Diversity of biological activities of crude venom extracted from five species of South China Sea anemones

Abstract

Developing novel, efficient, and safe peptide drugs from sea anemones has aroused great interest in countries around the world today. Sea anemones contain complex protein and peptide toxins, which determine the diversity of their biological activities. In this study, a variety of activities were assessed for crude venom extracted from five species of South China Sea anemones, including hemolytic, enzyme inhibition, anticancer, insecticidal, analgesic and lethal activities. The most toxic sea anemone was found to be Heteractis magnifica, which has high lethal activity in mice with an LD50 of 11.0 mg/kg. The crude venom of H. magnifica also exhibited a range of the most potent activities, including hemolytic, trypsin inhibitory, cytotoxic activity against U251 and A549 cells, insecticidal and analgesic activities. In addition, the crude venom of Stichodactyla haddoni was the most effective inhibitor of pepsin, and the crude venom of Heteractis crispa was extremely strong toxicity to HepG2 cells. These findings are of great significance for exploring the potential and application of South China Sea anemone resources, and are expected to provide new directions and possibilities for the development of novel anticancer drugs, analgesics and biopesticides.

Full text

Diversity of biological activities
of crude venom extracted
from five species of South
China Sea anemones
Panmin He
1
†
, Ming Li
1
†
, Jinxing Fu
1
, Yanling Liao
1
,
Bo Yi
2
*and Bingmiao Gao
1
*
1
Engineering Research Center of Tropical Medicine Innovation and Transformation of Ministry of
Education & International Joint Research Center of Human-machine Intelligent Collaborative for
Tumor Precision Diagnosis and Treatment of Hainan Province & Hainan Provincial Key Laboratory Of
Research and Development on Tropical Herbs, School of Pharmacy, Hainan Medical University,
Haikou, Hainan, China,
2
Department of Pharmacy, 928th Hospital of PLA Joint Logistics Support
Force, Haikou, Hainan, China
Developing novel, efficient, and safe peptide drugs from sea anemones has
aroused great interest in countries around the world today. Sea anemones
contain complex protein and peptide toxins, which determine the diversity of
their biological activities. In this study, a variety of activities were assessed for
crude venom extracted from five species of South China Sea anemones,
including hemolytic, enzyme inhibition, anticancer, insecticidal, analgesic and
lethal activities. The most toxic sea anemone was found to be Heteractis
magnifica, which has high lethal activity in mice with an LD
50
of 11.0 mg/kg.
The crude venom of H. magnifica also exhibited a range of the most potent
activities, including hemolytic, trypsin inhibitory, cytotoxic activity against U251
and A549 cells, insecticidal and analgesic activities. In addition, the crude venom
of Stichodactyla haddoni was the most effective inhibitor of pepsin, and the
crude venom of Heteractis crispa was extremely strong toxicity to HepG2 cells.
These findings are of great significance for exploring the potential and
application of South China Sea anemone resources, and are expected to
provide new directions and possibilities for the development of novel
anticancer drugs, analgesics and biopesticides.
KEYWORDS
sea anemones, crude venom, hemolytic activity, enzyme inhibitory activity, anticancer
activity, insecticidal activity, analgesic activity
Frontiers in Marine Science frontiersin.org01
OPEN ACCESS
EDITED BY
Roland Wohlgemuth,
Lodz University of Technology, Poland
REVIEWED BY
Rajesh Rajaian Pushpabai,
Chettinad University, India
Tan Suet May Amelia,
Chang Gung University, Taiwan
Chiara Lauritano,
Anton Dohrn Zoological Station Naples, Italy
*CORRESPONDENCE
Bo Yi
ddzj_yb@163.com
Bingmiao Gao
gaobingmiao@hainmc.edu.cn
†
These authors have contributed
equally to this work and share
first authorship
RECEIVED 14 August 2024
ACCEPTED 16 October 2024
PUBLISHED 05 November 2024
CITATION
He P, Li M, Fu J, Liao Y, Yi B and Gao B (2024)
Diversity of biological activities of crude
venom extracted from five species of South
China Sea anemones.
Front. Mar. Sci. 11:1480745.
doi: 10.3389/fmars.2024.1480745
COPYRIGHT
© 2024 He, Li, Fu, Liao, Yi and Gao. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication
in this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 05 November 2024
DOI 10.3389/fmars.2024.1480745

1 Introduction
Marine ecosystems are among the most biologically diverse and
marine biomass has become an effective source for the development
of innovative biotechnology, driving the discovery of new
therapeutic strategies and compounds, with venom from marine
animals being recognized as an emerging source of peptide-based
therapeutics (Liao et al., 2019b;Melendez-Perez et al., 2023). Since
the Food and Drug Administration approved the use of cytarabine
for leukemia treatment, an increasing number of scientists have
been motivated to invest significant efforts in the development of
marine drugs (Montaser and Luesch, 2011). Currently, ziconotide
extracted from Conus magus has been clinically approved for the
treatment of chronic pain, and ShK-186, an analogue of ShK from
Bunodosoma granulifera, now known as dalazatide, has successfully
entered Phase I trials for the treatment of autoimmune diseases
(Liao et al., 2019a;Coulter-Parkhill et al., 2021;Moovendhan,
2024). Despite the immense potential of marine organisms, there
remains a significant gap in the number of peptide drugs extracted
from non-marine species, such as eptifibatide, bivalirudin and
exenatide (Koh et al., 2018;Coulter-Parkhill et al., 2023;Tonin
and Klen, 2023). Therefore, more intensive and systematic research
and development are urgently needed.
Sea anemones (Cnidaria, Actiniaria) are marineinvertebrates and
one of the oldest surviving venomous species (Madio et al., 2019;
Ashwood et al., 2022). At present, approximately 1100 sea anemone
species have been recorded, most of which inhabit temperate regions
and some live in the tropics (Gomes et al., 2016;Fu et al., 2021). In
Chinese sea areas, the distribution of sea anemone resources indicates
that the South China Sea has the highest abundance, followed by the
Yellow Sea, and the East China Sea has the lowest. Sea anemones
survive in a variety of marine habitats, typically adhering to rocks and
other objects, displaying immobility, with only a few capable of slow
crawling (Kasheverov et al., 2022;Menezes and Thakur, 2022). Like
all other cnidarians, sea anemones possess structures that produce
microscopic venom transport, called nematocysts, venoms were
produced and stored by highly specialized nematocysts (Fautin,
2009;Moran et al., 2012;Rachamim et al., 2015;Menezes and
Thakur, 2022;Monastyrnaya et al., 2022). With no skeleton and
lacking the most basic brain foundation or central information
processing mechanism, they rely on the production of venoms to
capture prey, which also perform roles for defensing, intraspecific
aggressing and digesting (Frazao et al., 2012;Mitchell et al., 2021). For
example, grass shrimp are predators of Nematostella, and studies of
sea anemone venom proteomes revealed more families with
neurotoxic activity than those with other modes of action, and
studies have demonstrated that transgenically deficient and native
Nematostella strain lacking a major neurotoxin have reduced defense
to grass shrimp, suggesting that neurotoxins are essential in defense
against invertebrate predators (Ashwood et al., 2022;Smith et al.,
2023;Surm et al., 2024).
Venoms are usually a mixture of peptides, proteins and non-
proteinaceous compounds. Among them, the peptide components
are of great interest to scientists due to their high toxicity, diversity
and structural stability. Sea anemone peptides mainly form disulfide
bonds with multiple cysteine residues, forming stable structural
scaffolds. So far, at least 17 different molecular scaffolds have been
found in sea anemone peptides (Casewell et al., 2013;Madio et al.,
2018;Pinheiro-Junior et al., 2022). In addition to exhibiting a broad
range of biological activities, these venoms serve as valuable research
tools for different molecular targets, currently affecting at least 20
pharmacological targets (Prentis et al., 2018;Tajti et al., 2020;
Kasheverov et al., 2022;Da Silva et al., 2023). The most extensively
researched sea anemone toxins are those that regulate voltage-gated
sodium (Nav) and potassium (Kv) channels, acid-sensing ion
channels (ASICs), transient receptor potential (TRP) ion channels,
as well as toxins that inhibit polyfunctional proteases (mainly Kunitz-
type) and pore-forming toxins that can interact with the plasma
membrane of eukaryotic cells (Isaeva et al., 2012;Cardoso and Lewis,
2018;Leychenko et al., 2018;Prentis et al., 2018;Madio et al., 2019;
Wang et al., 2021;Zhao et al., 2024). Transcriptomic and proteomic
studies have shown that each sea anemone can produce
approximately 1000 transcripts, with 1174 sea anemone species
annotated in WORMS (https://www.marinespecies.org, accessed on
26 September 2024), it is estimated that sea anemones may be able
to produce about 1,200,000 natural peptides (Fu et al., 2021;Guo
et al., 2024;Li et al., 2024). However, with only about 492 sea
anemone toxins annotated in UniProtKB (https://www.uniprot.org,
accessed on 21 September 2024), there are still numerous
undiscovered sea anemone venoms that could be developed into
novel peptide medicines.
With advances in transcriptome sequencing technology, mass
spectrometry instrumentation, and bioinformatics tools, a detailed
overview of proteins and peptide toxins in sea anemones using
transcriptomic and/or proteomic approaches has revealed the
complexity of venoms (Mazzi Esquinca et al., 2023;Li et al.,
2024). It had been identified from transcriptomics that Heteractis
magnifica,Entacmaea quadricolor,Stichodactyla haddoni,and
Heteractis crispa expressed 728, 1251, 508, and 1049 toxin-like
transcripts, respectively (Madio et al., 2017;Guo et al., 2024;
Hoepner et al., 2024;Li et al., 2024). However, due to the huge
sequencing data, synthesizing peptides one by one has become
difficult, and rapidly evaluating the pharmacological activities of
synthesized peptides also faces challenges. Therefore, it is necessary
to verify the activities of the five sea anemones collected from the
South China Sea and select the most promising anemone resources
for further study. So far, sea anemones have played a crucial role in
the development of marine biotechnology. Some sea anemone
venoms have been shown to possess different activities with rich
medicinal value, promoting the development of marine medicines
and biologics. For example, venoms of H. magnifica,S. haddoni,
Paracodylactis sinensis and Stichodactyla helianthus induced
spontaneous hemolysis in erythrocytes (Ravindran et al., 2010;
Rivera-de-Torre et al., 2020). Moreover, enzyme inhibitors had
been found in sea anemone toxins such as Bunodosoma caissarum
and E. quadricolor (Mazzi Esquinca et al., 2023;Hoepner et al.,
2024). Notably, sea anemone researches had also identified several
venoms with anticancer activities. For example, H. magnifica and
E. quadricolor venoms were cytotoxic to human lung cancer cells
(A549) and toxins from H. crispa showed anticancer activities and
He et al. 10.3389/fmars.2024.1480745
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prevented HT-29 colorectal cancer cell migration (Ramezanpour
et al., 2012;Kvetkina et al., 2020;Moghadasia et al., 2020). It had
also been shown that ShK-like peptide produced by Steinernema
carpocapsae is toxic to Drosophila melanogaster, and crude venom
of Anthopleura elegantissima and S. haddoni anemones exhibited
toxic effects when tested against the rice weevil Sitophilus oryzae
(Frias et al., 2022;John, 2022). Peptides isolated from Metridium
senile and H. crispa were found to be analgesic (Andreev et al., 2008;
Logashina et al., 2017,2021;Maleeva et al., 2023).
In this study, five representative sea anemone species were
collected from the South China Sea and extracted crude venom,
which were Macrodactyla doreensis,H. magnifica,E. quadricolor,
S. haddoni, and H. crispa. The biological activity diversity of crude
sea anemone venom in hemolytic, enzyme inhibition, anticancer,
insecticidal, analgesic and lethal activities was evaluated, aiming to
screen the most potential sea anemone resource in the South China
Sea and conduct in-depth research to lay the foundation for the
development of novel peptide drugs.
2 Results
2.1 Characteristics of sea anemones and
component analysis of crude venom
Five species of sea anemones collected from the South China Sea
were identified by morphological characteristics as: M. doreensis
(Quoy and Gaimard, 1833), H. magnifica (Quoy and Gaimard,
1833), E. quadricolor (Rüppell and Leuckart, 1828), S. haddoni
(Saville-Kent, 1893), and H. crispa (Hemprich and Ehrenberg,
1834) (Figure 1A)(Raghunathan et al., 2014). M. doreensis is
reddish, with a pronounced and distinctly beige to yellow blotched
pedal disc, and fewer tentacles, concentrated on the margin of the oral
disc. Morphological characteristics of H. magnifica mainly include an
ochre-yellow body, reddish-brown pedal disc, column cylindrical,
and the tentacles spread over the surface of the oral disc, with purple
tips and moderate length of tentacles. E. quadricolor is usually light
green to green in color, has long, cylindrical and smooth tentacles,
which are usually bulbous below the tip with distinctive longitudinal
wrinkles, and has a flattened, pinkish oral disc. S. haddoni is
characterized by a flattened and deeply folded oral disc, extremely
short tentacles that are highly robust at periphery of disc, and a beige
color with purple spots distributed on the base. H. crispa is light ivory,
with long, slender and numerous tentacles, partially entwined, whose
tips are lavender in color. In addition, five lyophilized extracts of
crude venom have different morphologies and show different colors
when dissolved in deionized water (Figures 1B,C).
Protein and peptide components were detected by reversed-
phase high performance liquid chromatography (RP-HPLC) and
SDS-PAGE (Figures 1D,E). RP-HPLC analysis revealed
dissimilarities in both peak shapes and peak heights of crude
venom. Notably, H. magnificaandH. crispa exhibited two
significant peaks in chromatograms. The proteins in crude venom
were mainly distributed at 10-180 kDa in SDS-PAGE analysis. In
particular, the common bands at 17 kDa were observed in
H. magnifica,E. quadricolor,S. haddoni and H. crispa and the
electrophoretic band of H. magnifica was the most abundant.
2.2 Evaluation of the hemolytic and
enzyme inhibitory activities
The hemolytic and enzyme inhibitory activities of crude venom
were studied using rat erythrocytes and different enzymes,
respectively, and the results were shown in Table 1.The
hemolysis test showed that crude venom exhibited 100%
hemolysis of erythrocytes at concentrations of 1 to 150 mg/mL.
Specifically, the venom of H. magnifica (1 ± 0.27 mg/mL) and
E. quadricolor (10 ± 0.27 mg/mL) displayed potent hemolytic
activity compared to S. haddoni (40 ± 0.27 mg/mL), H. crispa
(100 ± 1.27 mg/mL) and M. doreensis (150 ± 0.53 mg/mL). For
enzyme inhibition assay, the trypsin, pepsin and a-galactosidase
were selected for the determination of enzyme activities. For trypsin
assay, with the exception of E. quadricolor, all other venoms were
found to inhibit trypsin and H. magnifica venom showed the
highest inhibitory activity with a value of 251.25 ± 1.43 TIU/mg.
Meanwhile, each of crude venom displayed an inhibitory effect on
pepsin, the values were in the range of 145.08-391.25 TIU/mg. In
contrast, none of these venoms inhibited on a-galactosidase.
2.3 Cytotoxic effects of crude venom on
human glioma, hepatocellular carcinoma
and lung cancer cells
Due to the rapid growth and poor prognosis of glioma, the
invasiveness of hepatocellular carcinoma, the lack of effective
treatment, and the high mortality rate of lung cancer, human
glioma cells (U251), human hepatocellular carcinoma cells (HepG2)
and human lung cancer cells (A549) were selected for cytotoxicity
assays and the effects of different concentrations of crude venoms on
the viability of the cells was assessed using a cell counting kit (CCK-8)
(Ahmad et al., 2024;Dong et al., 2024;Huang et al., 2024;Jin et al.,
2024;Lv et al., 2024;Zheng et al., 2024). As shown in Figure 2,cell
viability decreased in a concentration-dependent manner with
increasing concentrations of crude venom. The order of toxicities of
the crude venom extracts were H. magnifica >E. quadricolor >S.
haddoni >M. doreensis >H. crispa for U251, H. crispa >H. magnifica
>E. quadricolor >M. doreensis >S. haddoni for HepG2, and H.
magnifica >E. quadricolor >S. haddoni >M. doreensis >H. crispa for
A549. Specifically, the IC
50
values of H. magnifica,E. quadricolor,S.
haddoni,M. doreensis and H. crispa were 105.0 mg/mL, 108.3 mg/mL,
201.8 mg/mL, 767.9 mg/mL, and 986.4 mg/mL, respectively, for U251
cancer cells (Figure 2F). Strikingly, HepG2 cells showed high
sensitivity to crude venom, being most sensitive to H. crispa with an
IC
50
value of 10.02 mg/mL and least sensitive to S. haddoni with an
IC
50
value of 307.4 mg/mL (Figure 2G). For A549 cells, IC
50
values of
73.18 mg/mL, 170.9 mg/mL, 179.1 mg/mL, 1858 mg/mL and 2039 mg/
mL were observed for H. magnifica,E. quadricolor,S. haddoni,M.
doreensis and H. crispa,respectively(Figure 2H).
He et al. 10.3389/fmars.2024.1480745
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2.4 Crude venom exhibit inhibitory effects
on migration and invasion of U251 cells
in vitro
Scratch wound healing and transwell assays were performed to
access the effects of crude venom on the migration and invasion of
U251 cells. Comparing the migration of U251 cells in the control
and treated groups and the invasion images after 24 h of treatment
with 100 mg/mL crude venom, it was found that there were
reductions in the migration of cells to the scratch site and in the
number of invaded cells (Figure 3). As shown in Figure 4, the lowest
wound closure rates were observed after 24 h of migration, at
concentrations of 10 mg/mL, 0.2 mg/mL, 1 mg/mL, 0.2 mg/mL, and
10 mg/mL for M. doreensis,H. magnifica,E. quadricolor,S. haddoni,
and H. crispa venoms respectively, the closure rates were 17.67 ±
4.68%, 6 ± 1.53%, 6 ± 1.73%, 11.33 ± 0.67%, and 1.33 ± 2.33%,
respectively (Figures 4A–E). H. magnifica venom significantly
inhibited cell migration, while the venom of M. doreensis had the
weakest inhibitory effect on migration. In addition, the
enhancements of the migration capacity of the cells were
observed after 48 h compared to the 24 h. The transwell assay
showed that most crude venom inhibited cells invasion in a dose-
dependent manner (Figure 4F). Notably, at a concentration of 200
mg/mL, the invasion rates of U251 cells were 18.33 ± 1.76%, 0.33 ±
FIGURE 1
Samples of five species of sea anemones and analysis of crude venom using SDS-PAGE and RP-HPLC. (A) Macromorphology of five species of sea
anemones in the South China Sea. (B) Solid forms of crude venom extracted from sea anemones. (C) Solutions of crude venom dissolved in
deionized water. (D) SDS-PAGE analysis of crude venom. (E) RP-HPLC analysis of crude venom. Md, M. doreensis; Hm, H magnifica; Eq, E
quadricolor; Sh, S. haddoni; Hc, H crispa.
He et al. 10.3389/fmars.2024.1480745
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0.33%, and 24.33 ± 0.33% for M. doreensis,E. quadracolor, and
H. crispa venoms, respectively, and the venoms of H. magnifica and
S. haddoni completely suppressed the invasive ability of U251 cells.
2.5 Toxic effects on Tenebrio molitor and
SF9 cells
Evaluation of the insecticidal activity of crude venom using
toxicity assays on T. molitor and insect ovarian cells (SF9). The
results showed that crude venom significantly reduced the survival of
T. molitor in a dose- and time-dependent manner (Figures 5A–E),
and also reduced the viability of SF9 insect cells in a concentration
dependent manner (Figure 5F). After 24 h treatment, H. magnifica
venom demonstrated the most toxic to T. molitor (LD
50
= 11.44 mg/
kg), followed by E. quadricolor (LD
50
= 30.91 mg/kg), S. haddoni
(LD
50
= 45.98 mg/kg), M. doreensis (LD
50
= 50.41 mg/kg), and
H. crispa (LD
50
= 87.87 mg/kg) venoms. Notably, H. magnifica
venom resulted in 100% insects mortality at 60 h after 20, 40 and
100 ng/mg treatments, and E. quadricolor venom caused all T. molitor
death within 72 h. In the toxicity assay for SF9 cells, E. quadricolor
venom showed extremely strong toxicity with an IC
50
value of about
9.8 × 10
-31
mg/mL, the IC
50
values of S. haddoni,H. crispa,
M. doreensis and H. magnifica venom on SF9 cells were 39.27 mg/
mL, 39.85 mg/mL, 101.2 mg/mL and 110.7 mg/mL, and H. magnifica
venom was the weakest toxicity to SF9 cells.
2.6 The analgesic effect of crude venom
2.6.1 Assessment of locomotor behavior in
larval zebrafish
In order to determine the analgesic properties of crude venom
on zebrafish larvae, test was carried out using locomotor behavior.
As shown in Figure 6, with the exception of H. crispa venom, other
venoms gradually reduced the locomotor behavior of larvae with
increasing concentrations compared to the acetic acid group.
Statistics were conducted on the total distance traveled by
zebrafish larvae (Figure 7). Acetic acid group moved a total
distance of 6192.67 ± 247.30 mm in 20 min. The total distance of
M. doreensis,H. magnifica,E. quadricolor,S. haddoni, and H. crispa
venoms reached 2767.21 ± 190.44 mm, 2448.82 ± 304.48 mm,
2076.45 ± 195.08 mm, 2867.87 ± 191.91 mm, and 5225.97 ± 626.47
mm at concentrations of 10 mg/ml, 0.5 mg/ml, 2 mg/ml, 2 mg/ml, and
15 mg/ml, respectively (Figures 7A–E), the strongest analgesic effect
on zebrafish larvae was observed in H. magnifica.However,
H. crispa was not significantly different from the acetic acid
group, indicating no analgesic effect on zebrafish larvae. In
addition, after exposure to the crude venom, the four venoms
with analgesic effects significantly reduced the distance travelled
and remained stable thereafter (Figure 7F).
2.6.2 Hot plate test and crude toxicity lethal test
on mice
To assess the analgesic effects of crude venom, a hot plate test
was performed. The results were shown in Figure 8.After
intraperitoneal injection of 5 mg/kg venoms, crude venom except
H. crispa venom significantly increased the latency time of mice to
lick the hind paws at different times compared to the negative
control (Figure 8A). Furthermore, H. magnifica venom exhibited
the strongest analgesic effect at 90 min, with maximum possible
effect (MPE) value of 84.14 ± 8.85%. The strongest analgesic effect
of H. magnifica venom on zebrafish larvae and mice was selected
and injected intraperitoneally into mice at doses of 0.5, 1, 3, 5, 7 and
9 mg/kg. The results showed that after 120 min of injection, the
analgesic effect was strongest at 9 mg/kg, with a maximum possible
effect value of 55.68 ± 4.54%. The ED
50
value for the analgesic effect
of H. magnifica on mice was 8.95 mg/kg (Figure 8B). For lethal
assay with H. magnifica crude venom, intraperitoneal injection of
11.0 mg/kg of venom was induced 50% lethality of the mice.
3 Discussion
Sea anemones contain complex and abundant venoms that
determine the diversity of the biological activities, which provide
a broad research space for exploring the biological properties of sea
anemones, and also bring great opportunities for the development
of safe and efficient novel marine peptide drugs. The sea anemone
resources from the South China Sea are abundant and diverse,
but traditional methods for isolating and identifying their proteins
and peptides are time-consuming and laborious, and their
TABLE 1 The values of hemolysis, trypsin and pepsin inhibition assays for crude venom.
Species
Assay
Hemolysis Trypsin Pepsin
MC
100
SD SEM M SD SEM M SD SEM
Macrodactyla doreensis 150.00 0.92 0.53 14.32 0.83 0.48 279.11 3.63 2.09
Heteractis magnifica 1.00 0.47 0.27 251.25 2.48 1.43 145.08 1.91 1.10
Entacmaea quadricolor 10.00 0.46 0.27 Nd Nd Nd 161.94 9.42 5.44
Stichodactyla haddoni 40.00 0.47 0.27 133.46 1.07 0.62 391.25 4.09 2.36
Heteractis crispa 100.00 2.20 1.27 22.64 0.78 0.45 250.93 5.02 2.90
MC
100
, the minimum concentration of protein to cause 100% hemolysis of the red blood cells (mg/mL); SD, standard deviation; SEM, standard error of the mean; M, mean (TIU/mg); Nd,
not determined.
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pharmacological activities are complex and varied, making it
difficult to obtain effective lead compounds for new drug
development. Therefore, this study investigated the crude venom
of five representative sea anemones under various activity tracking
conditions. Although the pharmacological activity may be caused
by multiple components, the best active sea anemone samples were
obtained. For the best pharmacological activity of sea anemones,
modern omics technologies such as transcriptomics, proteomics,
and metabolomics can be used to systematically explore the protein
and peptide groups, and then predict their structure and function
through artificial intelligence (AI) driven by deep learning
techniques, laying a solid foundation for new drug development.
FIGURE 2
Effect of crude venom on the viability of U251, HepG2 and A549 cells. (A-E) Results of M. doreensis,H. magnifica,E. quadricolor,S. haddoni and H.
crispa venoms on the cell viability respectively. (F-H) Dose effect curves of crude venom on U251, HepG2 and A549 cells, respectively. *P< 0.05,
**P< 0.01, ****P< 0.0001 versus the 0 mg/mL group. U251, human glioma cells; HepG2, human hepatocellular carcinoma cells; A549, human lung
cancer cells. Md, M. doreensis; Hm, H. magnifica; Eq, E. quadricolor; Sh, S. haddoni; Hc, H. crispa.
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The protein and peptide fractions in crude venom of five species
of sea anemones from the South China Sea were firstly
comprehensively analysed using RP-HPLC for the detection of
fractions less than 10 kDa in venoms and combined with SDS-
PEGE method for detecting a wide range of proteins, and the
bioactivities of crude venom were explored using various methods.
In the chromatograms, differences in peak shapes and heights of
crude venom indicated variations in venom components, and the
two distinct peaks of H. magnifica and H. crispa venoms suggested
that the components were extremely high in content. The
distributions of crude venom in SDS-PAGE were in the range of
10-180 kDa, which were consistent with the existing studies on
crude venom (Hu et al., 2011;Alcaide et al., 2024). And there was a
common band at 17 kDa in four venoms and H. magnifica protein
band had the highest expression, suggesting that the four venoms
may contain the same protein component, which was better
represented in H. magnifica.
The cytolysins identified in sea anemones can be classified into
four groups based on molecular weight and function, one of which
was pore-forming proteins or actinoporins (Hoepner et al., 2019;
Ramı

rez-Carreto et al., 2019a). It had been shown that actinoporins
and pore-forming proteins had strong hemolytic effects, such as
equinatoxin III and FraA, FraB and FraD actinoporins isolated from
Actinia equina and Actinia fragacea, respectively, as well as a pore
FIGURE 3
The typical images of scratch wound healing and transwell invasion assays for crude venom on U251 cells. (A) Pictures of wound healing and
transwell invasion assays in the control group. (B-F) Pictures of wound healing assay using different concentrations of crude venom and treating
U251 cells for 0, 24, 48 h and pictures of transwell assay using 100 mg/mL crude venom for 24 h of U251 cells. Control, control group; Md, M.
doreensis; Hm, H magnifica; Eq, E quadricolor; Sh, S. haddoni; Hc, H crispa.
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forming toxin called sticholysin II extracted from Stichodactyla
helianthus, had strong hemolytic activity (Suputetal.,2001;
Celedon et al., 2013;Morante et al., 2019). This hemolysis mainly
occurs through the binding of toxins to the cell membrane,
oligomerization of the protein, followed by translocation of the
N-terminal a-helix through the lipid bilayer leading to
the formation of functional pores in the plasma membrane of the
erythrocytes (Valle et al., 2018;Morante et al., 2019). In this study,
all crude venom extracts showed hemolytic activity, indicating that
the hemolytic properties of venoms may be related to the presence
of pore-forming proteins or actinoporins. And the hemolytic
activities of H. magnifica,S. haddoni and H. crispa had been
reported to be 3.6 × 10
4
, 56.3 and 3.3 × 10
4
HU/mg, respectively
(Khoo et al., 1993;Subramanian et al., 2011;Leychenko et al., 2018).
The differences in these values compared to the hemolytic activity of
the same species collected in the South China Sea may be related to
the diversity of the environments in which the species live
(Monastyrnaya et al., 2010).
Protease inhibitors are a group of peptides and proteins with
potential applications in cardiovascular, inflammatory and even
immune diseases based on the control of proteolysis (Ramirez-
Carreto et al., 2019b). At present, several peptides with protease
inhibitory activity have been extracted from sea anemones. The
main function of most known Kunitz-type peptides is protease
inhibition, especially trypsin, and their structure enables the
formation of two loops responsible for protease inhibition
(Kvetkina et al., 2022;Mazzi Esquinca et al., 2023). HCIQ2c1,
HCIQ4c7, and HMIQ3c1 of Kunitz-type peptides had been found
FIGURE 4
Crude venom suppress migration and invasion of U251 cells. (A-E) The migration ratios of cells at 24 h and 48 h after treatment of M. doreensis,H.
magnifica,E. quadricolor,S. haddoni and H. crispa venoms, respectively. (F) Invasion rates of crude venom on U251 cells at 50, 100 and 200 mg/mL
concentrations. Md, M. doreensis; Hm, H. magnifica; Eq, E. quadricolor; Sh, S. haddoni; Hc, H. crispa. *p<0.05, **p<0.01, ***p<0.001, ****
p<0.00001 versus control group.
He et al. 10.3389/fmars.2024.1480745
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to interact with several serine proteases (Kvetkina et al., 2022). In
the present study, the specific inhibitory effect of sea anemone
venoms on different proteases speculates that Kunitz-type peptides
may be present in the crude venom, which provides a basis for
further studies on the isolation of Kunitz-type peptides with
pharmacological effects from these venoms.
Nowadays, Hmg 1b-2 and Hmg 1b-4 peptides from
H. magnifica at 0.1-1 mg/kg have significant anti-inflammatory
effects (Gladkikh et al., 2023). For the anticancer activity of South
China Sea anemones, it was found that each of crude venom was
cytotoxic to U251, HepG2, and A549 cancer cells, this result
enhances the values of sea anemones in anticancer research and
highlights the promising future of sea anemone toxin research, and
transcriptomics will be used to sequence the cells treated with crude
venom to verify the key targets of inhibitory effect on these cancer
cells. However, preliminary studies have also detected cytotoxic
activity of crude venom against normal cells, and it is hoped that
after the isolation of specific peptide toxins, peptide toxins with
specific inhibitory activity on cancer cells and low toxicity on
normal cells can be screened out. Currently, glioma is the most
common malignant brain tumor with high mortality and poor
outcome, and is considered one of the most intractable early-death
solid tumor to treat in neurosurgery (Anton et al., 2012;Guo et al.,
2022). The drugs available today for the treatment of glioma are
cisplatin, lomustine and temozolomide, but gliomas greatly limit
the efficacy of treatment due to resistance to these drugs (Ren et al.,
2014). Therefore, in this study, the inhibitory effect of crude venom
from five sea anemones on the migration and invasion of U251 cells
was verified by scratch wound healing and transwell invasion
assays, so the peptide drugs of five sea anemones from the South
China Sea may be an important source for use in the treatment of
gliomas. And more significant migration inhibition was observed at
24 h than at 48 h in the migration assay, which may be attributed to
the continued proliferation of surviving cells after 24 h. The
cytotoxicity, invasion and migration assays of the crude venom
from sea anemones have confirmed the potential presence of
anticancer peptides in sea anemones, which require solid phase
peptide synthesis (SPPS), RP-HPLC purification and activity
FIGURE 5
Survival of crude venom in insecticidal experiments on T. molitor and SF9 cells. (A-E) Determination of the toxicity of crude venom of M. doreensis,
H. magnifica,E. quadricolor,S. haddoni and H. crispa at different times to T. molitor, respectively. (F) The cell viability of SF9 cells for five venoms at
different concentrations in CCK-8 assay. Md, M. doreensis; Hm, H. magnifica; Eq, E. quadricolor; Sh, S. haddoni; Hc, H. crispa. *P< 0.05, **P< 0.01,
***p<0.001, ****P< 0.0001 versus control group;
###
P< 0.001,
####
P< 0.0001 versus the 0 mg/mL group, n = 100.
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verification. This provides a new way to develop novel and efficient
anticancer peptide drugs from South China Sea anemones.
Sea anemone venoms were the main research targets for the
development of insecticidal peptides of biotoxin origin (Yan et al.,
2013). The most common insecticide currently mainly act on the
insect nervous system, with common targets being Nav and Kv
channels, glutamate and nicotinic acetylcholine receptors (Windley
et al., 2012;Ren et al., 2018). Bunodosoma granulifera and H. crispa
have been reported to affect Nav channels and can act as insecticides
(Bosmans et al., 2002;Kalina et al., 2020). In this study, crude
venom showed excellent insecticidal activity against T. molitor and
SF9 cells, peptides that act on Nav channels may be present in crude
venom. However, it was noted that E. quadricolor showed extreme
toxicity on SF9 cells, while H. magnifica was the most toxic to
T. molitor, suggesting that in addition to evaluations at the cellular
level, insect experiments are needed to validate these results in order
FIGURE 6
Locomotor behavior of zebrafish larvae after crude venom treatment. (A) Behavioral trajectories of zebrafish larvae in blank, acetic acid and tramadol
groups. (B) Behavior of zebrafish larvae in different concentrations of crude venom. Motion trajectory was recorded every 5 s and represented as a
curve. Detected instantaneous velocity and displayed in different colors (black,< 2 mm/s; green, 2-8 mm/s; red, > 8 mm/s). Md, M. doreensis; Hm, H
magnifica; Eq, E quadricolor; Sh, S. haddoni; Hc, H crispa.
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to gain a comprehensive understanding of the effects of anemone
toxins in insects. These results further validate the potential of
natural biotoxins for insecticidal activity and allow further study of
toxin peptides in crude venom for the development of new
insecticidal peptide lead molecules.
M. doreensis,H. magnifica,E. quadricolor,andS. haddoni
venoms were found to have analgesic effects on zebrafish larvae
and mice in locomotor behavior and hot plate tests, which
supported earlier findings on crude extracts of some echinoderms
(Kanagarajan et al., 2008). Zebrafish, sharing approximately 70%
homology with human genes, is now an ideal model system for drug
and gene discovery and is becoming increasingly popular in the
direction of understanding the pharmacology of drugs, especially
toxicity and behavioral effects (Howe et al., 2013;Kirla et al., 2021).
Acetic acid was used as a model in behavioral experiments with
zebrafish larvae, which was added to water to cause pain as a
nociceptive stimulus. Combined with the hot plate test, the results
of this study found that H. magnifica venom had the best analgesic
effect on zebrafish and mice. At present, a variety of peptides with
analgesic effects have been found in H. crispa, such as HCRG21 and
APETx2, and the analgesic mechanisms of sea anemone peptides
were mainly classified as full antagonist of TRPV1 receptor and
FIGURE 7
Distance travelled by zebrafish larvae in different treatment groups. (A-E) Total distance travelled by zebrafish larvae within 20 min after exposure to
M. doreensis,H. magnifica,E. quadricolor,S. haddoni and H. crispa, respectively. (F) The variation of travel distance over time for zebrafish larvae
juveniles of different treatment groups within 20 min. Md, M. doreensis; Hm, H. magnifica; Eq, E. quadricolor; Sh, S. haddoni; Hc, H. crispa. **P<
0.01, ****P< 0.0001 versus acetic acid group, n = 6.
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selective blocker of the proton-sensitive channel acid-sensing ion
channel 3 (ASIC3) (Deval et al., 2008;Monastyrnaya et al., 2016;
Logashina et al., 2021). In this study, sea anemones collected from
the South China Sea demonstrated the strongest analgesic activity of
H. magnifica, whereas H. crispa crude venom had no analgesic
activity. It can be assumed that the different peptides of these
anemones lead to different inhibitory mechanisms involved or no
inhibitory effect, it is necessary to strengthen the study of peptides
with analgesic effects in H. magnifica from the South China Sea and
to use patch-clamp to record the targets that produce analgesic
effects (Salvage et al., 2023;Zhang et al., 2023), in order to develop
safe and efficient analgesic drugs with few side effects.
4 Materials and methods
4.1 Samples collection and crude
venom preparation
The species of five sea anemones used in this study were
collected from the South China Sea (18°N, 112°E) at a depth of 4
m in March 2023. Later, these sea anemones were grown in glass
aquariums filled with seawater. Following 1 to 2 weeks of
maintenance, crude venom extracts were obtained from fresh
samples adoption a homogenization method, which primarily
requires mincing and homogenizing sea anemones and then
freezing at 4°C, followed by centrifugation of the samples at
10000 rpm for 30 min and lyophilized. Then, the lyophilized
samples were dissolved and centrifuged, the supernatant was
desalted using dialysis, finally crude venom of sea anemones were
obtained through freeze-drying.
4.2 The reverse-phase HPLC
Preliminary detection of crude venom fractions was performed
by using RP-HPLC with a UV detector and C18 column
(Diamonsil, 250 × 4.6 mm, 5 mm). 10 mg of each crude venom
was weighed and dissolved in 1 mL of deionized water, and then
centrifuged at 4°C and 10000 rpm for 10 min. The supernatant was
filtered through a 0.45 mm microporous filtration membrane, and
then 100 mL sample was injected into the RP-HPLC system with the
aid of the auto-sample. The mobile phase used in this study A (0.1%
trifluoroacetic acid (TFA) acetonitrile solution) and B (0.1% TFA
ultra-pure water). Before use, the mobile phase solvents were
filtered through 0.45 mmfilter papers and degassed for 15 min on
an ultrasonicator. Separation at a constant flow rate of 1 mL/min for
65 min using a linear gradient (5-70% A) and monitored by
absorbance at 214 nm.
4.3 SDS-PAGE analysis
SDS-PAGE was performed essentially as described by Laemmli
(Laemmli, 1970). 10 mg of each crude venom was weighed and
dissolved in 1 mL of deionized water, and then centrifuged at 1000
rpm for 5 min. 16 mL of the supernatant was mixed with 4 mLofthe
sample loading buffer (0.01 M Tris-HCl, 10% (w/v) SDS, 10% (v/v)
glycerol, 0.1% (w/v) bromophenol blue, 2% b-mercaptoethanol) and
then boiled for 10 min. Samples were loaded onto gel slab consisting
of 12% separating gel (pH 8.8) and 4% stacking gel (pH 6.8), and
subjected to electrophoresis at 100 V for 1 h. The electrophoresis gels
were stained using Coomassie Brilliant Blue R-250.
4.4 Hemolytic activity assay
Hemolysis, as indicative of red blood cells (RBCs) membrane
destruction caused by interaction with substances, was evaluated by
measuring the released hemoglobin (Macczak et al., 2015;Almasi
et al., 2019). Hemolytic activity of crude venom was determined
according to the method of Zhang with minor modifications
(Zhang et al., 2010). Briefly, harvested rat blood samples were
collected in tubes containing heparin to prevent coagulation and
centrifuged at 1500 rpm for 10 min at 4°C. Then, the RBCs at the
bottom were collected and washed three times with PBS at PH7.4
and the RBCs were resuspended in saline to a final concentration of
1% (v/v). Crude venom solutions dissolved in deionized water were
FIGURE 8
Analgesic effects of crude venom and PBS negative and tramadol positive controls in mice. (A) The MPE of pain reaction in mice to injection of
crude venom at 5 mg/kg. (B) The MPE of H magnifica venom at different doses on analgesic in mice. Md, M. doreensis; Hm, H magnifica; Eq, E
quadricolor; Sh, S. haddoni; Hc, H crispa. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 versus the PBS group, n = 6.
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mixed with RBCs and incubated at 37°C for 1 h before
centrifugation at 1500 rpm for 10 min at 4°C. 100 mL of the
supernatants were pipetted into 96-well microtiter plate and the
absorbance was measured at 540 nm using a microplate reader
(Synergy HTX, Bio-Tek, USA). RBCs were treated with deionized
water for fully hemolytic and used as positive controls. Percent
hemolysis was calculated using the following formula:
percent hemolysis =½OD540(samples)=OD540(positivecontrol)
100 %
4.5 Trypsin inhibitory activity
N-a-benzoyl-D, L-arginine p-nitroaniline hydrochloride
(BAPNA) was a chromogenic substrate for trypsin, which
hydrolyses BAPNA to produce p-nitroaniline, and the absorbance
of the solution is measured at 410 nm (Erturk et al., 2016). Trypsin
inhibitory activity of crude venom was determined as per the
method of Erlanger using BAPNA as a substrate in Tris-HCl
buffer (pH 8.2, containing 0.1 M CaCl
2
)(Erlanger et al., 1961).
Each crude venom dissolved in deionized water was mixed with the
substrate and incubated at 37°C for 10 min, the reaction was then
terminated by the addition of 30% (v/v) acetic acid (100 mL).
Finally, substrate hydrolysis was determined at 410 nm. One unit
of trypsin activity can be defined as a 0.01 unit increase in
absorbance at 410 nm and trypsin inhibitory activity was defined
as inhibiting one unit of trypsin activity.
4.6 Pepsin inhibitory activity
Hemoglobin can be rapidly hydrolyzed by pepsin and the
inhibitory effect of pepsin was determined using bovine hemoglobin
as a substrate (Marks et al., 1973;Vaghefiet al., 2002). Pepsin inhibition
assay was performed using a mixture of pepsin, hemoglobin and crude
venom followed by incubation at 37°C for 10 min before the reaction
was stopped by the addition of 100 mL of 30% trichloroacetic acid. After
centrifugation at 10000 rpm for 10 min, the supernatants were pipetted
into cuvettes and absorbances were read at 660 nm.
4.7 Inhibitory activity assay of
a-galactosidase
a-Galactosidase interacted with p-nitrophenyl-a-D-
galactopyranoside (PNPG) to produce p-nitrophenol, which
absorbed at 405 nm (Sakharayapatna Ranganatha et al., 2021). To
determine the inhibitory activity of crude venom against a-
galactosidase using PNPG as substrate. The enzyme and crude
venom solutions were mixed in 0.1 M phosphate buffer and
reactions were incubated at 37°C for 30 min, then the substrate
was added. Finally, the reactions were terminated by the addition of
1MNa
2
CO
3
and absorbances at 405 nm were finally measured.
4.8 Cell cultures
Cancer cell lines used in this study included insect cells (SF9),
human glioma cells (U251), human hepatocellular carcinoma cells
(HepG2) and human lung cancer cells (A549), and were obtained
from Hainan Provincial Key Laboratory of Carcinogensis and
Intervention. SF9 insect cells were seeded in cell culture flasks
and cultured in SFM medium supplemented with 10% fetal bovine
serum (FBS) at 27°C, other cells were incubated in DMEM medium
supplemented with 10% FBS at 37°C and 5% CO
2
for the CCK-8
assay, wound healing and transwell invasion assays. Then, the spent
medium was removed, control wells received fresh medium without
crude venom, and treatment wells received medium with different
concentrations of crude venom.
4.9 CCK-8 assay
10 mg of each crude venom was weighed and dissolved in the
culture medium, then the crude venom solution was taken for
dilution. U251, HepG2, A549 and SF9 cells were seeded into 96-well
cell culture plates at a density of 1 × 10
4
cells/well and treated
U251, HepG2 and A549 cells with differing concentrations of M.
doreensis (50, 100, 500, 1000, 3000, 4000 mg/mL), H. magnifica (1, 5,
75, 500, 750, 1000 mg/mL), E. quadricolor (5, 10, 100, 500, 750, 1000
mg/mL), S. haddoni (10, 50, 100, 500, 750, 1000 mg/mL), and H.
crispa (10, 50, 750, 1500, 3000 and 4000 mg/mL) venoms for 24 h,
the E. quadricolor,S. haddoni and H. crispa venoms were
formulated into 10, 20, 50, 500, 750, and 1000 mg/mL and
solutions of 50, 100, 200, 500, 750 and 1000 mg/mL were
prepared using M. doreensis and H. magnifica venoms to treat
SF9 cells. Following treatment, 10 mL of CCK-8 solution was added
to each well and then the culture was continued incubation for 2 h
at 37°C. Absorbance values at 450 nm were measured using the
microplate reader.
4.10 In vitro wound healing study
Assessment of the migratory ability of U251 cells using the
wound healing assay. Cells were inoculated in 24-well plates at a
density of 1 × 10
5
cells/well, cultured for 24 h, and then scraped
perpendicular to the bottom of the wells with a 200 μL sterile pipette
tip. Following this, the cellular debris were washed twice with PBS,
and serum-free medium containing different concentrations of
crude venom were incubated for 48 h at 37°C in a 5% CO
2
incubator, using untreated cells as controls. Images were taken
after 0 h, 24 h, and 48 h under an inverted microscope
(magnification × 100) and measurements of wound area were
performed using ImageJ software. The reduction of wound-size
was calculated according to the following formula:
wound −size reduction( % ) = ½(originalscratcharea−the
pointscratcharea)=originalscratcharea100 %
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4.11 Transwell invasion assay
For transwell invasion assay, diluted matrigel (1:8) was applied
to the chambers of the transwell apparatus and these chambers were
incubated for 1 h at 37°C. A total of 5 × 10
4
U251 cells containing
50, 100 and 200 μg/mL of crude venom were evenly seeded in the
upper chamber of the transwell, using cells untreated with crude
venom as a control, and DMEM medium with 10% FBS was placed
in the lower chamber. After incubation at 37°C for 24 h, U251 cells
were fixed with 4% paraformaldehyde for 30 min and then stained
with 0.1% crystal violet for 30 min and observed under a
microscope (magnification × 400) and counted using ImageJ.
4.12 Toxicity assay on Tenebrio molitor
Each crude venom was dissolved with saline and formulated
into concentrations of 20, 40 and 100 ng/mg for assessing the
toxicity of the venom to T. molitor. Three replicate groups of
T. molitor were injected of per dose, and the number of survivor
insects were counted after 6, 12, 24, 36, 48, 60 and 72 h of
application. Then the survival rates of T. molitor were determined
after the addition of venoms. Control groups were applied
with saline.
4.13 Evaluation of the analgesic activity
4.13.1 Locomotor behavior of zebrafish larval
Zebrafish, which share a high genetic similarity with mammals
and possess a complex nervous system that allows for assessment of
the swimming response to nociceptive stimuli, were utilized as a
model to analyze pain responses by measuring total distance in
locomotor behavior (Park et al., 2021;Zaig et al., 2021). 10 mg of
each crude venom was weighed and dissolved in deionized water,
then the crude venom solution was taken for dilution. Zebrafish
larvae at 5-day post-fertilization (5-dpf) were introduced into the
wells of 24-well plates (1 larva per well), given 1 mL of different
concentrations of crude venom and observed for 20 min, using 6
zebrafish larvae for each concentration. Then 1 mL of acetic acid
was added to each well and placed in a zebrafish tracking system
(Viewpoint Life Sciences, Montreal, QC, Canada) for 20 min. Each
larva was recorded 10 times (2 min each) and the total distances
swum by the larva were finally recorded. An acetic acid group was
add to the water as a nociceptive stimulus to cause pain and 15 mg/
mL tramadol was used as a positive control to evaluate its analgesic
effect. The blank group did not ingest acetic acid, both the acetic
acid group and the experimental group ingested equal amounts of
acetic acid.
4.13.2 Hot plate test
The hot plate test was used to assess central antinociceptive
activity in mice, indicating the antinociceptive effect (Benmaarouf
et al., 2020;Vandeputte et al., 2022). Mice were placed on a hot plate
(maintained at 55 ± 0.5°C) and the time of licking the hind paws
were recorded within 60 s to prevent tissue damage. Selected
animals were injected intraperitoneally with a dose of 5 mg/kg
crude venom dissolved using saline. And pain thresholds were
assessed at 30, 60, 90, 120 and 180 min deescalation after treatment.
The negative control group by intraperitoneal injection of PBS, with
a dose of 50 mg/kg of tramadol as a positive control. The analgesic
effects of crude venom were quantified as a percentage of the
maximum possible effect (%MPE) to equalize the bases latency
time in different animals using the following formula:
%MPE =½(testlatency−basallatency)=
(cutofftime−basallatency)100 %
4.14 Determination of lethality in mice
High concentrations of H. magnifica crude venom were found
to cause rapid death in mice by hot plate test. Specifically, the mice
were divided into five groups and given 8.9 mg/kg, 9.8 mg/kg, 10.8
mg/kg, 11.8 mg/kg and 13.0 mg/kg of H. magnifica venom,
deaths were recorded after intraperitoneal injection of venom and
the LD
50
value was calculated using the trimmed Spearman-
Karber method.
4.15 Statistical analysis
All experiments were performed in triplicate, plotted and
statistically analysed using Adobe Photoshop 20.0.4, GraphPad
Prism 10.1.2 and SPSS 19.0 software. Results were presented as
mean ± SEM and analysed using one-way ANOVA, with P< 0.05
considered statistically significant.
5 Conclusions
In summary, studies on crude venom extracted from five types
of South China Sea anemones, including M. doreensis,H. magnifica,
E. quadricolor,S. haddoni, and H. crispa, have shown that the
successfully screened H. magnifica exhibits extremely strong
biological activity, mainly manifested in hemolysis, enzyme
inhibition, anticancer, insecticidal, and analgesic activities.
Especially, the crude venom of H. magnifica has been extensively
studied on U251 cells, and it has been found that the crude venom
can significantly inhibit the migration and invasion of U251 cells at
low concentrations. Therefore, sea anemone H. magnifica has been
identified as one of the highly promising and efficient therapeutic
drugs in the South China Sea, which requires the development of
novel anticancer and analgesic peptide drugs through sequencing
technology and subsequent peptide synthesis work to promote the
treatment of human diseases.
He et al. 10.3389/fmars.2024.1480745
Frontiers in Marine Science frontiersin.org14

Data availability statement
The original contributions presented in the study are included
in the article/supplementary material. Further inquiries can be
directed to the corresponding authors.
Ethics statement
Ethical approval was not required for the studies on humans in
accordance with the local legislation and institutional requirements
because only commercially available established cell lines were used.
The animal study was approved by the Ethics Committee of Hainan
Medical University. The study was conducted in accordance with
the local legislation and institutional requirements.
Author contributions
PH: Writing –original draft, Methodology, Formal analysis, Data
curation. ML: Writing –original draft, Methodology, Data curation.
JF: Writing –review & editing. YL: Writing –review & editing. BY:
Writing –review & editing, Conceptualization. BG: Writing –review
& editing, Funding acquisition, Conceptualization.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This
research was funded by National Natural Science Foundation of
China (no. 82060686), Hainan Provincial Key Point Research and
Invention Program (no. ZDYF2022SHFZ309), Hainan Province
Health Industry Research Project (22A200358), and Special
scientific research project of Hainan academician innovation
platform (no. YSPTZX202132).
Acknowledgments
The author acknowledge the support and assistance in terms of
instruments and facilities provided by the Zebrafish Platform of
Public Research Center at Hainan Medical University.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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