Accepted Manuscript

Antiparasitic effects of oxymatrine and matrine against Toxoplasma gondii in vitro and
in vivo

Xiaochuan Zhang, Lili Jin, Zhe Cui, Changhao Zhang, Xue Wu, Hyun Park, Hongmei
Quan, Chunmei Jin

PII: S0014-4894(16)30046-7
DOI: 10.1016/j.exppara.2016.03.020
Reference: YEXPR 7218

To appear in: Experimental Parasitology

Received Date: 16 August 2015

Revised Date: 7 February 2016
Accepted Date: 15 March 2016

Please cite this article as: Zhang, X., Jin, L., Cui, Z., Zhang, C., Wu, X., Park, H., Quan, H., Jin,
C., Antiparasitic effects of oxymatrine and matrine against Toxoplasma gondii in vitro and in vivo,
Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.03.020.

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 ACCEPTED MANUSCRIPT

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 ACCEPTED MANUSCRIPT
Antiparasitic effects of oxymatrine and matrine against Toxoplasma
gondii in vitro and in vivo
Xiaochuan Zhanga,†, Lili Jina,†, Zhe Cuib, Changhao Zhanga, Xue Wuc, Hyun Parkd,
Hongmei Quana,*, Chunmei Jina,*
a
Key Laboratory of Natural Resources of the Changbai Mountain & Functional Molecules, Affiliated
Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, PR China
b
Yanbian University, Hospital of Pharmacy, Yanji 133002, PR China

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c
Research Centre for Chemical Biology, Department of Chemistry, Yanbian University, Yanji 133002,
PR China

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d
Zoonosis Reserch Center, Wonkwang University, School of Medicine, 570-749, 344-2
Shinyong-Dong, Iksan, Jeonbuk, Korea
†
These authors contributed equally to this work

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* Corresponding author: Key Laboratory of Natural Resources of the Changbai Mountain & Functional
Molecules, Affiliated Ministry of Education, Yanbian University, Yanji 133002, PR China. Tel: +86

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433 2436017. Fax: +86 433 2435026. E-mail: cmjin@ybu.edu.cn (C.-M. Jin), lijm@ybu.edu.cn (H.-M.
Quan)
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Abstract
Toxoplasma gondii (T. gondii) is an important pathogen which can causes serious
public health problems. Since the current therapeutic drugs for toxoplasmosis present
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serious host toxicity, research on effective and new substances of relatively low
toxicity is urgently needed. This study was carried out to evaluate the anti-parasitic
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effect of oxymatrine (OM) and matrine (ME) against T. gondii in vitro and in vivo. In
our study, the anti-T. gondii activities of ME and OM were evaluated in vitro using
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cell counting kit-8 assay, morphological observation and trypan blue exclusion assay.
In vivo, mice were sacrificed four days post-infection and ascites were drawn out to
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determine the extent of tachyzoite proliferation. Viscera indexes and liver biochemical
parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase
(AST), glutathione (GSH) and malondialdehyde (MDA), were examined to evaluate
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the toxicity of compounds to mice. As a result, OM and ME showed anti-T. gondii

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activity but low selectivity toxicity to HeLa cells. Both compounds also significantly
decreased the number of tachyzoites in peritoneal cavity and recovered the levels of
ALT, AST, GSH and MDA in liver. Moreover, the mice treated with OM or ME
achieved better results in viscera index and survival rate than that of spiramycin.
These results suggest that OM and ME are likely the sources of new drugs for
toxoplasmosis, and further studies will be necessary to compare the efficacy of drug
combination, as well as identify its action of mechanism.
Key words: anti-Toxoplasma gondii effect; oxymatrine; matrine; toxicity; in vitro
and in vivo
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1. Introduction
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that infects
humans and other warm-blooded animals, causing zoonotic toxoplasmosis. It is
reported that approximately 30% of all people are infected with toxoplasma around
the world (Lüder et al., 2001). Toxoplasma infection is considered to be a leading
cause of death in patients with immunosuppression, organ transplantation and AIDS.
In pregnant women, T. gondii infection can lead to encephalitis, neonatal

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malformations, chorioretinitis and mental retardation in the fetus (Weiss and Dubey,
2009).

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The effective drugs used today to treat toxoplasmosis are sulfonamide and
pyrimethamine (Petersen and Schmidt, 2003). These drugs restrain or kill toxoplasma
mainly by blocking or destroying the toxoplasma folic acid metabolic pathways (Chio

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et al., 1996). In addition, current treatment of toxoplasmosis in pregnant women is
based on the administration of spiramycin, which reaches high concentrations in

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placental tissue, thus decreasing the risk of fetal transmission (Lopes et al., 2007).
However, Disadvantages of these drugs are related to limited effectiveness in parasite
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clearance and low penetration in fetal tissues for spiramycin and numerous toxicity
problems for sulphadiazine and pyrimethamine (Franco et al., 2011; Schmidt et al.,
2006).
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Studies of herbal medicine for the treatment of toxoplasmosis have been

conducted, but research on relatively effective and low toxic substances is still needed
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(Choi et al., 2013). Until now, the extracts of Artemisia annua L., Zingiber officinale,
Sophora flavescens Aiton, Eurycoma longifolia, Ginkgo biloba sarcotesta, Fraxinus
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rhychophylla, etc. have been shown to be effective against RH strain of Toxoplasma

gondii (Chen et al., 2008; Choi et al., 2013; Choi et al., 2008; Jiang et al., 2008;
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Kavitha et al., 2012; Oliveira et al., 2009). In addition, Some effective compounds of
herbal medicine were also used to carry out the experimental study of toxoplasmosis
and showed different research results. For example, artemisinin and β-carboline
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alkaloids can inhibit the replication of Toxoplasma gondii while phytoecdysteroids

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promote the multiplication of Toxoplasma gondii in cultures in vitro (Alomar et al.,

2013; D’Angelo et al., 2009; Dzitko et al., 2015; Jones-Brando et al., 2006). These
researches can provide rich theoretical basis for finding new anti-T. gondii drugs in
the natural plants. Sophora flavescens Aiton is a traditional Chinese medicine that has
been reported to have antibacterial (Du et al., 2010), antiviral (Ma et al., 2002),
antitumor (Li et al., 2002), anti-inflammatory (Hong et al., 2009) and antiparasitic
activity (Youn et al., 2004). The crude extract of Sophora flavescens roots had good
anticoccidial effect against Eimeria tenella (Youn and Noh, 2001). The methanolic
extract of Sophora flavescens had high anti-T. gondii activity and high selective
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toxicity (Choi et al., 2008). The ethanol extract of Sophora flavescens also
demonstrated good efficacy in reducing replication of Toxoplasma and Neospora
caninum, but the chemical composition and mechanism is not yet clear (Youn et al.,
2004).
In recent years, pharmacological research of Sophora flavescens has made great
progress and extended to the clinical application. The major actively functional
constituents of Sophora flavescens has been related to the presence of Sophora

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alkaloids, such as oxymatrine (OM) and matrine (ME) (Miao et al., 2001). It has
already been reported that Sophora alkaloid has multi-pharmacological effects,

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including antioxidation, antitumor, antihepatic fibrosis, anti-inflammatory,
antiarrhythmic and enhance immune function, etc. (Chang et al., 2013; Deng et al.,
2014; He et al., 2015; Lin, 2006; Yamazaki et al., 1984; Zhang et al., 1985). The

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antitumor effects of OM and MO have been demonstrated promoting apoptosis via
mitochondria, modulating the immune response, inhibiting

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EGF/VEGF-VEGFR1-Akt-NF-kB signaling etc. (Sun et al., 2012). Chen et al. (2008)
suggested that OM may attenuate pulmonary fibrosis partly through inhibition of
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inflammatory response and lipid peroxidation and reduction of fibrolast proliferation
and collagen synthesis. Sophora alkaloids reduced LPS-induced NF-κB nuclear
translocation and activity independently of IκBa status, prevented intestinal
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inflammation through blockade of inflammatory signaling and ameliorates overall

intestinal inflammation in vivo (Guzman et al., 2013). The roots of Sophora
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flavescens were showed that increased oxidative activities were consensus with the
elevation of the protein levels of CYP1A2, CYP2B1/2, CYP2C11, and CYP3A (Ueng
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et al., 2010).
However, there are fewer current studies that have analyzed the effect of
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Sophora alkaloid in the control of T. gondii replication in vitro or in vivo. And, as we

known, infection can increase free radicals, which induce human more serious
damages, some results already showed the scavenging free radicals effect of KuShen
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alkaloids and that to attribute liver protection and anti-T. gondii effects. Hence, in this
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work we have evaluated the therapeutic efficacy of two Sophora alkaloids, OM and
ME, for their potential to control T. gondii infection. The obtained results showed that
both compounds have unique properties against T. gondii tachyzoites in comparison
to spiramycin, providing new hope for the treatment of toxoplasmosis but further
studies are necessary and should focus on the mechanisms of their action, which
directly or indirectly influences the parasite growth.

2. Materials and methods

2.1 Materials
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Dulbecco's Modified Eagle Medium (DMEM, HyClone®, USA), Trypsin-EDTA
(Solarbio, Beijing, China), Penicillin-Streptomycin solution (Solarbio, Beijing, China)
and fetal bovine serum (FBS, Gibco®, NY, USA) were used for cell culture. OM
(FW264.37) and ME (FW248.37) were purchased from Guangrun Bio-technology Co.,
Ltd. (Nanjing, China) and were of the highest purity available (> 98%). Structures of
these two compounds are presented in Figure 1. Spiramycin (SPY, FW843.05) was
purchased from Aladdin Industrial Inc. (Shanghai, China). Each compound was

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dissolved in dimethyl sulfoxide (DMSO, Solarbio, Beijing, China) and diluted with
DMEM to different concentrations, with the final DMSO concentration at less than

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1% (v/v). All other chemicals were of reagent grade and purchased from Aladdin
Industrial Inc. (Shanghai, China).
2.2 Cells, parasites and animals

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HeLa cells were cultured in DMEM, supplemented with 0.01%
Penicillin-Streptomycin and 10% heat-inactivated FBS and maintained at 37℃ and

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5% CO2. Cells were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). Tachyzoites used in our study were from the virulent RH strain
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of T. gondii (generously donated by Zoonosis Research Center, Wonkwang
University School of Medicine, South Korea) and maintained by serial intraperitoneal
passage in KM female mice, which were purchased from Experiment Center, Yanbian
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University. All experimental procedures were conducted in conformity with

institutional guidelines for the care and use of laboratory animals in Yianbian
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University, Jilin, China, and conformed to the National Institutes of Health Guide for
Care and Use of Laboratory Animals (Number of license SCXK 2011-0007). All mice
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were kept in a central animal care facility with free access to water and rodent food
during the experiment.
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2.3 Anti-toxoplasma activity and cytotoxicity of OM and ME in vitro

The anti-T. gondii activity and cytotoxicity of tested compounds in vitro were
evaluated by the cell counting kit-8 (CCK-8) assay (BestBio, Shanghai, China) as a
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slightly modified procedure by Jin et al. (2009). This assay method was described to
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be very simple for the screen of anti-T. gondii compounds in vitro as it exhibited high
correlation with the conventional morphological assay. HeLa cells were seeded onto
the 96-well plates (3 × 103/well) for 24 h for obtaining the full monolayer, and then
the host cells were infected with T. gondii (parasite:cell = 5:1) in complete medium.
After 24 h, cells were washed to remove any extra parasites and then incubated with
different concentrations (10, 100, 200, 500 and 1000µM) of OM and ME, SPY served
as the positive control and DMSO as the negative control. After 24 h of treatment,
CCK-8 solution (10 µl) was added directly to the culture wells and then incubated for
2 h at 37℃. The optical absorbance was measured at a wavelength of 450 nm using a
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microplate reader (Corona SH-1000Lab, Japan).
2.4 Microscopic observation of T. gondii-infected HeLa cells
HeLa cells (2×105/well) were cultured in a 6 well plate for 24 h and then infected
with T. gondii (1× 106tachyzoites/well). After 24 h, the T. gondii-infected HeLa cells
were washed with medium and treated with 100 µM of OM, ME and SPY,
respectively. Following day, the morphological changes of HeLa cells and T.
gondii-infected HeLa cells were observed under an inverted microscope (OPTEC

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BDS200, China) (Choi et al., 2013) and the cell survival rates were determined by the
trypan blue (Leagene, Beijing, China) exclusion assay (Zhang and Azrak, 2009).

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2.5 Anti-toxoplasma activity of OM and ME in vivo
For the in vivo study, KM female mice were divided into six groups consisting of
5 animals each and then intraperitoneally injected with 2×103tachyzoites of the T.

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gondii RH strain. After four hours of infection, mice were orally treated or not with
different drugs: 0.2 ml normal saline (normal group and negative control group), 100

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mg/kg spiramycin (SPY1 group), 200 mg/kg spiramycin (SPY2 group), 100 mg/kg
oxymatrine (OM group) and 100 mg/kg matrine (ME group), respectively, once a day
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for 4 days. At the last day, heart blood samples were collected after anesthesia to
separate the serum, and then mice were sacrificed by cervical dislocation. Tachyzoites
were harvested from mice peritoneal cavities to determine the level of tachyzoites
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proliferation (Jiang et al., 2008).

2.6 Measurement of visceral weights and liver biochemical parameters
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The livers and spleens were weighed and alanine aminotransferase (ALT),
aspartate aminotransferase (AST), glutathione (GSH) and malondialdehyde (MDA)
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levels were measured (Choi et al., 2014). The GSH was measured according to the
metheod of (Beutler et al., 1963). The liver homogenate was mixed with half volume
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trichloroacetic acid (20%, w/v) and centrifuged at 4000 rpm for 10 min. Then,
phosphate buffer (phosphate 0.3 mol/L, pH7.5) and 5, 5-dithio-bis-(2-nitrobenzoic
acid) (0.04%, w/v) were added to the separated supernatant and mixed thoroughly.
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After 5 min at room temperature, the absorbance was measured at 412 nm.
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MDA was measured by the standard method (Ohkawa et al., 1979) with minor
modifications. The liver homogenate supernatant was mixed with thiobarbituric acid
(0.5%, w/v) and heated in boiling water bath for 1 h, then cooled quickly and
centrifuged at 6000 rpm for 10 min, the absorbance of pink colored supernatant was
measured at 532 nm. Tetraethoxypropane replaced the liver homogenate in the
standard sample.
Serum levels of AST and ALT were measured according to the method of
(Reitman and Frankel, 1957). The substrate reaction of ALT or AST and serum was
carried out under incubation at 37 ℃ for 30min, then added 2, 4-DNPH and held for
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20 min. Finally, NaOH was added and allowed to react for 5 min. The absorbance at
505 nm was measured.
2.7 The survival analysis of T. gondii-infected mice
Five groups of 6 mice each were intraperitoneally injected with 2×103tachyzoites
of T. gondii as above mentioned. After four hours, the negative control group was
orally treated with 0.2 ml normal saline, the mice in different drug groups were orally
administered with SPY (100 mg/kg and 200 mg/kg), OM (100 mg/kg) and ME (100

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mg/kg), respectively daily. The mice were observed every day and the percentages of
mice survived were calculated during 15 days.

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2.8 Statistical analysis
All data were expressed as mean ± standard deviation (S.D.) triplicate. Statistical
analyses and graphs in the study were performed using SPSS 16.0 software (SPSS

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Inc., Chicago, USA) and GraphPad Prism 5.0 (GraphPad Software Inc., San Diego,
USA). The method of Kaplan and Meier was used to compare the survival rates of the

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studied groups. A value of P < 0.05 was considered statistically significant.
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3. Results
3.1 Effect of compounds on the selectivity index of T. gondii-infected HeLa cells
and T. gondii
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In vitro, the cell viability was expressed as a percentage of the normal value, and
the anti-T. gondii activity was expressed as the selectivity index, which was calculated
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using formula: the ratio of the CC50 value for host cells non infected with
Toxoplasma to the IC50 for T. gondii cultivated in host cells (SI= CC50 / IC50) (Jiang
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et al., 2008; Jin et al., 2009). The all tested compounds (OM, ME and SPY) did not
show inhibition effect on the proliferation of HeLa cells below 200 µM, and when the
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concentration were more than 500 µM, the inhibition effect occurred (Fig.2). In our
study, the selectivity index (SI) was calculated after treated with the different
compounds and the results are summarised in Table 1. The SI values obtained for OM,
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ME and SPY were 1.28, 1.41 and 0.92, respectively. ME showed high anti-T. gondii
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activity (CC50 = 1433.79 µM, IC50 = 1014.01 µM) relative to OM (CC50 = 1079.00
µM, IC50 = 843.44 µM). However, the activity of SPY was relatively weaker than
those of OM and ME, with CC50 of 863.53 µM and IC50 of 936.31 µM, respectively.
3.2 Effect of compounds on T. gondii infection under microscope
Observed changes in cell morphology are typical for T. gondii infection. In order
to intuitive observation the effects of the changes in cell morphology on the host cell
for T. gondii infection by treatment with drugs, morphological observation and cell
survival analysis were applied. At the same time, to eliminate the toxic effects of
drugs on cells growth, the concentrations of 100 µM (>90% proliferation rate
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compared with normal cells) was used for this experiment, which could be used safely
for the following microscopic observation. As shown in Fig.3, the morphology of
HeLa cells were changed remarkably in those that were only treated with T. gondii,
including cell shrinkage and cell fragmentation (Fig.3B). However, after treatment
with 100 µM OM, ME or SPY, the number of tachyzoites in sight decreased markedly
and the cell morphology change was not obvious compared with normal cells
(Fig.3C-3E). Furthermore, compared with normal cells, the survival ratio reached

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74.6%, 84.6%, 86.4% and 85.1%, respectively when treated with T. gondii, OM, ME
and SPY. Compared with T. gondii-infected cells group, the survival rate increased by

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10.0%, 11.8% and 10.5% (P < 0.01) after treatment with OM, ME and SPY, the rate
was significantly increased, but there was no significant difference between the three
(Fig.3F).

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3.3 Effect of compounds on the infection of T. gondii in vivo
In order to verify whether OM and ME could have shown the anti-T. gondii

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effect in vivo, the numbers of tachyzoites in the peritoneal cavity were evaluated in
mice. As shown in Fig.4, after treated with 100 mg/kg of SPY, OM and ME, the
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numbers of tachyzoites in the peritoneal cavity of mice were significantly decreased,
the inhibition rates reached to 20.1%, 45.2% and 53.8% (P < 0.05), respectively.
Under the same concentration, the inhibitory effect of OM and ME was far more
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effective than the clinical medicine SPY. In order to facilitate comparison, we

designed to increase the concentration of the SPY to 200 mg/kg for this experiment.
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In contrast, the effect of anti-T. gondii effect of OM and ME was similar to high
concentration of SPY (48.6%). That was no significant differences were observed
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when T. gondii-infected mice were treated with 100 mg/kg of OM, 100 mg/kg of ME
or 200 mg/kg of SPY.
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3.4 Effect of compounds on liver and spleen weights

In order to evaluate the effect of compounds on viscera, the liver and spleen
index were calculated by following formula: the viscera index = viscera weight / body
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weight. As shown in Fig.5, for the toxoplasma infection group, the live index
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decreased slightly and the spleen index increased significantly compared with normal
group (P < 0.01). For the treatment with OM or ME group, only OM group caused
spleen index rose to a certain extent compared with normal group (Fig.5B), but did
not significantly change the liver index compared to the negative control group, which
was the toxoplasma infection mice (Fig.5A). However, for the SPY group, a dose of
200 mg/kg of SPY (SPY2 group) could be significantly reduced the mouse liver index
(P < 0.05), and also dramatically increased spleen index (P < 0.01), and there was no
significant difference in the liver and spleen index when treatment with 100mg/kg of
SPY groups.
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3.5 Effect of compounds on biochemical parameters in liver
To further investigate the compounds toxicity in mice, the liver biochemical
parameters were measured. As shown in Fig.6, the levels of ALT and AST in serum
from negative control group and SPY1 group mice were significantly higher than
those of normal group (P < 0.01). Mice treatment with either OM or ME significantly
reduced the levels of ALT and AST in serum (P < 0.05), which was also better than
SPY2 treatment group.

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The infection of T. gondii resulted in a significant decrease in the level of GSH
(P < 0.01), in addition to a slight increase in MDA level, as shown in Fig.6. However,

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when mice treated with OM and ME, the increase of MDA by infected with T. gondii
was reduced (Fig.6D) and the GSH level was increased compared to that in negative
control group (Fig.6C). Furthermore, the GSH level either OM or ME treatment was

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significantly higher than those in SPY1 or SPY2 group (P < 0.05), but no significant
difference to the MDA level.

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3.6 Effect of compounds on the survival rate of T. gondii-infected mice
To determine the effect of compounds on T. gondii control in mice, we next
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examined the survival rate of T. gondii-infected mice. As shown in Fig.7, compared
with the T. gondii-infection group on the seventh day started to death, mice of SPY,
OM and ME groups started to death on the ninth day and the tenth day, respectively.
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The treatment with OM and ME achieved better results in mice survival than
treatment with SPY. At the end of 15 days, 67% of mice treated with 100 mg/kg/day
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OM or ME continued alive versus 33% of mice treated with 100 mg/kg/day SPY.
Mice treated with a higher dose of SPY (200 mg/kg/day) also increased mouse
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survival compared to that of 100 mg/kg/day SPY, which protected 50% of mice from
death in the end, but the result was weaker than that of OM or ME. Although these
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drugs had enhanced mouse survival compared to untreated group, the differences
between these groups were not statistically significant (P > 0.05).
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4. Discussion
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OM and ME are the main alkaloid in sophora leguminous plants, both having
two fused rings piperidine shared one nitrogen atom in the chemical structure
(Bohlmann et al., 1958). Between them the difference only lies in the oxidation of
nitrogen atom in the OM and under certain conditions can they mutual transformation.
From the structural formula can we speculate that OM and ME maybe have a similar
pharmacological activities (Zhao et al., 2000). In our study, we evaluated the effects
of OM and ME in the anti-T. gondii effect in vitro and in vivo, both OM and ME
exhibited strong anti-T. gondii activity with low cytotoxic effects.
In vitro anti-T. gondii activity of different compounds was expressed as the
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selectivity index (SI = CC50 / IC50). According to (Jin et al., 2009), the system to
screen drugs in vitro involved using cell viability methods to calculate drug
selectivities, which could offer a solution to the technical difficulties of T. gondii
screening in vitro. This method utilized the cell viability of host cells to represent the
T. gondii infection ratio, indirectly. But it could not indicate the growth of T. gondii in
host cells. The selectivity index of tested compounds was observed in the following
order: ME > OM > SPY (table 1), indicating that both OM and ME had been shown

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high anti-T. gondii activity and low toxicity, especially to ME, it was more protective
against the T. gondii-infected HeLa cells than SPY treatment. In addition, T. gondii

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growth was the result of repeated cycles of host cell invasion, replication and egress,
which is destructive to cell monolayer and lead to host cell death (Alomar et al., 2013).
Observed changes in cell morphology are typical for T. gondii infection. Through the

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photos which directly observed the effects of the changes in cell morphology on the
host cell for T. gondii infection by treatment OM and ME, the morphology changes of

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host cells and a decrease in extracellular tachyzoites was observed, and at the same
time the number of drug-treated cells was increased significantly compared with T.
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gondii-infected cells. These results may as well indicate that compounds improve the
host cell condition and not only diminish the parasite activity. Since parasites
replicate inside the cells they tend to rupture the whole cell culture at the same time so
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the actual number of parasites at the end of the experiment (after complete destruction
of host cells) would show the actual antiparasitic activity of tested compounds.
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In our study, the effects of OM and ME in vivo were analyzed in acute T. gondii
infection model with virulent RH strain in KM mice. Both OM and ME could
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significantly decrease the number of tachyzoites in the peritoneal cavity of infected

mice, and these inhibition levels had reached that of high dose of the clinical medicine
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SPY. Moreover, the mice treated with OM and ME also achieved better effect in
survival than that of SPY. These results indicated that either OM or ME has anti-T.
gondii activity in both in vitro and in vivo experimental models even better than
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clinical drug SPY.

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The liver is a very important organ in metabolism system of organisms, which

plays a key role in the metabolism and detoxification of endogenous and exogenous
harmful material (Yang et al., 2010). Acute toxoplasmosis can attack the body’s
various tissues and organs, including the liver (Neves et al., 2009). In our study, it
could be seen that T. gondii infection had no evident effect on the change of liver
weights. However, comparison of the treatments showed that SPY could lead to great
influence on the weights of liver (as well as spleen) than OM and ME, compared with
normal group.
To further verify the difference of three compounds on hepatotoxicity in mice,
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we measured some of major biochemical parameters in liver. ALT and AST are
nonspecific intracellular functional enzymes, which widely exist in organs and tissues,
especially in liver (Lee et al., 2007). When the liver cells damaged, ALT and AST in
cytoplasm were released to blood, which enhanced the activities of two enzymes in
serum (Wang and Wang, 2001). Therefore, the contents of ALT and AST in serum can
evaluate the degree of liver damage after toxoplasma infection in mice. The present
study has shown that either OM or ME can significantly decreased the level of ALT

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and AST in T. gondii-infected mice serum, indicating that the degree of liver damage
can be prevented with OM or ME.

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In addition, GSH, a tripeptide containing a sulfhydryl group, plays a role in
detoxification mechanism by reacting with organic peroxides and the harmful
byproducts of metabolism (Park et al., 2005). MDA is the main product of lipid

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peroxidation, which can make macromolecular substances, such as protein and
nucleic acid, cross-linking polymerization and produce cytotoxicity (Domenicali et al.,

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2009). The levels of MDA and GSH in liver can reflect the degree of liver lipid
peroxidation in organism and indirectly reflect the degree of cells damage
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(Cederbaum et al., 2009). In the present study, Livers of mice infected with T. gondii
showed significantly lower content of GSH and higher level of MDA than that of
normal mice. However, the activity of GSH and MDA in livers remarkably restored
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after exposure to OM or ME, compared with SPY. This further proved that both
compounds can effectively reduce the degree of liver damage.
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OM and ME’s anti-toxoplasmic mechanism of action is not known. Toxoplasma

infection affects both liver and kidney functions (Amany et al., 2010). The actives of
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AST and ALT were consequence the degree of damage in the liver in the acute stage
of infection (Suzuki et al., 1973). Changes of AST and ALT varied according to the
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qualitative difference in intensity of inflammation by strains of Toxoplasma and host

(Khan et al., 1997). In our studies, the significant decreases in AST and ALT indicated
that the treatment improved the immune system and protected the hepatocyte
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metabolism, especially probably taking into account a very short time of T. gondii
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invasion.
Previous studies revealed that T. gondii infection can lead to oxidative stress and
immune suppression in blood donors (Elsheikha et al., 2009). Karaman et al. (2008)
suggested that one of the main reasons for high MDA levels in Toxoplasma
seropositive patients could be a decreased activity of the defense system protecting
tissues form free radical damage. Xu et al. (2012) showed that antioxidants have
potential as a therapeutic regimen for the treatment of T. gondii-related diseases. Our
consistent results in mice strongly suggested OM and ME’s anti-T. gondii activities
probably due to the antioxidants mechanism.
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And, it should be demonstrated cautiously that although IC50 concentrations data
were in mM ranges, might be thought the high concentration ranges in vitro for
medicine candidates, but in vivo rat experiments, ME and OM showed lower toxicity
and more effective anti-T. gondii activities compared with reference drug, SPY, which
widely used in clinically. These results might be explained that after administration
OM or ME, combining comprehensive biological effects in body (e.g. antioxidation,
anti-inflammatory, and enhance immune functions, etc) caused desirable results, and

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further investigation definitely should be carried on ME, OM as potentially anti-T.
gondii candidates before consideration of clinical trials.

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Moreover, in vivo rat acute-infection experiment, ME and OM showed delayed
onset time and increased survival rate, especially ME had significant anti-T. gondii
effects than OM and SPY. These results indicated that ME and OM showed

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low-toxicity properties and anti-T. gondii effect efficiently. During the later period of
15 days continuous administration, mice did not die and showed the increase of food

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intake and physical activities. Probably, OM and ME could be used for the chronic
toxoplasmosis infection, which important for human health and parasite food-borne
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transmission.
In conclusion, the naturally derive herbs and plants extracts as alternative
medicines are getting increasing interest in the world. In this paper, this is the first
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roundly demonstration of anti-T. gondii activity of OM and ME in vitro and in vivo.

Furthermore, both compounds showed effective protection to the liver damage caused
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by toxoplasma infection in comparison to spiramycin. Our findings demonstrate that

ME and OM possess low-toxicities, more-effective anti-T. gondii activities in vitro
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and in vivo, and acting at least in part through partial protection of liver and kidney
damage in mechanism. Further studies should be performed to compare the efficacy
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of drug combination, elucidate the deep-precise mechanism for their anti-toxoplasma

activity, and especially as well as we also hope whether OM and ME could have the
chronic toxoplasmosis effect, because its importance for human health and parasite
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food-borne transmission.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China
(No.81160409).

References
Alomar, M.L., Rasse-Suriani, F.O., Ganuza, A., Cóceres, V.M., Cabrerizo, F.M.,
Angel, S.O., 2013. In vitro evaluation of β-carboline alkaloids as potential
anti-Toxoplasma agents. BMC. Res. Notes. 6, 193-198.
Amany, M., Eid, R.A.A., Fahmy, B.G.A., 2010. Biochemical studies on the
 ACCEPTED MANUSCRIPT
effect of Toxoplasma infection on liver and kidney function in mice. Egypt. J. Comp.
Path & Clinic. Path. 23, 174-185.
Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the
determination of blood glutathione. J. Lab. Clin. Med. 61, 882-888.
Bohlmann, F., Rahtz, D., Arndt, C., 1958. Lupinen-alkaloide, XI. Die Alkaloide
aus Sophora Flavescens. Chem. Ber. 91, 2189-2193．
Cederbaum, A.I., Lu, Y.K., Wu, D.F., 2009. Role of oxidative stress in

PT
alcohol-induced liver injury. Arch. Toxicol. 83, 519-548.
Chang, C., Liu, S.P., Fang, C.H., He, R.S., Wang, Z., Zhu, Y.Q., Jiang, S.W.,

RI
2013. Effects of matrine on the proliferation of HT29 human colon cancer cells and
its antitumor mechanism. Oncol. Lett. 6, 699–704.
Chen, S.X., Wu, L., Jiang, X.G., Feng, Y.Y., Cao, J.P., 2008. Anti-Toxoplasma

SC
gondii activity of GAS in vitro. J. Ethnopharmacol. 118, 503-507.
Chen, X., Sun, R., Hu, J., Mo, Z., Yang, Z., Liao, D., Zhong, N., 2008.

U
Attenuation of bleomycin-induced lung fibrosis by oxymatrine is associated with
regulation of fibroblast proliferation and collagen production in primary culture. Basic
AN
Clin. Pharmacol. Toxicol. 103, 278-286.
Chio, L.C., Bolyard, L.A., Nasr, M., Queener, S.F., 1996. Identification of a class
of sulfonamides highly active against dihydropteroate synthase form Toxoplasma
M

gondii, Pneumocystis carinii, and Mycobacterium avium. Antimicrob. Agents

Chemother. 40, 727-733.
D

Choi, H.J., Yu, S.T., Lee, K.I., Choi, J.K., Chang, B.Y., Kim, S.Y., Ko, M.H.,
Song, H.O., Park, H., 2014. 6-Trifluoromethyl-2-thiouracil possesses
TE

anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity. Exp.
Parasitol. 143, 24-29.
EP

Choi, K.M., Gang, J.G., Yun, J.S., 2008. Anti-Toxoplasma gondii RH strain
activity of herbal extracts used in traditional medicine. Int. J. Antimicrob. Agents. 32,
360–362.
C

Choi, W.H., Jiang, M.H., Chu, J.P., 2013. Antiparasitic effects of Zingiber
AC

officinale (Ginger) extract against Toxoplasma gondii . J. Appl. Biomed. 11, 15-26.
D’Angelo, J.G., Bordón, C., Posner, G.H., Yolken, R., Jones-Brando, L., 2009.
Artemisinin derivates inhibit Toxoplasma gondii in vitro at multiple steps in the lytic
cycle. J. Antimicrob. Chemoth. 63, 146-150.
Deng, X., Shen, C., Meng, Q., 2014. Screening of herbal components for
attenuating amiodarone-induced hepatotoxicity on gel-entrapped rat hepatocytes.
Drug Chem. Toxicol. 37, 100–106.
Domenicali, M., Caraceni, P., Giannone, F., Baldassarre, M., Lucchetti, G.,
Quarta, C., Patti, C., Catani, L., Nanni, C., Lemoli, R.M., Bernardi, M., 2009. A novel
 ACCEPTED MANUSCRIPT
model of CCl4-induced cirrhosis with ascites in the mouse. J. Hepatol. 51, 991-999.
Du, S.M., Ma, L.Q., Sun, J.J., Yang, Z.X., 2010. Study on in vitro antibacterial
activity of extracts from Sophora flavescens Ait. Acta Chin. Med. Pharmacol. 38,
74–76.
Dzitko, K., Grzybowski, M.M., Pawełczyk, J., Dziadek, B., Gatkowska, J.,
Stączek, P., Długońska, H., 2015. Phytoecdysteroids as modulators of the Toxoplasma
gondii growth rate in human and mouse cells. Parasit. Vectors. 8, 1-13.

PT
Elsheikha, H.M., El-Motayam, M.H., Abouel-Nour, M.F., Morsy, A.T., 2009.
Oxidative stress and immune-suppression in Toxoplasma gondii positive blood donors:

RI
implications for safe blood transfusion. J. Egypt. Soc. Parasitol. 39, 421-428.
Franco, P.S., Gomes, A.O., Barbosa, B.F., Angeloni, M.B., Silva, N.M.,
Teixeira-Carvalho, A., Martins-Filho, O.A., Silva, D.A., Mineo, J.R., Ferro, E.A.,

SC
2011. Azithromycin and spiramycin induce anti-inflammatory response in human
trophoblastic (BeWo) cells infected by Toxoplasma gondii but are able to control

U
infection. Placenta. 32, 838-844.
Guzman, J.R., Koo, J.S., Goldsmith, J.R., Mühlbauer, M., Narula, A., Jobin, C.,
AN
2013. Oxymatrine prevents NF-κB nuclear translocation and ameliorates acute
intestinal inflammation. Sci. Rep. 3, 1629-1629.
He, X.R., Fang, J.C., Huang, L.H., Wang, J.H., Huang, X.Q., 2015. Sophora
M

flavescens Ait.: Traditional usage, phytochemistry and pharmacology of an important

traditional Chinese medicine. J. Ethnopharmacol. 172, 10-29.
D

Hong, M.H., Lee, J.Y., Jung, H., Jin, D.H., Go, H.Y., Kim, J.H., Jang, B.H., Shin,
Y.C., Ko, S.G., 2009. Sophora flavescens Aiton inhibits the production of
TE

pro-inflammatory cytokines through inhibition of the NF κB/lκB signal pathway in

human mast cell line (HMC-1). Toxicol. in Vitr. 23, 251–258.
EP

Jones-Brando, L., D’Angelo, J., Posner, G.H., Yolken, R., 2006. In vitro
inhibition of Toxoplasma gondii by four new derivatives of artemisinin. Antimicrob.
Agents Ch. 50, 4206-4208.
C

Jiang, J.H., Jin, C.M., Kim, Y.C., Kim, H.S., Park, W.C., Park, H., 2008.
AC

Anti-toxoplasmosis effects of oleuropein isolated from Fraxinus rhychophylla. Biol.

Pharm. Bull. 31, 2273-2276.
Jin, C.M., Kaewintajuk, K., Jiang, J.H., Jeong, W., Kamata, M., Kim, H., Wataya,
Y., Park, H., 2009. Toxoplasma gondii: a simple high-throughput assay for drug
screening in vitro. Exp. Parasitol. 121, 132–136.
Karaman, U., Celik, T., Kiran, T.R., Colak, C., Daldal, N.U., 2008.
Malondialdehyde, glutathione, and nitric oxide levels in Toxoplasma gondii
seropositive patients. Korean J. Parasitol. 46, 293-295.
Kavitha, N., Noordin, R., Chan, K.L., Sasidharan, S., 2012. In vitro
 ACCEPTED MANUSCRIPT
Anti-Toxoplasma gondii Activity of Root Extract/Fractions of Eurycoma longifolia
Jack. BMC. Complem. Altern M. 12, 91-98.
Khan, L.A., Schwartzman, J.D., Matsuura, T., Kasper, L.H., 1997. A
dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice.
Proc. Nati. Acad. Sci. 94, 13955-13960.
Lee, C.P., Shih, P.H., Hsu, C.L., Yen, G.C., 2007. Hepatoprotection of tea seed
oil (Camellia oleifera Abel.) against CCl4-induced oxidative damage in rats. Food

PT
Chem. Toxicol. 45, 888-895.
Li, J.L., Cheng, A.M., Jin, Y.H., Cai, F.Q., 2002. Experiment study on the

RI
antitumor effects of Sophora flavescens Ait. Chin. J. Clin. Oncol. Rehabil. 9, 28–29.
Lin, D.W., 2006. Analysis of the chemical constituents from Sophora flavescens
root. ChungYuan Christian University. pp. 1–10.

SC
Lopes, F.M., Gonçalves, D.D., Mitsuka-Breganó, R., Freire, R.L., Navarro, I.T.,
2007. Toxoplasma gondii infection in pregnancy. Braz. J. Infect. Dis. 11, 496-506.

U
Lüder, C.G., Bohne, W., Soldati, D., 2001. Toxoplasmosis: a persisting
challenge. Trends. Parasitol. 17, 460-463.
AN
Ma, S.C., Du, J., But, P.P.H., Deng, X.L., Zhang, Y.W., Ooi, V.E.C., Xu, H.X.,
Lee, S.H.S., Lee, S.F., 2002. Antiviral Chinese medicinal herbs against respiratory
syncytial virus. J. Ethnopharmacol. 79, 205–211.
M

Miao, K.L., Zhang, J.Z., Dong, Y., Xi, Y.F., 2001. Research progress on the
chemical compounds and pharmacology of sophora flavescens. Nat. Prod. Res. Dev.
D

13, 69-73.
Neves, E.S., Bicudo, L.N., Curi, A.L., Carregal, E., Bueno, W.F., Ferreira, R.G.,
TE

Amendoeira, M.R., Benchimol, E., Fernandes, O., 2009. Acute acquired

toxoplasmosis: clinical-laboratorial aspects and ophthalmologic evaluation in a cohort
EP

of immunocompetent patients. Mem. Inst. Oswaldo. Cruz. 104, 393-396.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal
tissue by thiobarbituric acid reaction. Anal. Biochem. 95, 351-358.
C

Oliveira, T.C., Silva, D.A.O., Rostkowsa, C., Béla, S.R., Ferro, E.A.V.,
AC

Magalhães, P.M., Mineo, J.R., 2009. Toxoplasma gondii: Effects of Artemisia annua
L. on susceptibility to infection in experimental models in vitro and in vivo. Exp.
Parasitol. 122, 233-241.
Park, J.C., Han, W.D., Park, J.R., Choi, S.H., Choi, J.W., 2005. Changes in
hepatic drug metabolizing enzymes and lipid peroxidation by methanol extract and
major compound of Orostachys japonicus. J. Ethnopharmacol.102, 313-318.
Petersen, E., Schmidt, D.R., 2003. Sulfadiazine and pyrimethamine in the
postnatal treatment of congenital toxoplasmosis: what are the options? Expert Rev.
Anti infect. Ther. 1, 175-182.
 ACCEPTED MANUSCRIPT
Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of
serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol.
28, 56-63.
Schmidt, D.R., Hogh, B., Andersen, O., Hansen, S.H., Dalhoff, K., Petersen, E.,
2006. Treatment of infants with congenital toxoplasmosis: tolerability and plasma
concentrations of sulfadiazine and pyrimethamine. Eur. J. Pediatr. 165, 19–25.
Sun, M., Cao, H., Sun, L., Dong, S., Bian, Y., Han, J., Zhang, L., Ren, S., Hu, Y.,

PT
Liu, C., Xu, L., Liu, P., 2012. Antitumor activities of kushen: literature review. Evid
Based Complement. Alternat. Med. 2012, 373219-373219.

RI
Suzuki, N., 1973. Pathophysiological observation on mice infected with Deen
strain of Toxoplasma gondii. Res. Bull. Obihiro. Univ. 8, 45-55.
Ueng, Y.F., Tsai, C.C., Lo, W.S., Yun, C.H., 2010. Induction of hepatic

SC
cytochrome P450s by the herbal medicine Sophora flavescens extract in rats: impact
on the elimination of theophylline. Drug Metab. Pharmacokinet. 25, 560-567.

U
Wang, Y.M., Wang, X.L., 2001. Molecular biological characteristics of VP2
protein of infectious bursa disease virus. Chin. J. Vet. Med. 37, 36-38.
AN
Weiss, L.W., Dubey, J.P., 2009. Toxoplasmosis: a history of clinical
observations. Int. J. Parasitol. 39, 895-901.
Xu, X., Liu, T., Zhang, A., Huo, X., Luo, Q., Chen, Z., Yu, L., Li, Q., Liu, L.,
M

Lun, Z.R., Shen, J., 2012. Reactive oxygen species-triggered trophoblast apoptosis is
initiated by endoplasmic reticulum stress via activation of caspase-12, CHOP, and the
D

JNK pathway in Toxoplasma gondii infection in mice. Infect. Immun. 80, 2121-2132.
Yamazaki, M., Arai, A., Suzuki, S., Takeuchi, T., 1984. Protective effects of
TE

matrine and oxymatrine on stress ulcer in relation to their effects on the central
nervous system. Yakugaku Zasshi. 104, 293–301.
EP

Yang, J.Y., Li, Y., Wang, F., Wu, C.F., 2010. Hepatoprotective effects of apple
polyphenols on CCl4-induced acute liver damage in mice. J. Agric. Food Chem. 58,
6525-6531.
C

Youn, H.J., Lakritz, J., Rottinghaus, G.E., 2004. Antiprotozoal efficacy of high
AC

performance liquid chromatography fractions of Torilis japonica and Sophora

flavescens extracts on Neospora caninum and Toxoplasrna gondii. Vet. Parasitol. 125,
409-414.
Youn, H.J., Noh, J.W., 2001. Screening of the anticoccidial effects of herb
extracts against Eimeria tenella. Vet. Parasitol. 96, 257-263.
Zhang, B.F., Song, L.Y., Zhu, L.G., Liu, Y.L., Li, X.Q., 1985. Antiarrhythmic
effect of total alkaloids from Sophora flavescens. Chin. J. Chin. Mater. Med. 10,
229–230.
Zhang, Q.L., Azrak, R.G., 2009. The effect of methylseleninic acid on paclitaxel
 ACCEPTED MANUSCRIPT
efficacy in A2780 ovarian cancer cells. J. Nanjing Med. Univ. 23, 111-116.
Zhao, B.Z., Rong, D.Q., Wang, X.J., Fu, Q., Meng, Z., 2000. Relationship of
activity with electron structure for matrine and oxymatrine. J. Mol. Sci. 16, 88-93.

Table and Figure legends

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Fig.1. Structures of OM and ME.

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Fig.2. Cytotoxicity of tested compounds on the viability of HeLa cells. HeLa cells were
treated with different concentrations (10—1000 µM) of OM, ME and SPY for 24h
respectively. Viable cell numbers were analyzed using cell counting kit-8 (CCK-8) assay. All

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data are presented as mean ± S.D. and the experiments were performed in triplicate. *P < 0.05
and **P < 0.01 compared with control.

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Fig.3. Morphology and the percentage of living cells of HeLa cells infected with T. gondii
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and treated with compounds. (A) Normal. (B) T. gondii-infected with no treatment. (C) T.
gondii-infected and treatment with 100 µM SPY. (D) T. gondii-infected and treatment with
100 µM OM. (E) T. gondii-infected and treatment with 100 µM ME. Arrows indicate
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tachyzoites inside the HeLa cells, as well as adhered to the plate, and the morphological
changes including cell shrinkage and cell fragmentation. (F) Effect of compounds on the
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percentage of living cells. The cells in every treatment groups were counted by typan blue
straining using a hemacytometer. Data are expressed as mean ± S.D. (n ≥ 3). *P < 0.01
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compared with control (T. gondii-infected HeLa cells with no treatment).

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Fig.4. Effect of compounds on the number of tachyzoites in mice peritoneal cavity. All mice
were sacrificed at four days post-infection and ascites were drawn out to count the number of
tachyzoites using a hemocytometer. N.C., Negative control (T. gondii-infected and treatment
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with normal saline); *P < 0.05 compared with N.C.

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Fig.5. Effect of compounds on relative organ weights in T. gondii-infected KM mice.

Liver index: Liver weight / body weight × 100; Spleen index: Spleen weight / body weight ×
100; N.C., Negative control group. Values are expressed as mean ± S.D. (n = 5). *P < 0.05,
**P < 0.01 compared with the normal group.

Fig.6. Effect of compounds on (A)ALT, (B)AST, (C)GSH and (D)MDA levels in T.

gondii-infected KM mice. N.C., treatment with normal saline; SPY1, treatment with 100
mg/kg spiramycin; SPY2, treatment with 200 mg/kg spiramycin; OM, treatment with 100
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mg/kg oxymatrine; ME, treatment with 100 mg/kg matrine. Values are expressed as mean ±
S.D. (n = 5). *P < 0.05, ***P < 0.01 compared with normal; **P < 0.05 compared with N.C.;
#
P < 0.05 compared with SPY1; ##P < 0.05 compared with SPY2.

Fig.7. Effect of compounds on the survival rate of KM mice acutely infected with
2×103tachyzoites of T. gondii RH strain. The animals were accompanied daily to mortality.

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Table 1. In vitro anti-toxoplasma activity of tested compounds by CCK-8 assay.
Compound CC50a (µM) IC50b (µM) SIc
OM 1079.00 843.44 1.28
ME 1433.79 1014.01 1.41
SPY 863.53 936.31 0.92
Results are expressed as mean ± S.D. of three different experiments.
a
CC50 = Concentration required to reduce HeLa cells growth by 50%.

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IC50 = Concentration required to inhibit T. gondii-induced cytopathic effect by 50%.
c
Selectivity index (SI) = CC50 / IC50.

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OM and ME are firstly proved to have anti-Toxoplasma gondii activity in vitro and in
vivo.

Both compounds showed effective protection to the liver damage caused by T. gondii
in comparison to spiramycin.

OM and ME are likely the sources of new drugs for toxoplasmosis.

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