Potential effects of alpha-pinene, a monoterpene commonly found in essential oils against Toxoplasma gondii infection; an in vitro and in vivo study

Abstract

This survey designed to assess the in vitro and in vivo activity of α-pinene, a monoterpene commonly originated in essential oils on Toxoplasma gondii. The in vitro effect of various concentration of α-pinene against tachyzoites of T. gondii Rh strain was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The activity of α-pinene on the stimulation of apoptosis in tachyzoites of T. gondii was also examined using the caspase 3 colorimetric activity assay. In vivo assay, mice were orally received α-pinene at 2 and 4 mg/kg/day for 14 days, then, pre-treated mice were daily tested and the rate of death was recorded. α-pinene meaningfully declined (p < 0.001) the tachyzoites viability with the IC50 value of 23.3 µg/mL. α-pinene induced the apoptosis through increasing the caspase-3 activity by 35.6%. Oral treatment with α-pinene significantly (p < 0.01) improved the survival rate infected mice with by 9th day. α-pinene + atovauone (50 mg/kg) significantly (p < 0.01) improved the survival rate infected mice up to 11 days compared with the control groups. α-pinene especially in combined atovaquone at 50 mg/kg for 2 weeks meaningfully (p < 0.05) declined oxidative stress. We found the promising in vitro anti-Toxoplasma effects of α-pinene on T. gondii RH strain. In addition, we found that α-pinene therapy particularly along with the reference drug declined the mortality rate of infected mice. Although, we just confirmed the stimulation of apoptosis and anti-inflammatory effects as the main anti-Toxoplasma mechanisms of α-pinene; however, more surveys concerning the accurate mechanisms, toxicity, and efficacy on other T. gondii strains are required to confirm these results.

Full text

ORIGINAL ARTICLE
Potential effects of alpha-pinene, a monoterpene commonly found
in essential oils against Toxoplasma gondii infection; an in vitro
and in vivo study
Ali Kharazmkia
1,2
•Hiba Riyadh Al-Abodi
3
•Javad Ghasemian Yadegari
1
•
Ashkan Vahidi
4
•Hossein Mahoudvand
1
Received: 5 May 2022 / Accepted: 13 June 2022
ÓIndian Society for Parasitology 2022
Abstract This survey designed to assess the in vitro and
in vivo activity of a-pinene, a monoterpene commonly
originated in essential oils on Toxoplasma gondii. The
in vitro effect of various concentration of a-pinene against
tachyzoites of T. gondii Rh strain was assessed by MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay. The activity of a-pinene on the stimulation of
apoptosis in tachyzoites of T. gondii was also examined
using the caspase 3 colorimetric activity assay. In vivo
assay, mice were orally received a-pinene at 2 and 4 mg/
kg/day for 14 days, then, pre-treated mice were daily tested
and the rate of death was recorded. a-pinene meaningfully
declined (p\0.001) the tachyzoites viability with the IC
50
value of 23.3 lg/mL. a-pinene induced the apoptosis
through increasing the caspase-3 activity by 35.6%. Oral
treatment with a-pinene significantly (p\0.01) improved
the survival rate infected mice with by 9th day. a-
pinene ?atovauone (50 mg/kg) significantly (p\0.01)
improved the survival rate infected mice up to 11 days
compared with the control groups. a-pinene especially in
combined atovaquone at 50 mg/kg for 2 weeks meaning-
fully (p\0.05) declined oxidative stress. We found the
promising in vitro anti-Toxoplasma effects of a-pinene on
T. gondii RH strain. In addition, we found that a-pinene
therapy particularly along with the reference drug declined
the mortality rate of infected mice. Although, we just
confirmed the stimulation of apoptosis and anti-inflamma-
tory effects as the main anti-Toxoplasma mechanisms of a-
pinene; however, more surveys concerning the accurate
mechanisms, toxicity, and efficacy on other T. gondii
strains are required to confirm these results.
Keywords Toxoplasmosis Alpha-pinene Tachyzoites 
Mortality rate Apoptosis Caspase-3
Introduction
Toxoplasma gondii is a forced protozoan parasite which
multiplies in all nucleated cells of vertebrate hosts and its
main host is the cat (Innes 2010). Routinely, humans being
infected by eating raw or cooked infected meat (especially
beef and pork), by eating oocysts in water and food con-
taminated with cat feces, and probably transmitted through
the placenta during pregnancy (Hill et al. 2005). Toxo-
plasmosis occurs in a variety of forms, from an asymp-
tomatic self-limiting infection to a fatal disease in patients
with congenital infections and patients with special con-
ditions (Saadatnia and Golkar 2012). In healthy people
with normal immune systems, infection with this protozoan
usually has no clinical symptoms and in some cases may be
dangerous due to the parasite tending to the host’s eyes and
brain and the formation of cysts in these organs (Weiss and
Dubey 2009); but in people with defective immune sys-
tems, and the same pregnant women, the parasite causes
severe complications (Wang et al. 2017). In immunocom-
promised individuals, chronic infection with T. gondii can
reactivate and cause encephalitis, chorioretinitis, or death
&Hossein Mahoudvand
dmhmodvand@gmail.com
1
Razi Herbal Medicines Research Center, Lorestan University
of Medical Sciences, Khorramabad, Iran
2
Department of Clinical Pharmacy, School of Pharmacy,
Lorestan University of Medical Sciences, Khorramabad, Iran
3
Department of Environment, College of Science, University
of Al-Qadisiyah, Al-Diwaniyah, Iraq
4
Student Research Committee, Lorestan University of Medical
Sciences, Khorramabad, Iran
123
J Parasit Dis
https://doi.org/10.1007/s12639-022-01514-1

(Wang et al. 2017; Fallahi et al. 2017; Hanifehpour et al.
2019). The same congenital toxoplasmosis that results from
the parasite passing through the placenta during primary
maternal infection can cause miscarriage, fetal death in the
womb, or severe congenital complications, e.g. hydro-
cephalus and chorioretinitis (Goldstein et al. 2008;
Kheirandish et al. 2019).
One of the main problems of toxoplasmosis is its ther-
apeutic limitation (Dunay et al. 2018). The drug of choice
for toxoplasmosis is a synergistic mixture of pyr-
imethamine and sulfonamide, which in addition to having
side effects, do not have the ability to kill parasites in the
cyst and eradicate the infection (Dunay et al. 2018). This
treatment regimen is usually associated with many com-
plications, e.g. blood poisoning, hypersensitivity, intoler-
ance, bone marrow suppression and teratogenic effects in
the first trimester of pregnancy (Smith et al. 2021).
One of the research priorities of toxoplasmosis is to
obtain an anti-Toxoplasma drugs with the desired effect
and with the least side effects (Arab-Mazar et al. 2017;
Cheraghipour et al. 2021). Medicinal plant products are
one of these options that have been mentioned in traditional
medicine about the antiparasitic effects of some of these
plants. The use of natural compounds to treat diseases has a
long history (Al-Snafi, 2016). Today, although most drugs
are of chemical origin, it is estimated that about one third
of all medicinal products are of plant origin or have been
deformed after extraction from the plant (Bauri et al. 2015).
Alpha-pinene (a-pinene, C
10
H
16
), an organic compound of
the terpenoid hydrocarbon, is broadly found in essential
oils a large number of medicinal herbs (Allenspach and
Steuer 2021). In modern medicine, a-pinene have been
displayed various therapeutic and pharmacological prop-
erties, e.g. anti-inflammatory, gastroprotective, neuropro-
tective, anticancer, anticoagulant, antinociceptive,
antioxidant, and antimicrobial effect (Allenspach and
Steuer 2021; Koziol et al. 2014; Santos et al. 2011). By
anti-parasitic effects, studies have displayed the favorable
effects of a-pinene against various parasite species such as
Leishmania spp, and Plasmodium spp, (Allenspach and
Steuer 2021). Based on the potent pharmacological prop-
erties of a–pinene, we intended to the in vitro and in vivo
effects of a-pinene against acute T. gondii infection.
Materials and methods
Drugs and reagents
Atovaquone (C98% purity), a-pinene (C98% purity), and
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide) powder was procured from Sigma
Chemical Co. (St. Louis, MO, USA).
Parasite and cells
T. gondii RH tachyzoites were kindly provided from
Department of Medical Parasitology, Khorramabad, Iran.
Tachyzoites collected from BALB/c mice peritoneum and
adjusted into 10,000 tachyzoites/mL for tests (Cher-
aghipour et al. 2022; Saadatmand et al. 2021). The Vero
cells were procured from the Pasteur Institute of Iran cell
flask and cultured in RPMI1640 fluid culture medium
containing 10% of inactivated bovine fetal serum (Merck,
Germany) and 1% of the antibiotics of penicillin and
streptomycin. The cells were incubated in sterile flasks at
37 °C in 5% CO2 and 95% humidity (Kareshk et al. 2015).
In vitro cell viability assay
To do this, 0.2 mL of tachyzoites (10
6
parasites per each
mL) were incubated with various concentrations of a-
pinene (12.5–100 lg/mL) in 96-well plates for 24 and
48 min at 37 °C. After incubation time, 50 ll of MTT
suspension (5 mg/ml) was added and incubated again at
37 °C for 4 h. After 4 h incubation, by adding the iso-
propanol (200 lL) formazan crystals were eluted. As the
last step of experiment, the solution absorbance was
assessed at 570 nm by ELISA reader. Tachyzoites treated
with the tween-80 aqueous solution were considered as the
negative control. The 50% inhibitory concentrations (IC
50
)
was also measured. To increase the validity of the tests, all
tests were accomplished in triplicate (Asgari et al. 2013).
Activity of a-pinene on the stimulation of apoptosis
The activity of a-pinene on the stimulation of apoptosis in
tachyzoites was examined using the kits of Caspase 3
colorimetric activity test (Sigma-Aldrich, Germany) based
on the dye obtained from the activity of caspase-3 enzyme.
Initially, T. gondii RH tachyzoites were treated with a-
pinene at 1/3 IC
50
, 1/2 IC
50
, and IC
50
. After centrifuging,
and lysing the pellet cells, they were then centrifuged at
16,000 rpm for 15 min. Next, 5 lL of superior was mixed
by 10 lL of caspase 3 substrate and 85 lL buffer solution
and the combination was incubated for two hours min at
37 °C. As the final step, the absorbance of mixture was
measured by the ELISA reader at 410 nm (Ezzatkhah et al.
2021).
In vivo effects on acute toxoplasmosis in mice
Animals
Sixty male BALB/c mice (40 to 60 days’ age) and weight
of 20–25 g purchased from Animal Breeding Center of
Pasteur Institute, Iran and were selected for in vivo
J Parasit Dis
123

evaluation on acute toxoplasmosis in mice. Storage con-
ditions were the same for all mice and were considered as
12 h of light / dark and an ambient temperature of
21 ±2°C in special cages and in a bed of straw. Diet and
water were also freely available. During the study, the
maintenance and testing of animals and the destruction of
animals were in accordance with standard methods of work
and principles of ethics with animals.
Ethics
The procedure was permitted by Lorestan University of
Medical Sciences Ethical Committee, Khorramabad, Iran
(IR.LUMS.1401.014).
Treatment of mice
After random division of mice into 5 groups (12 mice per
each), mice were orally received 500 lL of tween-80
aqueous solution, atovaquone 100 mg/kg, a-pinene 2 and
4 mg/kg, as well as a-pinene (2 mg/kg) ?atovaquone
(50 mg/kg), one time per day for two weeks. It should be
mentioned that the selection of these doses for in vitro and
in vivo tests was based on the primary experiments as well
as previous study (Felipe et al. 2019).
Induction of acute toxoplasmosis in mice
One day after 14 days’ treatment, all mice were intraperi-
toneally infected with 100 lLofT. gondii tachyzoites
(10,000 tachyzoites/mL) (Kareshk et al. 2015).
Evaluation of the oxidative stress markers
To study the effects of a-pinene therapy on reducing the
oxidative stress markers in infected mice, on the third day
after toxoplasmosis induction, six mice from each group
were euthanized using sodium pentobarbital. The serum
level of malondialdehyde (MDA) as a main marker of lipid
peroxidation (LPO) was measured according to the tech-
nique defined elsewhere (Hagege et al. 1990). In summary,
mice sera were separately added to the Thiobarbituric acid
(0.06%) and phosphoric acid (1%) and the suspension was
incubated in for 45 min. In the next step, after adding the
n-buthanol to the mixture, the serum level of MDA was
read at 532 nm by ELISA reader.
Assessment of mortality rate
Treated mice were everyday checked and the frequency of
death was studied for mice in tested groups (Mahmoudvand
et al. 2020).
Parasitological examination
To do this, three days’ post-infection, after collecting the
peritoneal fluids of mice in all groups, the number of the
collected tachyzoites of each mice were recorded by a light
microscopic (19).
Statistical analysis
Finally, the collected outcomes were analyzed using SPSS
software version 22.0 and one-way analysis of variance
(ANOVA). Tukey’s test was applied to compare the means
of the data. The difference in the level of probability was
considered to be less than 0.05.
Results
In vitro effects on cell viability of tachyzoites
Figure 1shows the results of in vitro cell viability assay
used to evaluate the effect of various concentrations of a-
pinene (12.5–100 lg/mL) on tachyzoites. The results of
MTT assay revealed that a-pinene significantly reduced
(p\0.001) the tachyzoites viability as dose-dependent
response, in comparision with the control group. The IC
50
value for a-pinene and atovaquone was 23.3 and 17.6 lg/
mL, respectively (Fig. 1).
Effects of a-pinene on the stimulation of apoptosis
The activity of a-pinene on apoptosis stimulation in
tachyzoites was examined using the Caspase 3 colorimetric
activity assay. Based on the obtained findings after treat-
ment of T. gondii RH tachyzoites with a-pinene at 1/3 IC
50
,
1/2 IC
50
, and IC
50
, induced the apoptosis through
increasing the caspase-3 activity by 16.3, 27.4, and 35.6%,
respectively (Fig. 2).
Fig. 1 Activity of some concentrations of a-pinene on the viability of
T. gondii tachyzoites. Mean ±SD (n = 3). * P\0.001
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123

In vivo effects on mortality rate of T. gondii mice
infected
Treatment with a-pinene at 2, and 4 mg/kg considerably
(p\0.01) improved the survival rate infected mice with
by the 8th, and 9th day p.i, respectively. In addition, pre-
treatment of mice infected with atovaquone increased
survival to 9 days. a-pinene at 4 mg/kg in combination
with atovauone (50 mg/kg) meaningfully (p\0.01)
improved the survival rate infected mice up to 11 days
compared with the control group and atovaquone at
100 mg/kg (Fig. 3).
Effect on the number of tachyzoites isolated
from infected mice
The results showed that treatment with a-pinene at doses of
2 and 4 mg/kg, the mean number of peritoneal tachyzoites
collected from infected mice was significantly (p\0.001)
decreased by 17.2 910
4
and 11.3 910
4
, respectively. In
addition, pre-treatment of infected mice with atovaquone at
a dose of 100 mg/kg considerably (p\0.001) declined the
mean number of peritoneal tachyzoites collected from
infected mice at 8.7 910
4
. The results also showed that a-
pinene (4 mg/kg) in combination with atovaquone (50 mg/
kg) markedly (p\0.001) declined the average number of
tachyzoites in infected mice up to 8.7 910
4
compared
with the control group and atovaquone (100 mg/kg)
(Fig. 4).
Evaluation of the oxidative stress markers
Figure 5indicates the effects of a-pinene therapy on the
oxidative stress markers in infected mice. The results
Fig. 2 The effects of a-pinene at 1/3 IC
50
, 1/2 IC50, and IC50 on the
caspase-3 like in tachyzoites. Mean ±SD (n = 3). *P\0.001
Fig. 3 The effect of oral
administration of a-pinene (AP)
at 2 and 4 mg/kg, a-pinene at
4 mg/kg ?atovaquone 50 mg/
kg for 14 days in comparison
with the atovaquone 100 mg/kg
and control group. Mean ±SD
(n = 6)
Fig. 4 The effect of oral
administration of a-pinene (AP)
for 14 days on the number of
tachyzoites isolated from
infected mice in comparison
with the atovaquone 100 mg/kg
and control group. Mean ±SD
(n = 6). *P\0.001. #
P\0.001 when compared with
the atovaquone 100 mg/kg
J Parasit Dis
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showed that in T. gondii infected mice the level of serum
MDA was significantly increased (p\0.001). On the other
hand, treatment of infected mice with a-pinene especially
in combined atovaquone at 50 mg/kg for 2 weeks mean-
ingfully (p\0.05) declined oxidative stress.
Discussion
Natural compounds are still considered an important
resources for drug discovery (Bauri et al. 2015; Rasoulian
et al. 2019). a-Pinene, as one of the famous secondary
metabolite (monoterpenes) derived from plants is of high
attention for medicinal, industrial, and commercial use
(Silva et al. 2012). Today, studies have reported various
pharmacological and therapeutic of a-pinene properties for
treating diseases (Allenspach and Steuer 2021; Koziol et al.
2014; Santos et al. 2011). Today, the main treatment reg-
imens for toxoplasmosis are frequently accompanying with
various side effects such as blood poisoning, hypersensi-
tivity, intolerance, bone marrow suppression and terato-
genic effects in the first trimester of pregnancy
(Cheraghipour et al. 2020; Keyhani et al. 2020; Smith et al.
2021). Therefore, finding an anti-Toxoplasma agent with
the desired effect and with the least side effects especially
from natural products seems necessary for researchers and
clinicians (Cheraghipour et al. 2021). Based on the potent
pharmacological properties of a–pinene, we intended to
evaluate the effects of a-pinene against acute T. gondii
infection.
By in vitro assay, our results showed that that a-pinene
significantly reduced (p\0.001) the viability of tachy-
zoites as dose-dependent response. The IC
50
value for a-
pinene and atovaquone was 23.3 and 17.6 lg/mL, respec-
tively. Here, we studied the effect of a-pinene on the
apoptosis stimulation in tachyzoites of T. gondii RH strain
was examined using the caspase 3 colorimetric activity
assay. Our findings exhibited that after treatment of T.
gondii RH tachyzoites with a-pinene at 1/3 IC
50
, 1/2 IC
50
,
and IC
50
, provoked the apoptosis through increasing the
caspase-3 activity by 16.3, 27.4, and 35.6%, respectively.
In line with our findings, Hou et al. (2019) and Matsuo
et al. (2011) exhibited that a-pinene significantly elevated
the level of caspase-3 in murine melanoma cell line and
human ovary cell lines.
In view of the antimicrobial effects, investigations
showed the relevant antibacterial effects of a-pinene
against various pathogenic bacteria (e.g. Staphylococcus
aureus, S. pyogenes, Streptococcus pneumonia, Klebsiella
pneumonia, Haemophilus influenza) and fungal pathogenic
strain (e.g. Candida spp., and Aspergillus spp.) with min-
imum inhibitory concentration (MIC) values 1.024 to
256 lg/mL (No
´brega et al. 2021; Utegenova et al. 2018;
Yang et al. 2015). da Franca Rodrigues et al. (2015)
reported that a-pinene had potent anti-parasitic activity
against promastigote, axenic amastigotes, and intracellular
amastigote forms of Leishmania amazonensis, after 48 h
incubation with IC
50
values of 19.7, 16.1, and 15.6 lg/mL,
respectively. In another study, Van Zyl et al. (2006) have
revealed the considerable antimalarial effects of a-pinene
against Chloroquine-resistant Plasmodium falciparum
(FCR-3) with the IC
50
value of 1.2 lM. Wang et al. (2019)
have also reveal that after incubation of Bursaphelenchus
xylophilus (pinewood nematode) with a-pinene (98% pur-
ity) at 4, 8, and 16 mL, the mortality rate of nematode was
considerably increased by 51.1, 60.6, and 60.2%, respec-
tively. Concerning the antimicrobial mechanisms of action
of monoterpenes compounds, investigations reported that
these compounds presented their antimicrobial efficacy
through some cellular mechanisms, e.g. cell wall inter-
ruption, disruption of cellular oxygen consumption,
induction of apoptosis, and deactivation of pathogenic
virulence factors (Anand et al. 2019). Other study also
showed that a-pinene displayed its antimicrobial mecha-
nisms by structural changes in cell morphology, disruption
of protein, DNA, and RNA synthesis, and the reactive
oxygen species (ROS) creation (Li et al. 2014; Melkina
et al. 2021). These reasons indicate the direct antiparasitic
effects of a-pinene on the viability of tachyzoites of T.
gondii RH strain by the mentioned mechanisms.
By in vivo assay, our findings revealed that oral
administration of a-pinene at 2, and 4 mg/kg/day, espe-
cially along with atovaquone (50 mg/kg) significantly
improved the survival rate infected mice; whereas, the
mean number of peritoneal tachyzoites was significantly
decreased. Previous studies showed that a-pinene had
Fig. 5 The effect of oral administration of a-pinene (AP) for 14 days
on the serum level of malondialdehyde (MDA) in infected mice in
comparison with the atovaquone 100 mg/kg and control group.
mean ±SD (n = 6). *P\0.001. # P\0.001 when compared with
the atovaquone (AVQ) 100 mg/kg
J Parasit Dis
123

promising anti-inflammatory, antioxidant, neuroprotective
through some mechanisms such as suppressing of inflam-
matory markers, reducing the inducible cyclooxygenase-2
and nitric oxide synthase, and inhibition of lipid peroxi-
dation and oxidative stress, and inhibition of acetyl-
cholinesterase (Karthikeyan et al. 2018; Kim et al. 2015;
Miyazawa and Yamafuji, 2005). These reasons suggest that
a-pinene, through its anti-inflammatory, antioxidant, and
neuroprotective effects has been able to increase survival
rate of infected mice.
It has been proven one of the frequent mechanisms of
complicated in the pathogenesis of hepatic damage during
acute toxoplasmosis is increasing the oxidative stress
through the producing free radicals (Karaman et al. 2008).
LPO is as an indicator of oxidative stress, which results in
destruction of the cell membrane and subsequently dis-
charge of indicator enzymes of hepatotoxicity (Tonin et al.
2014). Our results revealed that in T. gondii infected mice
the level of serum MDA was considerably elevated
(p\0.001). On the other hand, treatment of infected mice
with a-pinene especially in combined atovaquone at
50 mg/kg for 2 weeks meaningfully (p\0.05) declined
oxidative stress. Consequently, it may be claimed that a-
pinene therapy by its anti-inflammatory activity protects
the liver from injuries provoked with T. gondii. Consider-
ing the toxicity of a-pinene, in the study conducted by
Felipe et al. (2019), the results showed that 50% lethal dose
(LD
50
) for both monoterpenes of a-pinene and b-pinene
was determined higher than 2000 mg/kg in tested mice,
where oral administration of a-pinene and b-pinene at the
doses of 100, 200 and 400 mg/kg had no toxicity in mice;
indicating that a-pinene at the doses used in the present
study had minimal toxicity in tested mice.
Conclusion
Here, we revealed the promising the in vitro anti-Toxo-
plasma activity of a-pinene on T. gondii tachyzoites. In
addition, we found that treatment of T. gondii infected mice
with a-pinene especially in combination with the reference
drug markedly declined the mortality rate of infected mice.
Although, we just confirmed the induction of apoptosis and
anti-inflammatory activity as the main anti-Toxoplasma
mechanisms of a-pinene; however, more surveys con-
cerning the accurate mechanisms, toxicity, and efficacy on
other T. gondii strains are necessary to confirm these
findings.
Declarations
Conflict of interest The author declares that they have no competing
interests.
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