EFFECTS OF GLYCOALKALOIDS FROM IMMATURE, GREEN Lycopersicon

esculentum (TOMATO) ON NEURODEVELOPMENT OF CHICK EMBRYOS

Karen Paula V. Manalo

March 3, 2018

Graduate Studies Department, College of Science and Computer Studies, De La Salle University -
Dasmariñas, City of Dasmariñas, Cavite, Philippines

ABSTRACT

Alkaloids are cyclic nitrogen-containing phytochemicals from several

plant species including unripe, green tomato fruits. Many studies have shown
that glycoalkaloids have teratogenic effects on different embryos. This special
problem focuses on the possible teratogenic effects of various concentrations of
alkaloids from immature, green tomatoes on neurodevelopment of chick
embryos. Methods conducted by Guvenc et al. (2016) and Farjadmand, Eslami
and Sadighara (2011) on induction and observation will be followed.

INTRODUCTION determine if different concentrations of

The initially proposed title of the special
problem was “Effects of Tomatine from
Immature, Green Lycopersicon esculentum
(Tomato) on Neural Tube Development of
Chick Embryos.” However, due to equipment
and time constraints, the research will now
focus on alkaloids where tomatine belongs to. In
addition Taveira et al. (2012) stated that the
determination of compounds like tomatine via
HPLC which was also initially included in the
research is complex, tedious, and not readily
available in most laboratories due to its high
price. This was the exact encounter upon
development of the research. The
neurodevelopment will also be considered as a
whole and not just the neural tube development
as only gross microanatomy will be observed.
Histological preparations and observations need
to be done to better observe and characterize the
neural tube.
This research aims to determine and
characterize the effects of the different
concentrations of glycoalkaloids from immature
Lycopersicon esculentum (tomato) on
neurodevelopment of chick embryos and to
glycoalkaloids from immature Lycopersicon
esculentum (tomato) have terataogenic effects
on the neurodevelopment of chick embryos.
ALKALOIDS
According to Zulak et al. (2006 in
Kennedy & Wightman, 2011), alkaloids are a
diverse group of “over 12,000 cyclic nitrogen-
containing compounds that are found in over
20% of plant species.” They are known as
secondary metabolites or phytochemicals
produced by plants to protect themselves from
outside stress and pathogens (Rafiqkhan, et al.,
2014).
Zulak et al. (2006 in Kennedy &
Wightman, 2011) stated that alkaloids are
primarily toxic to insects and herbivores as their
targets are the nervous system. Kennedy and
Wightman (2011) described that alkaloids can
already be tolerated by herbivores; however, are
still toxic to mammals.
Alkaloids can be categorized into certain
groups depending on their chemical structures.
Some of these groups are the purines which

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include xanthines, caffeine, theobromine, and
theophylline; the steroid alkaloids like tomatine
which has a steroid skeleton in a nitrogen
containing structure; solanum alkaloids from
potato and tomato such as solanidine, solanine,
and chaconine; and pyridine alkaloids like
nicotine (Kennedy & Wightman, 2011).
ALKALOIDS IN TOMATO
Alkaloids found in tomato are known as
steroidal glycoalkaloids (SGAs). They occur in
all plant parts with the highest concentrations in
leaves, unripe fruits, and flowers (Ventrella et
al., 2016). SGAs are said to be toxic by Koffi et
al. (2016).
The previous literature presented focused
on the glycoalkaloid tomatine. Another
glycoalkaloid found in tomato is solanine.
Solanine is described as a poison occurring in
plants. It is also said to be very toxic even in
small amounts (Kakhia, 2012). Kakhia (2012)
discussed that solanine poisoning is mainly
observed in gastrointestinal and neurological
ailments. He further added that ripe tomatoes
(red or yellow) do not have notable amounts of
solanine; however, is high in concentration in
unripe (green) tomatoes.
This is in line with Choi et al. (2013)
who stated that green tomato has high
concentrations of glycoalkaloids like tomatine
and dehydrotomatine as well as Cichon and
Schwartz (2015) who said that tomatine is most
concentrated in green tomatoes and is
enzymatically broken down upon ripening.
Friedman and Levin (1995 in Cichon &
Schwartz, 2015) reported that α-tomatine in
unripe green tomatoes is approximately 1.6
mg/100 g and approximately 0.03 mg/100 g in
ripe, red.
EFFECTS OF GLYCOALKALOIDS ON
THE NEURODEVELOPMENT OF
CHICKS
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Bjørnstad et al. (2015) said that the chick
embryo has been used as a vital model in
developmental biology and neurodevelopment.
The 46-stage chick development, that is still
used nowadays, was thoroughly described by
Hamburger and Hamilton (1951 in Bjørnstad et
al. (2015) with the characterization of the
neurodevelopmental stages.
Doshi et al. (2016) also described the
neurodevelopment of the chick based on
Hamburger – Saunders. According to them,
development starts from the neuroectoderm at
about 20-21 hours of incubation. The neural
folds will then be visible in the region of head at
23-26 hours. Neural folds will then meet at the
midbrain after 26hrs and three major brain
vesicles will be observed by 33-38 hours. After
40-45 hours, the cranial flexure takes place, five
neuromeres of the hindbrain are observable and
the anterior neuropore closes. It is by 48 hours
when the posterior neuropore closes. At 52-64
hours, the forebrain is elongated and
compressions between brain parts are more
distinct.
As already presented in the previous
related literature presented on this topic,
tomatine, as an example of a glycoalkaloid has
both positive and negative effects on other
organisms. Lather et al. (2011) described the
brain defects found in cattle and sheep due to
exposure to glycoalkaloids from L. esculentum.
Silva et al. (2017) also illustrated the
neurotoxicity of glycoalkaloids from immature
tomatoes.
Since such previous studies described
the neurotoxicity of alkaloids from unripe, green
tomatoes, their effects on the neurodevelopment
of chick embryos is still considered in the
present study. As also discussed in the initial
report, Pamir et al. (2006 in Doshi et al., 2016)
stated that one of the early and major events in
central nervous system development is the
neural tube closure. They said that deformities
in the closure result to malformations which can
pose complications in fetal development.
 This is in line with Mete et al. (2017)
who said that some disorders and physical
abnormalities that happen in later stages of life
are because of teratogens that affect neural tube
development. They further added that
neurulation that is vital for central nervous
system (CNS) development, needs to be totally
accomplished for several organs and tissues
related to the CNS to develop normally in terms
of structure and function.
Several studies have shown alkaloids as
teratogenic to chick embryos. Pamir et al.
(2006) discussed that caffeine caused neural
tube defects in chick embryos. This is is
supported by Cetinkal et al. (2010) who showed
the same results. These are also supported by
Ma et al. (2012) who concluded that
concentrations of caffeine resulted to
teratogenesis and even death of the fetus when
induced on chicken eggs. They were able to
observe prominent defect in the prosencephalon
of the chick embryo. They, however, also said
that the actual events of caffeine on
neurodevelopment is yet to be clarified.
Other alkaloids that were discussed to
have caused neural tube defects on chick
embryos are papaverine and cotinine (Cetinkal
et al., 2010).
MATERIALS AND METHODS
The previously presented related
literature showed the first report of
malformation in chick embryos injected with
tomatine from tomato leaves (Farjadmand,
Eslami & Sadighara, 2011). They administered
0.1, 1, and 10 mg of extract per egg to their
three experimental groups. They reported that
those injected with 10 mg of extract resulted to
100% mortality. The concentrations that they
used in their study will also be applied in the
current special problem. The only difference is
that instead of tomatine from tomato leaves,
alkaloids from immature, green tomatoes will be
extracted and then induced to chicken eggs.
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Immature tomatoes will be purchased
from Kadiwa market prior to the extraction of
alkaloids that will be based on Friedman et al.
(2009). The number and/or weight of needed
tomatoes will still be confirmed with POG2C
Lab and Training Center, Imus, Cavite. This is
the same laboratory where alkaloid extraction
and phytochemical tests will be conducted.
Tomato fruit samples with uniform size
will be combined and chopped with a knife.
They will then be weighed and blended in a
homogenizer with 2% acetic acid in methanol
(100 mL). The resulting mixture will then be
concentrated to 2-3 mL with the aid of a rotary
evaporator. The concentrate will be dissolved in
0.2 N HCl (40 mL) and centrifuged at 18000g
for 5 min at 5C. The residue will be rinsed
twice with 0.2 N HCl (10 mL) and then
centrifuged again. Concentrated NH4OH (20
mL) will be added to the supernatant to
precipitate the glycoalkaloids. The basic
solution will be placed in a 65C water bath for
50 min and then refrigerated overnight. The
precipitate will then be collected after
centrifugation at 18000g for 10 min at 5C and
washed twice with 2% NH4OH. The ammonia
will be dissipated, and the resulting pellet will
be dried at 30C under reduced pressure and
then dissolved in 2% acetic acid in methanol (2
mL) and centrifuged at 18000g for 10 min at
5C. All extractions and will be subjected to
phytochemical analysis for the determination of
alkaloids (Friedman et al., 2009).
It is important to do phytochemical tests
in order to confirm the different classes of
compounds from the extract. The Liebermann’s
test for glycosides from Gul et al. (2017) will be
followed. 2.0 ml of acetic acid and 2 ml of
chloroform will be added with whole aqueous
plant crude extract. The mixture will then be
cooled and added with concentrated H2SO4.
Green color will show the entity of aglycone,
steroidal part of glycosides.
 The Wagner’s test for alkaloids will also
be performed. Wagner’s reagent (iodine in
potassium iodide) will be added to the extract.
The appearance of reddish brown precipitate
will indicate the presence of alkaloids (Gul et
al., 2017).
Induction methods will be done based on
the methods of Guvenc et al. (2016) and
Farjadmand, Eslami & Sadighara (2011) for the
concentrations.
A total of 30 fertile eggs will be
purchased from Sustamina Agri-Industrial
Corporation. They will be incubated at a
constant temperature range of 37.5 ± 0.2C and
humidity of around 60–70% at the DLSU-D
Biology Laboratory until the embryos reach
stage 8 of development, or at the 28th hour of
incubation based on Hamburger and Hamilton.
The eggs will then be divided into five (5)
groups. Six eggs in A will be incubated for 48
hours without any more procedure. The
remaining 24 eggs will be further divided into
four groups. Eggs in group B will be injected
with 0.1 ml of saline solution. Eggs in group C
will be injected with 0.1 mg of alkaloid extract
per egg. Eggs in groups D and E will be injected
with 1 mg and 10 mg of alkaloid extract
respectively, per egg. The embryos will then be
removed from the egg at the 48th hour or 12th
stage based on Hamburger-Hamilton. All
embryos will be mixed with 10% buffered
formalin for 48 hours. The embryos be
examined under a dissecting or
stereomicroscope to observe gross anatomy.
SUMMARY
The effects of glycoalkaloids from
immature, green tomato on chick
neurodevelopment will be determined in the
study. Several studies have shown that alkaloids
from different sources posed neural tube defects
on chick embryo. Some studies also showed
some other morphological deformities of a chick
that could be attributed to teratogenic exposure
upon central nervous development in chicks.
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This study will follow the methods conducted
by Guvenc et al. (2016) and Farjadmand, Eslami
and Sadighara (2011).
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