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Safety evaluation and antiobesogenic effect of Sargassum liebmannii
J. Agardh (Fucales: Phaeophyceae) in rodents
Jorge Tapia-Martinez
1
&Karina Hernández-Cruz
2
&Margarita Franco-Colín
1
&Luz Elena Mateo-Cid
2
&
Catalina Mendoza-Gonzalez
2
&Vanessa Blas-Valdivia
3
&Edgar Cano-Europa
1
Received: 13 April 2018 / Revised and accepted: 29 January 2019
#Springer Nature B.V. 2019
Abstract
Sargassum has been used as a supplement diet in domestic animals with a hypolipidemic effect. However, Sargassum is a marine
alga that bioaccumulates heavy metals. Marine forests of Sargassum liebmannii develop on the Mexican coasts (North Pacific),
and it could be employed as a functional food. Nevertheless, it is necessary to prove its safety regarding intake. This study aimed
to examine S. liebmannii for chemical composition, heavy-metal quantification, acute and subchronic toxicities, and its
antiobesogenic effect. Sargassum liebmannii provides 790.24 kJ (100 g)
−1
and it bioaccumulates higher levels of arsenic
(11.2165 ± 0.2793 ppm) compared to zinc, nickel, chromium, copper, lead, cadmium, and mercury (0.0059–0.0437 ppm). The
acute toxicity was evaluated in C57BL/6J male mice, obtaining LD
50
>10gkg
−1
, and it did not produce any sign of toxicity
within 7 days of feeding without histological damage in the stomach, intestine, liver, and kidneys. For the subchronic toxicity and
antiobesogenic effect, a diet with 20% S. liebmannii was used in Sprague Dawley male rats for 11 weeks. During the study, the
animals fed the Sargassum diet did not show toxicity signs, but body weight gain and energy intake were reduced and insulin
sensitivity increased. During the end treatment, the adipose tissue decreased 31.5% from the control. The hematology, clinical
biochemistry, and the oxidative stress and cellular damage in the stomach, intestine, liver, and kidneys did not show alterations.
These results suggest that a S. liebmannii–supplemented diet (Sls-d) is safe and that it has an antiobesogenic effect in rodents.
Keywords Sargassum liebmannii .Phaeophyceae .Acute toxicity .Subchronic toxicity .Antiobesogenic effect
Introduction
Sargassum liebmannii J. Agardh (Fucales: Phaeophyceae) is a
brown seaweed that grows in exposed intertidal areas. It is
abundant on rocky coasts, where it grows in the littoral and
sublittoral zone, occupying intertidal or subtidal benthic areas
between 25 and 30 m deep, especially in cold, agitated, and
well-aerated waters. It develops in blooms of high biomass
along the coast of Guerrero state in Mexico (North Pacific).
Sargassum liebmannii is an important primary producer that
forms a true underwater forest that constitutes a favorable
environment for many marine animals (Phillips 1995).
Currently, S. liebmannii iscommonlyusedbycoastal
Mexican populations in Guerrero State as fertilizer, fodder,
and livestock feed, and to produce alginates, agar, or potassi-
um salts (Rodríguez-Montesinos et al. 2008; Marín et al.
2009).
Additionally, Sargassum spp. constitutes a potential func-
tional food source, as it produces secondary metabolites as
polyphenols, terpenoids, alkaloids, fatty acids, and sulfated
polysaccharides, among others, with pharmacological action
on lipid and carbohydrate metabolism, as well as body weight
regulation (Lee and Han 2018;Lietal.2018;Sanjeewaetal.
2018). Experimentally, when crustaceans and laying hens
consumed a Sargassum spp.–supplemented diet, the choles-
terol and saturated fatty acid levels were reduced. These
hypocholesterolemic and hypolipidemic effects improve the
nutritional value of the resulting food products (Casas-Valdez
et al. 2006; Carrillo et al. 2012). Also, Sargassum hystrix
extract can reduce cholesterol and triglyceride levels in dia-
betic rats (Gotama et al. 2018). With respect to lipid
*Edgar Cano-Europa
edgarcanoeuropa@yahoo.com.mx
1
Laboratorio de Metabolismo I, Departamento de Fisiología, Instituto
Politécnico Nacional, Mexico City, Mexico
2
Laboratorio de Ficología, Departamento de Botánica, Instituto
Politécnico Nacional, Mexico City, Mexico
3
Laboratorio de Neurobiología, Departamento de Fisiología. Escuela
Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,
Mexico City, Mexico
Journal of Applied Phycology
https://doi.org/10.1007/s10811-019-1752-y
metabolism in mammals, Sargassum spp. could prevent car-
diovascular disease, metabolic syndrome, and obesity
(Cardoso et al. 2015; Wan-Loy and Siew-Moi 2016).
Additionally, S. liebmannii can cause an antiobesogenic action
in mammals due to its high dietary fiber content which re-
duces calorie absorption in the intestines. Furthermore, the
fiber in the stomach causes a vagal–vagal response that delays
gastric emptying (Mohamed et al. 2012). With respect to
carbohydrate-based metabolism, Sargassum hystrix extract
has been demonstrated to avoid hyperglycemic and pancreatic
necrosis in diabetic rats (Gotama et al. 2018). Due to all the
benefits mentioned, S. liebmannii could be a promising alter-
native as a functional food with antiobesogenic effects.
However, it is crucial to be careful of consumption due to its
heavy-metal bioaccumulation property. Seaweeds
bioaccumulate heavy metals in their tissues through the ab-
sorption of contaminants in the water. Bioaccumulation oc-
curs because the cell wall contains polysaccharides such as
alginates, which contain anionic groups as sulfate, carboxyl,
or phosphate groups. All these chemical groups act as metal-
binding sites favoring accumulation in the seaweed (Bilal
et al. 2018; Poo et al. 2018). This seaweed’s biological prop-
erties have been employed in the bioremediation process of
some contaminated water bodies (Poo et al. 2018).
For all of the abovementioned information, it is necessary
to prove whether S. liebmannii is safe for domestic or human
intake as a functional food. This study aimed to examine
S. liebmannii collectedintheMexicanNorthPacific
(Guerrero, Mexico) for chemical composition, heavy-metal
quantification, acute and subchronic toxicities, and its
antiobesogenic effect.
This is the first study of the chemical proximal composition
and toxicological evaluation of S. liebmannii and that the re-
sults of this research provide relevant information for its pos-
sible application as a functional food.
Materials and methods
Sampling
Sargassum liebmannii was collected in the Majahua locality
in Guerrero State in the North Pacific (17° 48′08″N and 101°
44′55″W) in May 2013. It was collected using spatulas and
field knives, then it was washed twice with seawater to re-
move epibionts attached to the thallus. A total of 50 kg of
wet weight seaweed was collected and it was washed again
with double-distilled water to remove salts; it was subsequent-
ly dehydrated under an artificial drying light. Once dry, the
seaweed was ground in a Hammer Mill (M 20S3, IKA
Labortechnik) at 20,000 cycles for 10 min to obtain the pow-
dered algae. After that, the powder was passed through a
N100 sieve with a mesh size of 150 μm.
Sargassum liebmannii was taxonomically identified by
Angela Catalina Mendoza-Gonzalez and Luz Elena Mateo-
Cid from the Escuela Nacional de Ciencias Biologicas-
Instituto Politecnico Nacional by using relevant dichotomous
keys (Setchell 1924;Taylor1945). A voucher sample of
S. liebmannii was deposited in the herbarium collections of
the ENCB from I.P.N., Mexico City, with the accession num-
ber 19533.
Bromatological analysis
The bromatological analysis was performed following the
method established by the official Mexican standards for qua-
druplicate for moisture (NOM-116-SSA1-1994), lipids
(NMX-F-615-NORMEX-2004), saturated lipids (PROY-
NMX-Y-348-SCFI-2006), crude fiber (NOM-051-SCFI/
SSA1-2010), protein (NMX-F-608-NORMEX-2011), ash
(NMX-F-607-NORMEX-2002), total carbohydrates (NOM-
086-SSA1-1994), sugar (NOM-086-SSA1-1994), sodium
(NMX-F-150-S-1981), and energy supply (NOM-051-SCFI/
SSA1-2010).
Moisturecontentwasdeterminedbydrying1gof
S. liebmannii powder at 100° ±2 °C for 4 h using the constant
weight technique.
Lipids were extracted from 2 g of S. liebmannii powder
with 50 mL hexane in a Soxhlet system for 5 h. The satu-
rated lipids in 10 g of the sample were measured by titra-
tion with sodium thiosulfate (Asakai et al. 2007). A total of
10gofS. liebmannii powder was added to 100 mL of
chloroform, and it was shaken for 1 h. After that, the mix-
ture was filtered through 12.5-cm filter paper. Two aliquots
of 20 mL were taken. One aliquot was added to a crystal-
lizer. The solvent was evaporated in the crystallizer and
driedat130±2°Cfor1hbeforeitwascooledand
weighed. The weight obtained was used to calculate the
iodine index. The other aliquot was placed in an iodine
flask with 25 mL of Hanus reagent (Norris and Buswell
1943). The mixture was stirred for 1 h and then 10 mL of
15% KI solution and 100 mL of cold water were added.
Then, the previous solution containing 2 mL of the starch
solution was titrated with 0.1 N of Na
2
S
2
O
3
until the blue
coloration completely disappeared.
Crude fiber was obtained after treatment of 2 g of
S. liebmannii powder with 200 mL of boiling 1.25% H
2
S0
4
for 30 min. Then the sulfuric acid was neutralized with
200 mL of boiling 1.25% NaOH. The residue was filtered
using filter paper (Whatman 1001-185) and was dried at
130 ± 2 °C for 2 h using the constant weight technique.
Proteins were determined according to the Kjeldahl method
(Bradstreet 1954). A total of 1 g of S. liebmannii powder was
hydrolyzed with 25 mL concentrated H
2
SO
4
containing two
copper catalysts (2 g CuSO
4
and 10 g Na
2
SO
4
) in a heat block
(Kjeltec system 2020 digestor, Tecator, Inc.) at 420 °C for 2 h
JApplPhycol
until all of the material was charred. Then 450 mL of water
was added to the charred material and 4 zinc granules and
50 mL 25 N of NaOH were added. Immediately after this,
the flask was connected to a distillation system. In the refrig-
erant outlet, an Erlenmeyer flask containing 50 mL of 2%
H
3
BO
4
and 3 drops of Shiro Tashiro reagent (Short 1954)
received the product of distillation. This new solution was
distilled until all the ammonia had been removed; as a result,
a few drops of distillate did not show alkalinity with litmus
paper (about 300 mL). The first drops of distillate should have
turned the color of the indicator from violet to green. The
receiving flask and titrate the distillate with 0.1 N of HCl were
removed. The formula below was used to calculate the nitro-
gen percentage:
%Nitrogen ¼ðmL of HCl 0:1 N HCl 14:007
2gofSargassum powder 100
where the 14.007 is the molecular weight of nitrogen.
Ash was obtained by incinerating 1 g of S. liebmannii pow-
der at 550 ± 2 °C in a muffle furnace and only inorganic matter
(ashes) remains. The ash contents were measured by measur-
ing the weight of inorganic matter remaining.
The total carbohydrates were estimated using the following
formula:
Carbohydrate ¼100−percentage of protein;lipids;moisture;ash;and fiberðÞ
The sugars were determined using the Lane and Eynon
method (Alexander et al. 1989) for reducing sugars in 10 g
of a sample. A total of 10 g of the Sargassum powder was
added to 200 mL of hot water (56–60 °C). It was stirred for
30 min after that; 4 mL of 1.18M of zinc acetate solubilized in
3% of acetic acid and 4 mL of 0.25 M of K
4
Fe (CN)
6
·3H
2
O
were then added. The mixture was adjusted to 250 mL in a
volumetric flask, then it was filtrated, and 25 mL of the solu-
tion was added to 30 mL of 1 N of HCl. The mixture was
incubated at 96 °C for 15 min and then cooled, and 3 drops of
1% of phenolphthalein were added. It was then neutralized
with 30 mL of 1 N of NaOH until the pink hue disappeared.
The S. liebmannii mixture was titrated in a solution containing
0.6 M of sodium and potassium tartrate, 0.02 M of Cu
2
SO
4
,
0.125 M of NaOH, and 1% methylene blue until the blue hue
disappeared. The following formula was used to calculate
sugar content:
Sugar ¼250 100 Fehling factor
mL sample 10 g of Sargassum powder
This method employed 0.27 M of sucrose solubilized in
5.6% HCl as a standard. The standard was used to calculate
the Fehling factor through titration, an indicating solution con-
taining 0.6 M of sodium and potassium tartrate, 0.02 M of
Cu
2
SO
4
, 0.125 M of NaOH, and 1% methylene blue. The
Fehling factor was obtained as follows:
Fehling factor ¼mL sucrose standard
dilution of sucrose mgðÞ
Sodium was determined using 0.15 g of S. liebmannii pow-
der in 75 mL of water; then, the mixture was boiled at 96 °C
for 15 min. The mixture was cooled to 56 °C, and it was added
to1mLof5%ofK
2
CrO
4
as an indicator solution. The mix-
ture was titrated with 0.1 N of AgNO
3
until it turned orange.
The %sodium was calculated using the equation below:
%sodium ¼0:0585 mEq NaCl ððVsVbÞ
0:15g Sargassum powder 100
where V
s
=volumeofAgNO
3
in sample titration (mL) and
V
b
= volume of AgNO3 in titration of the blank.
Energy supply was calculated using the below formula:
Energy supply kJðÞ¼%lipids 37:65 kJðÞ
þ%proteins 16:73 kJðÞ
þ%total carbohydrates 16:73 kJðÞ
Heavy metal quantification
Heavy metals were assessed in S. liebmannii at the Centro
Interdisciplinario de Investigaciones y Estudios Sobre el
Medio Ambiente y Desarrollo (CIIEMAD, I.P.N). A total
of1gofS. liebmannii powder by triplicate was used and
was digested with 1:3:10 (v/v) HN0
3
–HCl–H
2
O
2
using a
microwave digestor (Paar Physica Multiwave Six Place)
at300Wfor5minandthenat600Wfor10min,which
proved satisfactory. After digestion, the samples were
allowed to cool at room temperature for 20 min and then
they were diluted to 10 mL with deionized water. Digests
were stored in labeled polyethylene vials at 0–5°Cuntil
the metal concentration analysis took place. The concen-
trations of As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn were
determined using an atomic absorption spectrophotometer
(Perkin Elmer Analyst 100) and using the direct flame
aspiration technique. All the metals used for the measures
employed its respective standard.
Ethical consideration
All experimental procedures described in this study were in
accordance with the guidelines of the laws and codes ap-
proved in the seventh title of the General Law of Health re-
garding Health Research of the Mexican Government (NOM-
062-ZOO-1999) which details the technical specifications for
the production, care, and use of laboratory animals. The ani-
mals were housed in a room with controlled lighting (8:00–
JApplPhycol
20:00, lights on), temperature (21° ± 1 °C), and relative hu-
midity (40–60%). Three mice were housed in an acrylic cage
withavolumeof20×30×12cm.Theratsweresingly
housed in a metallic cage (20× 30 × 18 cm). The cages were
arranged in racks to maintain visual, auditory, and olfactory
contact. Animals received a pelletized commercial rat chow
diet (Laboratory Rodent Diet 5001, LabDiet) and water ad
libitum. The internal bioethical committee approved this
protocol.
LD
50
acute toxicity assay
LD
50
of S. liebmannii were designed in accordance with the
method provided by the Organization for Economic
Cooperation and Development (OECD 2001)andLorke
method (Lorke 1983). Twelve male C57BL/6J mice were ran-
domly divided into four groups:
1) Vehicle (0.9% saline solution (SS) oral gavage route
(o.g.r.))
2) 0.5 g kg
−1
of S. liebmannii powder o.g.r., which was
administrated from an 0.008 g mL
−1
mixture of the pow-
der suspended in 0.9% SS
3) 1gkg
−1
of S. liebmannii powder o.g.r., which was ad-
ministrated from a 0.016 g mL
−1
mixture of the powder
suspended in 0.9% SS
4) 10 g kg
−1
of o.g.r. S. liebmannii powder, which was ad-
ministrated from a 0.16 g mL
−1
mixture of the powder
suspended in 0.9% SS
We did not administrate doses higher than 10 g kg
−1
of
S. liebmannii powder because the suspension was gelled and
could not be administrated using the o.g.r.
Themicewereobservedduringthefirst30minafter
administration.Thentheywereobservedevery3hforthe
first day, and then every day until the seventh day after
administration. The toxicity signs observed included
piloerection, eye and mucous membrane irritation, motor
abnormalities, convulsions, tremors, lethargy, salivation
alteration, coprophagy, respiratory alteration, diarrhea, co-
ma, and death.
The animals that received the S. liebmannii powder were
euthanized 7 days after administration by cervical dislocation,
and the stomach, intestine, liver, and kidneys were dissected
for histological studies.
Subchronic toxicity test
The subchronic toxicity assay was based on the method de-
scribed by the WHO guide (WHO 2000)andtheOECD
guidelines for chemical tests (OECD 1998).
Fourteen Sprague Dawley male rats were randomly divid-
ed into two groups:
1) Control group
2) 20% Sargassum liebmannii–supplemented diet (Sls-d)
Briefly, the Sls-d was prepared using an 80% ground com-
mercial diet (Laboratory Rodent Diet 5001, LabDiet), which
was mixed with 20% of S. liebmannii powder. The mixture
was homogenized with 10% distilled water and oval pellets
were formed (2.5× 1 ×1.6 cm). They were dried at 50 °C for
12 h. Table 1shows the dietary composition of the control and
Sls-d groups.
The rats were observed daily for 77 days for mortality or
clinical toxicity. Observation included signs, such as
piloerection, eye and mucous membrane irritation, motor ab-
normalities, convulsions, tremors, lethargy, salivation alter-
ation, coprophagy, respiratory alteration, diarrhea, mucosal
cyanoses, phalanges cyanoses, increased capillary refill, leth-
argy, coma, and death. In addition, body weight and energy
intake were monitored on a weekly basis. Animals were eu-
thanized using 35 mg kg
−1
i.p. of monosodic pentobarbital
before the heart stopped beating; blood was collected for all
the hematological and biochemical analyses. Additionally, the
stomach, duodenum, liver, and kidneys were preserved for
biochemical and histological studies.
Hematology and biochemical evaluations
Blood was analyzed to determine hematocrit level, hemoglo-
bin concentration, and lymphocytes number (mm
3
).
Hemoglobin levels were evaluated using the Randox kit. In
the serum samples, the aspartate aminotransferase (glutamic-
oxaloacetic transaminase or GOT), alanine aminotransferase
(glutamic pyruvic transaminase or GPT), total and direct bil-
irubin, uric acid, creatinine, cholesterol, HDL, LDL, and
VLDL levels were measured enzymatically using a commer-
cial assay kit (Randox).
Oxidative stress markers assay
Oxidative stress markers were measured in the stomach, in-
testine, liver, and kidneys of rats used for the subchronic tox-
icity test. The samples were homogenized in 3 mL of 10 mM
phosphate buffer with a pH of 7.4 using a manual tissue ho-
mogenizer. Protein concentration in the homogenates was
measured using the Bradford method (Bradford 1976).
Lipid peroxidation (LP) was determined by the formation
of a soluble fluorescent lipid, and 1 mL of the homogenates
was added to 7 mL of chloroform:methanol (2:1, v/v), stirred
for 15 s, and cooled on ice for 15 min to allow for phase
separation. The chloroformic phase was measured in an
RF5000U Shimadzu Spectrofluorometer at 370- nm
(excitation) and 430- nm (emission) wavelengths. The spec-
trophotometer sensitivity was adjusted to 140 units of fluores-
cence with a quinine sulfate solution containing 1 μgmL
−1
JApplPhycol
and 0.005 M of H
2
SO
4
. Results were expressed as relative
fluorescence units (RFU) per milligram of protein.
Reactive oxygen species were determined by the formation
of 2′,7′-dichlorofluorescein (DCF). A total of 10 μLofthe
homogenates were added to 1940 μL of Tris:Hepes (18:1)
and incubated in the presence of 50 μLof2′,7′-
dichlorofluorescein diacetate (DCFH-DA) for 1 h at 37 °C;
freezing stopped the reaction. The fluorescence was measured
in a RF5000U Shimadzu Spectrophotometer at 488- nm
(excitation) and 525- nm (emission) wavelengths. The results
were expressed as picomole of DFC formed per milligram of
protein per hour (Rodríguez-Sánchez et al. 2012; Memije-
lazaro et al. 2018).
Histological analysis
The stomach, intestine, liver, and kidneys from the mice
(acute toxicity) and rats (subchronic toxicity) were fixed in a
10% buffered formalin for 48 h, and they were embedded in
paraffin. Five-micrometer section slices were obtained with a
standard microtome (LEICA RM 2145, Germany), and the
slices were then stained with hematoxylin-eosin (H–E) and
examined by an expert pathologist blinded to the treatment
groups using an optical microscope.
Effect of a 20% S. liebmannii–supplemented diet on body
weight regulation
The effect of a Sls-d on body weight regulation was evaluated
for 11 weeks in the rats used in the subchronic toxicity study.
Body weight and energy intake were determined on a weekly
basis. One day prior to being euthanized, the animals fasted
for 6 h and were tested for glucose tolerance using 1.8 g kg
−1
o.g.r. dextrose (J. T Baker) to determine the glucose curve at 0,
30, and 60 min using glucose strips (Abbot) and a glucometer
(Optium Xceed). Upon being euthanized atweek 11, the lead-
ing stores of adipose tissue (mesenteric, retroperitoneal, and
epididymal) were dissected to determine the adiposity levels
with respect to the body weight of each animal (total adipose
tissue × 100 g
−1
of body weight).
Statistical analysis
To test the bromatological analysis and for heavy metals, data
were expressed as the mean ± standard error (SE). All vari-
ables studied were described using the mean and standard
error. A Mann–Whitney Utest was used to analyze hematocrit
and the area under curve (AUC). A Student ttest was used to
test hemoglobin, lymphocytes, GOT, GPT, total bilirubin, di-
rect bilirubin, uric acid, creatinine, cholesterol, HDL, LDL,
VLDL, oxidative stress markers, blood glucose, and adiposity
levels. A two-way RM ANOVA test and Student–Newman–
Keuls post hoc test were used to analyze body weight and
energy intake. It was considered a statistically significant dif-
ference when P<0.05.
Results
In Table 2, the bromatological analysis and heavy concentra-
tion are presented. This table shows an energy supply of
790.24 kJ (100 g)
−1
of S. liebmannii. Meanwhile, the heavy-
metal analysis shows a concentration (in decreasing order) of
As, Zn, Ni, Cr, Cu, Hg, Pb, and Cd of between 11.2165 and
0.0059 ppm.
Table 1 Dietary composition of control (Laboratory Rodent Diet 5001,
LabDiet) and 20% Sargassum liebmannii supplemented-diet
Diet
component
Control
(LabDiet) (%)
20% Sargassum liebmannii–
supplemented diet (%)
Protein 23.90 20.75
Carbohydrates 48.70 47.34
Fat 5.00 4.08
Crude fiber 5.10 8.48
Minerals 6.6 9.72
Sodium 0.4 0.66
Moisture 10.30 8.97
Energy supply (kJ g
−1
) 16.73 15.15
Table 2 Proximate chemical composition, gross energy and heavy
metals contents of S. liebmannii
Determination Values g (100 g)
−1
Drying loss 16.6 ± 0.086
Lipids 0.35 ± 0.023
Saturated lipids 0.10 ± 0.001
Crude fiber 18.59 ± 0.03
Protein 10.30 ± 0.08
Ash 18.76 ± 0.061
Total carbohydrates 35.35 ± 0.055
Sugars 1.00 ± 0.001
Sodium 1.47 ± 0.026
Energy supply 790.24± 0.68 kJ (100 g)
−1
Heavy metals Concentration (ppm)
As 11.2165 ± 0.2793
Cd 0.0059 ± 0.0001
Cr 0.0217 ± 0.0068
Cu 0.0192 ± 0.0005
Hg 0.0138 ± 0.0005
Ni 0.0229 ± 0.0017
Pb 0.0098 ± 0.0011
Zn 0.0437 ± 0.0027
Data are expressed as mean ± SE, (n=3)
JApplPhycol
Fig. 1 Acute toxicity of 10 g kg
−1
of Sargassum liebmannii.
Photomicrography of the
stomach, intestine, liver, and
kidney from C57BL/6J male
mice. × 40. Horizontal line
represents 50 μm. In the
Sargassum liebmannii,itwas
observed the epithelium of the
stomach mucosa or in the gastric
glands (GG) without abnormal
changes. The intestine does not
present changes in the mucosa or
in the submucosa (SM), the
integrity of the epithelial intestinal
villi (E) and the intestinal glands
is conserved (IG), also, arrows
showing goblet cells. In the liver,
it is presented the typical
formation of hepatocyte cords
arranged (HC) around a
centrilobular vein (CV). In
respect to the kidney, maintains
the normal cytoarchitecture in the
nephrons, glomerulus (G), and the
proximal (PCT) and distal
convoluted tubules (DCT)
Table 3 Effect of S. liebmannii on hematological parameters, hepatic and renal functions
Vari a b l e G r o u ps
Control 20% Sargassum liebmannii
Hematocrit (%) 47.69 ± 2.032 51.24 ± 1.501
Hemoglobin (g dL
−1
) 9.45 ± 0.005 9.45 ± 0.001
Lymphocyte number (cells mm
−3
) 12,430 ± 372.9 13,930 ± 660.8
GOT (UI L
−1
) 115.08 ± 7.55 110.23 ± 8.12
GPT (UI L
−1
) 42.21 ± 0.81 47.56 ± 1.54
Total bilirubin (mg dL
−1
) 1.71 ± 0.32 1.6 8 ± 0.28
Direct bilirubin (mg dL
−1
) 0.48 ± 0.07 0.5 9 ± 0.06
Uric acid (mg dL
−1
) 3.82 ± 0.13 3.9 3 ± 0.23
Creatinine (mg dL
−1
) 1.2 5 ± 0.03 1.30 ± 0.09
Cholesterol (mg dL
−1
) 86.23 ± 11.35 70.42 ± 14.39
HDL (mg dL
−1
) 26.95 ± 10.19 27.98 ± 2.92
LDL (mg dL
−1
) 29.93 ± 11.69 20.77 ± 18.33
VLDL (mg dL
−1
) 16.21 ± 2.62 17.45 ± 0.85
Data are expressed as mean ± SE, (n=7).Mann–Whitney Utest for hematocrit percentage and Student’sttest for the rest of determinations
JApplPhycol
Furthermore, S. liebmannii acute toxicity assay did not
show any signs of toxicity in the mice. Also, no death oc-
curred in any of the doses evaluated. Thus, the LD
50
>
10 g kg
−1
. There was no effect on the histological analysis
in the S. liebmannii group with the higher dose (10 g kg
−1
).
No histological damage in the epithelium of the stomach mu-
cosa or in the gastric glands was observed. The mucosa and
submucosa intestine did not show any histological changes,
and the integrity of the intestinal villi and the glands was
conserved. In the liver photomicrographs, typical hepatocyte
formation appeared in the cords arranged around a
centrilobular vein, and the kidneys showed normal
cytoarchitecture in all nephron parts (Fig. 1).
The subchronic toxicity test of 20% Sls-d showed no signs
of toxicity or death throughout the experiment. No changes in
the hematocrit, hemoglobin, or lymphocyte counts were ob-
served. To evaluate whether Sls-d administration had a bio-
chemical effect, the GOT, GPT, total and direct bilirubin, uric
acid, creatinine, cholesterol, LDL, and VLDL plasmatic con-
centrations were determined. None of these variables were
affected (Table 3).
Figure 2shows the histological analysis of the stomach,
intestine, liver, and kidneys of those with the Sls-d, in addition
to the quantification of reactive oxygen species and lipid per-
oxidation. The histological analysis does not show any toxic
effects on the tissues evaluated, and the oxidative stress
markers were not affected; an inclusive Sls-d decreases ROS
values in the stomach (73.99%) and kidney (41.68%) with
respect to the control group.
Figure 3shows the antiobesogenic effects of a Sls-d. The
groups with Sls-d showed a lower body weight gain (17.64%),
lower energy intake (17.24%), higher glucose tolerance
(23.91%), and lower levels of adiposity (31.48%) with respect
to the control group.
Discussion
The S. liebmannii employed in this study was collected in the
Majahua locality in Guerrero State in the North Pacific (17°
48′08″N and 101° 44′55″W) near the Petacalco thermal
power station (17° 59′01.14″N, 102° 06′55.58″Win
Guerrero, Mexico). This oceanic zone (Majahua locality) pre-
sents water circulation coming from the ocean currents, such
as the North Equatorial Current, the California Current, and
the Costa Rica Current. For this reason, the water of the algae
collection area could contain high concentrations of arsenic
and metal traces (Cd, Pb, Hg, Cu, Cr, Ni, Zn) from an anthro-
pogenic source (Petacalco thermal power station). This could
result in S. liebmannii bioaccumulating heavy metals (De la
Lanza-Espino et al. 2004). Currently, no regulations are in
place for heavy-metal limits in macroalgae used to feed do-
mestic animals or for human consumption. However, we use
CODEX 193-1995 as a reference to compare the heavy-metal
limits in S. liebmannii, because algae and marine bivalves
have similar heavy-metal chelation and adsorption to the bio-
accumulation process (Bryan and Langston 1992; Codex
Alimentarious 2009). Our results show that the heavy-metal
content in S. liebmannii powder is within the allowable limits
(Cd < 2 ppm, Pb < 0.3 ppm, Hg < 5 ppm), except for arsenic,
whose values exceed the allowed limit (3 ppm). The
macroalgae phytochelatin (a polypeptide of (γ-glu-cys-)
n
-
gly) sequesters heavy inorganic metals in the water and con-
tributes to the bioaccumulation process. However, the heavy
metals there are not bioavailable because the algal alginate
covalently binds with heavy metals. Meanwhile, the complex
alginate containing heavy metals could be excreted through
feces because mammals do not have an enzymatic complex
with which to degrade it (Gekeler et al. 1988; Shanura et al.
2018). With respect to arsenic, the seaweed can also bind
arsenic to cell walls as arsenosugars, which are not bioavail-
able for mammals because gastric acid and gastric and intes-
tine enzymes do not hydrolyze them (Chávez-Capilla et al.
2016). Despite having found the presence of arsenic in
S. liebmannii, the values found were below those reported in
other species of the genus Sargassum,suchasS. pallidum,
S. fusiforme,S. thunbergii,andS. vachellianum,asshownin
Table 4(Miao et al. 2014; Pan et al. 2018). The other
Sargassum species that we compared heavy-metal content to
were collected in China because all the metals that we mea-
sured in North American Sargassum species were not report-
ed. Based on the above, S. liebmannii could be used for human
consumption or to feed domestic animals, but we sought to
ensure this idea by assessing acute and subchronic toxicity
assays to evaluate the safety of S. liebmannii.
LD
50
was obtained in the acute toxicological study, and it
was higher than 10 mg kg
−1
without causing clinical signs of
toxicity or cellular damage in organs, such as the stomach,
intestine, liver, and kidneys. Thus, the acute administration of
high doses of S. liebmannii powder was considered slightly toxic
because it has been reported that LD
50
between5and15gkg
−1
of a new product or chemical substance is considered class 4
(Hodge and Sterner 2005). We did not prove a higher
S. liebmannii concentration because the alginate avoids solubi-
lizing the powder, but the cellular damage assay in the higher
doses of S. liebmannii powder (10 g kg
−1
) demonstrated the
safety of the seaweed used. The histological study of the stom-
ach, intestine, liver, and kidneys showed normal cytoarchitecture
without damage compared to the control group.
To provide further certainty to the study and to ensure the
seaweed’s safety, a subchronic toxicity test was performed on
the rats. Experimental animals were fed 20% Sls-d over the
course of 11 weeks, and they did not show any toxicity clinical
signs or die. Also, the hematologic, hepatic, and renal func-
tions did not differ from the control group. The rats fed a Sls-d
were assessed at the end of the treatment for oxidative stress,
JApplPhycol
and a histological test was used to increase the toxicological
study’s sensitivity. A toxical substance generates an imbalance
between the production of reactive oxygen species and the
capacity of the biological systems to compensate for the oxi-
dative process to avoid cellular damage during the
toxicokinetic and toxicodynamic processes (Albert et al.
2012). When the system does not compensate for the bio-
chemical imbalance alterations, membrane lipoperoxidation,
protein nitrosylation, and DNA damage occur. All these bio-
chemical events are carried out through cellular damage and
tissue dysfunction (Valko et al. 2005; Jomova and Valko
2011). We showed that oxidative stress markers are reduced
in 20% Sls-d animals. This reduction in oxidative stress could
be because the seaweed has antioxidants, such as polyphenols,
which inhibit cell oxygenases and reduce oxidizing agents in
the body (Conner and Grisham 1996), thus acting as antioxi-
dants. Thus, we could suggest that a 20% Sls-d is safe.
However, some parameters would be necessary to completely
ensure the safety of this algae, such as measuring the concen-
tration of the heavy metals eliminated in feces and urine and
contained inthe biological tissues of the animals fed the Sls-d.
Furthermore, the antiobesogenic effect of a Sls-d is due to
the high presence of dietary fiber in the algae, such as the
alginic acid that is present in the cell walls of brown algae
(Rhein-Knudsen et al. 2017). This soluble fiber becomes hy-
drated in the intestinal tract, and it has been reported that this
ef
ij
mn
0.00
0.05
0.10
0.15
0.20
Control
Slsd
*
c
ROS (pmoles DCF formed
mg protein
-1
h
-1
)
0.00
0.05
0.10
0.15
0.20
*
d
Lipidperoxidation
(FRU mg proteín
-1
)
0.0
0.5
1.0
1.5
2.0
g
ROS (pmoles DCF formed
mgprotein
-1
h
-1
)
0.00
0.05
0.10
0.15
0.20
h
Lipidperoxidation
(FRU mg proteín
-1
)
0.0
0.5
1.0
1.5
2.0
k
ROS (pmoles DCF formed
mg protein
-1
h
-1
)
0.00
0.05
0.10
0.15
0.20
l
Lipid peroxidation
(FRU mg proteín
-1
)
0.0
0.5
1.0
1.5
2.0
o
ROS (pmoles DCF formed
mg protein
-1
h
-1
)
*
0.00
0.05
0.10
0.15
0.20
Lipid peroxidation
(FRU mg proteín
-1
)
p
Stomach
Intestine
Liver
Kidney
ab
Fig. 2 Determination of subchronic toxicity of a 20%
S. liebmannii–supplemented diet in Sprague Dawley male rats.
Photomicrography of the stomach (A, B), intestine (E, F), liver (I, J),
and kidney (M, N), ×40 and oxidative stress (ROS: C, G, K, O, and
lipid peroxidation: D, H, L, P) markers. Data represent the mean ± SE
(n=7),*P< 0.05. Student’sttest. Photomicrography of the control group
and 20%. S. liebmannii–supplemented diet, for the stomach: submucosa
(SM), gastric glands (GG). Intestine: submucosa (SM), intestinal glands
(IG), arrows showing goblet cells. Liver: centrilobular vein (CV),
hepatocyte cords (HC). Kidney: glomerulus (G), proximal convoluted
tubules (PCT), distal convoluted tubules (DCT). Horizontal line
represents 50 μm
JApplPhycol
hydrocolloid delays gastric emptying and intestinal absorp-
tion, reducing the vago-vagal reflex and causing satiety
(Clark and Slavin 2013). Moreover, the hydrocolloid formed
in the gastrointestinal tract slows the digestion and absorption
of lipids and carbohydrates. This process reduces the caloric
intake in the intestine (Slavin 2005; Howarth et al. 2009).
S. liebmannii presented a higher fiber content (18.5%) than
was reported in other species of Sargassum (10.2–11.4%), so
the effect on body weight regulation could be more noticeable
(Marín et al. 2009).
Time (Weeks)
Body weight (g)
1234567891011
150
200
250
300
350
400
450
Control
Slsd
a
**
*
1234567891011
100
200
300
400
500
Tim e (W eeks)
Energy intake (KJ d
-1
)
b
****
*
**
*
***
03060
0
50
100
150
200
250
Time (min)
Blood glucose (mg dL
-1
)
c
*
0
1
2
3
4
Adiposity levels (%)
*
d
Control Slsd
20000
30000
40000
50000
*
AUC
Control Slsd
2000
3000
4000
5000
*
AUC
Control Slsd
4000
6000
8000
10000
AUC
*
Control Slsd
Fig. 3 Effect of 20% S. liebmannii–supplemented diet on body weight
(a), energy intake (b), glucose tolerance (c), and adiposity levels (d). Data
represent the mean ± SE (n=7); *P< 0.05. RM two-ANOVA and
Student–Newman–Keuls post hoc to body weight. Energy supply and
glucose tolerance, and total adipose tissue and adiposity levels were de-
termined by Student’sttest and the AUC by Mann–Whitney Utest
Table 4 Heavy-metal concentrations in different species of the Sargassum genus
Metal Sargassum
liebmannii
Sargassum fusiforme
(Pan et al. 2018)
Sargassum thunbergii
(Pan et al. 2018)
Sargassum vachellianum
(Pan et al. 2018)
Sargassum pallidum
(Miao et al. 2014)
As (ppm) 11.2165 ± 0.2793 57.71 ± 13.44 49.08 ± 2.46 23.77 ± 3.88 122.3 ± 32.6
Cd (ppm) 0.0059 ± 0.0001 1.71 ± 0.17 5.63 ± 0.12 8.48 ± 0.29 ND
Cr (ppm) 0.0217 ± 0.0068 0.85 ± 0.11 3.84 ± 0.47 1.21 ± 0.12 ND
Cu (ppm) 0.0192 ± 0.0005 7.93 ± 0.89 12.81 ± 0.83 8.59 ± 0.23 3.94 ± 1.12
Hg (ppm) 0.0138 ± 0.0005 ND ND ND ND
Ni (ppm) 0.0229 ± 0.0017 2.87 ± 0.38 6.04 ± 0.53 3.11 ± 0.38 ND
Pb (ppm) 0.0098 ± 0.0011 1.50 ± 0.62 2.00 ± 0.24 1.90 ± 0.39 7.74 ± 4.10
Zn (ppm) 0.0437 ± 0.0027 20.87 ± 6.69 40.69 ± 4.67 81.03 ± 12.90 ND
ND not determined. Data are expressed as mean ± SE
JApplPhycol
Additionally, it has been reported that algae of the
Sargassum genus present with high amounts of fucoxanthin,
which is a carotenoid with an antiobesogenic effect (Maeda
et al. 2005;Pengetal.2011). Fucoxanthin reduces body
weight because it has a hypocholesterolemic and hypolipemic
effect on plasmatic and hepatic lipid concentrations.
Fucoxanthin has been determined to reduce the lipolysis in
adipocytes and regulate cholesterol-regulating enzymes in he-
patocytes (Gammone and D’Orazio 2015). The animals eating
a20%Sls-d had a higher glucose tolerance because fucoxan-
thin improves insulin sensitivity and reduces plasma glucose
levels (Aeda et al. 2007).
Conclusion
Toxicological studies and their antiobesogenic effect show the
potential of S. liebmannii as a functional food and its possible
use for anti-obesity therapies. However, due to the high con-
centration of arsenic found, it would be necessary to carry out
bioavailability studies to analyze the content of arsenic elim-
inated in feces and urine and if accumulation occurs in organs
and tissues.
Acknowledgements This study was partially supported by CONACyT
(221057) and SIP-IPN (20181115; 20180911, 20181768). We thank
INSTITUTO POLITÉCNICO NACIONAL, CONACyT for the financial
support and CIIEMAD for the technical support. The researchers are
fellows of EDI, COFAA, and SNI. Edgar Cano-Europa thanks
COTEBAL for its support this year.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’snoteSpringer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
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