In vitro evaluation of the antiparasitic activity of Syzygium aromaticum against adult and larval stages of Trichinella spiralis

Abstract

Abstract. Benzimidazole is the most commonly used therapeutic drug for trichinellosis clinical treatment, but it
has many drawbacks. The quest for alternative natural compounds, such as essential oils, is, therefore, a target
for researchers. The present work is intended to test the in vitro anthelmintic effect of clove oil (Syzygium
aromaticum) against adult and muscle larva of Trichinella spiralis. Adult forms and muscle larvae of Trichinella
spiralis were incubated with clove oil at concentrations ranging from 5 to 500 μg/ml to analyze the lethal
effective concentrations on the parasite and to track the changes occurred on the cuticle by scanning electron
microscopy. At 50 μg/ml, a 100% death rate on the adult worms of T. spiralis was observed only at 24 hours.
However, at concentrations of 100 and 500 μg/ml, the lethal effect started at 16 and 8hours respectively. Clove
oil killed the total larvae at the concentrations of 100 and 500 μg/ml at 24 and 16 hours of in vitro incubation
respectively. Adult worms and muscle larvae of T. spiralis incubated with 100 µg/ml of clove oil exhibiting
marked morphological changes, multiple vesicles, and blebs, sloughing of some areas of the cuticle with fissures,
loss of normal annulation, and destruction of the cuticle. Our results suggested that clove oil has the potential as
a therapeutic agent and an alternative drug against adults and larvae stages of Trichinella spiralis.
Keywords: Trichinellosis; Clove oil; Anthelmintic activity; Electron microscopy.

Full text

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In vitro evaluation of the antiparasitic activity of Syzygium
aromaticum against adult and larval stages of Trichinella spiralis
Azza Fahmy1, Rabab Zalat1, Asser Rabei2
1 – Parasitology department, Theodor Bilharz Research Institute, Giza, Egypt.
2 – Faculty of Medicine, Menoufia University, Menoufia, Egypt.
Correspondence: Tel. 00201004335729, E-mail azzafhmy@gmail.com
Abstract. Benzimidazole is the most commonly used therapeutic drug for trichinellosis clinical treatment, but it
has many drawbacks. The quest for alternative natural compounds, such as essential oils, is, therefore, a target
for researchers. The present work is intended to test the in vitro anthelmintic effect of clove oil (Syzygium
aromaticum) against adult and muscle larva of Trichinella spiralis. Adult forms and muscle larvae of Trichinella
spiralis were incubated with clove oil at concentrations ranging from 5 to 500 μg/ml to analyze the lethal
effective concentrations on the parasite and to track the changes occurred on the cuticle by scanning electron
microscopy. At 50 μg/ml, a 100% death rate on the adult worms of T. spiralis was observed only at 24 hours.
However, at concentrations of 100 and 500 μg/ml, the lethal effect started at 16 and 8hours respectively. Clove
oil killed the total larvae at the concentrations of 100 and 500 μg/ml at 24 and 16 hours of in vitro incubation
respectively. Adult worms and muscle larvae of T. spiralis incubated with 100 µg/ml of clove oil exhibiting
marked morphological changes, multiple vesicles, and blebs, sloughing of some areas of the cuticle with fissures,
loss of normal annulation, and destruction of the cuticle. Our results suggested that clove oil has the potential as
a therapeutic agent and an alternative drug against adults and larvae stages of Trichinella spiralis.
Keywords: Trichinellosis; Clove oil; Anthelmintic activity; Electron microscopy.
Evaluarea in vitro a activității antiparazitare a Syzygium aromaticum față de stadiile larvare și adulte a
lui Trichinella spiralis
Rezumat. Benzimidazolul este medicamentul cel mai frecvent folosit în tratamentul clinic al trichinelozei, dar cu
multiple dezavantaje. Căutarea compușilor naturali alternativi, cum ar fi uleiurile esențiale, este, prin urmare, un
scop important pentru cercetători. Lucrarea de față este menită să testeze efectul antihelmintic in vitro al uleiului
de cuișoare (Syzygium aromaticum) împotriva adulţilor și larvelor musculare de Trichinella spiralis. Formele
adulte și larvele musculare ale Trichinellei spiralis au fost incubate cu ulei de cuișoare la concentrații cuprinse
între 5 și 500 μg/ml, pentru a analiza concentrațiile letale eficiente și pentru a urmări modificările survenite în

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cuticule, prin microscopie electronică cu baleiaj. La 50 μg/ml, s-a observat o rată de mortalitate de 100% la
adulții de T. spiralis, dar acest efect a apărut doar la 24 de ore post expunere. Cu toate acestea, la concentrații de
100 și 500 μg/ml, efectul letal a început la 16 și respectiv 8 ore. Uleiul de cuişoare a omorât toate larvele la
concentrații de 100 și 500 μg/ml la 16, respectiv 24 ore de incubare in vitro. Paraziţii adulți și larvele musculare
de T. spiralis incubate cu 100 μg/ml de ulei de cuișoare au prezentat modificări morfologice marcate, vezicule
multiple și hemoragii, slăbirea unor zone ale cuticulei cu fisuri, pierderea anulației normale și distrugerea
cuticulei. Rezultatele noastre au sugerat că uleiul de cuișoare are potențial terapeutic și poate fi folosit ca un
medicament alternativ împotriva adulților și a stadiilor de larvare ale Trichinellei spiralis.
Cuvinte cheie: Trichineloză; Ulei de cuișoare; Activitate antihelmintică; Microscopie electronică.
Received 09.05.2020. Accepted 13.08.2020.
Introduction
Trichinellosis is a zoonotic parasitic disease
caused by Trichinella spiralis (T. spiralis), a
nematode of the genus Trichinella (Bai et al.,
2017). These parasites have the capacity of
infecting a wide range of mammals by eating
improperly cooked or raw meat containing
the infective larvae of Trichinella (Abou Rayia
et al., 2017). It infects around 11 million
individuals around the world (Luis Muñoz-
Carrillo et al., 2019). In Egypt, it was
diagnosed in a man (Abdel-Hafeez et al., 2015)
and pigs slaughtered in Cairo Abattoirs (Dyab,
2019). All phases of development of T. spiralis,
adult, migratory, and encysted stages are
found in the same host, so, this parasite has
been normally utilized as a trial model to
assess the effectiveness of numerous
anthelmintic agents (Yadav and
Temjenmongla, 2012).
Currently, benzimidazole derivatives are the
main anthelmintic therapeutic drugs used for
the clinical treatment of trichinellosis.
However, they have many drawbacks (Huang
et al., 2020), as none of these medications is
compelling to kill encapsulated and newborn
larvae (Nassef et al., 2018), due to their low
bioavailability (Caner et al., 2008) and drug
resistance (Shalaby et al., 2010). Likewise,
most of them are contraindicated in pregnant
women and children below two years of age.
(Yadav and Temjenmongla, 2012). Therefore,
finding a new, secure and efficient anthelmintic
agent against T. spiralis is a target for scientific
researchers. The World Health Organization
encourage researches on medicinal plants to
produce new, easy-to-use anthelmintic
compounds with lower side effects in the battle
against diseases affecting people in
underdeveloped countries. The wider
acceptance of medicinal plants as treatments is
due to the pharmacological activities
attributable to their phytoconstituents, the
lesser side effects and improved viability than
their synthetic counterparts (Batiha et al.,
2020).
Syzygium aromaticum (clove oil) is worldwide
used as a food flavoring agent. It has been
widely used in traditional medicine to treat a
variety of diseases since ancient times;
additionally, its major constituent, eugenol,
showed a potential lethal efficacy against
various parasites including Giardia lamblia,
Fasciola gigantica, Haemonchus contortus, and
Schistosoma mansoni (Machado et al., 2011; El-
Kady et al., 2019). Clove oil has been
traditionally utilized in inhibiting food-borne
pathogens (Bhowmik et al., 2012), and has
been recorded as a “Generally Regarded As
Safe” substance by the United States Food and
Drug Administration, and the World Health
Organization (WHO) Expert Committee on
Food Additives which had established the
acceptable daily admission of clove oil at 2.5
mg/kg body weight for humans (Anderson et
al., 1997). Considering the aforementioned
reasons, an attempt has been made to assess
the anthelmintic efficacy of clove oil (Syzygium
aromaticum) against adult worms and muscle

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larvae of T. spiralis. The study is also motivated
by the lack of scientific data in the literature
regarding in vitro activity of clove oil against
this parasite.
Materials and methods
Parasite and animals
The strain of T. spiralis was obtained from
infected pork meat collected from Cairo
abattoir and kept in the lab of Parasitology,
Theodor Bilharz Research Institute (Giza,
Egypt) by consecutive passages on rats and
mice. Male Swiss albino mice, 6–8 weeks old,
weighing 25–30 g each were utilized. The
animals were housed in proper cages and fed
with commercial rodent chow and tap water ad
libitum, as per the institutional and national
guidelines. Mice have been orally infected with
200 T. spiralis larvae (Abou Rayia et al., 2017).
After 48 hours (h), the animals were killed, the
small intestine detached, cut into pieces, and
kept in phosphate-buffered saline for 4 hours
of incubation at 37°C to recover the adult
worms of T. spiralis. To obtain the muscle
larvae, the infected mice were sacrificed after
35 days of infection and the muscles digested
in pepsin-HCL as indicated by the technique of
Jiang et al. (2012).
In vitro experimental design
The collected T. spiralis adults and muscle
larvae were added to a 48-well microtiter plate
prepared with RPMI-1640 medium (containing
200 U/ml penicillin, 200 μg/ml streptomycin,
and 20% fetal bovine serum). Clove oil was
purchased from El-Captain Company in the
local market, Cairo, Egypt, dissolved in
dimethyl sulfoxide (DMSO) and diluted in
RPMI-1640 medium. The final concentrations
of clove oil against adults and muscle larvae
ranged from 5 to 500 µg/ml at time intervals of
1, 2, 4, 8, 16 and 24 h. Every determination was
performed in triplicate and the summation of
wells for each concentration were calculated.
The plates were incubated at 37°C and 5% CO2
for 24 h, and the survival of T. spiralis stages
was observed utilizing a microscope. Control
parasites were incubated in RPMI-1640
medium having 1% DMSO. Worm viability rate
(%) was calculated using the formula: number
of viable worms/total number of worms ×100.
The viability of adult parasites was made by
assessing their shape and mobility, the dead
worms being characterized by C-shaped or
linear body and lack of mobility. The same
sequence was handled for muscle larvae.
Samples of worms and larvae were taken after
24 h of incubation and handled for scanning
electron microscopy.
Preparation for scanning electron microscopic
examination
The adults or larvae of T. spiralis were added
to a fixed solution of 2.5% glutaraldehyde
and incubated medium at 4°C. The parasites
were washed in 0.1M sodium cacodylate
buffer at pH 7.2 for 5 minutes, post-fixed in a
2% (w/v) osmium tetroxide in sodium
cacodylate buffer for 1 hour. Post-fixed
specimens were then dehydrated in
ascending concentrations of alcohol and
dried utilizing a critical point of carbon
dioxide drying. The parasites thus prepared
were examined by scanning electron
microscopy (Hitachi SU8040, Japan). Photos
were recorded on electron image plates.
Statistical analysis
Graph drawing and statistical analysis were
performed using Excel Software 2013. The data
were expressed as means ± SD, and the
Student’s 𝑡-test was used to determine the
significance of differences between mean
values.
Results
The activity of clove oil on adult worms and
muscular larvae of T. spiralis
Clove oil markedly affected both adult worms
and muscular larvae of T. spiralis. The lethal
effect of clove oil on the adult worms is
revealed in figure 1. At 50 μg/ml, a 100 %
death rate on the adult worms of T. spiralis was
observed only at 24h. However, in 10 up to 500
μg/ml concentrations, the clove oil significantly
influenced the adult worms from the first hour
of in vitro exposure at concentrations from 10
μg/ml (p<0.01) to 500 μg/ml (p<0.001) (table
1).

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Clove oil only killed all larvae at concentrations
of 100 and 500 μg/ml at 24 h of in vitro
incubation, while the viability rates of larvae
diminished with prolonged exposure at lower
concentrations (figure 2). At concentrations of
50, 100 and 500 μg/ml, the larvicidal impact of
clove oil was significantly higher beginning in
the first hour of incubation (P<0.001)
compared with controls (table 2). The worms
and larvae treated with 100 μg/ml were
chosen for scanning electron microscopy
(SEM) examination due to the early lethal
effect when compared with the other low
concentrations.
SEM assessment of the adult worms revealed
that culturing adult worms with clove oil (100
μg/ml) for 24 hours caused serious alterations.
The cuticle of the indicated areas showed
marked swellings, numerous large blebs,
fissures and vesicles accompanied by loss of
the normal creases, ridges and annulations
(figures 4, 5). The sloughing of certain
territories of the cuticle was also noticed. The
body collapsed, and there was a lot of carrions.
While culturing adult worms in the incubation
medium only saved the normal morphology of
the cuticle with the characteristic annulations
and ridges well-arranged as vertical lines
between the stripes on the surface of the
parasite (figure 3). Examining of T. spiralis
larvae incubated in culture medium only by
scanning electron microscope demonstrating
normal cuticle with transverse creases and
longitudinal ridges (figure 6). Incubation of T.
spiralis Larvae at 100 µg/ml clove oil indicated
marked morphological changes, multiple
vesicles and blebs, sloughing of some areas of
the cuticle with fissures, loss of normal
annulation, destruction of the cuticle, and
collapsing of the body of Trichinella larvae was
seen (figures 7, 8).
Figure 1. In vitro effect of Syzygium aromaticum on viability rates (%) of T. spiralis adult worms
Table 1. In vitro effect of Syzygium aromaticum (clove oil) on viability rates (%) of T. spiralis adult worms
Cloves oil dose (μg/ml)
Time intervals
1 hours
2 hours
4 hours
8 hours
16 hours
24 hours
Parasite control
97.67
±0.67
96.83
±0.40
96.83
±1.08
95.33
±0.76
95.33
±1.02
91.50
±0.76
5
94.33
±1.71
91.33
±2.50
88.50
±3.60
89.33
±3.53
83.33
±5.38
77.33
±6.88
10
90.00
±1.93**
84.83
±2.24***
77.17
±4.85**
69.50
±3.87***
63.83
±1.74***
54.33
±1.78***
50
85.50
±1.36***
58.50
±3.02***
43.33
±2.72***
30.67
±2.98***
22.00
±2.98***
0.00
±0.00***
100
81.67
±1.67***
67.17
±2.43***
54.00
±2.18***
30.50
±2.55***
0.00
±0.00***
0.00
±0.00***
500
79.67
±1.74***
44.67
±1.87***
26.67
±1.86***
0.00
±0.00***
0.00
±0.00***
0.00
±0.00***
Data were expressed as the mean ± SD **P<0.01 and ***p<0.001 compared with the corresponding parasite controls.

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Figure 2. In vitro effect of Syzygium aromaticum on viability rates (%) of T. spiralis muscular larvae
Table 2. In vitro effect of Syzygium aromaticum (clove oil) on viability rates (%) of T. spiralis muscular larvae
Cloves
oil dose
(μg/ml)
Time intervals
1h
2h
4h
8h
16h
24h
Parasite
control
98.17±0.79
98.17±0.60
99.00±0.37
98.83±0.48
96.50±0.50
93.83±1.11
5
94.83±1.64
96.33±0.71
94.67±2.48
95.00±1.73
91.83±2.20
81.33±5.74
10
95.33±1.56
94.00±1.83
85.67±2.92**
88.50±2.85**
73.33±2.79***
62.33±1.67***
50
88.50±1.38***
86.83±1.08***
73.83±1.22***
68.67±1.09***
63.17±2.02***
51.17±1.40***
100
86.33±1.93***
82.00±0.58***
62.33±1.56***
52.00±1.93***
27.50±1.91***
0.00±0.00***
500
82.83±1.49***
60.33±2.95***
49.83±2.63***
26.67±1.86***
0.00±0.00***
0.00±0.00***
Data were expressed as the mean ± SD **P<0.01 and ***p<0.001 compared with the corresponding parasite controls.
Figure 3. Higher magnification of T. spiralis adult worm
showing the cuticle with intact annuli (black arrow) and
hypodermal gland (white arrow). The cuticle is lined up in
neat rows
Figure 4. Adult worm incubated with 100 µg/ml clove oil,
large blebs (white arrows) and areas with small vesicles
(black arrow) are seen

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Figure 5. The cuticle of the adult worm after incubation with
1oo µg/ml clove oil was severely damaged, and there was a
large amount of carrion and blebs (black arrow). Intact
annuli and the vertical lines disappeared and completely
degenerated cuticle (white arrow).
Figure 6. Isolated muscular infective larvae, demonstrating
their typical coiled appearance, the cuticle is normal,
annulated with transverse creases (black arrow) and
longitudinal ridges (white arrow)
Figure 7. Muscle larva treated with clove oil (100 µg/ml)
showing fissures of the cuticle (white arrow)
and collapsing of the body (black arrow)
Figure 8. muscular larva incubated in 100 µg/ml clove oil
showing sloughing of some areas of the cuticle (white arrow)
with swelling and blebs (black arrows). Multiple fissures
(headless arrow) and loss of the normal annulations
Discussion
The in vitro test with helminths is one of the
useful tools to explore the anthelmintic
properties of a certain agent and is also helpful
to analyze its mode of action. In vitro tests are
preferred to in vivo methods due to their low
cost, simplicity, and rapid turnover. Our analysis
has shown that clove oil has lethal action on
adult stage and muscle larvae of T. spiralis in
vitro. This remarkable effect was shown to occur
in a dose- and time-dependent manner (figures
1, 2). Many experiments studied the in vitro
effect of clove oil and other extracts against
various nematodes such as Haemonchus
contortus (Charitha et al., 2017), Cotylophoron
cotylophorum (Manoj Dhanraj and Veerakumari,
2015), and Meloidogyne incognita (Meyer et al.,
2008). As was shown in these experiments, the
lethal effect of clove oil for these parasites was
expressed at various concentrations.
In the present study, SEM demonstrated the
detrimental effects of clove oil against the
incubated adult worms and larvae of T. spiralis.
These effects were described as swelling,
sloughing and damage to the cuticle of the adult
worms and muscle larvae of T. spiralis associated
with the drug exposure. Previous studies have
investigated the effects of clove oil on nematodes.
(Charitha et al., 2017) proved that, Syzygium
aromaticum is very effective in killing the
Haemonchus contortus worms where 100%
mortality was attained within minutes of
exposure and attributed the wormicidal activity
of clove to its strong corrosive action on cuticle
and tegument of helminths. These results agreed
with our results which characterized in table 1

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and figures 4, 5 for T. spiralis worms also
represented in table 2 and figures 7, 8 for muscle
larvae. The difference in time of mortality was
due to the variance in concentrations used in the
two studies. Similarly, the study of Manoj Dhanraj
and Veerakumari (2015) revealed anthelmintic
property of Syzygium aromaticum ethanol extract
against Cotylophoron cotylophorum worms, and
the authors accredited this property to the
disruption involved in the metabolic pathway of
carbohydrates that may be fatal to the parasites.
Our research focused on exploring the impact of
clove oil on the structure of the T.spiralis cuticle
because tegumental and/or cuticular structures
are the vital parasite-host interfaces that are both
nutritional and defensive roles as well as retain
their form (Roy et al., 2010). The lethal action of
clove oil against adults and T. spiralis muscle
larvae may be attributed to its deleterious effect
on the parasite cuticle. This effect facilitating the
diffusion of active oil constituents within the
organism due to its lipid solubility, causing
dramatic changes in the internal structural
features of helminths (Roy et al., 2010). In
addition, clove oil was reported to disrupting
metabolism (Manke et al., 2015) and/or DNA
synthesis or folic acid cycle of the parasite,
triggering cellular damage. Such results were not
surprising, as transcuticular passive diffusion is
the key mechanism of drug entry into the
helminths (Roy et al., 2010).
The observed in vitro anthelmintic activity of
clove oil against the adult worm and larva stages
of T. spiralis may be also attributable to the
enrichment of clove oil with tannins (10-13%)
(Mittal et al., 2014). Tannins have been reported
to induce anthelmintic activities through binding
to the free protein available in the wells for
parasite nutrition; decreased supply of nutrients
in the wells, resulting in malnutrition and death
of Trichinella adults and larvae (Chandrashekhar
et al., 2008). Tannins may also bind to the cuticle
of the parasite, which is rich in glycoprotein,
triggering its death. Clove oil contains also,
monoterpenes and sesquiterpenes (Plata-Rueda
et al., 2018), these constituents allows
compounds to permeate the cell membrane that
affects the metabolic pathways or organelles
destroying the parasites. β-caryophyllene
constitutes about 13% of clove oil and was
identified for several important pharmacological
activities, including anti-inflammatory action
(Sharma et al., 2016; Machado et al., 2018). As we
know, pharmacotherapy used in trichinellosis
includes the use of anti-parasitic drugs which are
directed against the parasite (Gottstein et al.,
2009) and steroidal anti-inflammatory drugs,
whose purpose is to alleviate the signs and
symptoms of the inflammatory response
produced by T. spiralis infection (Shimoni et al.,
2007). Therefore, β-caryophyllene may add
another dimension for using clove oil as anti-
parasitic plus anti-inflammatory in treating
T. spiralis.
In conclusion, clove oil has a promising in vitro
anthelmintic activity and can be considered an
effective and safe alternative drug against adult
worms and muscle larvae of T. spiralis. However,
further in vivo testing will be imperative to
investigate the main active components of clove
oil in studies that will establish the doses,
administration routes, bioavailability, and
metabolic biotransformation. Studying
combinations of clove oil with standard
antitrichinellosis drugs may also be used to
improve the treatment effect and to reduce the
toxicity associated with these drugs and to
supress resistance.
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