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Vol. 11 No. 1 (2019)

Egyptian Academic Journal of Biological Sciences is the official English language journal of
the Egyptian Society of Biological Sciences, Department of Entomology, Faculty of Sciences
Ain Shams University.
The Journal publishes original research papers and reviews from any zoological discipline or
from directly allied fields in ecology, behavioral biology, physiology & biochemistry.
www.eajbs.eg.net

Citation: Egypt. Acad. J. Biolog. Sci. (B. Zoology) Vol. 11(1)pp 109-115 (2019)
 Egypt. Acad. J. Biolog. Sci., 11(1): 109- 115 (2019)
Egyptian Academic Journal of Biological Sciences
B. Zoology
ISSN: 2090 – 0759
www.eajbsz.journaks.ekb.eg

Toxic and Teratogenic Effects of Sampa-Sampalukan

(Phyllanthus niruri) Leaves Extract Using Danio rerio Embryo Assay

I. Q. Lamban1, E. S. Balbeuna1, M. Y. S. Lee2, M. Sacdalan2, A. P. Carpio JR.2

AND Kevin Smith P. Cabuhat3*
1
Assemblywoman Felicita G. Bernardino Memorial Trade School, Lias Marilao,
Bulacan, Philippines
2
Department of Biology, College of Science, Bulacan State University, City of
Malolos, Bulacan, Philippines
3
AD-DRUGSTEL Pharmaceutical Laboratories Incorporation, First Bulacan Industrial
City, Tikay, Malolos, Bulacan, Philippines
E-mail: [email protected]

ARTICLE INFO ABSTRACT

Article History This paper established toxic and teratogenic effects of P. nururi
Received:12 /1/2019 leaves extract to the embryonic development of D. rerio. After 12
Accepted:8/4/2019 hours of exposure to various treatment concentrations, 100%
_________________ coagulated embryos were observed in 1% and higher concentrations.
Keywords: Meanwhile, mortality in lower concentrations was found to be as
Danio rerio, time and dose-dependent. Coagulation was the most remarkable toxic
embryo-toxicity, effect of the plant leaves extract. On the other hand, heartbeat and
Phyllanthus niruri, hatchability rate of zebrafish embryo was affected in a dose-
Sampa-sampalukan, dependent manner. In teratogenicity testing, tail malformation was
teratogenicity, the most evident teratogenic effect of the plant leaves extract. Taken
zebrafish together, P. niruri leaves extract was embryo-toxic and teratogenic to
D. rerio.
INTRODUCTION

Phyllanthrus niruri, commonly known as “Sampa-sampalukan”, belonging to

the family Phyllantaceae, is commonly found in the tropical and sub-tropical region of
the world. It grows as a weed in moist abandoned land. It has various applications in
tradition and folk medicine for treatment of various diseases such as hepatitis B, HIV,
microbial infections, plamodiasis, nematode infestation, hepatoxicity, cough, diuretic,
menstruation problem and dysentery (Danladi et al., 2018). However, embryo-toxicity
and teratogenicity of this invasive weed are still unexplored.
Toxicity can be defined as the degree to which a substance can harm organisms
(Kasper et al., 2015). Teratogenicity, on the other hand, is the ability of any substances
(teratogens) to cause malformations in developing organisms (Duong et al., 2011).
However, Blagosklonny (2005) stated that most of the anticancer drugs are teratogenic
in nature and teratogens can be developed as anticancer drugs.
Nowadays, zebrafish (Danio rerio) becomes a trending model when it comes to
toxicity and teratogenicity due to its high fecundity, transparent embryos and larvae, no
pain model, similarity to mammalian embryonic development (David et al., 2016) and
physiological responses (Dulay & De Castro, 2017).

Citation: Egypt. Acad. J. Biolog. Sci. (B. Zoology) Vol. 11(1)pp 109-115 (2019)
110 I. Q. Lamban etal.

Herein, toxicity and teratogenicity of P. niruri leaves extract were evaluated

using D. rerio embryo in return to the assessment of its bio-potentialities in drug development.
MATERIALS AND METHODS

Source of Plant Leaves Specimen:

The leaves of P. niruri were collected from Lias Marilao Bulacan, Philippines.
The botanical specimen was brought to the Institute of Biology of the University of the
Philippines, Diliman, Quezon City, Philippines for verification and authentication. The
remaining leaves were air-dried for seven days, milled using a blender and prepared for
hot water extraction.
Preparation for Plant Leaves Extraction:
The plant leaves extract was obtained after Eguchi et al. (1999), with minor
modifications. Thirty grams (30) of the pulverized plant sample were extracted in 300
mL hot water at 80-90oC in the water bath for 2 hours. Afterward, the extract was
filtered using Whatman filter paper No. 2. The filtrates were used for the preparation of
different treatment concentrations by diluting to the embryo medium (Thomas, 2000).
Eight treatment concentrations were formulated such as 10%, 5%, 3%, 1%, 0.5%,
0.1%, 0.05% and the control (embryo medium).
Acclimatization and Spawning of Zebrafish:
Following the procedure established by Nagel (2002), with minor
modifications, 10 female and 20 male zebrafish were acclimatized in the aquarium
with water saturated by oxygen for one (1) week. The zebrafish were fed using dry
flakes twice a day and the water quality was maintained by removing excess feeds out
of the aquarium after 1 hour (h). To initiate spawning, zebrafish were confined in the
breeding tank with plastic mesh to avoid cannibalism of the released eggs. The
breeding tank was enclosed using a black trash bag for 12 h. After 12 h, the trash bag
was removed and allowed the eggs to be fertilized for another 12 h. typically,
fertilization occurs after 30 minutes of exposure to the light condition. At 12 hours post
fertilization, embryos were siphoned out using a hose and then washed using distilled
water thrice. The embryos were sorted out using a simple compound microscope.
Fertilized eggs were used in the assay. Meanwhile, unfertilized and coagulated eggs
were discarded.
Toxicity and Teratogenicity Assay:
Two (2) mL of different treatment concentrations were doled out into each well
of 24-well ELISA plate. Each treatment was triplicated. Four (4) embryos at
segmentation period were exposed into each well of the plate. Afterward, the plate was
maintained at room temperature (26 ± 1ºC) (Reneses et al., 2016). The mortality of
zebrafish embryo was assessed at 12, 24, 36 and 48 hours post-treatment application
(hpta). On the other hand, heartbeat was monitored at 36 hpta while hatchability was
observed at 48 hpta. To evaluate the teratogenicity of the plant leaves extract, protocol
of Nagel (2002) was used: lethal (coagulation, no heartbeat, tail not detached and no
somites), teratogenic (malformation of the head and tail, scoliosis, limited movement,
stunted tail, light pigmentation and growth retardation or delayed growth) and normal.
Malformations, if any, were captured using a mobile android phone with 8 megapixels.
Statistical Analyses:
The collected data were analyzed using SPSS program (17.0 versions). Data
were run in One-Way ANOVA followed by Duncan’s Multiple Range Test, to
compare the means at 5% level of significance.
 Toxic and Teratogenic Effects of Sampa-Sampalukan Leaves Extract 111

RESULTS

In this study, zebrafish embryo was used to determine the toxicity and
teratogenicity of the Phylanthus niruri leaf extract. The assay was conducted from
segmentation period up to hatching period of zebrafish.
Mortality of Zebrafish Embryo:
Mortality refers to no visible heartbeat and coagulation. Herein, mortality of
zebrafish embryo was observed after 12, 24, 36 and 48 hours of exposure to various
treatment concentrations (Table 1).
As early as 12 h of exposure in 1% and higher concentrations, 100% mortality
was recorded. Meanwhile, embryos exposed in 0.05% had 58.33% mortality. However,
0% mortality was noted in 0.05% and 0.1%. At 24 hours post-treatment application
(hpta), all embryos treated in 0.5% have died. After 36 h of exposure in 0.1%, 16.67%
dead embryos were observed. At 48 hpta, an increase of mortality was recorded in
0.05% and 0.1% with 8.33% and 25%, respectively. Nevertheless, even the mortality
of embryo in 0.05% increases, it was still comparable to the control at 5% level of
significance.

Table1. Mortality rate of zebrafish embryo after exposure to different treatment concentrations.

Treatment
12 hpta 24 hpta 36 hpta 48 hpta
Concentrations
Control 0.00a 0.00a 0.00a 0.00a
0.05% 0.00a 0.00a 0.00a 8.33a
0.1% 0.00a 0.00a 16.67b 25.00b
0.5% 58.33b 100.00b 100.00c 100.00c
1.0% 100.00c 100.00b 100.00c 100.00c
3% 100.00c 100.00b 100.00c 100.00c
5% 100.00c 100.00b 100.00c 100.00c
10% 100.00c 100.00b 100.00c 100.00c

Means that do not share a superscript in a column are significantly different at 5% level of significance

In this result, it is clearly observed that the survivability of the embryo was
affected as the number of concentration increases and as the time of exposure is
prolonged. Coagulation was the most marked lethal or toxic effect of the plant leaves
extract.
In the study of Paithankar et al. (2011), aqueous extract of P. niruri shows an
inhibition to Human Immunodeficiency Virus (HIV) on MT-4 cells culture. Alkaloid
was found as the responsible agent for this effect. Similarly, Limonene, a kind of
terpenes, extracted from this plant shows an inhibition of liver tumor models
(Bagalkotkar et al., 2006). Also, even other plants extract exhibit toxic effect to D.
rerio embryo. Particularly, the aqueous extract of Artocarpus heterophyllus stem-bark
exhibited 100% mortality at 0.1% and higher concentrations while the leaves extract
exhibited 100% mortality at lower concentration (0.5%) and higher concentrations
after 48 hours of exposure (Meman et al., 2016). Likewise, fruit rind extract of Annona
muricata, Annona squamosa and Garcinia mangostana exhibited a toxic effect to D.
rerio embryo in time and dose-dependent manner (Palambergo et al., 2018). These
results strongly suggested that plants can be a source of bioactive constituents
112 I. Q. Lamban etal.

specifically, P. nururi. Thus, continuous identification of those active phytochemical

components is indeed necessary for pharmacological purposes.
Cardio-toxicity of P. niruri Leaves Extract to D. rerio Embryo:
Heartbeat rate is one of the important parameters to determine the toxicity of
natural compounds or substances. Many Philippine plant studies have shown that
zebrafish was one of the suitable models to determine cardio-toxicity. Usually, the
effect to the heartbeat of zebrafish embryo was monitored after 36 hours of exposure to
plant extract due to transparency and the visibility of its heartbeat (Cabuhat et al.,
2018). Mably & Childs (2010) reported that the heartbeat of zebrafish is closely the
same to the heartbeat of the human with 120-180 beats per minute (bpm). In the
present study, the results on the effect of P. niruri leaves water extract to the heartbeat
zebrafish embryos after 36 h of exposure were presented in Table 2.

Table 2. Heartbeat rate and hatchability rate of P. niruri to D. rerio embryo.

Treatment Concentrations Heartbeat

Control 152.00a
0.05% 147.17a
0.1% 150.33a
0.5% Coagulated
1.0% Coagulated
3% Coagulated
5% Coagulated
10% Coagulated

Means that do not share a superscript in a column are significantly different at 5% level of significance

In 0.5% up to 10%, no heartbeat was recorded due to coagulation at early

developmental stages of zebrafish. However, embryos treated in the control obtained
the highest heartbeat with 152 beats per minute (bpm) while those embryos exposed to
0.05% and 0.1% register 147.17 and 150.33 bpm, which shows comparability to the
control at 5% level of significance.
Accordingly, cardiac function may have been affected due to underdeveloped
heart and pericardium, which could induce an abnormal heartbeat and circulation
failure and subsequently result in body growth retardation via insufficient nutrients
(Yamauchi et al., 2005 as cited in Reyes et al. (2016). Thus, this deficiency to
zebrafish will eventually lead to death.
Hatchability of Zebrafish Embryos Treated in Different Treatments:
Hatchability of zebrafish embryo defines its normal and successful
development. Usually, it takes place between 48-72 h after exposure to the test sample.
In this study, the hatchability rate was evaluated after 48 hours of exposure to different
treatment concentrations (see Table 3).
Apparently, no hatched embryos treated in 0.5% up to 10% was observed due
to early arrest of embryos. On the other hand, embryos treated in 0.1%, 41.67%
hatched embryos were recorded. However, 83.33%-hatched embryos was noted in
0.05%, which shows no significant difference to the control at 5% level of
significance.
 Toxic and Teratogenic Effects of Sampa-Sampalukan Leaves Extract 113

Table 3. Hatchability rate of P. niruri to D. rerio embryo.

Treatment Concentration Hatchability

Control 100.00a
0.05% 83.33a
0.1% 41.67b
0.5% Coagulated
1.0% Coagulated
3% Coagulated
5% Coagulated
10% Coagulated

Means that do not share a superscript in a column are significantly different at 5% level of significance

Teratogenicity of P. niruri Leaves Extract to D. rerio Embryo:

Teratogenicity assay is a desirable property because many anticancer drugs are
teratogenic in nature and teratogens can be developed as anticancer drugs
(Blagosklonny, 2005). Morphological endpoints of the plant leave extract was based on
the parameters established by Nagel (2002). The teratogenic effects of P. niruri leaves
extract was observed at 72-84 hpta. Tail malformation was the most marked
teratogenic effect of the plant extract (see Figure 1).
In early developmental observation (12-48 hpta), most of the embryos were
coagulated. However, those embryos treated in lower concentrations exhibited
teratogenic effects. Particularly, embryos exposed in 0.05%, head malformation, yolk
deformities, bent body tail and the loop-like tail was observed. On the other hand,
those embryos treated in 0.1% showed scoliosis, hook-like tail, malformed head, yolk
deformities and bent tail. These results suggested that plant extract contains important
teratogenic component/s that can be developed as an anticancer drug.

Fig. 1.Teratogenic effects of plant extract to D. rerio at 72-84 hpta. (A). Larva with head
malformation, yolk deformities and bent body tail (observed in 0.05%) (B). Larva with the
loop-like tail (observed in 0.05%) (C). Larva with scoliosis and hook-like tail (observed in
0.1%) (D). Larva with malformed head, yolk deformities and bent tail (observed in 0.1%). (E).
Normal hatched embryo (control)
114 I. Q. Lamban etal.

These observed teratogenic effects were similar to the effect observed in other
Philippine medicinal plants. In the study of Jose et al. (2016), tail malformation was
evident particularly in 0.01% leaf extract of Garcinia mangostana (Mangosteen). Also,
this plant can cause head malformation to zebrafish embryo. Similarly, the result of
this study conforms to the outcome obtained in Trinidad et al. (2017), wherein the
extract of Lantana camara (Stink grass), a weed, exhibited tail malformation (hook
and bent tail), scoliosis and head malformation. On the other hand, yolk deformities
were also observed in the extract of Moringa oleifera (Malunggay)(David et al., 2016).
Conclusion
Based on the collected findings, P. niruri leaves extract was toxic and
teratogenic to D. rerio embryo. Thus, plant leaves contain phytochemicals that can be
developed as anticancer drugs. Since, many anticancer drugs are teratogenic and
teratogens can be developed as anticancer drugs. Identification of the specific
phytochemical component(s) is highly recommended for future studies.

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