Aqueous Bark Extract of Ceiba speciosa (A. St.-Hill) Ravenna Protects against Glucose Toxicity in Caenorhabditis elegans

Abstract

Plants are widely used in folk medicine because of their pharmacological properties. Ceiba speciosa, popularly known as paineira-rosa or tree-of-wool, is a species found in the Northwest of Rio Grande do Sul, being native of the upper Uruguay River, Brazil. The tea obtained from the stem bark is employed in folk medicine to reduce cholesterol, triacylglycerides, and glucose levels. However, there are no studies in the literature proving its efficacy or the safety of its use. For this study, we used Caenorhabditis elegans as an animal model considering its advantages for risk assessment and pharmacological screenings. For the toxicological tests, C. elegans N2 (wild type) was treated with the aqueous extract of the stem bark of C. speciosa (ECE) at the first larval stage (L1) at concentrations of 5, 25, 50, and 250 μg/mL. To evaluate biological activities, we challenged the extract for oxidative stress resistance in the presence of paraquat (0.5 mM), H2O2 (1 mM), and against glucose-induced toxicity. Our results demonstrated that ECE did not alter survival rate, pharyngeal pumping, and reproduction of the nematodes. The extract was not able to protect the nematodes against the toxicity induced by prooxidants. Notably, ECE protected against glucotoxicity by increasing worms' life span and by reducing glucose levels. On the other hand, ECE treatment did not reduce lipid accumulation induced by exogenous glucose feeding, as observed in worms which lipid droplets were tagged with GFP. Based on our results, we believe that the extract is indeed promising for further studies focusing on carbohydrates metabolism; however, it needs to be carefully evaluated since the extract does not seem to modulate lipid accumulation.

Full text

Research Article
Aqueous Bark Extract of Ceiba speciosa (A. St.-Hill) Ravenna
Protects against Glucose Toxicity in Caenorhabditis elegans
Fabrine Bianchin dos Santos,
1
Caroline Brandão Quines ,
1
Luiz Eduardo Ben Pilissão,
1
Ana Helena de Castro Dal Forno,
1
Cristiane Freitas Rodrigues,
1
Cristiane Casagrande Denardin ,
1
Fabiane Moreira Farias,
2
and Daiana Silva Ávila
1
1
Laboratório de Pesquisa em Bioquímica e Toxicologia em Caenorhabditis elegans, Programa de Pós-Graduação em Bioquímica,
Universidade Federal do Pampa, Uruguaiana, Brazil BR 472-Km 592-Caixa Postal 118, CEP 97500-970
2
Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Pampa, Uruguaiana, Brazil BR 472-Km 592-
Caixa Postal 118, CEP 97500-970
Correspondence should be addressed to Daiana Silva Ávila; avilads1@gmail.com
Received 29 April 2020; Revised 1 July 2020; Accepted 25 July 2020; Published 8 October 2020
Academic Editor: Márcio Carocho
Copyright © 2020 Fabrine Bianchin dos Santos et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Plants are widely used in folk medicine because of their pharmacological properties. Ceiba speciosa, popularly known as paineira-
rosa or tree-of-wool, is a species found in the Northwest of Rio Grande do Sul, being native of the upper Uruguay River, Brazil. The
tea obtained from the stem bark is employed in folk medicine to reduce cholesterol, triacylglycerides, and glucose levels. However,
there are no studies in the literature proving its efficacy or the safety of its use. For this study, we used Caenorhabditis elegans as an
animal model considering its advantages for risk assessment and pharmacological screenings. For the toxicological tests, C. elegans
N2 (wild type) was treated with the aqueous extract of the stem bark of C. speciosa (ECE) at the first larval stage (L1) at
concentrations of 5, 25, 50, and 250 μg/mL. To evaluate biological activities, we challenged the extract for oxidative stress
resistance in the presence of paraquat (0.5 mM), H
2
O
2
(1 mM), and against glucose-induced toxicity. Our results demonstrated
that ECE did not alter survival rate, pharyngeal pumping, and reproduction of the nematodes. The extract was not able to
protect the nematodes against the toxicity induced by prooxidants. Notably, ECE protected against glucotoxicity by increasing
worms’life span and by reducing glucose levels. On the other hand, ECE treatment did not reduce lipid accumulation induced
by exogenous glucose feeding, as observed in worms which lipid droplets were tagged with GFP. Based on our results, we believe
that the extract is indeed promising for further studies focusing on carbohydrates metabolism; however, it needs to be carefully
evaluated since the extract does not seem to modulate lipid accumulation.
1. Introduction
Glucose toxicity refers to the biological effects of its excessive
levels in cells and tissues due to metabolic alterations and
production of advanced glycation end products (AGEs) [1].
AGES interact with lipids, proteins, and other molecules
causing cell damage by inducing oxidative stress and cell
death [2]. With the growing interest in finding natural anti-
oxidants and drugs with beneficial action on glucose metab-
olism, many extracts from different plants and their
different parts have been tested [3, 4]. However, the indis-
criminate use of natural products has led to cases of intoxica-
tion [5]. Therefore, a safety assessment of natural products
should be performed previously to medicinal use [4].
The scientific literature describes some medicinal proper-
ties for several species of the genus Ceiba. A study conducted
by Leal et al. [6] reported a considerable variety of secondary
compounds in the bark extract and antimicrobial activity
against strains of Staphylococcus aureus which may be related
to the presence of phytochemicals such as tannins and phe-
nols. In addition, Ceiba pentandra (L.) Gaertn, commonly
known as silk cotton tree, has been used by practitioners of
traditional medicine in Northern and Eastern Nigeria for dia-
betes control.
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2020, Article ID 1321354, 9 pages
https://doi.org/10.1155/2020/1321354

In this same pharmacological context, Ceiba speciosa (A.
St.-Hil.) has been used by the southern Brazilian population
[7]. The species is found in the Northwestern Rio Grande
do Sul, Brazil, being native in the Upper Uruguay vegetation
[8]. Regarding its use in folk medicine, there are records for
the use of barks and flowers for the treatment of heart dis-
ease, hypertension, and diabetes [9]. With the increasing
interest in the bioprospection of natural products, especially
of vegetal material originating from the Pampa Biome, the
validation and application of alternative models for the eval-
uation of these natural products becomes relevant.
Among the animal models that have been used to inves-
tigate the toxicology and pharmacology of medicinal plants is
the nematode Caenorhabditis elegans. This worm has a num-
ber of characteristics that make it not only relevant but also
quite powerful as a model for this type of research [10]. C. ele-
gans is easy and cheap to maintain in the laboratory with an
Escherichia coli diet, has a short reproductive cycle (3 days)
and a large number of descendants (~200), which allow the
large-scale production of animals within a short period of
time. The transparent body allows a clear observation of all
cells in these animals. Notably, C. elegans shows a strong con-
servation in molecular and cellular pathways with respect to
mammals and humans [11], including those signaling related
to glucose and lipid metabolism [12, 13].
Notably, this nematode does not have specialized storage
tissues such as adipocytes, and the energetic excess is stored
in lipid droplets in enterocytes, hypodermis, and gonads
[14, 15]. On the other hand, the absence of adipocytes
removes central components in the control of energetic
metabolism in mammals, which may be advantageous by
facilitating the interpretation of other conserved factors and
mechanisms involved in energy homeostasis [16, 17]. Thus,
this study is aimed at evaluating the safety of the aqueous
bark extract from Ceiba speciosa (ECE) as well as at verifying
the glucose-reducing levels action attributed to this plant
using C. elegans as an animal model.
2. Materials and Methods
2.1. Extract Preparation. The stem bark of C. speciosa was
collected in the municipality of Santo Antônio das Missões,
Rio Grande do Sul, Brazil, on April 2012. The plant material
was identified by a botanist, and an exsiccata or botanical
voucher was deposited in the Zoobotanic Herbarium
Augusto Ruschi at the University of Passo Fundo, under
the code RSPF 12637. C. speciosa barks were ground, and
the aqueous extract was prepared by decoction in distilled
water according to Malheiros et al. [18]. Since we have used
the exact same batch of lyophilized extract, the phytochemi-
cal composition has been already reported [18]. To expose
worms, the lyophilized material was diluted in water to
obtain a stock solution at 10 mg/mL that was subsequently
used for further dilutions.
2.2. Caenorhabditis elegans Maintenance. The C. elegans
strains used in this study were N2 (wild type) and VS29
(hjSi56 [vha-6p::3xFLAG::TEV::GFP::dgat-2::let-858 3′
UTR]) [19] which were obtained from Caenorhabditis
Genetics Center (CGC) and maintained in NGM (nematode
growth medium) seeded with E. coli OP50 at 20
°
C [20]. First
larval stage worms were obtained by a synchronization pro-
cess, which consists of exposing pregnant worms to the lysis
solution (0.45 N NaOH, 2% HOCl p/v) to separate the eggs
from the worms. After 14 h, the eggs hatched and released
the L1 larvae, used for exposures. All experiments were per-
formed at 22
°
C in a controlled humidified environment.
2.3. Treatment Protocol and LC
50
Determination. A total of
2,000 L1 nematodes were exposed to different concentrations
of C. speciosa bark extract (ECE-5 μg/mL, 25 μg/mL,
50 μg/mL, and 250 μg/mL) for 30 min at 22
°
C, in a homoge-
nizer. After treatment, the nematodes were washed with
85 mM NaCl solution for 3 times in order to terminate the
exposure, and then, nematodes were plated on NGM seeded
with E. coli OP50. Twenty-four hours after exposure, the
number of surviving nematodes was counted, and a survival
curve was drawn [21]. The experiments were performed in
duplicates. The results were expressed as % of control.
2.4. Brood Size. The nematodes were treated as described
above and were maintained on NGM/E. coli OP50 plates
until they reached larval stage L4. To evaluate progeny size,
one nematode from each ECE treatment was transferred to
a new plate containing NGM medium, and the total number
of progeny was counted during the whole reproductive
period [22]. The experiments were performed in triplicates
and independently repeated three times. The results were
expressed as % of control.
2.5. Pharyngeal Pumping. This assay is used to verify the die-
tary intake of the nematodes. 24 h after the treatment
described previously, 5 nematodes from each experiment
were transferred to new plates without bacteria. The pharyn-
geal pumps were counted for 1 min at the stereomicroscope
[23]. The experiments were repeated in three independent
assays. The results were expressed as % of control.
2.6. Oxidative Stress-Resistance Assays. Nematodes were pre-
treated at the L1 stage as described previously. After the last
wash, worms were posttreated with the prooxidants paraquat
(0.5 mM) or hydrogen peroxide (1 mM) for 30 min. Subse-
quently, nematodes were washed four more times and then
transferred to NGM/E. coli OP50 plates. The number of sur-
viving nematodes on each plate 24 h postexposure was scored
to determine stress resistance [10]. The experiments were
performed in duplicates and repeated at least 3 times. The
results were expressed as % of control.
2.7. Glucose Toxicity. In the pilot test, we have used 2 glucose
concentrations (2 and 4%) to verify whether they would
reduce worms’life span. Plates containing NGM medium
were inoculated with E. coli OP50 which were UV-
inactivated. Thereafter, glucose solution was added on top
of the dried E.coli. Based on pilot results, we have chosen glu-
cose 4% for the next assays. Worms were pretreated with dif-
ferent concentrations of th e extract (ECE 5 μg/mL or
250 μg/mL) for 30 min, washed to remove the treatments,
and then transferred to NGM/UV-inactivated E.coli plates
2 Oxidative Medicine and Cellular Longevity

containing glucose 4% or vehicle. Then, 20 live worms at the
same developmental stage were collected at the late L4 stage
and transferred daily to new plates NGM UV-inactivated
E.coli + glucose 4% or vehicle in order to prevent contamina-
tion or progeny. Live animals were scored until all animals
died. The experiments were performed in triplicates, and
three independent experiments were conducted. The results
were expressed as % of control.
2.8. Glucose Levels. The glucose content was quantified by
glucose oxidase method. Worms (N2) were treated as
described in Section 2.7 and kept in glucose 4% until they
reached the L4 stage. After these 48 h, worms were washed
offwith M9 buffer from the plates and centrifuged at
2500 rpm for 2 min. Worms were washed until removing all
existing bacteria. Then, worms were frozen and thawed 3
times, sonicated with lysis buffer, and centrifuged. The super-
natant (50 μL) was transferred to a 96-well plate, and glucose
levels were measured using a Labtest glucose colorimetric
assay kit (Labtest Diagnostica S.A., Minas Gerais, Brasil).
The optical density of each of the incubated samples
(10 min at 37
°
C) was measured at 505 nm. For normalization
of the data, protein was measured using Bradford colorimet-
ric method and expressed as mg glucose/μg protein. The
experiments were performed in duplicates and normalized
as % of control.
2.9. Glucose-Induced Lipid Accumulation. N2 and VS29 nem-
atodes were treated as described in Section 2.7, and worms
were kept on NGM/UV-inactivated E.coli OP50 plates
containing or not glucose 4% until reaching the larval
stage L4. After these 48 hours of glucose exposure, worms
were transferred to slides containing levamisole, and
images were acquired in a fluorescent microscope to
observe the effect of glucose exposure on lipid accumula-
tion. VS29 has a transgene which DGAT2 (acyl CoA:dia-
cylglycerol O-acyltransferase 2) is fused to GFP. This
enzyme transfers acyl groups and is localized in the lipid
particles; then, lipid droplets can be observed. N2 was
used as control to verify lipofuscin or autofluorescence
production caused by glucose treatment.
2.10. Statistical Analysis. Statistical analysis was carried out
by one-way analysis of variance (ANOVA), followed by post
hoc Tukey test when the overall p<0:05. For life span assay,
a repeated measure two-way analysis of variance (ANOVA)
followed by post hoc Tukey test was applied.
3. Results
3.1. Toxicity Evaluation of C. speciosa Aqueous Bark Extract.
Figure 1(a) shows that acute exposure to different concentra-
tions of ECE did not alter C. elegans survival rate (up to
250 μg/mL), thus suggesting that no toxic effects occurred
under the experimental conditions tested (acute). Based on
that, we decided to use the concentrations of 5 μg/mL,
25 μg/mL, 50 μg/mL, and 250 μg/mL for further assays. In
addition, we did not find any toxic effect of C. speciosa bark
extract on worms’reproduction, as brood size did not change
(Figure 1(b)). In order to verify whether ECE would affect
food ingestion per se, we analyzed pharyngeal pumping,
which was not changed as well (Figure 1(c)).
3.2. Stress Resistance. To verify a possible stress resistance
conferred by ECE, we exposed worms to different prooxi-
dants: paraquat (a pesticide, Figure 2(a)) or hydrogen perox-
ide (H
2
O
2
, Figure 2(b)). Our results demonstrate that ECE
did not protect against the damage induced by these prooxi-
dants at any of the tested concentrations.
3.3. ECE Treatment Protected against Glucose Toxicity. In
order to verify the protection of ECE treatment against glu-
cose toxicity, we analyzed life span. Post hoc analysis of the
data depicted in Figure 3 has revealed that 4% glucose
induced a reduction in worms’life span from day 5 until
day 14. The treatment with ECE at both concentrations (5
and 250 μg/mL) was effective against the glucose-induced
reduction in life span starting on day 6 until day 12. Two-
way ANOVA demonstrated a significant interaction between
treatment ×days (Fð54,162Þ=4:418;p<0:001), thus indicat-
ing a protection conferred by ECE throughout the life span.
In addition, glucose 4% administration significantly
increased glucose levels, and the treatment with ECE was
effective by reducing this elevation (Figure 4(b)). Notably,
ECE per se treatment did not alter glucose levels
(Figure 4(a)).
Furthermore, we took advantage of a GFP-tagged strain
and observed lipid accumulation induced by glucose vs.
ECE pretreatment. In wild-type (N2) worms, no increase or
decrease in fluorescence intensity caused by lipofuscin pro-
duction was evidenced, even in those worms exposed to
high glucose levels (Figures 5(a)–5(d)). On the other hand,
in VS29 mutants, a stronger fluorescence around the gut
following glucose 4% exposure was detected, therefore
indicating an increase in lipid droplets accumulation
(Figure 6(b)). However, ECE treatment did not reduce lipid
accumulation at any of the concentrations tested since no
significant reduction in GFP fluorescence was evidenced
(Figures 6(c) and 6(d)).
4. Discussion
In the present work, the safety and pharmacological activity
of C. speciosa aqueous bark extract were studied, since little
scientific knowledge about its effects are described in the lit-
erature. The consumption of this plant occurs in the North-
western region of the State of Rio Grande do Sul, Brazil,
which consumes it as a tea due to its alleged beneficial effects
in humans [9]. Our work verified that ECE is safe and pro-
tected against glucose-induced toxicity by reducing its levels
and the associated consequences in C. elegans life span.
The antioxidant potential of the extract has been previ-
ously demonstrated in vitro through the DPPH radical scav-
enging capacity and demonstrated a good scavenging
potential [18, 24]. The phytochemical analysis of the extract
used in this study was published by Malheiros and cols and
revealed that flavonoids (quercetin, rutin, and kaempferol)
and phenolic acids (gallic, chlorogenic, ellagic, and caffeic)
are present [18, 25].
3Oxidative Medicine and Cellular Longevity

Survival (% of control)
Control Paraquat 5 25 50 250
0
50
100
150
Paraquat+Ceiba
speciosa
(𝜇g/mL)
⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎
(a)
Survival (% of control)
Control H2O25 25 50 250
0
50
100
150
200
H2O2+Ceiba speciosa (𝜇g/mL)
⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
(b)
Figure 2: Effect of different concentrations of ECE treatment in oxidative stress resistance. (a) Response to oxidative stress in paraquat
posttreated nematodes (0.5 mM) and pretreated with ECE. Values are expressed as mean ± S:E:M:of six experiments. (b) Response to
oxidative stress in post-H
2
O
2
treated nematodes (1 mM) and pretreated with the ECE. Values are expressed as mean ± S:E:M:of three
experiments. Data were analyzed by using one-way ANOVA following by the Tukey post hoc test. ∗indicates p<0:05;∗∗ indicates
p<0:01;∗∗∗ indicates p<0:001 when compared to the control group.
Control 5 25 50 250
0
50
100
150
Ceiba speciosa (𝜇g/mL)
Survival (% of control)
(a)
0
100
200
300
Ceiba speciosa (𝜇g/mL)
Brood size (% of control)
Control 5 25 50 250
(b)
Ceiba speciosa (𝜇g/mL)
Control
52550250
0
50
100
150
Pharyngeal pumping
(% of control)
(c)
Figure 1: Toxicological evaluation of different concentrations of C. speciosa aqueous extract in (a) nematodes survival, (b) brood size, and (c)
pharyngeal pumping. Values are expressed as mean ± S:E:M:of four experiments. Data were analyzed by using one-way ANOVA following
by the Tukey test.
4 Oxidative Medicine and Cellular Longevity

⁎⁎⁎
⁎⁎⁎
##
⁎⁎⁎
⁎⁎⁎
⁎⁎
⁎⁎
###
⁎⁎⁎
⁎⁎
⁎⁎⁎
###
###
###
⁎⁎⁎
⁎⁎⁎
### ###
⁎⁎⁎
#
⁎⁎
#
⁎
5101520
0
20
40
60
80
100
Days
Longevity (% of control)
### ###
Control
Glucose
Glucose+5 𝜇g/mL
Glucose+250 𝜇g/mL
Figure 3: Effect of different concentration of ECE on glucose-induced toxicity in worms’life span. Values are expressed as mean ± S:E:M:of
four experiments. Data were analyzed by using a repeated measures two-way ANOVA followed by the Tukey posttest. ∗indicates p<0:05;
∗∗ indicates p<0:01;∗∗∗ indicates p<0:001 when compared to the control group.
#
indicates p<0:05;
##
indicates p<0:01;
###
indicates
p<0:001 when compared to the glucose group.
Ceiba speciosa (𝜇g/mL)
N2 5 25 50 250
0
50
100
150
Glucose/
𝜇
g of protein
(% of control)
(a)
Glucose/
𝜇
g of protein
(% of control)
N2 Glucose 5 25 50 250
0
50
100
150
200
250
Glucose+Ceiba speciosa
(𝜇g/mL)
⁎
###
#
#
(b)
Figure 4: Glucose levels in (a) following ECE treatment per se and (b) worms treated with ECE and exposed to glucose 4%. Values are
expressed as mean ± S:E:M:of four experiments. Data were analyzed by using one-way ANOVA following by the Tukey post hoc test.
∗indicates p<0:05 when compared to the control group.
#
indicates p<0:05;
###
indicates p<0:001 when compared to the glucose group.
5Oxidative Medicine and Cellular Longevity

In vivo, however, the extract did not show the same anti-
oxidant performance. We challenged ECE against two pow-
erful prooxidants, paraquat and H
2
O
2
, which have
significantly increased nematodes mortality. Paraquat is an
herbicide widely used in agriculture, but its biochemical
mechanism responsible for its toxicity is not fully
(a) (b)
(c) (d)
Figure 5: Representative images of N2 worms following glucose-induced lipid-accumulation and ECE treatment to observe autofluorescence:
(a) untreated worms; (b) worms treated with glucose 4%; (c) worms treated with ECE 5 μg/mL+ 4% glucose; (d) worms treated with ECE
250 μg/mL+ 4% glucose.
(a) (b)
(c) (d)
Figure 6: Representative images of VS29 worms following glucose-induced lipid-accumulation and ECE treated worms: (a) untreated worms;
(b) worms exposed to glucose 4%; (c) worms treated with ECE 5 μg/mL+ 4% glucose; (d) worms treated with ECE 250 μg/mL+ 4% glucose.
6 Oxidative Medicine and Cellular Longevity

understood. It is known that paraquat is a superoxide rad-
ical generator (O2·), which can produce other reactive spe-
cies such as hydrogen peroxide (H
2
O
2
) and hydroxyl
radical (·OH), which are unstable and react quickly with
fatty acids, causing lesions in the membranes, proteins,
and DNA. H
2
O
2
is one of the most versatile oxidants in
existence, superior to chlorine, chlorine dioxide, and
potassium permanganate; through catalysis, H
2
O
2
can be
converted to hydroxyl radical (·OH). It can easily pass
through cell membranes [26, 27]. Our results demon-
strated that despite the antioxidant action in vitro, the
treatment with ECE was not able to reduce the toxicity
induced by these prooxidants. In vivo, molecules can be
transformed, sometimes losing activities presented in some
in vitro assays, particularly those without tissue.
C. elegans contains many key metabolic-related com-
ponents such as oxidative stress and insulin signaling
pathways, making it a useful platform to improve our
understanding of complex diseases such as metabolic syn-
drome [13]. Glucose is the main energy source and key
regulator of animal metabolism; however, a chronic abun-
dance of glucose will have deleterious effects on cellular
and tissue functions [28]. Carbohydrate excess activates
several lipogenic enzymes contributing to deregulation in
lipid metabolism and insulin resistance development [29].
Furthermore, high levels of glucose induce cell injury of
mammalian hepatocytes and pancreatic cells through
molecular mechanisms of endoplasmic reticulum stress,
oxidative stress, and mitochondrial impairment [29, 30].
Moreover, chronic hyperglycemia can deteriorate cognitive
function and impair memory [31].
C. elegans is a suitable model organism to study glucose
toxicity, once high glucose conditions impair their life span
by increasing ROS formation and AGEs modification of
mitochondrial proteins in a DAF-2 independent manner
[32]. Furthermore, glucose-enriched diet significantly
decreases worms’life span by inhibiting the translocation of
DAF-16 and HSF-1 transcription factors that are involved
in antioxidant response [33]. On the other hand, glucose
restriction extends C. elegans life span by increasing the
expression of proaging and antioxidant genes [34].
We have found promising results when we challenged
ECE-treated worms against high glucose levels. The extract
was able to protect from the reduced longevity and to dimin-
ish whole body glucose levels induced by high glucose expo-
sure. A similar effect has been observed in the alloxan-
induced diabetic rats treated with B. ceiba bark methanolic
extract [35]. Furthermore, the alcoholic extract of C. pentan-
dra, a species taxonomically related to C. speciosa which is
commonly used in North Africa, has been used to control
hyperglycemia in diabetic patients [36]. Notably, its alcoholic
extract has been tested in rats, and it has been proven that
besides safe, decreases plasma glucose levels following oral
administration [37].
In relation to mechanism of action, the treatment with C.
pentandra decreased glucose levels in diabetic rats through
enhanced glucose utilization and regulation in insulin levels
[38, 39]. Similar to C. pentandra, we believe that the treat-
ment with ECE could enhance glucose utilization and stimu-
late another form of energy storage, like glycogen or
trehalose. Notably, C. elegans can synthesize saturated and
unsaturated fatty acids and is capable of storing energy as
lipids (triglycerides, phospholipids, sphingolipids, etc.). Up
to 35% of C. elegans dry body mass is lipids, being triacylgly-
ceride fat deposits the major energy storage molecules,
depending on diet and life stage [16]. To analyze that, we
have used a transgenic strain which acyl CoA:diacylglycerol
O-acyltransferase 2 is fused to GFP. This enzyme transfers
the acyl groups and is localized in the lipid particles; then,
lipid droplets can be observed in vivo. As expected, our
results have shown that high glucose administration
increased lipid accumulation in worms. However, ECE treat-
ment failed to reduce this storage, therefore indicating that
glucose levels reduced by the extract may have been con-
verted to lipids.
Finally, we observed that acute exposure at different
concentrations of ECE did not cause toxicological effects
on worms, as analyzed by survival rate, brood size, and
pharyngeal pumping. These findings confirm that worms
were ingesting the extract and feeding normally, indicat-
ing that the use of these concentrations is safe, consider-
ing the experimental conditions tested. Previous in vitro
data have also demonstrated that this same extract did
not reduce leukocytes viability and did not cause geno-
toxicity [18]. C. speciosa is a plant widely used by south-
ern Brazilians; therefore, the safety of its use by this
population is germane. Thus, for the first time, it was
demonstrated that the aqueous bark extract of C. speciosa
has low toxicity and has potential against glucose toxic-
ity, thus suggesting that ECE may be useful against
hyperglycemia in mammals. Therefore, additional studies
will be carried out by our group with the purpose of
expanding the hypoglycemic effects of C. speciosa and
its mechanisms.
Data Availability
All authors declare the availability of the use of data from this
manuscript by the journal Antioxidants and Prooxidants:
Effects on Health and Aging 2020.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
The authors are thankful to PROPPI/Universidade Federal
do Pampa (UNIPAMPA) for providing financial assistance.
DSA is recipient of CNPq researcher scholarship and sup-
ported by FAPERGS/PqG # 18/2551-0000434-0. AHCDF
and CFR were recipients of scholarships from Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 001. Some strains were provided
by the CGC, which is funded by NIH Office of Research
Infrastructure Programs (P40 OD010440).
7Oxidative Medicine and Cellular Longevity

Supplementary Materials
The supplementary material includes the graphical abstract
of the manuscript. (Supplementary Materials)
References
[1] X. Luo, J. Wu, S. Jing, and L. J. Yan, “Hyperglycemic stress and
carbon stress in diabetic glucotoxicity,”Aging and Disease,
vol. 7, no. 1, pp. 90–110, 2016.
[2] A. Pinkas and M. Aschner, “Advanced glycation end-products
and their receptors: related pathologies, recent therapeutic
strategies, and a potential model for future neurodegeneration
studies,”Chemical Research in Toxicology, vol. 29, no. 5,
pp. 707–714, 2016.
[3] S. Surveswaran, Y. Z. Cai, J. Xing, H. Corke, and M. Sun,
“Antioxidant properties and principal phenolic phytochemi-
cals of Indian medicinal plants from Asclepiadoideae and
Periplocoideae,”Natural Product Research, vol. 24, no. 3,
pp. 206–221, 2010.
[4] A. M. Pisoschi, A. Pop, C. Cimpeanu, and G. Predoi, “Antiox-
idant capacity determination in plants and plant-derived
products: a review,”Oxidative Medicine and Cellular Longev-
ity, vol. 2016, Article ID 9130976, 36 pages, 2016.
[5] K. Boubaker, M. Ounissi, N. Brahmi et al., “Acute renal failure
by ingestion of Euphorbia paralias,”Saudi Journal of Kidney
Diseases and Transplantation, vol. 24, no. 3, pp. 571–575,
2013.
[6] I. C. R. Leal, K. R. N. dos Santos, I. I. Júnior et al., “Ceanothane
and lupane type triterpenes from Zizyphus joazeiro–an anti-
staphylococcal evaluation,”Planta Medica, vol. 76, no. 1,
pp. 47–52, 2010.
[7] E. Beleski-Carneiro, J. A. Sugui, and F. Reicher, “Structural and
biological features of a hydrogel from seed coats of Chorisia
speciosa,”Phytochemistry, vol. 61, no. 2, pp. 157–163, 2002.
[8] L. Cappelatti and J. L. Schmitt, “Caracterização da flora
arbórea de um fragmento urbano de floresta estacional semi-
decidual no Rio Grande do Sul, Brasil, Pesquisas,”Botânica,
vol. 60, pp. 341–354, 2009.
[9] R. F. P. de Lucena, V. T. do Nascimento, E. de Lima Araújo,
and U. P. de Albuquerque, “Local uses of native plants in
an area of Caatinga vegetation (Pernambuco, NE Brazil),”
Ethnobotany Research and Applications, vol. 6, pp. 003–
014, 2008.
[10] J. H. An and T. K. Blackwell, “SKN-1 links C. elegans mesen-
dodermal specification to a conserved oxidative stress
response,”Genes & Development, vol. 17, no. 15, pp. 1882–
1893, 2003.
[11] C. Weinhouse, L. Truong, J. N. Meyer, and P. Allard, “Caenor-
habditis elegans as an emerging model system in environmen-
tal epigenetics,”Environmental and Molecular Mutagenesis,
vol. 59, no. 7, pp. 560–575, 2018.
[12] J. Zheng and F. L. Greenway, “Caenorhabditis elegans as a
model for obesity research,”International Journal of Obesity,
vol. 36, no. 2, pp. 186–194, 2012.
[13] J. L. Watts and M. Ristow, “Lipid and carbohydrate metabo-
lism in Caenorhabditis elegans,”Genetics, vol. 207, no. 2,
pp. 413–446, 2017.
[14] K. Ashrafi,“Obesity and the regulation of fat metabolism,”
WormBook: The Online Review of C. Elegans Biology, pp. 1–
20, 2007.
[15] K. Palikaras, M. Mari, B. Petanidou, A. Pasparaki,
G. Filippidis, and N. Tavernarakis, “Ectopic fat deposition
contributes to age-associated pathology in Caenorhabditis ele-
gans,”Journal of Lipid Research, vol. 58, no. 1, pp. 72–80, 2017.
[16] K. Ashrafi, F. Y. Chang, J. L. Watts et al., “Genome-wide RNAi
analysis of Caenorhabditis elegans fat regulatory genes,”
Nature, vol. 421, no. 6920, pp. 268–272, 2003.
[17] S. Hashmi, Y. Wang, R. S. Parhar et al., “AC. elegans model to
study human metabolic regulation,”Nutrition & Metabolism,
vol. 10, no. 1, p. 31, 2013.
[18] C. K. C. Malheiros, J. S. B. Silva, T. C. Hofmann et al., “Prelim-
inary in vitro assessment of the potential toxicity and antioxi-
dant activity of Ceiba speciosa (A. St.-Hill) Ravenna
(Paineira),”Brazilian Journal of Pharmaceutical Sciences,
vol. 53, no. 2, 2017.
[19] N. Xu, S. O. Zhang, R. A. Cole et al., “The FATP1-DGAT2
complex facilitates lipid droplet expansion at the ER-lipid
droplet interface,”The Journal of Cell Biology, vol. 198, no. 5,
pp. 895–911, 2012.
[20] S. Brenner, “The genetics of Caenorhabditis elegans,”Genetics,
vol. 77, no. 1, pp. 71–94, 1974.
[21] D. Avila, K. Helmcke, and M. Aschner, “The Caenorhabiditis
elegans model as a reliable tool in neurotoxicology,”Human
& Experimental Toxicology, vol. 31, no. 3, pp. 236–243, 2012.
[22] Y. Guo, Y. Yang, and D. Wang, “Induction of reproductive
deficits in nematode Caenorhabditis elegans exposed to metals
at different developmental stages,”Reproductive Toxicology,
vol. 28, no. 1, pp. 90–95, 2009.
[23] D. Raizen, B.-m. Song, N. Trojanowski, and Y.-J. You,
“Methods for measuring pharyngeal behaviors,”2005.
[24] M. N. Yesmin, S. N. Uddin, S. Mubassara, and M. A. Akond,
“Antioxidant and antibacterial activities of Calotropis procera
Linn,”American-Eurasian Journal of Agricultural & Environ-
mental Sciences, vol. 4, pp. 550–553, 2008.
[25] C. K. C. Malheiros, Avaliação Preliminar In Vitro do Potencial
Antioxidante e da Toxicidade de Ceiba Speciosa (a. St.-hill)
Ravenna (paineira), 2014.
[26] J. Azadmanesh and G. E. O. Borgstahl, “A review of the cata-
lytic mechanism of human manganese superoxide dismutase,”
Antioxidants, vol. 7, no. 2, p. 25, 2018.
[27] H. Younus, “Therapeutic potentials of superoxide dismutase,”
International Journal of Health Sciences, vol. 12, no. 3, pp. 88–
93, 2018.
[28] M. Soto-Garcia, M. Rosales-Castro, G. N. Escalona-Cardoso,
and N. Paniagua-Castro, “Evaluation of hypoglycemic and
genotoxic effect of polyphenolic bark extract fromQuercus
sideroxyla,”Evidence-Based Complementary and Alternative
Medicine, vol. 2016, Article ID 4032618, 7 pages, 2016.
[29] M. Mota, B. A. Banini, S. C. Cazanave, and A. J. Sanyal,
“Molecular mechanisms of lipotoxicity and glucotoxicity in
nonalcoholic fatty liver disease,”Metabolism, vol. 65, no. 8,
pp. 1049–1061, 2016.
[30] C. Liu, Y. Hao, F. Yin, Y. Zhang, and J. Liu, “Geniposide pro-
tects pancreatic βcells from high glucose-mediated injury by
activation of AMP-activated protein kinase,”Cell Biology
International, vol. 41, no. 5, pp. 544–554, 2017.
[31] M. J. Overman, N. Pendleton, T. W. O'Neill et al., “Glycemia
but not the metabolic syndrome is associated with cognitive
decline: findings from the European Male Ageing Study,”
The American Journal of Geriatric Psychiatry, vol. 25, no. 6,
pp. 662–671, 2017.
8 Oxidative Medicine and Cellular Longevity

[32] A. Schlotterer, G. Kukudov, F. Bozorgmehr et al., “C. elegans as
model for the study of high glucose- mediated life span reduc-
tion,”Diabetes, vol. 58, no. 11, pp. 2450–2456, 2009.
[33] S. J. Lee, C. T. Murphy, and C. Kenyon, “Glucose shortens the
life span of C. elegans by downregulating DAF-16/FOXO
activity and aquaporin gene expression,”Cell Metabolism,
vol. 10, no. 5, pp. 379–391, 2009.
[34] T. J. Schulz, K. Zarse, A. Voigt, N. Urban, M. Birringer, and
M. Ristow, “Glucose restriction extends Caenorhabditis ele-
gans life span by inducing mitochondrial respiration and
increasing oxidative stress,”Cell Metabolism, vol. 6, no. 4,
pp. 280–293, 2007.
[35] R. Zahan, L. Nahar, M. Haque, M. L. Nesa, and Z. Alam, “Anti-
oxidant and antidiabetic activities of Alangium salvifolium and
Bombax ceiba,”Dhaka University Journal of Pharmaceutical
Sciences, vol. 12, no. 2, pp. 159–163, 2015.
[36] A. Majeed, A. A. El-Sayed, T. Khoja, R. Alshamsan, C. Millett,
and S. Rawaf, “Diabetes in the Middle-East and North Africa:
an update,”Diabetes Research and Clinical Practice, vol. 103,
no. 2, pp. 218–222, 2014.
[37] C. K. Fofie, E. P. Nguelefack-Mbuyo, N. Tsabang, A. Kamanyi,
and T. B. Nguelefack, “Hypoglycemic properties of the aque-
ous extract from the stem bark of Ceiba pentandra in
dexamethasone-induced insulin resistant rats,”Evidence-
Based Complementary and Alternative Medicine, vol. 2018,
Article ID 4234981, 11 pages, 2018.
[38] R. Satyaprakash, M. Rajesh, M. Bhanumathy et al., “Hypogly-
cemic and antihyperglycemic effect of Ceiba pentandra l.
gaertn in normal and streptozotocin-induced diabetic rats,”
Ghana Medical Journal, vol. 47, no. 3, pp. 121–127, 2013.
[39] O. Ladeji, I. Omekarah, and M. Solomon, “Hypoglycemic
properties of aqueous bark extract of Ceiba pentandra in
streptozotocin-induced diabetic rats,”Journal of Ethnophar-
macology, vol. 84, no. 2-3, pp. 139–142, 2003.
9Oxidative Medicine and Cellular Longevity