of time [7].
According to the results of the present study 1% E. globulus
EO, 10% povidone iodine and 0.5% silver nitrate demon-
strated 100% scolicidal power in a short time (one min). Fast
effectiveness of these agents could be considered as a gold
standard for an ideal scolicidal agent.
Various concentrations of hypertonic saline solution have
been used as scolicidal agent in recent 50 years all around
the world [8]. In the present study, 5% hypertonic saline
showed very low scolicidal power after 1 and 3 min of appli-
cation. It has been postulated that hypertonic saline in
concentrations lower than 20% could not be an effective
scolicidal agent within 5 min. On the other hand, previ-
ous experimental studies have shown that hypertonic saline
solution may cause mild to severe adverse effects on biliary
tract and liver depending on its concentration [8].
Hosseini et al. [9] injected 0.3 mL of 10% povidone
iodine into the gall bladders of guinea pigs, four months
after injection; they observed sclerosing cholangitis in
80% of animals. They concluded that povidone iodine
could not be an ideal protoscolicidal agent. Rajabi [10]
reported dyspnoea, cyanosis, renal failure, liver insuffi-
ciency, hypotension, intraoperative shock, cardiovascular
collapse, methaemoglobinaemia and death after the injec-
tion of 0.5% silver nitrate into the hydatid cysts in human
beings.
With regard to the results of the present study, even
though 10% povidone iodine and 0.5% silver nitrate exhib-
ited a powerful scolicidal action in a short period of time, in
the point of view of safety, 1% E. globulus EO has a consider-
able superiority to them, since it is a herbal product that is
more likely to be a safe scolicidal agent. In our study, 0.5% E
.globulus EO also demonstrated 100% scolicidal activty after
three minute of application.
One hundred percent scolicidal activity has been
reported for the essential oils of Mentha pulegium
(10 ␮g/mL) after 18 days [11], Salvia officinalis (1 mg/mL)
after 8 days [12], Thymus vulgaris (1 mg/mL) after 7
days [12], Trachyspermum ammi (10 mg/mL) after 10 min
[13], Bunium persicum (25 ␮l/mL) [14], Myrtus communis
(100 ␮l/mL) [15] and Pistacia vera (200 ␮l/mL) [16] after
5 min.
It has been reported that thymol as the main chemical
compound of Z. multiflora EO has 100% scolicidal power
at 50 ␮g/mL after 5 days [12]. Based on the manufacture’s
description, the main component of E. globulus EO that we
used in this study, was 1, 8-cineole or eucalyptol (79.32%).
According to the US Food and Drug Administration (FDA),
eucalyptol is a safe herbal product and can be used for
addition in foods [17].
Conclusion
Owing to the side effects of chemical scolicidal agents such
as povidone iodine and silver nitrate, E .globulus EO could be
considered as a potent natural scolicidal agent that could be
used before and during surgery of cystic echinococcosis. Fur-
ther experimental and clinical trials are required to confirm
the safety of different concentrations of this herbal product
in laboratory animals and human beings.
Acknowledgments
The authors would like to thank the parasitology department
in Shiraz University for experimental assistance.
Disclosure of interest
The authors declare that they have no competing interest.
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