ORIGINAL RESEARCH
published: 09 May 2022
doi: 10.3389/fnut.2022.888360
Antioxidant and Anti-inflammatory
Extracts From Sea Cucumbers and
Tunicates Induce a Pro-osteogenic
Effect in Zebrafish Larvae
Alessio Carletti 1,2† , Carlos Cardoso 3,4† , Jorge Lobo-Arteaga 5,6 , Sabrina Sales 3 ,
Diana Juliao 3 , Inês Ferreira 3 , Paula Chainho 6 , Maria Ana Dionísio 6 , Maria J. Gaudêncio 5 ,
Cláudia Afonso 3,4 , Helena Lourenço 3 , M. Leonor Cancela 1,2,7,8 , Narcisa M. Bandarra 3,4
and Paulo J. Gavaia 1,2*
Edited by:
Marcello Iriti,
University of Milan, Italy
Reviewed by:
Massimo Mariotti,
University of Milan, Italy
Sutapa Biswas Majee,
NSHM Knowledge Campus, India
*Correspondence:
Paulo J. Gavaia
[email protected]
† These authors have contributed
equally to this work and share first
authorship
Specialty section:
This article was submitted to
Food Chemistry,
a section of the journal
Frontiers in Nutrition
Received: 02 March 2022
Accepted: 31 March 2022
Published: 09 May 2022
Citation:
Carletti A, Cardoso C,
Lobo-Arteaga J, Sales S, Juliao D,
Ferreira I, Chainho P, Dionísio MA,
Gaudêncio MJ, Afonso C,
Lourenço H, Cancela ML,
Bandarra NM and Gavaia PJ (2022)
Antioxidant and Anti-inflammatory
Extracts From Sea Cucumbers
and Tunicates Induce
a Pro-osteogenic Effect in Zebrafish
Larvae. Front. Nutr. 9:888360.
doi: 10.3389/fnut.2022.888360
Frontiers in Nutrition | www.frontiersin.org
1
Faculty of Biomedical Sciences and Medicine (FCBM), University of Algarve, Faro, Portugal, 2 Centre of Marine Sciences,
University of Algarve, Faro, Portugal, 3 Division of Aquaculture, Upgrading and Bioprospection, Portuguese Institute
for the Sea and Atmosphere (IPMA), Algés, Portugal, 4 Interdisciplinary Centre of Marine and Environmental Research
(CIIMAR/CIMAR), University of Porto, Porto, Portugal, 5 Division of Environmental Oceanography, Portuguese Institute
for the Sea and Atmosphere, Algés, Portugal, 6 Marine and Environmental Sciences Centre (MARE), NOVA University
of Lisbon, Lisbon, Portugal, 7 Algarve Biomedical Center (ABC), University of Algarve, Faro, Portugal, 8 Centre for BioMedical
Research (CBMR), University of Algarve, Faro, Portugal
Bone metabolic disorders such as osteoporosis are characterized by the loss of mineral
from the bone tissue leading to its structural weakening and increased susceptibility
to fractures. A growing body of evidence suggests that inflammation and oxidative
stress play an important role in the pathophysiological processes involved in the
rise of these conditions. As the currently available therapeutic strategies are often
characterized by toxic effects associated with their long-term use, natural antioxidants
and anti-inflammatory compounds such as polyphenols promise to be a valuable
alternative for the prevention and treatment of these disorders. In this scope, the
marine environment is becoming an important source of bioactive compounds with
potential pharmacological applications. Here, we explored the bioactive potential of
three species of holothurians (Echinodermata) and four species of tunicates (Chordata)
as sources of antioxidant and anti-inflammatory compounds with a particular focus on
polyphenolic substances. Hydroethanolic and aqueous extracts were obtained from
animals’ biomass and screened for their content of polyphenols and their antioxidant
and anti-inflammatory properties. Hydroethanolic fractions of three species of tunicates
displayed high polyphenolic content associated with strong antioxidant potential and
anti-inflammatory activity. Extracts were thereafter tested for their capacity to promote
bone formation and mineralization by applying an assay that uses the developing
operculum of zebrafish (Danio rerio) to assess the osteogenic activity of compounds.
The same three hydroethanolic fractions from tunicates were characterized by a strong
in vivo osteogenic activity, which positively correlated with their anti-inflammatory
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Carletti et al.
Osteoactivity of Holothurians and Tunicates Extracts
potential as measured by COX-2 inhibition. This study highlights the therapeutic
potential of polyphenol-rich hydroethanolic extracts obtained from three species of
tunicates as a substrate for the development of novel drugs for the treatment of bone
disorders correlated to oxidative stress and inflammatory processes.
Keywords: osteoporosis, natural bioactives, polyphenols, sea cucumbers, tunicates, anti-inflammatory,
antioxidants, osteogenic
INTRODUCTION
properties, and have an appreciable market all around the world
(17, 18). Sea cucumbers were previously demonstrated to be able
to synthesize, among others, anti-inflammatory and antioxidant
compounds (19, 20).
Ascidians, tunicates belonging to the Class Ascidiacea, are
another group of marine invertebrates believed to have bioactive
potential. Overall, more than a thousand secondary metabolites
have been isolated from this group of organisms and several
were reported to have biological activity, including antioxidant
(21, 22).
Although these two groups of marine animals have been
demonstrated to hold great pharmacological potential, a relative
scarcity of literature explored their capacity to promote
bone formation and mineralization. In the present work,
we evaluated the bioactivity of extracts produced from 3
species of sea cucumbers belonging to the Genus Holothuria –
Holothuria (Roweothuria) arguinensis (Koehler and Vaney, 1906),
H. (Panningothuria) forskali (Delle Chiaje, 1823), and H.
(Holothuria) mammata (Grube, 1840), and from 4 species of
ascidians belonging to the Families Styelidae – Styela plicata
(Lesueur, 1823), Botrylloides diegensis (Ritter and Forsyth, 1917);
Polyclinidae – Aplidium sp.; and Cionidae – Ciona robusta
(Hoshino and Tokioka, 1967) collected from the western coast of
Portugal. We extracted the water- and ethanol-soluble phases and
explored their antioxidant potential, focusing on their content of
polyphenolic compounds, and their anti-inflammatory capacity.
Because of the high content of polyphenols and in vitro biological
activities observed, the extracts were also tested for their capacity
to induce bone formation and mineralization in vivo by using
a screening system based on zebrafish [Danio rerio, Hamilton
(1882)] larvae, which was previously developed to assess the bone
anabolic and pro-osteogenic activity of molecules and extracts, as
validated by Tarasco et al. (23).
Non-communicable diseases, also known as chronic diseases, are
the utmost burden of the global health systems being by far
the major cause of morbidity and deaths (1). Metabolic bone
disorders characterized by reduced bone mineral density (BMD),
i.e., osteoporosis and osteopenia, are by far the most common
diseases related to bone, with a global incidence of 40% in
people over the age of 50 (2, 3). The etiology of osteoporosis
has been extensively studied over the past decades and a full
picture of the underlying molecular mechanisms is being drawn.
As such, a growing body of evidence has stressed the importance
of inflammation and oxidative stress. The most common
type of osteoporosis, for instance, type 1 or postmenopausal
osteoporosis, is considered to have pathophysiological roots in
the dysregulation of inflammatory processes prompted by the
age-related decrease of estrogen levels (4–6).
Nevertheless, current therapeutic approaches for the
treatment of osteoporosis do not target to re-equilibrate
ROS unbalance nor resolve chronic inflammation. Instead,
they rely on anti-resorptive drugs that target osteoclastsinduced bone resorption, or on bone anabolic drugs, which
act by pharmacologically increasing osteoblasts-assisted
mineral deposition. Despite being successfully implemented in
osteoporotic patients, these therapies are still characterized by
long-term use-associated side effects and do not lend themselves
to the treatment of lifelong chronic conditions like the case of
osteoporosis (7).
In this context, natural compounds with antioxidant and
anti-inflammatory properties may represent alternative tools
for the prevention and treatment of osteoporosis and other
chronic diseases (8, 9). In this regard, polyphenols such as
flavonoids, and phenolic acids are among the better known
naturally occurring antioxidant and anti-inflammatories and
have raised the interest of the pharmaceutical industry due to
their therapeutic potential for the treatment of chronic disorders
(10–12).
In the last decades, the marine environment has emerged as an
interesting opportunity to discover new bioactives, and the wide
metabolic diversity characterizing marine organisms has drawn
the attention of the pharmaceutical industry (13–15). Different
classes of marine organisms yielded the discovery of bioactive
compounds, with marine invertebrates being the most prolific
group (16).
Among these, sea cucumbers, a group of echinoderms
belonging to the Class Holothuroidea, are particularly promising.
They have long been used in traditional medicine by the
communities of Eastern Asia, believed to have health-beneficial
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MATERIALS AND METHODS
Animal Collection and Identification
Sea Cucumbers
Fresh samples of sea cucumbers were captured by scuba
diving in the coastal zone between Sesimbra and Sado Estuary
(Setúbal, Portugal; 38◦ 25′ 23.50′′ N; 9◦ 0′ 45.06′′ W), from January
to July 2019. A total of 208 animals were captured, Holothuria
(Roweothuria) arguinensis (n = 62), H. (Panningothuria) forskali
(n = 64) and H. (Holothuria) mammata (n = 82). After dissection
and removal of internal organs and celomic fluid, specimens
were cleaned under running water, minced, and stored at –
80◦ C until analysis.
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Osteoactivity of Holothurians and Tunicates Extracts
were homogenized with a UNIDRIVE X1000D Homogenizing
System (CAT, Deerfield, United States) at 30,000 rpm for 1 min
and then left on an orbital shaker (VWR International, Radnor,
United States) overnight (15 h) to extract phenolic compounds.
The next day, extracts were centrifuged at 1,000 g for 5 min to
allow the insoluble material to precipitate and the supernatant
was transferred into new 50 mL Falcon tubes. Centrifugation
was repeated 3 times. HE and AQ extracts were aliquoted
and a half was immediately used for polyphenol determination
and antioxidant activity, while the other half was evaporated
and completely dried before testing anti-inflammatory activity
and osteogenic activity. Fractions were then evaporated using
a rotatory evaporator RV 10 digital (IKA-Werke GmbH & Co.,
Staufen, Germany), setting a temperature of 40◦ C for HE extracts
and 50◦ C for AQ extracts, and then stored at –80◦ C. Before
exposure to the fish, extracts were resuspended in their vehicle,
ethanol (Merck, Darmstadt, Germany) and Milli-Q water (pH
7.4), respectively.
Tunicates
Samples of 4 different tunicates species—Styela plicata, Aplidium
sp., Botrylloides diegensis, and Ciona robusta—were collected by
hand as epibionts of mussels cultured in the mussel aquaculture
rafts of the Albufeira lagoon. The Albufeira lagoon is a semienclosed lagoon located 20 km south of Lisbon on a mesotidal
area with a NE-SW orientation to the coast. The mussel rafts are
located in the main water bodies of Lagoa Grande with an average
depth of 4–5 m.
Phylogenetic Identification
Specimens were identified to the lowest possible taxonomic
level through integrative taxonomy, combining morphological,
and genetic approaches, when necessary. For the morphological
approach, a stereomicroscope and identification keys were used.
For the genetic approach, a small piece of tissue of each
organism was used to extract total DNA using the E.Z.N.A
Mollusc DNA Kit (Omega Bio-Tek, Norcross, United States),
following the manufacturer’s instructions. An iCycler (Bio-Rad,
Hercules, United States) thermal cycler was used to amplify
the fragment of the mitochondrial gene cytochrome c oxidase
subunit I gene (COI-5P), using a pre-made PCR mix (Invitrogen,
Waltham, United States). Each PCR reaction contained 2.5 µL
of 10× Taq polymerase buffer, 0.75 µL of 50 mM MgCl2,
0.5 µL of 10 mM dNTP mixture, 0.1 µL of 5 U/µL of Taq
DNA polymerase, 1.5 µL (10 µm) of each primer (LoboF1
5′ -KBTCHACAAAYCAYAARGAYATHGG-3′ and LoboR1 5′ TAAACYTCWGGRTGWCCRAARAAYCA-3′ ) (24), 1.25 µL of
1% W-1, 4 µL of DNA template and sterile Milli-Q water to
make up a total volume of 25 µL. The conditions of the PCR
thermal cycling were as follows: (1) 5 min at 94◦ C; (2) 5 cycles:
30 s at 94◦ C, 90 s at 45◦ C, 60 s at 72◦ C; (3) 45 cycles: 30 s
at 94◦ C, 90 s at 54◦ C, 60 s at 72◦ C; (4) 5 min at 72◦ C. The
amplified PCR products were purified using magnetic beads
and subsequently sequenced bidirectionally with the BigDye
Terminator 3 kit on an ABI 3730XL DNA analyzer (Applied
Biosystems, Waltham, United States) by StabVida. Trace files
obtained were carefully analyzed and sequences were aligned
using MEGA version X. GenBank BLASTn search (25) and
BOLD Identification System tool (BOLD-IDS) (26), were used
for matching sequences. The specimens and sequence data
obtained in this study is compiled in the Barcode of Life Data
Systems project titled “SCUTU—Properties of extracts from
sea cucumbers and tunicates.” GenBank accession numbers are
presented in Supplementary Table 1.
Total Polyphenols Content
Polyphenols content was determined in the AQ and HE
extracts by colorimetry determination with phosphomolybdicphosphotungstic acid with a protocol adapted from Singleton
and Rossi (27). The assay is based on the reactivity of the
Folin-Ciocalteu reagent, which consists of a yellow acidic
solution containing complex polymeric ions formed from
phosphomolybdic and phosphotungstic acids. In an alkaline
solution, this reagent oxidizes phenolic compounds and is
reduced producing a complex molybdenum-tungsten blue
pigment, which can be spectrophotometrically detected by
reading the absorbance at λ = 750 nm. The total polyphenols
content is calculated relatively to the reactivity of gallic acid
(3,4,5-trihydroxy benzoic acid), a known phenolic compound,
under the same conditions. Folin-Ciocalteu reagent (Merck) was
diluted 1:1 in Milli-Q water before utilization. For a volume
of 100 µL of HE or AQ extract, 600 µL of Milli-Q water and
150 µL of Folin-Ciocalteu reagent were added to test tubes.
The mixture was then vortexed and incubated in the dark
for 5 min at room temperature. Subsequently, 750 µL of 2%
sodium carbonate (Na2 CO3 ) prepared in water were added to
each test tube. Mixtures were then vortexed and incubated
in the dark for 90 min at room temperature. Each reaction
was performed in triplicate for each extract. Absorbance was
then detected at 750 nm using a UV-Vis spectrophotometer
Helios Alpha-model (Unicam, Cambridge, United Kingdom).
The calibration curve was calculated by using standard solutions
of gallic acid (Merck) —0.025, 0.05, 0.1, 0.2, 0.3 mg/mL—
obtained by serial dilutions from a stock solution (1 mg/mL)
prepared in ethanol for both AQ and HE extracts. Total
polyphenols content was assessed in triplicates and calculated
from the gallic acid calibration curve as milligrams of gallic
acid-equivalent (GAE) polyphenols per gram of dry biomass
(mg GAE/100 g dw).
Hydroethanolic and Aqueous Extraction
The samples of sea cucumbers and tunicates were freeze-dried in
a Heto PowerDry PL3000 Freeze Dryer (Thermo Fisher Scientific,
Waltham, United States) for 72 h. Freeze-dried biomass was
grounded into a fine powder with a Retsch GM200 ball mill
(Retsch GmbH, Haan, Germany). Hydroethanolic (HE) and
aqueous (AQ) extracts were prepared through liquid extraction.
Biomass powder was weighed and transferred into 50 mL Falcon
tubes covered with aluminum foil to prevent photo-oxidative
processes to take place and solubilized in distilled water and
96% ethanol-water mixture, respectively. A biomass-solvent ratio
of 30 mL of solvent per 1 g of biomass was used. Solutions
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Biological Activities
Antioxidant Activity
Antioxidant potential was assessed for the HE and AQ extracts
from sea cucumbers and tunicates. Free radical scavenging
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in Milli-Q water and incubating the mixture in the dark for 16 h
at room temperature. Because ABTS and potassium persulfate
reacts stoichiometrically at a ratio of 1:0.5, this results in
incomplete oxidation of the ABTS and production of ABTS+• .
After the incubation, the ABTS+• solution was diluted by adding
5 mM phosphate-buffered saline (PBS), pH 7.4 until obtaining a
final absorbance of 0.7 ± 0.02 at 734 nm. For a volume of 20 µL
of HE or AQ extract, 2.0 mL of ABTS+• solution was added to
test tubes. The mixture was then vortexed and incubated in the
dark for 6 min at 37◦ C in a water bath. Absorbance was detected
again at 734 nm using a UV-Vis spectrophotometer Helios
Alpha-model (Unicam). The calibration curve was calculated
by using standard solutions of Trolox (Merck) (0, 100, 250,
500, 1,000, and 2,000 µM) obtained by serial dilution from a
stock Trolox solution (3 mM) prepared in 96% ethanol for AQ
and HE. ABTS+• radical scavenging capacity was calculated as
micromoles of equivalents of Trolox per 100 g of dry biomass
(µmol eq TROLOX/100 g dw).
capacity was assessed with two assays based on a single electron
transfer reaction (FRAP and ABTS+ methods), and one assay
based on a hydrogen atom transfer reaction (DPPH method),
which are considered complementary methods for the evaluation
of antioxidant capacity (28, 29). Antioxidant activities were
analyzed in triplicate for all the tests performed.
Ferric Reducing Antioxidant Power Assay
The ferric reducing antioxidant power (FRAP) was evaluated
for HE and AQ extracts with a protocol based on the method
developed by Benzie and Strain (30) and adapted by Martins
et al. (31). The test is based on a redox reaction occurring
between the substrate (electron donor) and Fe3+ ions (electron
acceptor), producing Fe2+ ions. This reduction is monitored
spectrophotometrically by the change in the color of the solution
of Fe3+ with TPTZ [2,4,6-tris(2-pyridyl)-s-triazine], which turns
blue and absorbs electromagnetic radiation at λ = 595 nm. The
ferric reducing antioxidant power of the extracts is calculated
as related to the absorbance of a Fe2+ standard solution of
iron sulfate (FeSO4 ), tested in parallel. The FRAP reagent was
prepared by mixing acetate buffer (0.3 M), TPTZ (10 mM) (AlfaAesar, Cityward Hill, United States), and FeCl3 ·6H2 O (20 mM)
(Merck) in the ratio 10:1:1. For a volume of 100 µL of HE
or AQ extract, 3.0 mL of the FRAP reagent was added to
test tubes and the mixture was then incubated in the dark
for 30 min at 37◦ C in a water bath. The absorbance of the
samples was read in comparison to the blank (prepared by
adding the FRAP reagent to 96% ethanol and water for AQ and
HE extracts, respectively) at a wavelength of 595 nm, using a
UV-Vis spectrophotometer Helios Alpha-model (Unicam). The
calibration curve was prepared using standard solutions of FeSO4
(0.25, 0.5, 1, 1.5, 2 mM) obtained by serial dilution from a stock
solution (2 mM). All measurements were conducted in triplicate
and the results were expressed as micromoles of equivalents of
iron sulfate per g of dry biomass (µmol eq FeSO4 /g dw).
DPPH Assay
Radical scavenging activity of AQ and HE extracts against the
stable radical DPPH• (2,2-diphenyl-2-picrylhydrazyl hydrate)
was determined spectrophotometrically by following a protocol
adapted from Miliauskas et al. (33). When DPPH• reacts with
an antioxidant compound able to donate a hydrogen proton,
it is reduced to DPPH-H, and the color change which results
can be detected by measuring the reduction of absorbance at
λ = 517 nm. The scavenging capacity against the stable DPPH•
radical is determined relative to the reactivity of ascorbic acid
as an antioxidant standard, under the same conditions. DPPH
solution (0.15 mM) was prepared by dissolving 11.8 mg of
DPPH reagent (2,2-diphenyl-1-picrylhydrazyl, 95%, Alfa-Aesar)
into 200 mL of methanol in a volumetric flask. For 1 mL of
HE or AQ extract, 2.0 mL of the DPPH solution was added to
test tubes. Solutions were vortexed and incubated for 30 min
in the dark at room temperature. Reduction in the absorbance
at 527 nm was detected with a UV-Vis spectrophotometer
Helios Alpha-model (Unicam). The absorption of a blank
sample containing the same amount of DPPH• solution and
ethanol 96% or water was measured. The experiment was
carried out in triplicate for each extract and the controls
(water and 96% ethanol for AQ and HE extracts, respectively).
A calibration curve with ascorbic acid (Merck) was drawn by
reading absorbance (517 nm) of standard concentrations of
ascorbic acid (5, 10, 15, and 20 mg/L) obtained by serial dilution
from the higher concentration (20 mg/L) and a calibration
curve equation was calculated for both HE and AQ extracts.
Radical scavenging capacity was calculated as milligrams of
equivalents of ascorbic acid per 100 g of dry biomass (mg
eq AA/100 g dw).
ABTS+ Assay
The antiradical capacity of HE and AQ extracts was evaluated
by following their effect on the stable free cation radical
ABTS+• [2,2′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic
acid)], following the protocol used by Re et al. (32). The assay
is based on the scavenging capacity of antioxidants against
ABTS+• radical cation, a blue/green chromophore, which has
multiple absorbance maxima at 645 nm, 734 nm, and 815 nm.
ABTS+• is firstly produced by the reaction between ABTS and
potassium persulfate (K2 S2 O8 ). The addition of antioxidants
reduces it to ABTS resulting in the decolorization of the solution
which is monitored via reading the reduction of the absorbance
at λ = 734 nm. The scavenging capacity of the ABTS+• radical
cation is determined as a function of concentration and time
and calculated relative to the reactivity of Trolox (6-hydroxy2,5,7,8-tetramethychroman-2-carboxylic acid) as antioxidant
standard, under the same conditions. ABTS, [2,2′ -azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt,
and potassium persulfate (K2 S2 O8 ) were obtained from Merck.
ABTS+• solution (7 mM) was prepared by dissolving 10 mg of
ABTS into a potassium persulfate solution (2.45 mM) prepared
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Anti-Inflammatory Activity
Anti-inflammatory activity was evaluated by the cyclooxygenase2 (COX-2) inhibitory assay, an established biomarker of
inflammation processes and target of common non-steroidal
anti-inflammatory drugs (NSAID) (34). HE and AQ extracts
were subjected to heat treatment (80◦ C during 1 h) and
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Osteoactivity of Holothurians and Tunicates Extracts
centrifuged (3,000 g at 4◦ C for 10 min). The supernatant
was collected and the solvent was evaporated using a vacuum
rotary evaporator with the water bath temperature at 65◦ C.
The residue was directly dissolved in 100% dimethyl sulfoxide
(DMSO, Merck) to prepare a stock solution with a concentration
of 10 mg·mL−1 . Extracts were tested at 1 mg·mL−1 using
a commercial COX-2 inhibitory screening assay kit, Cayman
test kit-560131 (Cayman Chemical Company, Ann Arbor,
United States). A volume of 10 µL of each of the tested extracts or
DMSO (blank) was used. Results were expressed as a percentage
of inhibition of COX-2. Anti-inflammatory activity was analyzed
in quadruplicate.
images were analyzed using ImageJ software version 2.0.0-rc69/1.52p. For morphometric analysis, the images were processed
by using the toolbox available within the “ZFBONE” macro
toolset for Fiji (35). The area of the operculum and the area
of the head were digitally measured with the support of an
Intous M drawing tablet (Wacom, Ōtone, Japan). The ratio of
the operculum area on the head area was calculated as the
final value as the area of the head is proven to be the most
accurate morphological parameter to normalize inter-specimen
size variations (23).
Statistical Analysis
For all the experiments, normality was tested with a D’AgostinoPearson omnibus normality test or with an Anderson-Darling
test (p < 0.05). Homoscedasticity was tested through the BrownForsythe test (p < 0.05). When the distribution of the data of
all the experimental groups resulted normal and homogeneous,
statistical differences between the control and the extracts were
tested with a one-way ANOVA followed by Dunnett’s multiple
comparison test (p < 0.05). If the distribution of the data of
any of the experimental conditions resulted non-normal or nonhomogeneous, statistical differences between the control and
the extracts were tested with a non-parametric test followed by
Dunn’s multiple comparison test (p < 0.05). Statistical analyses
were performed using Prism version 8.00 (GraphPad Software,
Inc. La Jolla, United States).
Acute Toxicity and Osteogenic Activity
Adult zebrafish (AB wild-type strain) were crossed by using an
in-house breeding program. Fertilized eggs were transferred into
plastic 1 L tanks with static water conditions and maintained
until hatching, at 3 days post-fertilization (dpf), with the
following conditions: temperature 28 ± 0.1◦ C, pH 7.5 ± 0.1,
conductivity 700 ± 50 µS, NH3 and NO2 lower than 0.1 mg/L,
NO3 at 5 mg/L and a photoperiod of 14:10 h light-dark. Fish
water was prepared by adding a salt mixture (Instant Ocean,
Blacksburg, United States) and sodium bicarbonate to reverse
osmosis treated water in order to maintain stable pH and
conductivity. The osteogenic activity was evaluated for HE and
AQ extracts as described by Tarasco et al. (23). The process
is schematized in Figure 1. Briefly, at 3 dpf, hatched embryos
(n = 15) were transferred into 6 well-plates and treated with
each extract and their vehicle as the control group (ethanol
0.1% for HE and water for AQ extracts). Calcitriol (1α,25dihydroxy vitamin D3 , Sigma-Aldrich, St. Louis, United States),
was used as a positive control at a concentration of 10 pg/mL in
ethanol. Extracts were tested in repetitive experiments at different
concentrations and developmental toxicity was evaluated in
order to calculate the maximum non-lethal dose (MNLD) over
a 72 h period. All extracts were initially solubilized in either
ethanol or water at the higher concentration possible. For all
extracts, the upper limit of solubility was 200 µg/mL. Toxicity
was tracked daily by monitoring the mortality induced in
each condition. Larvae were considered not viable when one
of the following observations occurred: all or part of larval
body degraded, severe growth retardation, a developmental
anomaly with severity non-consistent with larval survival.
Whenever the extracts resulted in toxicity, the concentration
was reduced to 100 µg/mL and the experiment was repeated.
At 6 dpf, larvae were sacrificed with a lethal dose of MS222 (0.6 mM, pH 7.0, Sigma-Aldrich), stained for 20 min at
room temperature with 0.03% alizarin red S (AR-S) prepared
in Milli-Q water (pH 7.4), and washed twice with Milli-Q
water for 5 min. Euthanized larvae were placed in a lateral
position on top of a 2.5% agarose gel plate and imaged
using a Leica MZ10F fluorescence stereomicroscope (Leica,
Wetzlar, Germany) equipped with a green fluorescence filter
(λex = 546/10 nm), a barrier filter (λem = 590 nm), and
a DFC7000T camera (Leica). Images were acquired using the
following parameters: exposure time 300 ms, gamma 1.00, image
format 1,920 × 1,440 pixels, binning 1 × 1. Fluorescence
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R
RESULTS
Total Polyphenols Content
Tunicate extracts displayed a high content of polyphenolic
compounds, with higher values for the HE fractions. The extracts
with the highest content were the ones obtained by HE fractions
of Botrylloides diegensis, 669 ± 11 mg GAE/100 g dw, Aplidium
sp., 365 ± 21 mg GAE/100 g dw and Ciona robusta, 206 ± 14 mg
GAE/100 g dw (Table 1). Polyphenol contents from the extracts
of holothurians were generally lower than ascidians, never
exceeding 100 mg GAE/100 g dw, and were higher in the AQ
extraction compared to the HE (Table 1).
Antioxidant Activity
The antioxidant activity was measured by the use of three
alternative methodologies (ABTS, FRAP, and DPPH) in the
extracts from the studied tunicates and sea cucumbers (Table 2).
Three species of tunicates—Aplidium sp., Botrylloides diegensis,
and Ciona robusta—displayed the highest antioxidant activities
with consistent results across the different methodologies
used. When measured by DPPH and FRAP assays, the
HE extracts were the ones displaying higher antioxidant
activity. However, when tested with ABTS assay, the AQ
extract also displayed high antioxidant activity, surpassing the
ones of the HE extracts. Moderate antioxidant activity was
reported also for the AQ extracts from H. arguinensis and
H. mammata when assessed by ABTS assay, but always lower
compared to tunicates.
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FIGURE 1 | Scheme of the protocol used for the evaluation of the osteogenic activity.
to 33.2 ± 5.2% for Ciona robusta. However, the HE fractions
from the four species of tunicates showed high anti-inflammatory
activity, three of which in the 70.2–76.7% interval, while
the highest activity was measured for the extracts from
Ciona robusta, showing 92.2 ± 8.5% inhibition of COX2 activity.
Anti-inflammatory Activity
The anti-inflammatory activity was measured by the percentage
of inhibition of COX-2 (Table 3). First, HE extracts generally
showed higher anti-inflammatory activity than the AQ extracts.
HE fractions from sea cucumbers exhibited relatively low
values of COX-2 inhibition ranging from 16.4 to 41.8%, while
the AQ extracts did not present any detectable activity. AQ
extracts from tunicates presented a moderate anti-inflammatory
activity, ranging from 8.9 ± 3.7% in of Botrylloides diegensis
Developmental Toxicity and
Establishment of Maximum Non-lethal
Dose
Acute developmental toxicity was evaluated in zebrafish larvae
and the Maximum Non-Lethal Dose (MNLD) of each extract
was determined during the operculum assay and results are
summarized in Table 4. None of the extracts resulted in
toxicity at the higher concentration tested (200 µg/mL) but
the HE extracts of H. forskali, which induced mortality.
Because of this observation, all HE extracts were also tested at
100 µg/mL. Control groups—Calcitriol (10 pg/mL), water, and
ethanol 0.1% (v/v) never resulted in mortality in any of the
assays performed.
TABLE 1 | Polyphenol content (mg GAE/100 g dw) in aqueous and ethanolic
extracts of the studied tunicates and sea cucumbers.
Polyphenol content (mg GAE/100 g dw)
Extract
Species
Aqueous
(n = 3)
Ethanolic
(n = 3)
Styela plicata
31 ± 6aA
40 ± 7bA
Aplidium sp.
260 ± 13dA
365 ± 21dB
Botrylloides diegensis
428 ± 10eA
669 ± 11eB
Osteogenic Activity
8cA
206 ± 14cB
To explore the pro-osteogenic potential of the extracts studied,
the effect of HE and AQ extracts on bone formation during
zebrafish development was assessed using the operculum assay
method (23). None of the extracts significantly affected the area
of the head, indicating that there was not a significant variation
in growth among treatments (Supplementary Figure 1). Overall,
hydroethanolic extraction was able to isolate compounds with
pro-osteogenic bioactivity. In particular, HE fractions from three
tunicates species (Aplidium sp., Botrylloides diegensis, and Ciona
Ciona robusta
103 ±
H. (Roweothuria) arguinensis
84 ± 4bcA
9 ± 5abB
H. (Holothuria) mammata
79 ± 1bA
21 ± 17abB
H. (Panningothuria) forskali
48 ± 2aA
ndaB
Values are presented as average ± standard deviation. For all groups, n = 3. Nd,
not detected. Different lowercase letters within a column correspond to statistical
differences (p < 0.05) between different species and same extract type (ethanolic
or aqueous, respectively). Different uppercase letters within a row correspond to
statistical differences (p < 0.05) between aqueous and ethanolic extracts from the
same species.
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TABLE 2 | Antioxidant activity as measured by ABTS (µmol eq TROLOX/100 g dw), FRAP (µmol eq FeSO4/g dw), and DPPH (mg eq AA/100 g dw) in aqueous and
ethanolic extracts of the studied tunicates and sea cucumbers.
Species
ABTS (µ mol eq TROLOX/100 g dw)
FRAP (µ mol eq FeSO4 /g dw)
DPPH (mg eq AA/100 g dw)
Extract
Extract
Extract
Aqueous(n = 3)
Ethanolic(n = 3)
Aqueous(n = 3)
Ethanolic(n = 3)
Aqueous(n = 3)
Ethanolic(n = 3)
Styela plicata
1,126 ± 81aA
552 ± 60bB
2.9 ± 0.5aA
5.5 ± 0.1bB
6.9 ± 10.9abA
ndaB
Aplidium sp.
6,206 ± 180fA
5,020 ± 20dB
34.3 ± 0.4eA
34.2 ± 0.1dA
ndaA
102.0 ± 1.6bB
Botrylloides diegensis
6,735 ± 11gA
5,886 ± 83eB
54.6 ± 0.4fA
56.5 ± 0.0eB
16.7 ± 4.1bcA
179.0 ± 1.7cB
4,271 ±
109eA
56cB
19.7 ±
0.6dA
15.6 ±
0.6cB
4.4eA
162.7 ± 2.5cB
H. arguinensis
2,234 ±
136cA
7.0 ±
0.2bA
2.0 ±
0.1aB
H. mammata
3,089 ± 158dA
33 ± 57aB
6.2 ± 0.0bA
H. forskali
1,883 ± 81bA
ndaB
8.9 ± 0.5cA
Ciona robusta
3,027 ±
166 ±
35aB
151.7 ±
ndaA
13.5 ± 12.7aB
1.5 ± 0.2aB
31.8 ± 6.2cA
5.7 ± 9.8aB
2.1 ± 0.6aB
106.1 ± 4.7dA
ndaB
Values are presented as average ± standard deviation. nd, not detected. For all groups, n = 3. Different lowercase letters within a column correspond to statistical
differences (p < 0.05) between different species and same extract type (ethanolic or aqueous, respectively). For each antioxidant methodology, different uppercase letters
within a row correspond to statistical differences (p < 0.05) between aqueous and ethanolic extracts from the same species.
robusta) were characterized by high osteogenic activity, inducing
an increase in the mineralized area of the opercular bone by
41.7 ± 16.6%, 31.1 ± 13.8%, and ± 20.0 12.7%, respectively,
all at the concentration of 200 µg/mL (Figure 2C). Statistical
differences between the two concentrations tested for the HE
extracts were reported for Aplidium sp. (P = 0.0186) and Ciona
robusta (P < 0.0001) indicating that a dose-dependent effect was
present, but not for Botrylloides diegensis (P = 0.0975).
In contrast, the AQ extracts did not display any evident
osteogenic activity at the concentrations tested. Interestingly, also
the HE extracts from two species of sea cucumbers, H. arguinensis
and H. mammata, induced a potent pro-osteogenic effect by
increasing the area of the opercular bone in 33.0 ± 19.98%,
and 38.8 ± 22.8%, respectively (Figure 3). For holothurians HE
extracts, when differences between 200 vs. 100 µg/mL were tested
through Student’s t-test (P < 0.05), there were no statistical
differences for H. arguinensis (P = 0.1942), while these were
present for H. mammata (P = 0.0014).
DISCUSSION
Given the compelling need for novel therapies for the treatment
of bone disorders associated with mineral loss, and considering
the rising interest of the pharmaceutical industry toward
marine bioactive compounds, we decided to explore the
bioactive potential of two somewhat poorly studied groups
of marine invertebrates—Sea cucumbers (Holothuroidea) and
ascidians (Ascidiacea). Aqueous (AQ) and hydroethanolic (HE)
extracts from three species of marine holothurians—Holothuria
arguinensis, H. forskali, and H. mammata and 4 species of
ascidians: Styela plicata, Aplidium sp., Botrylloides diegensis,
and Ciona robusta were produced. As the increasing body of
evidences suggest that oxidative stress and inflammation are
related to the etiology of osteoporosis (4), we screened the
extracts for their antioxidant and anti-inflammatory activities by
applying several in vitro assays. Being polyphenolic compounds
one of the most relevant classes of naturally occurring
compounds, with recognized antioxidant and anti-inflammatory
properties, we determined their content in the extracts.
Concerning the sea cucumbers, previous reports described
the presence of compounds with antioxidant activity, including
polyphenols, in extracts of sea cucumbers (36–38). A significant
presence of phenolic substances was observed in species closely
related to the ones studied in the present work including
Holothuria atra (36), H. scabra (37), and one species analyzed in
the present work, H. arguinensis (38). In the study by Roggatz
et al. (38), a cold-water extract of H. arguinensis was found to
have a polyphenol content of 14.2 mg GAE/100 g dw, while no
polyphenols were detected in the ethanolic extract. Similarly, in
our study a higher content of polyphenols was found in the AQ
extracts compared with the HE. However, the extracts from sea
cucumber showed lower content of polyphenolic compounds,
when compared with the ones from tunicates. Accordingly, a
lower antioxidant and anti-inflammatory potential were also
reported for these species. Results from ABTS assay highlighted
a moderate antioxidant potential for the AQ extracts of
the three holothurians studied, but this observation was not
consistent with the antioxidant capacity when tested though
TABLE 3 | Anti-inflammatory activity (% inhibition of COX-2) in aqueous and
ethanolic extracts of the studied tunicates and sea cucumber species.
Anti-inflammatory activity (% inhibition COX-2)
Extract
Species
Aqueous (n = 4)
Ethanolic (n = 4)
Styela plicata
19.6 ± 5.8cA
76.7 ± 3.4cB
Aplidium sp.
27.8 ± 2.5dA
70.2 ± 6.3cB
Botrylloides diegensis
8.9 ± 3.7bA
75.9 ± 3.0cB
Ciona robusta
33.2 ± 5.2dA
92.2 ± 8.5dB
H. (Roweothuria) arguinensis
ndaA
40.0 ± 7.6bB
H. (Holothuria) mammata
ndaA
41.8 ± 4.9bB
H. (Panningothuria) forskali
ndaA
16.4 ± 9.7aB
Values are presented as average ± standard deviation. nd, not detected. For
all groups, n = 4. Different lowercase letters within a column correspond to
statistical differences (p < 0.05) between different species within same type of
extract. Different uppercase letters within a row correspond to statistical differences
(p < 0.05) between aqueous and ethanolic extracts from the same species.
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TABLE 4 | Acute developmental toxicity of the AQ and HE extracts from sea cucumbers and tunicates assessed in zebrafish larvae.
Species
Extract
Concentration
(µ g/mL)
S72
h
Concentration
(µ g/mL)
S72
h
Styela plicata
AQ
200*
15/15
–
–
Styela plicata
HE
200*
15/15
100
15/15
Aplidium sp.
AQ
200*
15/15
–
–
Aplidium sp.
HE
200*
15/15
100
15/15
Ciona robusta
AQ
200*
15/15
–
–
Ciona robusta
HE
200*
15/15
100
15/15
Botrylloides diegensis
AQ
200*
15/15
–
–
Botrylloides diegensis
HE
200*
15/15
100
15/15
H. (Panningothuria) forskali
AQ
200
15/15
–
–
H. (Panningothuria) forskali
HE
200
11/15
100*
15/15
H. (Roweothuria) arguinensis
AQ
200*
15/15
–
–
H. (Roweothuria) arguinensis
HE
200*
15/15
100
15/15
H. (Holothuria) mammata
AQ
200*
15/15
–
–
H. (Holothuria) mammata
HE
200*
15/15
100
15/15
Two concentrations have been tested for each extract. Final survival after 72 h of exposure (S7 2 h ) was calculated. Depending on the results of the experiment, the
concentrations were either increased to a maximum of 200 µg/mL or decreased following a half-logarithmic dilution (3.16, 10, 31.6, 100 µg/mL). Maximum non-lethal
dose (*, bold); concentration not tested (–).
FIGURE 2 | Osteogenic activity of aqueous (A) and hydroethanolic (B) extracts from four species of tunicates in zebrafish larvae. Results are displayed as corrected
operculum area (operculum area/head ratio) expressed as percentage of increase over the control. Representative image (C) of a fish treated with the negative
control (ethanol), the positive control (Calcitriol 10 pg/mL) and the most powerful osteogenic extracts among tunicates (Aplidium sp. HE200). Statistical differences
among the means were tested through one-way ANOVA followed by Dunnett’s multiple comparison test (p < 0.05) or, whenever normality and homoscedasticity
weren’t met, through a non-parametric test followed by Dunn’s multiple comparison test (p < 0.05). P-values are indicated as follow: 0.0021. (**), < 0.0001 (****).
HE, hydroethanolic extracts, AQ, aqueous extracts, 100–100 µg/mL, 200–200 µg/mL.
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FIGURE 3 | Osteogenic activity of aqueous (A) and hydroethanolic (B) extracts from three species of sea cucumbers in zebrafish larvae. Results are displayed as
corrected operculum area (operculum area/head ratio) expressed as percentage of increase over the control. Representative image (C) of a fish treated with the
negative control (ethanol), the positive control (Calcitriol 10 pg/mL) and the most powerful osteogenic extracts among holothurians (H. mammata HE200). Statistical
differences among the means were tested through one-way ANOVA followed by Dunnett’s multiple comparison test (p < 0.05) or, whenever normality and
homoscedasticity weren’t met, through a non-parametric test followed by Dunn’s multiple comparison test (p < 0.05). P-values are indicated as follow: 0.0332 (*),
0.0002 (***), < 0.0001 (****). HE, hydroethanolic extracts, AQ, aqueous extracts, 100–100 µg/mL, 200–200 µg/mL.
both aqueous and hydroethanolic extractions, but with the
HE extracts yielding higher contents. The same three species
were also characterized by the higher antioxidant activities,
as measured by different methodologies. In this regard, a
positive correlation was found between polyphenol content and
antioxidant potential measured with all the three methodologies
used, namely, through the DPPH (R2 = 0.29, P = 0.0311),
ABTS (R2 = 0.70, P < 0.0001), and FRAP (R2 = 0.32,
P = 0.0212), supporting the hypothesis that the antioxidant
potential observed for the tunicates HE extracts may be
related to their high content in polyphenolic compounds.
HE fractions from all the four species of tunicates also
presented the highest anti-inflammatory potential among all
the extracts. No previous studies investigated the presence
of polyphenolic compounds with anti-inflammatory activity
in tunicates, but other compounds with anti-inflammatory
properties were already described. For instance, it was previously
reported the presence of a dermatan sulfate, similar in structure
to the mammalian heparin, isolated from Styela plicata that was
shown to display anti-inflammatory activity (39). Moreover, two
new tricyclic thiazine-containing quinolinequinone alkaloids,
ascidiathiazones A and B, that were isolated from Aplidium sp.
FRAP assay, which is based on a similar mechanism used to
determine the presence of potential electron donors. On the
other side, the DPPH assay, which is used for the determination
of compounds with proton (H• -) donor potential, did not
reveal a high antioxidant activity when compared with the
other species studied. The presence of polyphenolic compounds
and a moderate antioxidant potential reported for the AQ
extracts from holothurians may indicate that these species of
sea cucumbers deserve further attention as natural sources of
polyphenolic compounds.
Tunicates, on the other side, yielded the most interesting
results in terms of all bioactivities analyzed. In literature,
very few studies investigated the presence of antioxidant
compounds in tunicate species. A radical scavenging potential
for hot water extracts of Styela clava measured by ABTS
assay was previously reported (22). Consistently, we observed
antioxidant activity on both AQ and HE extracts for the
closely related species Styela plicata studied in this work,
measured by ABTS, DPPH, and FRAP assays. However, the
species showing higher antioxidant potential were three tunicates
(Aplidium sp., Botrylloides diegensis, and Ciona robusta). These
species presented the highest content of polyphenols, for
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this process was widely reviewed by Weitzmann and Pacifici
(4). Estrogens regulate both osteoblastic and osteoclastic cells
through the estrogen receptor (ER), and elevated levels of
estrogens during young age act in both a bone anabolic and
anti-resorptive manner, contributing to the maintenance of
the equilibrium between bone formation and bone resorption,
paramount for a functional bone remodeling. Adequate estrogen
levels prolong osteoblast and osteocyte life-span (50), while
reducing it in osteoclasts (8, 51). Furthermore, the depletion in
circulating estrogens following menopause leads to an increased
thymic function, otherwise inhibited by estrogens, which results
in an elevated production of pro-inflammatory cytokines
including IL-1, IL-6, IL-7, and TNF-α, and to the overall
increase of TNF-producing T cells populations within bone
marrow, thymus and peripheral lymphoid organs (4). Activated
T lymphocytes release TNF, that has been shown to inhibit
the differentiation of osteoblastic cells (52). Additionally, it
increases osteoclast differentiation by stimulating the production
of RANKL by osteoblasts and bone marrow stromal cells (53),
and increase the sensitivity to RANKL signaling by upregulating
the expression of RANK receptor in pre-osteoclastic cells (54).
The abnormally increased osteoclastogenesis that results from
this process leads to an overregulated and then to a slowered
yet continuous bone resorption, which is not compensated by an
equally increased bone formation (4).
The age-related increase in oxidative stress within the
bone tissue contributes to exacerbate the pathophysiology of
postmenopausal osteoporosis (55). ROS are known to stimulate
the activation of T cells (56), increase osteoclastic differentiation
(57) and induce osteoblast apoptosis (58, 59). A sufficient
estrogen level is believed to prevent the instauration of oxidative
stress in bone either by directly inhibiting peroxidative processes,
upregulating the expression of antioxidant enzymes, or by
suppressing the production of ROS (60). Oxidative stress has
been recently proposed as major factor in the development of
postmenopausal osteoporosis (61).
The use of novel approaches such as the application of
treatments with natural-derived products that promote boneanabolic effects, by counteracting the pro-oxidative and proinflammatory processes, without inducing undesired side effects,
is paramount for the development of next-generation antiosteoporotic drugs.
Overall, the in vivo pro-osteogenic effect combined with the
high content of polyphenols, antioxidant, and anti-inflammatory
activities of these species led us to formulate the hypothesis
that the pro-osteogenic activity observed for the tunicates HE
extracts can be due to polyphenolic compounds. Previous reports
showed that polyphenols can stimulate osteoblast function by
directly interacting with different molecular pathways involved
in osteoblast differentiation (62, 63), by attenuating detrimental
effects from pro-inflammatory signals (64) and by exerting a
protective effect against reactive oxygen species on osteoblastic
cells (65). Interestingly, a positive correlation was found between
the pro-osteogenic and the anti-inflammatory bioactivities for the
extracts from tunicates (R2 = 0.59, P = 0.0266). This may indicate
that the presence of an anti-inflammatory activity can be directly
involved in the pro-osteogenic effect reported for the extracts.
collected from the coast of New Zealand, were reported to inhibit
the production of superoxide by human neutrophils (40).
Following the promising results obtained with antioxidant
and anti-inflammatory activities, we decided to test the extracts
for their osteogenic potential. In this scope, zebrafish offers
an interesting opportunity to explore the potential of bioactive
compounds in vivo, presenting several advantages in comparison
to commonly used mammalian models. Small size, high
fecundity, short life span, and a relatively high genetic homology
with humans, are some of the technical advantages zebrafish
has to offer that persuaded researchers in the field of drug
discovery to implement it as an animal model for pre-clinical
trials (41, 42). Moreover, the translucency of the early larval
stages has proved to be very useful for microscopy applications
in bone biology and other fields, allowing the observation of the
skeletal structures following the staining with dyes with calciumbinding affinity (43). As result, several zebrafish-based assays
were developed in recent years for the screening of drugs and
natural compounds with the capacity to induce bone formation
and mineralization (23, 44) and were successfully used for the
screening of osteoactive compounds, including the zebrafish
operculum assay (45, 46).
For the AQ extracts from holothurians, the antioxidant
activity reported was not translated into an appreciable effect in
terms of induction on bone formation and mineralization in vivo.
Conversely, HE fractions from two species of holothurians—
H. mammata and H. arguinensis displayed a pro-osteogenic effect
in zebrafish larvae. However, these extracts were characterized
by lower polyphenolic content, antioxidant activities, and antiinflammatory activities, compared with the tunicates, suggesting
that non-phenolic compounds in holothurians may be involved
in the effect observed. Nevertheless, holothurians have been
described to be able to synthesize compounds with proosteoblastogenic potential, as observed by increased viability
and activity of alkaline phosphatase in a human osteoblastic
cell line (47) and in rat bone marrow mesenchymal stem
cells (48). These observations highlight the need for further
investigating these two species of holothurians as potential
sources of osteogenic bioactives.
Concerning the tunicates, while AQ extracts did not induce
any osteogenic effect, the HE extracts from three species –
Aplidium sp., Botrylloides diegensis, and Ciona robusta – were
characterized by an increase in mineralized area of the opercular
bone in zebrafish larvae. This increase was in a similar extent to
the positive control (Calcitriol), and in the case of Aplidium sp. at
200 µg/mL (Aplidium sp. HE200), even surpassing the positive
control value. These observations indicate that pro-osteogenic
compounds were isolated through hydroethanolic extraction
from the three tunicates species. It is important to notice that
at the stage of zebrafish development used for analysis, neither
active osteoclasts nor bone resorption occurs (23, 49), therefore
the effect here observed for the extracts is due to pro-osteogenic
rather than anti-resorptive mechanisms.
The causes for osteoporosis have been extensively studied
including the involvement of oxidative stress and inflammation
that are linked to altered bone metabolism and reduction of
bone mineral density. The molecular landscape associated with
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sea cucumbers, these generally showed a lower antioxidant
activity, although moderate anti-inflammatory and strong
pro-osteogenic activities were yielded by the hydroethanolic
extraction. These findings provide an evidence of the presence of
bioactive compounds with potential for therapeutic applications
in the treatment of metabolic bone disorders, opening the
opportunity for further investigation.
Due to their richness in polyphenolic compounds, antioxidant
and anti-inflammatory activities associated with their high proosteogenic potential, we identified potential candidates for the
discovery of compounds with anti-osteoporotic potential in
three species of tunicates Aplidium sp., Botrylloides diegensis,
and Ciona robusta. Nevertheless, further investigation aimed at
evaluating the toxicity and safety of the studied extracts in other
models must be conducted and the chemical characterization
of these extracts will be necessary to identify phenolic
compounds responsible for the effect in the animal model.
With this aim, a good strategy may involve the application
of Liquid Chromatography and Mass Spectrometry (LC-MS).
Furthermore, the use of a transcriptomic approach would
be suitable to elucidate the mechanisms of action of the
extracts, which may be acting through a synergy of different
mechanisms. The application of extracts to zebrafish reporter
lines for osteoblastic cells already available (66, 67), may help
investigate the involvement and dynamics of specific cellular
types modulated by the extracts. Moreover, by testing the extracts
on juvenile stages, when osteoclasts are active, it will allow to
determine potential effects on bone resorption. Subsequently, it
will be necessary to validate whether the activity, as here observed
in the zebrafish ontogenetic model, could efficiently translate
into the rescue of disease phenotypes in osteoporotic models. In
this regard, a medaka (O. latipes) model of genetically induced
osteoporosis (68) was previously developed and it would be
suitable for this purpose. The application to mammalian models
of osteoporosis will be important to validate the translational
applications of these promising set of extracts to mammalians.
In this context, ovariectomized rat is a well-established animal
model that can be used for the validation of anti-osteoporotic
compounds (69, 70). Overall, the present study provides a
first validation of the pro-osteogenic activity of extracts rich
in polyphenols, antioxidant and anti-inflammatory compounds,
highlighting the suitability of the studied species for the discovery
of novel compounds with anti-osteoporotic potential.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The animal study was reviewed and approved by the Direção
Geral de Alimentação e Veterinária approval number
012769 from 2021.
AUTHOR CONTRIBUTIONS
AC, CC, MC, NB, and PG: conceptualization and investigation.
AC, CC, DJ, IF, JL-A, SS, PC, and MD: methodology. CC, MC,
NB, PG, and MG: resources. AC and CC: data curation. AC, CC,
NB, and PG: writing. AC, CC, NB, PG, JL-A, and SS: review and
editing. CC, NB, PG, MC, and MG: supervision. CC, NB, MC, and
PG: funding acquisition. All authors contributed to the article and
approved the submitted version.
FUNDING
This project has received funding from the European Union’s
Horizon 2020 Research and Innovation Programme under
the Marie Skłodowska-Curie grant agreement no. 766347; and
from the Portuguese National funds from FCT—Foundation
for Science and Technology through grants UIDB/04326/2020,
2021.05406.BD, and UIDB/04292/2020.
CONCLUSION
In the present work we explored the potential of extracts from
three species of sea cucumbers and four species of tunicates as
source of bioactive compounds to be used toward the prevention
and treatment of chronic bone disorders characterized by mineral
loss, such as osteoporosis and osteopenia. A total of 14 aqueous
and hydroethanolic extracts were produced and screened for their
content of polyphenols and their antioxidant, anti-inflammatory,
and osteogenic potentials. Overall, three species of ascidians
showed the strongest antioxidant activities associated with
high polyphenols content. These polyphenol-rich extracts were
also characterized by the strongest anti-inflammatory activity
and a high osteogenic activity by promoting bone formation
in zebrafish larvae. We hypothesize that the positive effect
on bone formation observed for the HE extracts from the
tunicates studied can be due to the antioxidant and antiinflammatory properties of polyphenolic compounds isolated
through hydroethanolic extraction. Regarding the extracts from
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SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnut.2022.
888360/full#supplementary-material
Supplementary Figure 1 | Effect of the treatment with different extracts on the
area of the head of zebrafish larvae for holothurians hydroethanolic (A) and
aqueous extracts (B) and for tunicates hydroethanolic (C) and aqueous extracts
(D) respectively. Statistical differences among the means were tested through
One-way ANOVA followed by Dunnett’s multiple comparison test (p < 0.05) or,
whenever normality and homoscedasticity weren’t met, through a non-parametric
test followed by Dunn’s multiple comparison test (p < 0.05). HE – hydroethanolic
extracts, AQ – aqueous extracts, 100 – 100 µg/mL, 200 – 200 µg/mL.
Supplementary Table 1 | GenBank accession numbers for the sequence data
obtained for the identification of four species of Tunicates.
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