life

Article
Toxicity and Anti-Proliferative Properties of Anisomeles indica
Ethanol Extract on Cervical Cancer HeLa Cells and
Zebrafish Embryos
Nguyen T. Bich-Loan 1,2 , Kieu Trung Kien 1 , Nguyen Lai Thanh 1 , Nguyen T. Kim-Thanh 1 , Nguyen Quang Huy 1 ,
Pham The-Hai 1 , Marc Muller 3 , Amandine Nachtergael 2 , Pierre Duez 2 and Nguyen Dinh Thang 1, *

1 Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi 100000, Vietnam;
nguyenthibichloan.iph@gmail.com (N.T.B.-L.); kieukien1@gmail.com (K.T.K.);
nguyenlaithanh@hus.edu.vn (N.L.T.); kimthanh_bio@yahoo.com (N.T.K.-T.); huy_nq@hus.edu.vn (N.Q.H.);
hai.phamthe@gmail.com (P.T.-H.)
2 Unit of Therapeutic Chemistry and Pharmacognosy, University of Mons (UMONS), 7000 Mons, Belgium;
Amandine.NACHTERGAEL@umons.ac.be (A.N.); Pierre.DUEZ@umons.ac.be (P.D.)
3 Laboratory for Organogenesis and Regeneration, GIGA-R, Department Life Sciences, University of Liege,
4000 Liege, Belgium; m.muller@uliege.be
* Correspondence: ndthang@hus.edu.vn

Abstract: In this study, we showed that crude extract of Anisomeles indica (AI-EtE) expressed its
toxicity to HeLa cells with an IC50 dose of 38.8 µg/mL and to zebrafish embryos with malformations,





 lethality and hatching inhibition at 72-hpf at doses higher than 75 µg/mL. More interestingly,
flow cytometry revealed that AI-EtE significantly promoted the number of cells entering apoptotic.
Citation: Bich-Loan, N.T.; Kien, K.T.;
Accordingly, the transcript levels of BAX, CASPASE-8, and CASPASE-3 in the cells treated with
Thanh, N.L.; Kim-Thanh, N.T.; Huy,
AI-EtE at IC50 dose were 1.55-, 1.62-, and 2.45-fold higher than those in the control cells, respectively.
N.Q.; The-Hai, P.; Muller, M.;
Nachtergael, A.; Duez, P.; Thang, N.D.
Moreover, treatment with AI-EtE caused cell cycle arrest at the G1 phase in a p53-independent
Toxicity and Anti-Proliferative manner. Particularly, percentages of AI-EtE-treated cells in G1, S, G2/M were, respectively 85%, 6.7%
Properties of Anisomeles indica and 6.4%; while percentages of control cells in G1, S, G2/M were 64%, 15% and 19%, respectively.
Ethanol Extract on Cervical Cancer Consistent with cell cycle arrest, the expressions of CDKN1A and CDNK2A in AI-EtE-treated cells
HeLa Cells and Zebrafish Embryos. were up-regulated 1.9- and 1.64-fold, respectively. Significantly, treatment with AI-EtE also decreased
Life 2021, 11, 257. https:// anchorage-independent growth of HeLa cells. In conclusion, we suggest that Anisomeles indica can be
doi.org/10.3390/life11030257 considered as a medicinal plant with a possible use against cervical cancer cells; however, the used
dose should be carefully monitored, especially when applying to pregnant women.
Academic Editor: Balazs Barna

Keywords: medicinal plant; Anisomeles indica; HeLa cell; zebrafish; apoptosis; cell cycle arrest; gene
Received: 27 February 2021
expression; colony formation
Accepted: 18 March 2021
Published: 20 March 2021

Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in
published maps and institutional affil- A recent survey showed that 64% of new drugs discovered between 1981 and 2010
iations. were natural products or at least inspired by them [1,2]. Several products/medicines
used for treatments of different cancers had been extracted from medicinal plants such
as paclitaxel from the bark of western yew tree Taxus brevifolia [3], capsaicin from the hot
pepper of the genus Capsicum [4], and resveratrol from the skin of grapes [5]. Thus, the
Copyright: © 2021 by the authors.
importance of putting new efforts into development and use of traditional medications has
Licensee MDPI, Basel, Switzerland. been highlighted in recent recommendations [6,7].
This article is an open access article Vietnam represents a rich source of endemic and potentially novel natural medicines.
distributed under the terms and Many plant extracts have been used as traditional medicines for thousands of years.
conditions of the Creative Commons However, so far, the use of traditional medicines has been relying mostly on empirical
Attribution (CC BY) license (https:// observations. Toxicities, as well as the biological and pharmaceutical properties of the
creativecommons.org/licenses/by/ medicinal plant extracts have not been thoroughly investigated yet. Thus, administrations
4.0/).

Life 2021, 11, 257. https://doi.org/10.3390/life11030257 https://www.mdpi.com/journal/life

Life 2021, 11, 257 2 of 15

of these natural products for a long period of time may also lead to more or less adverse
effects on human health.
Cervical cancer is the fourth most common cancer in women. Recent statistics re-
vealed that cervical cancer caused about 7.5% of all female cancer deaths; of those deaths,
approximately 90% occurred in the developing countries [8,9]. In Vietnam, it was similarly
estimated that cervical cancer was the fourth most common cancer in Vietnamese women
with more than 5000 new cases and about 3000 deaths in 2018 [8].
Cell cycle arrest and apoptosis are the two main reasons for cell death, therefore they
are always considered as endpoints for screening of drugs for cancer treatments. Apoptosis
is initiated by intrinsic and/or extrinsic pathways, which finally promote the activities
of caspases to damage the cellular DNA and cause cell death [10–12]. On the other hand,
the cell cycle is regulated by Cyclin-Dependent Kinases (CDKs), which are activated at
specific points during progression of the cell cycle. Arresting the cell cycle at any phase
of G1, S, and G2/M of the cell cycle inhibits cell division and may ultimately cause cell
death [13–18].
Anisomeles indica is one of the medicinal plants displaying different biological activities
including antioxidant [19,20], antibacterial [20,21] and anticancer [22,23]. Previous studies
demonstrated that the products originating from Anisomeles indica could induce the death
of lung cancer cells [22,23], oral cancer cells [24], and liver cancer cells [10] by inducing
cell cycle arrest and activating the apoptosis pathway. Anisomeles indica was also able
to inhibit the migration and invasion activities of breast cancer cells [25]. However, the
molecular mechanisms underlying these actions have not been thoroughly investigated.
In addition, so far, no study addressed the anticancer activity of Anisomeles indica on
cervical cancer cells nor its putative toxicity on human embryos. An effective and popular
in vivo experimental model for drug screening and, in particular, embryotoxicity, is the
zebrafish [26,27]. Therefore, in this study, we examined the biological and pharmaceutical
properties, focusing on cell-targeted toxicity (cultured cells), organism-targeted toxicity
(zebrafish embryos) and anticancer activity on cervical cancer in vitro, of the crude extracts
of the medicinal plant Anisomeles indica, which has been used as traditional medicine in the
North-West of Vietnam for a long time.

2. Materials and Methods

2.1. Plant Material
Medicinal plants (Table 1) were selected based on the recipes of traditional medicines
and collected from the North-Western mountainous areas of Vietnam in the dry season in
2019. They were identified, coded (with Vietnamese and Scientific names) and placed at
the Museum of Biology, Faculty of Biology, VNU University of Science, Vietnam National
University, Hanoi, Vietnam.

Table 1. Medicinal plants used for cytotoxicity screening.

Vietnamese Name Scientific Name Code in Museum Place for Collection Part Used
1 Phòng phong thảo Anisomeles indica HNU 024777 Bát xát, Lào Cai Dry stem leaves
2 Hoàng liên ôro lá dày Mahonia bealei HNU 024779 Bát xát, Lào Cai Dry stem
3 Ða lá lệch Ficus semicordata HNU 024780 Vị Xuyên, Hà Giang Dry leaves
4 Gắm núi Gnetum montanum HNU 024781 Vị Xuyên, Hà Giang Dry stem
5 Náng hoa trắng Crinum asiaticum HNU 024783 Cẩm Phả, Quảng Ninh Dry leaves
6 Bùm bụp Mallotus barbatus HNU 024784 Lạc Sơn, Hòa Bình Dry root
7 Mạn mân Aganope balansae HNU 024785 Bắc Quang, Hà Giang Dry stem
8 Dạ cẩm Hedyotis capitellata HNU 024786 Bắc Quang, Hà Giang Fresh leaves

2.2. Preparation of Plant Extracts

Fresh medicinal plants were cleaned and washed thoroughly with water and re-
washed with distilled water. Washed fresh leaves were shade-dried, powdered mechan-
ically, and sieved by using a mesh. For preparation of organic solvent extracts, 100 g of
 chanically, and sieved by using a mesh. For preparation of organic solvent extracts, 100 g
of powdered material was mixed with 1000 mL of ethanol, sonicated for 15 min in a
sonication bath (with set-up conditions of 35 °C; 40 kHz frequency and 150W ultrasonic
power) [28], before extraction for overnight with stirring at room temperature. The re-
Life 2021, 11, 257 3 of 15
sulting extract was removed, and the procedure was repeated two additional times on
the solid residue. All extracts were filtered, pooled, and the solvent removed under re-
duced pressure at 40 ± 5 °C using a rotary flash evaporator. The scheme of the extraction
procedure is presented
powdered material in Figure
was mixed with1. ThemL
1000 obtained extract
of ethanol, was for
sonicated dissolved in aDMSO
15 min in for
sonication
further analysis and exposure on cells
◦ and zebrafish eggs.
bath (with set-up conditions of 35 C; 40 kHz frequency and 150W ultrasonic power) [28],
before extraction for overnight with stirring at room temperature. The resulting extract was
2.3. Cell Culture
removed, and the procedure was repeated two additional times on the solid residue. All ex-
tractsCervical
were filtered,
cancerpooled, and the CCL-2)
HeLa (ATCC: solvent removed
and breastunder reduced
cancer MCF-7pressure 40 ± 5 ◦ C
(ATCC:atCRL-3435)
using
cell a rotary
lines flash evaporator.
were provided The
by Health scheme
Science of the extraction
Research Resources procedure
Bank, Japanis and
presented in
cultured
Figure
in DMEM 1. The
andobtained
RPMI-1640extract was dissolved
(respectively), in DMSO forwith
supplemented further
10%analysis and exposure
Fetal Bovine Serum
on cells
(FBS) andand
1%zebrafish eggs.
Penicillin/Streptomycin at 37 °C in 5% CO2.

Figure 1.
Figure Extraction scheme
1. Extraction scheme for
for plant
plant powder.
powder.

2.4.
2.3. Embryo Zebrafish Toxicity Test
Cell Culture
Adult
Cervicalzebrafish wild (ATCC:
cancer HeLa type strain
CCL-2) ABand
(Danio rerio)
breast (ZIRC,
cancer MCF-7 Eugene,
(ATCC:OR) [26] were
CRL-3435) cell
maintained in the zebrafish
lines were provided facility
by Health of the
Science AnimalResources
Research Laboratory. TheJapan
Bank, fish were
and cultured
cultured in
in
glass
DMEM rectangular pools measuring
and RPMI-1640 40 cm
(respectively), (wide) × 50with
supplemented cm (long) × 30Bovine
10% Fetal cm (high).
SerumSeveral
(FBS)
and 1%
pools of Penicillin/Streptomycin at 37 ◦ C in 5%
adult fish were bred individually for CO 2 . assay. After sorting, embryos from
each
pools with high fertility (facil were mixed and used for subsequent experiments. Ex-
2.4. Embryowere
periments Zebrafish Toxicity
validated Test
only when the survival rate of the controls was ≥ eri at 4 days
Adult zebrafish wild type strain
post fertilization (dpf). Fish were reared in a rerio)
AB (Danio (ZIRC, Eugene,
Techniplast OR) [26]
recirculating wereunder
system main-
tained in
14:10-h the zebrafish
light/dark facility Before
photocycle. of the Animal
use, eggsLaboratory.
were screenedThe fish
andwere cultured
sorted under in glass
a stere-
rectangular
oscope pools measuring
to remove 40 cmand/or
the unfertilized × 50 cm (long)
(wide) abnormal ones.× 30 cm (high).
Healthy embryos Several pools of
that showed
adult fish
normal were bred
cleavage wereindividually
distributed for
intoeach assay.
6-well After
plates at sorting, embryos for
25 embryos/well from pools
the with
embryo-
high fertility
toxicity tests. (facil were mixed and used for subsequent experiments. Experiments were
validated only when the survival rate of the controls was ≥ eri at 4 days post fertilization
(dpf). Fish were reared in a Techniplast recirculating system under 14:10-h light/dark
photocycle. Before use, eggs were screened and sorted under a stereoscope to remove the
unfertilized and/or abnormal ones. Healthy embryos that showed normal cleavage were
distributed into 6-well plates at 25 embryos/well for the embryotoxicity tests.

2.5. Cellular Toxicity MTT Assay

MTT (Sigma Chemical Co., St. Louis, MO, USA) assays were conducted as previously
described [29] with some modifications. Briefly, cells were seeded into 96-well plates. The
MTT stock solution of 5 mg/mL was prepared in PBS (pH 7.2) and filtered through 2 µm
Life 2021, 11, 257 4 of 15

pore-size membrane. At the end of the treatment period with extracts (12, 24 or 48 h), 20 µL
of MTT solution were added to each well and incubated for 4 h at 37 ◦ C. Then, 100 µL of
solubilizing buffer (prepared by dissolving of 10% sodium dodecyl sulfate SDS in 0.01 N
HCl) were added to each well and incubated overnight. After that, the 96-well plate was
read using an ELISA reader at 570 nm for absorbance density values. Viable cells produce
a dark blue formazan product, whereas no such staining was formed in dead cells. The
percentage of viable cells was calculated.

2.6. Annexin V Apoptosis Assay

The annexin V apoptosis assay was performed as described by Schutte [30]. It is
based on the detection of phosphatidylserine, present on the extracellular side of the cell
membrane only in apoptotic cells, by FITC-conjugated annexin V. The cells were seeded at
30 to 40% confluence in 6-cm plates. After overnight incubation, medium was aspirated
and replaced with medium with or without extract. Treatment with paclitaxel was used
as a positive control for apoptosis induction. After 36 h, medium was collected. The
cells were washed with PBS and the collected cells were resuspended in annexin binding
buffer (Thermo Fisher Scientific, Singapore) at 1 × 106 cells/mL. Cells were stained with
propidium iodide (Invitrogen, Thermo Fisher Scientific, Singapore) to label dead or late
apoptotic cells, and annexin V-FITC according to the manufacturer’s protocol and assayed
by flow cytometry on a FACSCanto II (BD Biosciences). The percentage of apoptotic cells
was measured as the percentage of annexin V-positive cells.

2.7. Cell Cycle Analysis

Cells were stained with propidium iodide (PI) (BD Biosciences) to measure the DNA
content using a flow cytometer (FACs CANTO system) [31]. Briefly, cells collected by
trypsinizing were washed twice with cold PBS buffer (pH 7.4) and two volumes of cold
100% ethanol were added. After ethanol fixation, the cells were centrifuged at 400 g for
5 min and washed once in the PBS buffer. Cells were then re-suspended at 106 cells/mL.
Fifty µg/mL of RNase A (Sigma Chemical Co., St. Louis, MO, USA) was added to each
sample before incubating at 37 ◦ C for 30 min. After incubation, 20 µg of PI was added
to each tube and kept for at least 30 min to provide the nuclear signal for fluorescence-
activated cell sorting. After staining the cells with PI, the tubes were transferred to ice,
protected from light, and used for cell cycle analysis on the flow cytometer.

2.8. Real-Time PCR Analysis

Total RNA was isolated from cells or zebrafish larvae using the RNA Isolation Kit
(Thermo Fisher Scientific, Singapore), according to the protocol of the Kit. Then cDNA was
synthesized from total RNA by reverse transcription, according to the protocol of the cDNA
Synthesis Kit (Thermo Scientific, USA). Real-time quantitative RT-PCR with SYBR green
was performed using power SYBR1 green PCR master mix in a LightCycler@96 Instrument
(Roche Diagnostics GmbH, Mannheim, Germany). The transcript expression levels of
BAX, CASPASE-3, CASPASE-8, p53, CDKN1A (p21) and CDKN2A (p16) were measured
by quantitative RT-PCR (real-time PCR) were standardized relative to the transcript level
of TBP. Real-time PCR was carried out using 10 µL of power SYBR1 green PCR master
mix containing 900 nM forward primer and 900 nM reverse primer in a final volume of
20 µL. The sequences of the primers are presented in the Supplementary Materials, Table S1.
After running, the amplification curve, melting peak, and melting curve were analyzed
to confirm the reliability of the results and to ensure that there are no noise signals which
may come from amplifications of primer dimer and/or other side reactions.

2.9. Colony Formation Assay

A colony formation assay was performed to assess the development of tumor in vitro,
according to the previous report [32]. Briefly, after pre-treating with the AI-EtE or drug
for 24 h, 5 × 104 cells were mixed with 2 mL of 0.36% soft agar in RPMI medium, poured
 2.9. Colony Formation Assay
A colony formation assay was performed to assess the development of tumor in
vitro, according to the previous report [32]. Briefly, after pre-treating with the AI-EtE or
Life 2021, 11, 257 5 of 15
drug for 24 h, 5 × 104 cells were mixed with 2 mL of 0.36% soft agar in RPMI medium,
poured onto slightly solid 0.72% hard agar in RPMI medium pre-seeded on the 6-well
plate, and then cultured for 3 weeks. Medium was added on the surfaces of the agar layer
every two days.
onto slightly Colonies
solid wereagar
0.72% hard examined under
in RPMI microscope
medium everyonthree
pre-seeded days. At
the 6-well dayand
plate, 21,
colonies exceeding
then cultured 50 µm Medium
for 3 weeks. in diameter
waswere counted
added on the and presented
surfaces as anlayer
of the agar activity of two
every an-
chorage-independent growth. under microscope every three days. At day 21, colonies
days. Colonies were examined
exceeding 50 µm in diameter were counted and presented as an activity of anchorage-
2.10. Statisticalgrowth.
independent Analysis
Statistical analysis in this study was performed as previously described [33]. Results
2.10. Statistical
from Analysis experiments in each group were statistically analyzed by a Stu-
three independent
dent’sStatistical analysis
t-test. The in this study
SPSS (version was performed
18) software packageas previously
(SPSS described
Japan Inc., Tokyo,[33]. Results
Japan) was
from three independent experiments in each group were statistically analyzed
used for statistical analyses, and the significance level was set to p < 0.05. by a Stu-
dent’s t-test. The SPSS (version 18) software package (SPSS Japan Inc., Tokyo, Japan) was
used
3. for statistical analyses, and the significance level was set to p < 0.05.
Results
3.1. Anisomeles Indica Extract Displayed the Highest Cytotoxicity on Cervical Cancer HeLa
3. Results
Cells
3.1. Anisomeles Indica Extract Displayed the Highest Cytotoxicity on Cervical Cancer HeLa Cells
Ethanol
Ethanol extracts
extracts ofof eight medicinal plants,
eight medicinal plants, including
including Anisomeles
Anisomeles indica,
indica, Mahonia
Mahonia
bealei, Ficus semicordata, Gnetum montanum, Crinum asiaticum, Mallotus
bealei, Ficus semicordata, Gnetum montanum, Crinum asiaticum, Mallotus barbatus, barbatus,
Aganope
Aganope balansae,
balansae, and
and Hedyotis
Hedyotis capitellata
capitellata were
were used
used for
for screening
screening of
of their toxicity on
their toxicity on
cervical
cervical cancer
cancer HeLa
HeLa and
and breast
breast cancer
cancer MCF-7
MCF-7 cells.
cells. Treatment
Treatment with paclitaxel was
with paclitaxel used
was used
as
as a positive toxicity control. The observed toxicity curves in HeLa and MCF-7 cells are
a positive toxicity control. The observed toxicity curves in HeLa and MCF-7 cells are
shown
shown inin Figures
Figures 22 and
and 3,
3, respectively.
respectively.

Figure 2. HeLa cells were treated with the indicated extracts at various concentrations (1, 5, 10, 50, 100, 250, 500 and
1000 µg/mL) and MTT survival tests were performed. The toxicity curves of the ethanol extracts of Mahonia bealei, Ficus
semicordata, Gnetum montanum, Crinum asiaticum, Mallotus barbatus, Aganope balansae, Hedyotis capitellata, Anisomeles indica
and Paclitaxel are presented. The cell viability is shown as % relative to untreated control. Cell viability curves were drawn
using GraphPad Prism software.
 Figure 2. HeLa cells were treated with the indicated extracts at various concentrations (1, 5, 10, 50, 100, 250, 500 and 1000
µg/mL) and MTT survival tests were performed. The toxicity curves of the ethanol extracts of Mahonia bealei, Ficus semi-
cordata, Gnetum montanum, Crinum asiaticum, Mallotus barbatus, Aganope balansae, Hedyotis capitellata, Anisomeles indica and
Life 2021, 11, 257
Paclitaxel are presented. The cell viability is shown as % relative to untreated control. Cell viability curves were drawn6 of 15
using GraphPad Prism software.

Figure 3.
Figure TheMCF-7
3. The MCF-7cells
cells were
were treated
treated with
with thethe extracts
extracts at various
at various concentrations
concentrations of 1,of5,1,10,5,50,
10,100,
50, 250,
100, 500
250,and
5001000
and
µg/mL. The toxicity
1000 µg/mL. curves curves
The toxicity of the ethanol extracts extracts
of the ethanol of Mahonia bealei, Ficus
of Mahonia semicordata,
bealei, Gnetum montanum,
Ficus semicordata, Crinum asiaticum,
Gnetum montanum, Crinum
Mallotus
asiaticum,barbatus,
MallotusAganope balansae,
barbatus, AganopeHedyotis capitellata,
balansae, HedyotisAnisomeles
capitellata, indica and Paclitaxel
Anisomeles indica andwere presented.
Paclitaxel were The cell viability
presented. is
The cell
shown
viabilityasis%shown
relative
asto
%untreated
relative tocontrol.
untreatedThecontrol.
curves The
werecurves
drawnwereby using
drawn GraphPad
by using prism
GraphPad software.
prism software.

Based
Based on on these
these dose-response
dose-response curves,curves, the
the 50%
50% inhibitory
inhibitory concentration
concentration (IC50)
(IC50) and
and
correlation
correlationcoefficient
coefficient(R(R2)2values
) values forfor
allall
extracts were
extracts weredetermined
determined as summarized
as summarized in Ta-in
ble 2. Among
Table 2. Among these eight
these plant
eight plantextracts,
extracts, the
theethanol
ethanolextract Anisomelesindica
extractofofAnisomeles indica(AI-EtE)
(AI-EtE)
displayedthe
displayed thehighest
highesttoxic
toxicactivity
activityonon HeLa
HeLa cells,
cells, withwith
an an
IC50IC50 value
value of 38.8
of 38.8 µg/mL,µg/mL,
fol-
followed by the ethanol extract of Crinum asiaticum (IC50 = 69.5
lowed by the ethanol extract of Crinum asiaticum (IC50 = 69.5 µg/mL) and Mahonia bealei µg/mL) and Mahonia
bealei =(IC50
(IC50 = 101 µg/mL)
101 µg/mL) (Figure(Figure
2, Table2, Table
2). A 2).
veryA low
verytoxicity
low toxicity was observed
was observed for allforthe
all ex-
the
extracts
tracts on on
thethe MCF-7
MCF-7 cells,
cells, again
again theAI-EtE
the AI-EtEwas wasthethemost
mosttoxic
toxicwith
with IC50
IC50 of
of 133 µg/mL
µg/mL
(Figure3,
(Figure 3, Table
Table 2).
2). Paclitaxel
Paclitaxel (taxol),
(taxol), aa drug
drug approved
approved by by FDA
FDA forfor cancer
cancer treatment
treatment and and
usedas
used as aa positive
positive control,
control, had its IC50 values at 13.5 13.5 ng/mL
ng/mL andand 15 ng/mL
ng/mLon onHeLa
HeLa andand
MCF-7 cells, respectively (Figures 2 and 3, Table 2). Generally, a substance
MCF-7 cells, respectively (Figures 2 and 3, Table 2). Generally, a substance with IC50 with IC50 value
in thein
value range of 0–50ofµg/mL
the range 0–50 µg/mLis normally considered
is normally as a toxic
considered as aagent;
toxic therefore the AI-EtE
agent; therefore the
was used for further experiments.
AI-EtE was used for further experiments.

Table 2. IC50 values of different ethanol extracts on HeLa and MCF-7 cells.
Life 2021, 11, 257 7 of 15

Table 2. IC50 values of different ethanol extracts on HeLa and MCF-7 cells.

No Sample HeLa MCF-7

IC 50 (µg/mL) R2 IC 50 (µg/mL) R2
1 Anisomeles indica 38.8 0.92 133 0.99
2 Mahonia bealei 101 0.84 254 0.90
3 Ficus semicordata 358 0.96 188 0.86
4 Gnetum montanum 150 0.93 257 0.96
5 Crinum asiaticum 69.5 0.93 215 0,95
6 Mallotus barbatus 498 0.98 276 0.84
7 Aganope balansae 228 0.71 310 0.91
8 Hedyotis capitellata 173 0.99 184 0.94
Paclitaxel 13.5 (ng/mL) 0.96 15 (ng/mL) 0.98

3.2. Toxicity of Anisomeles Indica Ethanol Extract on Zebrafish Embryos

Experiments were conducted to evaluate the effect of AI-EtE on the development
of zebrafish embryos at different stages. AI-EtE at various doses of 0, 5, 10, 12.5, 25,
50, 75, 100, 150, 200 and 400 (mg/L) were applied. At each time point (24, 48, 72, and
96 h post-fertilization (hpf)), the presence of developmental defects and lethality of the
zebrafish embryos (larvae) were assessed (Figure 4A,B). The specific endpoint of hatching
was also assessed at 48, 72 and 96 hpf (Figure 4C). At concentrations up to 75 mg/L,
AI-EtE had almost no effect on the development of defects or death of zebrafish larvae
with malformation of 5%, lethality of 2.5%, and hatching efficiency of 70% (Figure 4A–C).
However, at 100 mg/L, a sharp increase in developmental malformations (Figure 4A) with
haemovascular defects and heart/yolk sac oedema on 75% larvae at 72 hpf and 90% larvae
at 92 hpf (Figure 4G,H). Similarly, at doses above 100mg/L, a sharp increase in embryo
and larva death was observed at each observation time point (Figure 4B). At 150 mg/L,
200 mg/L and 400 mg/L, AI-EtE was very toxic to zebrafish embryos because of the death
of all embryos at very early time (Figure 4H–J). In addition, we monitored the hatching
percentage of the embryos, which normally takes place between 48 and 72 hpf, as illustrated
in the control fish (Figure 4C). Treatment with AI-EtE had a negative effect on the hatching
ability in a dose-dependence manner. Specifically, at 75 mg/L, AI-EtE strongly decreased
the hatching percentage of zebrafish embryos to around 30% at both 72 hpf and 96 hpf
(Figure 4C).

3.3. The Anisomeles Indica Ethanol Extract Induced HeLa Cell Apoptosis
We also tested the effect of drug/extract on cell apoptosis by treating HeLa cells
with AI-EtE and paclitaxel for 24 h at the IC50 doses. Flow cytometry of the cells after
staining with propidium iodide (PI) and annexin V-FITC revealed the percentage of cells in
necrosis (Q1), late apoptosis (Q2), early apoptosis (Q3), and living cells (Q4) (Figure 5A–C).
We observed that the percentage cells entering apoptosis (Q2) was 3.0-fold higher in AI-
EtE-treated cells compared to control cells (Figure 5A,B), while paclitaxel increased the
percentage of apoptotic cells up to 3.3-fold (Figure 5A,C).
Cell apoptosis may be initiated by intrinsic and/or extrinsic signaling pathways.
Therefore, we investigated the transcript levels of genes that play important roles in apop-
tosis pathways in HeLa cells by real-time PCR analysis. The obtained results demonstrate
that the transcript levels of BAX, CASPASE-8, and CASPASE-3 were up-regulated in the
AI-EtE-treated HeLa cells in a dose-dependent manner (Figure 5D–F). After 12 h and 24 h
treatment with AI-EtE, the expression levels of BAX, CASPASE-8, and CASPASE-3 genes
were up-regulated 1.34-, 1.23- and 2.33-fold (at 12 h) and 1.55-, 1.62- and 2.45-fold (at 24 h),
respectively (Figure 5D–F). It was also revealed that the effects of AI-EtE and paclitaxel
on apoptosis-related gene expressions had similar patterns; however, paclitaxel was more
effective on the intrinsic pathway than on the extrinsic pathway, as shown by the massive
increase of the BAX transcript level (Figure 5A).
Life 2021, 11, 257 8 of 15
Life 2021, 11, x FOR PEER REVIEW 8 of 15

FigureFigure 4. Effect
4. Effect of AI-EtE
of AI-EtE on development
on development andand survivalof
survival of zebrafish
zebrafish embryos
embryosatatdifferent time
different points.
time The effects
points. of
The effects of
AI-EtE at various doses (0, 5, 10, 12.5, 25, 50, 75, 100, 200 and 400 mg/L) on defects during development (A), lethality (B),
AI-EtE at various doses (0, 5, 10, 12.5, 25, 50, 75, 100, 200 and 400 mg/L) on defects during development (A), lethality (B),
and hatching percentage (C) of zebrafish embryos at 24, 48, 72 and 92 hpf, respectively, are presented as dose-response
and hatching percentage (C)
curves. Representative of zebrafish
images of 96 hpf embryos at 24,presenting
zebrafish larvae 48, 72 andvarious
92 hpf, respectively,defects
developmental are presented as dose-response
upon AI-EtE exposure
curves.atRepresentative images
different doses, (D) of (negative
0 mg/L 96 hpf zebrafish larvae
control), (E) presenting
25 mg/L, various
(F) 50 mg/L, (G) developmental
75 mg/L, (H) 100 defects
mg/L, (I)upon AI-EtE
200 mg/L, andexposure
(J)
400 mg/L.
at different Illustrated
doses, defects(negative
(D) 0 mg/L on larvaecontrol),
include yolk-sac oedema(F)
(E) 25 mg/L, (e),50
haemovascular
mg/L, (G) 75defect (h),(H)
mg/L, and100
necrosis
mg/L,(n)(I)
as 200
indi-mg/L,
and (J)cated with arrows.
400 mg/L. Illustrated defects on larvae include yolk-sac oedema (e), haemovascular defect (h), and necrosis (n) as
indicated with arrows.
3.3. The Anisomeles Indica Ethanol Extract Induced HeLa Cell Apoptosis
We also tested the effect of drug/extract on cell apoptosis by treating HeLa cells
3.4.with
The AI-EtE
Anisomeles Indica Ethanol Extract Induced HeLa Cell Cycle Arrest at the G1 Phase
and paclitaxel for 24 h at the IC50 doses. Flow cytometry of the cells after
HeLa with
staining cellspropidium
were treated with
iodide (PI) AI-EtE or paclitaxel
and annexin at the the
V-FITC revealed IC50 doses for
percentage of 24 h. The
cells
cells
in were stained
necrosis withapoptosis
(Q1), late propidium (Q2),iodide that directly
early apoptosis (Q3),binds to thecells
and living DNA (Q4)in(Figure
the nucleus
5A–C). We observed
of ethanol-fixed that thecontents
cells. DNA percentage cells
were entering apoptosis
measured (Q2) was 3.0-fold
by flow cytometry high- the
to determine
er in AI-EtE-treated
proportion cellsS,compared
of cells in G1, and G2/M to control
phases.cells
The(Figure 5A,B), while
DNA content paclitaxel
histograms in- that
reveal
creased
in the the percentage
negative control of apoptotic
batch cells6A),
(Figure up tothe
3.3-fold (Figure 5A,C).
percentages of HeLa cells distributed in
the G1, S, and G2/M phases were around 64%, 15% and 18%, respectively; while, in the
AI-EtE-treated batch (Figure 6B), the percentages of HeLa cells in the G1, S, and G2/M
phases were 85%, 7% and 6.5%, respectively. These results indicate that AI-EtE significantly
induced cell cycle arrest in the G1 phase and consequently decreased the number of cells
entering the S and G2/M phases. In contrast, paclitaxel arrested the cell cycle at the G2/M
phase, as indicated by the percentages of cells distributed in the G1, S, and G2/M phases
of 26%, 29% and 45%, respectively (Figure 6C).
 in necrosis (Q1), late apoptosis (Q2), early apoptosis (Q3), and living cells (Q4)
5A–C). We observed that the percentage cells entering apoptosis (Q2) was 3.0-fold
er in AI-EtE-treated cells compared to control cells (Figure 5A,B), while
Life 2021, 11, 257
paclita
9 of 15
creased the percentage of apoptotic cells up to 3.3-fold (Figure 5A,C).

Figure 5. HeLa cells were treated with DMSO (negative control), AI-EtE at IC50 dose of 38.8 mg/L or paclitaxel (positive
control) at IC50 dose of 13.5 ng/mL for 24 h before collecting, fixing, staining with propidium iodide (PI)/annexin V-FITC
and subjecting to flow cytometry system to measure of apoptosis. Representative data sets of apoptotic pattern of HeLa cells
upon DMSO treatment (A), AI-EtE treatment (B), and paclitaxel treatment (C) are presented. Transcript levels of BAX (D),
CASPASE-8 (E), CASPASE-3 (F) in HeLa cells treated with AI-EtE and paclitaxel for 12 h and 24 h. *, ** and ***, significant
differences with p values < 0.05, <0.01 and <0.001, respectively. TBP was used as internal control gene. NC: negative control,
PC: positive control (paclitaxel).

Finally, to define the molecular events causing the cell cycle arrest, the transcript
levels of genes coding for proteins regulating the cell cycle, such as CDKN1A (p21CIP1 ),
CDKN2A (p16INK4A ) and p53 were investigated. Interestingly, treatment with either AI-EtE
or paclitaxel did not affect the transcript levels of p53 in HeLa cells (Figure 6F). However,
consistent with G1-phase retardation, the transcript levels of CDKN1A and CDKN2A in the
AI-EtE-treated cells were up-regulated 1.64-fold at 24 h and 1.90-fold at 12 h, compared
with those in the control cells, respectively (Figure 6D,E). On the other hand, paclitaxel
strongly increased the expression levels of both CDKN1A and CDKN2A genes at the time
of 24 h of 25.9- and 2.57-folds, respectively (Figure 6D,E).

3.5. AI-EtE Decreased Anchorage-Independent Growth Ability of HeLa Cells

We next performed a colony formation assay to investigate anchorage-independent
growth of HeLa cells. The obtained results demonstrated that exposure to AI-EtE at IC50
dose of 38.8 mg/mL decreased colony formation ability as well as reduced the size of
colonies of cells in soft agar (Figure 7B,D) compared with control cells (Figure 7A,C).
Particularly, number of colonies in case of control cells was 3.5-fold higher than that in
AI-EtE-treated cells (Figure 7E). Meanwhile, treatment with paclitaxel at the IC50 dose of
13.5 ng/mL totally inhibited colony formation of cells (Figure 7E).
Life 2021, 11, 257 10 of 15
Life 2021, 11, x FOR PEER REVIEW 10 of 15

Figure
Figure 6. 6. HeLa
HeLa cells
cells were
were treated
treated with DMSO (negative
with DMSO (negative control),
control), AI-EtE
AI-EtE at
at IC50
IC50 dose
dose of
of 38.8
38.8 mg/L
mg/L oror paclitaxel
paclitaxel (positive
(positive
control)
control) at IC50 dose of 13.5 ng/mL for 24 h before collecting, fixing, staining with propidium iodide and
at IC50 dose of 13.5 ng/mL for 24 h before collecting, fixing, staining with propidium iodide and subjecting
subjecting to
to
flow cytometry system to measure of cellular DNA content. Representative data sets of cell cycle pattern of HeLa cells
flow cytometry system to measure of cellular DNA content. Representative data sets of cell cycle pattern of HeLa cells
upon DMSO treatment (A), AI-EtE treatment (B), and paclitaxel treatment (C) were presented. Transcript levels of
upon DMSO treatment (A), AI-EtE treatment (B), and paclitaxel treatment (C) were presented. Transcript levels of CDKN1A
CDKN1A (D), CDKN2A (E) and p53 (F) in HeLa cells treated with AI-EtE and paclitaxel for 12 h and 24 h. *, ** and ***,
(D), CDKN2A
significant (E) and p53
differences with(F)
p in HeLa<0.05,
values cells treated with
<0.01 and AI-EtE
<0.001, and paclitaxel
respectively. TBPfor 12 used
was h andas24internal
h. *, ** and ***, significant
control gene. NC:
differences
negative with
control,p values <0.05, <0.01 and <0.001, respectively. TBP was used as internal control gene. NC: negative control,
Life 2021, 11, x FOR PEER PC: positive control (paclitaxel).
REVIEW 11 of 15
PC: positive control (paclitaxel).
Finally, to define the molecular events causing the cell cycle arrest, the transcript
levels of genes coding for proteins regulating the cell cycle, such as CDKN1A (p21CIP1),
CDKN2A (p16INK4A) and p53 were investigated. Interestingly, treatment with either AI-EtE
or paclitaxel did not affect the transcript levels of p53 in HeLa cells (Figure 6F). However,
consistent with G1-phase retardation, the transcript levels of CDKN1A and CDKN2A in
the AI-EtE-treated cells were up-regulated 1.64-fold at 24 h and 1.90-fold at 12 h, com-
pared with those in the control cells, respectively (Figure 6D,E). On the other hand,
paclitaxel strongly increased the expression levels of both CDKN1A and CDKN2A genes
at the time of 24 h of 25.9- and 2.57-folds, respectively (Figure 6D,E).

3.5. AI-EtE Decreased Anchorage-Independent Growth Ability of HeLa Cells

We next performed a colony formation assay to investigate anchorage-independent
growth of HeLa cells. The obtained results demonstrated that exposure to AI-EtE at IC50
dose of 38.8 mg/mL decreased colony formation ability as well as reduced the size of
colonies of cells in soft agar (Figure 7B,D) compared with control cells (Figure 7A,C).
Particularly, number of colonies in case of control cells was 3.5-fold higher than that in
AI-EtE-treated cells (Figure 7E). Meanwhile, treatment with paclitaxel at the IC50 dose of
13.5 ng/mL totally inhibited colony formation of cells (Figure 7E).
Figure
Figure 7. Anchorage-independent
Anchorage-independent growthgrowthof ofHeLa
HeLacells.
cells.Colonies
Coloniesformed
formedonon
thethe soft
soft agar
agar in case
in case of control
of control cellscells
(A,C)(A,C)
and
and AI-EtE-treated
AI-EtE-treated cellscells
(B,D)(B,D) at magnifications
at magnifications of 2Xof(A,B)
2X (A,B)
and and 5X (C,D).
5X (C,D). The The differences
differences in colony
in colony formation
formation ability
ability on
on soft
soft
agaragar of HeLa
of HeLa cellscells are presented
are presented in a in a graph
graph (E).
(E). *, *, significant
significant differences
differences withwith p values
p values < 0.05.
< 0.05.

4. Discussion
Nowadays, natural products or medicines originating from natural sources are at-
tracting the interest of scientists all over the world. Thus, in a recent report, WHO in-
sisted on the importance to develop and use traditional medications [6]. However, the
effects of traditional medicines on human health are not always supported by hard sci-
entific evidence and the molecular mechanisms they rely on are rarely assessed. In this
study, we screened the toxicity and the anticancer activity of crude extracts from eight
Life 2021, 11, 257 11 of 15

4. Discussion
Nowadays, natural products or medicines originating from natural sources are at-
tracting the interest of scientists all over the world. Thus, in a recent report, WHO insisted
on the importance to develop and use traditional medications [6]. However, the effects
of traditional medicines on human health are not always supported by hard scientific
evidence and the molecular mechanisms they rely on are rarely assessed. In this study,
we screened the toxicity and the anticancer activity of crude extracts from eight medicinal
plants, which have been used as traditional medicines in North-Western Vietnam, on the
cervical cancer HeLa cell line. We found that an ethanolic extract from Anisomeles indica
(AI-EtE) was the only one presenting a high toxicity on HeLa cells, with an IC50 value of
38.8 mg/L (Figure 2).
Previous studies indicated that extracts and products isolated from Anisomeles indica
(AI) had many biological effects in vitro. Particularly, aqueous extract of AI expressed
anticancer activity via inhibition of metastatic activity of breast cancer cells [25], different
AI extracts had antimicrobial and antioxidant properties [20–23]. Further, chemical com-
positions of AI had also been investigated, and ovatodiolide, a macrocyclic diterpenoid
compound, was one of the most important chemicals that possessed various biological
activities [22–25,34,35]. Ovatodiolide expressed its anticancer properties by initiation of
apoptosis via a ROS-dependent ATM/ATR signaling pathways, induction cell cycle arrest
at G2/M phase of breast, lung and oral squamous carcinoma cancer cells [22,24,25,35].
However, so far, the effects of AI extracts on cervical cancer cells have not been examined
yet, thus, in this study, we focused our investigation on the anticancer effects of this AI-EtE
on HeLa cancer cells.
Apoptosis induces distinct biochemical and morphological changes in the cell and
can be triggered by internal and/or external factors. The intrinsic pathway is initiated
by activation of the pro-apoptotic (BAX) leading to the release of cytochrome C from the
mitochondria [10–12]. The extrinsic pathway, triggered mainly by extracellular signals,
activates CASPASE-8. Both pathways will finally lead to activation of the execution caspase
CASPASE-3, which in turn degrades the cellular DNA and causes cell death [10–12]. Our
results show that AI-EtE could induce cell apoptosis up to 2.7-fold (Figure 5A) via activating
both BAX (Figure 6A) and CASPASE-8, as well as increase the expression of the down-
stream CASPASE-3. Thus, AI-EtE activates both the intrinsic and extrinsic apoptotic
pathways. Similarly, paclitaxel triggered apoptosis by both intrinsic and extrinsic pathways;
however, its effect was much stronger on the intrinsic pathway as shown by the more
intense increase in expression of BAX as compared to CASPASE-8) (Figures 5C and 6A,B).
In addition, it had been revealed that AI-EtE could induce the transcript expressions of
CASPASE-3 at the early time (12 h); on the other hand, although paclitaxel had almost
no effect on transcription levels of BAX, CASPASE-8 and CASPASE-3 at 12 h, it strongly
increased the expressions of these genes at 24 h, especially in the case of BAX (Figure 6A–C).
These results might also support for the fact that AI-EtE induced cell arrest at the early
time (G1 phase) while paclitaxel promoted cell arrest at the late time (G2/M phase).
Although, previous studies found that ovatodiolide isolated from n-hexane extract of
Anisomeles indica caused the cell arrest at the G2/M phase of the cell cycle in cases of lung
cancer cells [22], breast cancer [25,35] and oral cancer cells [24]. In this study, we found that
AI-EtE might promote the HeLa cell arrest at the G1 phase rather than at the G2/M phase
of the cell cycle (Figure 5B). This means that there may be other compounds in the ethanol
extract of Anisomeles indica, which can act on cell cycle arrest at G1 or it may be because of the
different behavior of HeLa cells compared with other cancer cells. We further investigated
the expression levels of genes encoding proteins involved in regulation of the cell cycle,
including p53, CDKN1A (p21CIP1 ), and CDKN2A (p16INK ). p53 is generally involved in
cell cycle arrest induced by DNA damage [13], and can also stimulate the transcription of
CDKN1A and BAX [14,15]. However, in this study, the transcript expression of p53 in HeLa
cells was not affected by treatment with ether AI-EtE or paclitaxel (Figure 6F), suggesting
that neither of them induced DNA damage. However, both AI-EtE and paclitaxel strongly
Life 2021, 11, 257 12 of 15

increased the transcript levels of CDKN1A, which mainly responds for G1/S phase and
CDKN2A, which normally has dominant expression at the G2/M phase (Figure 6D,E).
Previously, p21 has been reported as a key molecule, which plays an important role in
regulation of the critical G1 to S phase transition of the cell cycle, senescence and apoptosis,
and to be positively regulated by p16 [16]. Moreover, previous studies demonstrated that
p21 could be up-regulated and resulted in cell cycle arrest at G1 phase in a p53-independent
manner [17,18,36]. In addition, it had been also revealed that paclitaxel could induce the
cell apoptosis with a p53-independent way [37]. Normally, the expressions of p53 and
p16 are in a motive balance, a decrease of p53 leading to an increase of p16 [38]. Thus,
in this study, the obtained results suggested that the AI-EtE contributed in regulation of
cell cycle via activating the expressions of cyclin-dependent kinase inhibitors p21 and p16.
Moreover, CDK also contribute in regulating the cyclin protein levels rise and fall during
the cell cycle [39]. Different cyclins are required at different phases of the cell cycle [40–42],
particularly, mitosis of cell is regulated by cyclins in complex with CDK1 [43,44]. So,
consistent with previous report, this study suggested that the induction of p21 and p16
might have inactivated CDK and resulted in cell cycle arrest. Along with invasion activity,
anchorage-independent growth is a hallmark of cancer cells. It represents the proliferative
ability of cancer cells in the absence of adhesion to extracellular matrix proteins and
correlates closely with tumorigenesis [32,45,46]. To investigate the anchorage-independent
growth, colony formation (tumor formation in vitro) assay had been carried out. The ability
in inhibiting the colony formation of cancer cells is one of the most important effects of drug
for cancer treatment, especially for cancers with solid tumors [32,45,46]. In this study, we
found that AI-EtE was not only significantly decreased in the number of colony formation
of HeLa cells on soft agar but also strongly reduced the size of colonies. Although there
had been reports that products extracted from AI were able to decrease invasion activity of
several cancer cells [25,35], there is no report about the effect of AI originated products on
anchorage-independent growth of cancer cells.
Zebrafish is an effective in vivo model for whole animal screening for developmental
defects because of its advantageous properties such as high throughput, high similarity
of the genome with the human one, rapid development, transparency of the embryos
making them easy to observe and manage [26,27]. In this study, the effects of AI-EtE on
zebrafish embryos at 24, 48, 72 and 96 hpf were examined. We found that AI-EtE at the
concentrations below 80 mg/L had almost no effect on zebrafish embryo development
and survival. Only a decrease in hatching ability was observed at a dose of 75 mg/L. In
contrast, however, at higher concentrations starting at 100 mg/L, AI-EtE strongly induced
developmental defects and death of the larvae. Further investigations about the effect of
AI-EtE in vivo are needed.
Based on the chemical properties, ovatodiolide is mainly distributed into n-hexane,
however, the extraction protocol presented in previous study [47] was described with
following steps: firstly, ethanol was used for crude extraction, and after ethanol evaporation,
the pellet was collected for next extraction steps in different solvents such as n-hexane,
and ethyl acetate to collect several fractions [47]. This means that the ethanol crude
extract surely contains an amount of ovatodiolide. Moreover, other phytochemicals such
as phenolics and flavonoids are easily dissolved in ethanol/methanol [20,28,47–50], and
therefore they certainly present in the ethanol extract. In fact, previous studies found
that ovatodiolide (isolated from Anisomeles indica) induced cell cycle at G2/M arrest in
lung cancer cells [22] and squamous carcinoma cancer cells [24]; while, in this study, we
revealed that the Anisomeles indica ethanol extract induced HeLa cell arrest at G1. It
implies that there is not only ovatodiolide but also other phytochemicals could effect on
HeLa cancer cell and/or zebrafish embryos. Although the obtained results from this study
demonstrated that the AI-EtE has possible use in traditional medicine against cervical
cancer, in the future studies, chemical compositions of AI-EtE should be addressed by
using HPLC and/or HPTLC methods [51,52] to identify the phytochemicals that are able
to be used as fingerprints for quality control of the AI-EtE.
Life 2021, 11, 257 13 of 15

5. Conclusions
Taken together, we suggest that Anisomeles indica can be considered as a medicinal
plant and that Anisomeles indica ethanol extract is able to use as a traditional medicine
against cervical cancer cells; however, the real impact of this extract on cancer remain to
be evaluated through epidemiological data and pharmacokinetic studies coupled with
clinical trials. Moreover, a cautious approach is advised with the used dose, especially
when applying for pregnant women, because of its ability in causing hatching inhibition,
the development of defects and death of zebrafish larvae.

Supplementary Materials: The following are available online at https://www.mdpi.com/2075-172

9/11/3/257/s1. Table S1. Primer sequences of genes used in the study.
Author Contributions: Conceptualization, N.D.T. and N.T.B.-L.; writing—original draft preparation,
N.D.T. and N.T.B.-L.; writing—review and editing, N.D.T., M.M., A.N., P.D., N.L.T. and N.Q.H.;
data processing, N.T.B.-L., N.D.T., N.L.T., N.T.K.-T. and K.T.K.; sample collecting and preparation,
N.T.K.-T., N.Q.H., N.T.B.-L. and K.T.K.; funding acquisition, M.M. and P.T.-H. All authors contribute
to the writing—review and editing. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by ARES (Académie de Recherche et d’Enseignement Supérieur)
and the Ministry of Cooperation (DGD (Direction Générale de la Coopération au Développement)) in
Belgium. M. M. is a “Maître de Recherche” at the FNRS (Fonds National de Recherche Scientifique).
Institutional Review Board Statement: Fish Embryo Acute Toxicity (FET) was determined according
to OECD test guidelines of OECD [27]. Experiments were performed under the authorization
LA1610002, ethical protocols 13-1506 and 19-2135.
Informed Consent Statement: Not applicable.
Data Availability Statement: Publicly available datasets were analyzed in this study.
Conflicts of Interest: The authors declare no conflict of interest.

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