PHYTOCHEMICALS AND TERATOGENIC EFFECTS OF WATER EXTRACTS OF RIND OF SELECT FRUITS
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IJBPAS, May, 2018, 7(5): 938-952
ISSN: 2277–4998
PHYTOCHEMICALS AND TERATOGENIC EFFECTS OF WATER
EXTRACTS OF RIND OF SELECT FRUITS
ROBERT I. PALAMBERGO, RICH MILTON R. DULAY AND EDEN S. DAVID*
Department of Biological Sciences, College of Arts and Sciences, Central Luzon State
University, Science City of Munoz, Nueva Ecija, Philippines
*Corresponding Author, E-mail: eusdavid@yahoo.com
Received 20 Jan. 2018; Revised 24th Feb. 2018; Accepted 23rd March 2018; Available online 1st May 2018
th
DOI: https://doi.org/10.31032/IJBPAS/2018/7.5.4452
ABSTRACT
This paper reported the phytochemicals and teratogenic activities of fruit rind extracts of Annona
muricata, Annona squamosa, and Garcinia mangostana. Zebrafish (Danio rerio) embryos at
segmentation phase were exposed to various concentrations of the three extracts. Mortality,
heartbeat rate, hatchability, delayed growth and malformations of zebrafish were determined.
Phytochemical screening revealed that tannins, saponins, flavonoids, terpenoids, and cardiac
glycosides were present in A. squamosa, and A. muricata. However, only flavonoid was not
detected in G. mangostana. The toxic effects of the extracts were found dependent on the time of
exposure, and concentrations of extracts. The most common toxic effect of the three extracts was
coagulation. No heartbeat rate was observed to embryos in 1% and higher concentrations of
extracts of A. muricata and G. mangostana and 0.5% and higher concentrations of A. squamosa
extract. The 0.1% of A. muricata extract-treated embryos had 33.33% hatchability, while no
hatched was recorded in those exposed to all concentrations of extracts of A. squamosa and G.
mangostana. Embryos showed 100% delayed growth when exposed in 0.5% of both A. muricata
and A. squamosa extracts, and in 0.1% of G. mangostana extract. Bent-tail tip embryos were the
most common abnormalities and were observed at 0.1% of both extracts of A. muricata and A.
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Robert I. Palambergo et al Research Article
squamosa. Altogether, rind extracts of the three fruits contain bioactive compounds and exhibit
embryo-toxic and teratogenic effects in developing embryos of zebrafish.
Keywords: Annona muricata, Annona squamosa, Garcinia mangostana, zebrafish, phytochemicals
INTRODUCTION
Plant-based medicine as valuable remedy for purgative. Its fruit peel extract contains
several diseases has been used over the past alkaloids, flavonoids, phenols, saponins,
years. Plants contain various phytochemicals steroids, terpenoids, coumarin [3], and
such as alkaloids, tannins, flavonoids, acetogenins [4]. Annona muricata Lin., on
terpenes, saponins, coumarins, glycosides, the other hand, is commonly known as
phenolics, essentials oils and others which guyabano in the Philippines. The fruit is used
essentially contribute to their biological to treat dysentery, mouth sores, fever, and
effects. Phytochemicals have shown an diabetes. It contains alkaloids, flavonoids,
important role in preventing chronic diseases carbohydrates, glycosides, saponin,
like cancer, diabetes, and coronary heart terpenoids, and protein [5]. With the
disease [1]. Therefore, many plant based important compositions and significant
products have been developed and available biological activities of the three medicinal
not only in the local but also in the plants, their toxic and teratogenic effects, if
international market. any, have not yet been studied, hence, this
Garcinia mangostana Lin., commonly study.
known as mangosteen, is a fruit-bearing tree Zebrafish (Danio rerio), a member of
that is cultivated in Philippines. It is used to Cyprinidae family, is being used in
treat fever, skin disease, diarrhea, diabetes, toxicological and teratological studies
stomach ache and intestinal ailments. It is primarily due to their various advantages
also reported to contain a variety of such as transparency of embryo, high
secondary metabolites, such as oxygenated fecundity, easy to handle, and inexpensive to
and prenylated xanthones [2]. Annona raise. Teratogens are substances that may
squamosa Lin., commonly called as atis by cause defects in normal embryonic
many Filipinos, was already a part of human development but not necessarily being toxic
diet due to its nutritional and medicinal in adults [6]. Some teratogens such as
values. In folkloric medicine, the unripe fruit thalidomide [7] and arsenic [8] are presently
is used as astringent, and the root is a drastic used as anticancer drug.
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Robert I. Palambergo et al Research Article
Herein, we investigated the phytochemical placed into a double boiler water bath at 80
compositions and the toxic and teratogenic to 90°C for 2 hours. Extracts were filtered
effects of the hot water extracts of fruit rinds using a Whatman filter paper No. 2 and the
of select medicinal plants in developing filtrates were diluted to embryo water [12] to
embryos of D. rerio. prepare 10 ml of the different concentrations
MATERIALS AND METHODS (3%, 2%, 1%, 0.5%, 0.1%,and 0%, control)
Source of Plant Materials which served as treatments.
The ripe fruits of A. squamosa, A. muricata, Maintenance and Acclimatization of
and G. mangostana were purchased from Zebrafish
Science City of Munoz, Nueva Ecija, An aquarium comprising of untreated and
Philippines. The ripe fruits were washed and clean water with continuous aeration at
peeled off, then the fruit rinds were cut into 26±1°C was maintained for female and male
small pieces and air-dried for 5 days. The adult zebrafish at a ratio1:2, respectively.
dried fruits were pulverized using blender Fish were then acclimatized for one week
and their powders were subjected to before spawning and fertilization. They were
extraction. nourished daily with a high protein diet
Phytochemical Screening flakes. The high quality of water was
The bioactive components of each fruit rind maintained.
were determined based on the chemical test Zebrafish Spawning and Fertilization
described by Sofowora [9], Trease and Evans Zebrafish were confined in a plastic mesh to
[10], and Harborne [11]. Three replicates prevent cannibalism. After which, the
were prepared in each test. The results were zebrafish were subjected in dark condition by
determined based on color and intensity of wrapping the aquarium with black plastic bag
the action and were noted as present or not to allow spawning. After 12 hours in the dark
detected. condition, the aquarium was exposed to light
Hot Water Extraction condition for another 12 hours. Fertilization
The plant materials were separately extracted occurs 30 min after the light was turned on.
through hot water extraction protocol. In Twelve hour post fertilized embryos were
each fruit rind, 300 ml of distilled water was siphoned out from the aquarium using a hose
added to 10 grams of powdered fruit rind in a and transferred in a beaker. Embryos were
1000 ml capacity flask. The mixtures were rinsed three times with distilled water and
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Robert I. Palambergo et al Research Article
placed in a Petri plate to check the uniformity (DMRT) was carried out to compare the
and normal conditions of embryos using a treatment effects at 5% level of significance.
compound microscope. Unfertilized or RESULTS AND DISCUSSION
coagulated eggs were not considered. Phytochemical Constituents of Fruit Rinds
Teratogenicity and Toxicity Assay A number of phytochemicals are present in
Two ml of each treatment concentration of plants. In this study, five phytochemicals
the extract was dispensed into each well of were screened using qualitative analysis in
the 12-well ELISA plate. Triplicate was the three samples and the results are
prepared each treatment concentration. Four presented in Table 1. Apparently, all
embryos at segmentation phase were phytochemicals screened were present in A.
transferred into each well containing the squamosa, and A. muricata. However,
different treatments. The plate was incubated flavonoid was not detected in G.
at 26°C±1°C. Mortality, heartbeat, and mangostana. These phytochemicals are
hatchability rates were recorded. responsible to several biological activities.
Teratogenic activity was examined under These qualitative results of A. muricata, A.
40X magnification using a compound squamosa and G. mangostana extracts were
microscope. Morphological endpoint correspond to findings from previous
evaluation of treated zebrafish was based on reported studies. Ethanol extract of pericarp
the parameters established by Schulte and of A. muricata showed the presence of
Nagel [13] and Nagel [14]: lethal saponins, terpenoid, tannins, flavonoids, and
(coagulation, tail not detached, no somites, cardiac glycosides [15]. Using petroleum
and no heartbeat), teratogenic (malformation ether, ethyl acetate, and alcohol extraction, A.
of head and tail, scoliosis, growth retardation, squamosa detected almost all the
stunted tail, and limited movement), and phytochemicals screened [16]. However, in
normal. Percentage delayed growth and the study of Kaladhar et al. [3], the pericarp
malformation were determined. of A. squamosa showed positive result of
Statistical Analysis terpenoid. Moreover, the pericarp of G.
Experiment was laid out in a Completely mangostana also contains some
Randomized Design (CRD). Data were phytochemicals. The chloroform extract of
analyzed using analysis of variance the pericarp showed positive results of
(ANOVA). Duncan’s Multiple Range Test terpenoids and flavonoids [17]. However,
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saponins and tannins were not detected in the gymnemagenol from Gymnema sylvestre
extract. In the study of Alvarez et al. [18], the exhibited cytotoxic activity (73%) on HeLa
methanol extract of the pericarp showed cells after 96 hours [24]. Saponins also
positive result of tannins and cardiac possessed significant antimicrobial and
glycoside. However, some phytochemicals antifungal activities against pathogenic
including flavonoid was detected in bacteria and fungi [25]. There were some
chloroform extracts but not in aqueous studies exhibiting the activities of saponin in
extract. These differences are due to the medicinal applications such as
solvent used in the extraction of bioactive anticardiovascular [26], and anti-
components. inflammatory activities [27]. They can also
Tannins are bioactive compounds that are interfere the replication of cellular DNA and
naturally present in plant. This bioactive prevent the proliferation of cancer cells [28].
compound was detected in the three extracts Another bioactive compound present in
as indicated by brownish green or blue plants is flavonoid and its presence in plant
coloration. This compound is widely extracts can be distinguished by yellow
distributed in higher plants and members of coloration. This coloration was observed in
polyphenol chemical family. This suggests A. muricata and A. squamosa. This naturally
that the presence of tannins in the extracts is occurring compound possesses several
tend to be acidic because of the phenolic biological activities such as anti-
groups and could possibly be responsible for inflammatory, antimicrobial, anticancer, high
its antimicrobial activity [19]. Tannins could antioxidant, and anti-tumor activities [29, 30,
inhibit the growth of fungi, yeast, bacteria, 31, 32, 33].
and viruses [20] and has anti-cancer activity The terpenoid is also present in the three
[21, 22]. extracts and is considered as one of many
The three extracts showed the presence of secondary metabolites. This bioactive
saponins through formation of emulsion or compound was detected in the three plant
stable frothing. This is due to the mixture of extracts by exhibiting a color of reddish
the non-polar sapogenin and the water- brown.Terpenoids exhibited potential use in
soluble side chain compound producing pharmacological activities such as antiviral,
visible foam [23]. Saponins have shown antibacterial, antimalarial, anti-inflammatory,
biological activities. The isolated saponin,
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inhibition of cholesterol synthesis, and anti- different concentrations of A. muricata, A.
cancer activities [20]. squamosa, and G. mangostana fruit rinds hot
The last bioactive compound that was water extract. The toxic effects of the
screened is the cardiac glycoside and this was extracts were found dependent on the time of
detected in all extracts. The brown ring exposure and concentrations of extracts. The
formation indicates the presence of cardiac most common toxic effect of the three
glycosides.This bioactive compound has extracts was coagulation (Figure 1 ACE).
been used in therapy for many years in heart After 12 hours of exposure, 100% mortality
failure [34]. Moreover, cardiac glycosides was observed in 2% and higher concentration
can inhibit cell proliferation and has of A. muricata and G. mangostana. However,
cytotoxic activity by inducting apoptosis although lower than 100% mortality,
[35]. Therefore, the presence of bioactive embryos exposed in 2% of A. squamosa
compounds in the plants indicates their showed statistically comparable with 3%.
medicinal values. These findings support the Apparently, the mortality of embryos had
traditional use of the plants in the increased as the time of exposure prolonged
management of diseases because of their and this evidently observed to embryos at 1%
varied pharmacological effects. concentration of all extracts. At 48 hours post
Toxic Effects of Fruit Rind Extracts treatment exposure, a 100% mortality was
This study evaluated the toxic and observed in embryos exposed to 0.50% and
teratogenic effects of the three fruit rind higher concentrations of both A. muricata
extracts in zebrafish embryos as animal and A. squamosa and to those at 1.0% and
model. The embryos were exposed in various higher concentrations of G. mangostana. In
treatment concentrations and observed at contrast, no mortality was observed to those
different stages of development from treated 0.10% of all extracts.
segmentation phase (12 hours), pharyngula It is clearly seen that all extracts at 0.5% and
stage (24-36 hours), and hatching period (48- higher concentrations significantly affect the
72 hours). To determine the toxic effects, survival of D. rerio embryos. Coagulation
coagulation and absence of heartbeat were was the most observed lethal effects on
notably observed in treated embryos. Table 2 embryos of the three plant extracts. These
presents the percentage mortality of embryos toxic effects of plant extracts can be
after 12, 24, 36, and 48 hours of exposure at attributed to their bioactive component
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detected in phytochemical screening. These of A. squamosa exhibited cytotoxic effect
compounds have major impact on the against in vitro bel-7402 and CNE2 human
embryos causing death as early as 12 hours tumor cell lines [39]. The panaxanthone
and prolonged time of exposure to the isolated from the pericarp of G. mangostana
extracts. For instance, Wahyuni [36] reported suppressed the tumor volumes treated with
that the compound saponins can disturb the panaxanthone and also significantly lower
development of Aedes egypti by causing the metastasis of a mouse model [40].
retardation of development and weight Moreover, the xanthone of G. mangostana
reduction. Increasing of mortality level of the which reported previously to have anti-
larvae was also attributed by this compound proliferating effect on cancer cells was
that can cause hemolysis in the blood vessels evaluated its toxic effects in D. rerio. The
causing death of the larvae. Joy and Remani embryos were dead at 250 μg/mL and 125
[37] isolated new compound from pericarp of μg/mL after 48 hours of incubation. Thus
A. squamosa as (-)–ent-kaur-16-en-19-oic embryos at 62.5 μg/mL and higher
acid and 16a,17-dihydroxy-ent-kauran-19-oic concentrations were considered toxic and
acid showed cytotoxicity against different lethal to embryo. However, no malformation
cell lines (He La cervical cancer cell, DLA was observed to all the embryos survived
cells). However, the root of A. muricata [41]. Some plants such as Artocarpus
demonstrated high cytotoxic effect against heterophyllus and Tinospora cordifolia also
HL-60 cells by disrupting the mitochondrial demonstrated toxic effects in D. rerio
membrane to arrest the G0/G1 phase and embryos [42, 43].
inhibit cell proliferation at different Heartbeat Rate of Zebrafish
concentrations after 24 hours of treatment Heartbeat is an important parameter to
[38]. Similarly, this activity of A. muricata determine the viability of developing D. rerio
was also observed in embryos of D. rerio by embryos. The normal heartbeat rate of D.
demonstrating growth retardation as the time rerio embryos ranges from 120-180 per
of exposure prolonged. Acetogenins is minute [44]. The heartbeat rates of all
another compound abundant among embryos at pharyngula stage were monitored
Annonaceae family. This has been reported and the results are presented in Table 3.
to possess anticancer properties. The Apparently, no heartbeat rate was observed
acetogenin, squamostolide isolated from seed to embryos in 1% and higher concentrations
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of extracts of A. muricata and G. mangostana than that of the control embryos. No hatched
and 0.5% and higher concentrations of A. was recorded in those exposed to all
squamosa extract due to coagulation at early concentrations of extracts of A. squamosa
stage of embryonic development. The zero and G. mangostana. This is due to early
values of heartbeat rate means no visual heart coagulation and obvious delayed growth of
beat during the observation period which embryos. Delayed of hatching process could
indicate delayed growth.However, only be attributed possibly to the inhibition of the
embryos at 0.1% of extracts of A. muricata release of chorionase, an enzyme responsible
and A. squamosa showed obvious heartbeat for hatching [47] by the plant extracts.
but lower than those in control. These results Another reason for delay or failure to hatch
suggest that the three plant extracts are was due to the abnormalities which resulted
cardiotoxic to D. rerio embryos. This effect to the limited movement of treated embryos,
could be attributed to cardiac glycoside thus unable to break the chorion.
present in the three plant extracts. According Teratogenic Effects of Fruit Rind Extracts
to Poindexter et al. [45] as cited by Jose et al. Delayed growth is one of the distinct
[46], the cardiac glycoside was found to teratogenic effects of the three extracts. The
inhibit the myocardial Na-K ATPase enzyme percentage delayed growth of embryos
that causes an increase in contraction force of exposed in varying concentrations of the
the heart which can lead to cardiac arrest. extracts after 48 hours was observed and the
Hatchability of Zebrafish results are shown in Table 3. Embryos
An indicative of successful developmental showed 100% delayed growth when exposed
process is hatching. The mean percentage in 0.5% and higher concentrations of both A.
hatchability of the embryos exposed to muricata and A. squamosa extracts, and in
various concentrations of A. muricata, A. 0.1% and higher concentrations of G.
squamosa, and G. mangostana extracts is mangostana extract. The 0.1%
also presented in Table 3.Control embryos concentrations of the first two extracts
completed their hatching period at 48 hours registered low percentage delayed growth but
post treatment exposure. The 0.1% of A. found statistically comparable to those in
muricata extract-treated embryos control. Aside from delayed growth, the
demonstrated mean percentage hatchability different morphological abnormalities were
of 33.33% which was significantly lower also observed, and the most common
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IJBPAS, May, 2018, 7(5)Robert I. Palambergo et al Research Article
abnormality was tail malformation (Figure were still in advanced segmentation phase
1). Bent-tail tip embryos were observed at (Figure 1F) during the last observation
0.1% of both extracts of A. muricata and A. period. In the previous work of Noordin et al.
squamosa. This abnormality is probably due [41], the survived embryos of zebrafish did
to the disturbance of embryonic development not show any malformation when exposed to
of D. rerio. However, embryos treated with xanthone from the pericarp of G.
G. mangostana extract did not show any mangostana.
malformations, which probably due to the
very obvious delayed growth. These embryos
Table 1: Phytochemical composition of fruit rinds of A. muricata, A. squamosa, and G. mangostana
Phytochemical A.muricata A.squamosa G.mangostana
Tannin Present Present Present
Saponin Present Present Present
Flavonoid Present Present Not detected
Terpenoid Present Present Present
Cardiac Glycoside Present Present Present
Table 2: Mortality of D. rerioembryo after 12, 24, 36, and 48 hours of exposure at the different concentrations of
fruit rind extracts of A. muricata, A. squamosa, and G. mangostana
Fruit Rind Extract (%) Mortality (%)
12 h 24 h 36 h 48 h
A. muricata
3.00 100.00a 100.00a 100.00a 100.00a
2.00 100.00a 100.00a 100.00a 100.00a
1.00 75.00a 100.00a 100.00a 100.00a
0.50 0.00b 8.25b 16.75b 100.00a
0.10 0.00b 0.00b 0.00bc 0.00b
0.00 0.00b 0.00b 0.00bc 0.00b
A. squamosa
3.00 100.00a 100.00a 100.00a 100.00a
2.00 91.75a 100.00a 100.00a 100.00a
1.00 58.25b 83.25ab 100.00a 100.00a
0.50 0.00c 0.00c 100.00a 100.00a
0.10 0.00c 0.00c 0.00b 0.00b
0.00 0.00c 0.00c 0.00b 0.00b
G. mangostana
3.00 100.00a 100.00a 100.00a 100.00a
2.00 100.00a 100.00a 100.00a 100.00a
1.00 0.00b 41.75b 100.00a 100.00a
0.50 0.00b 0.00c 8.25b 75.00ab
0.10 0.00b 0.00c 0.00b 0.00c
b c b
0.00 0.00 0.00 0.00 0.00c
Values are expressed as mean of three replicates each concentration of extracts. Means with the same letter of
superscript are not significantly different at PRobert I. Palambergo et al Research Article
Table 3: Heartbeat rate, percentage hatchability, and percentage delayed growth of D. rerio
embryos at the different concentrations of three fruit rind extracts
Fruit Rind Extract (%) Heartbeat rate Hatchability Delayed growth (%)
(per minute) (%)
A. muricata
3.0 NBH 0.00c 100.00a
c
2.0 NBH 0.00 100.00a
c
1.0 NBH 0.00 100.00a
b c
0.5 0.00 0.00 100.00a
a b
0.1 127.00 33.33 8.33b
a a
0.0 137.67 100.00 0.00b
A. squamosa
3.0 NBH 0.00b 100.00a
b
2.0 NBH 0.00 100.00a
b
1.0 NBH 0.00 100.00a
b
0.5 NBH 0.00 100.00a
b b
0.1 106.67 0.00 16.67b
a a
0.0 145.33 100.00 0.00b
G. mangostana
3.0 NBH 0.00b 100.00a
b
2.0 NBH 0.00 100.00a
b
1.0 NBH 0.00 100.00a
b b
0.5 0.00 0.00 100.00a
b b
0.1 0.00 0.00 100.00a
a a
0.0 150.67 100.00 0.00b
Values are expressed as mean of three replicates each concentration of extracts. Means with the same letter
of superscript are not significantly different at PRobert I. Palambergo et al Research Article
CONCLUSION muricata pericarp. Fitoterapia, 2000, 183-
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of the three extracts, respectively. 07.
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