Extensive research on marine algae, especially on their health-promoting properties, has been conducted. Various ingredients with potential biomedical applications have been discovered and extracted from marine algae. Alginate oligosaccharides are low molecular weight alginate polysaccharides present in cell walls of brown algae.
Trang 1Available online 8 July 2021
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Potential applications of alginate oligosaccharides for biomedicine – A
mini review
Mingpeng Wanga, Lei Chena,*, Zhaojie Zhangb
aCollege of Life Science, Qufu Normal University, Qufu 273100, China
bDepartment of Zoology and Physiology, University of Wyoming, Laramie, Wyoming, USA
A R T I C L E I N F O
Keywords:
Alginate oligosaccharides
Brown algae
Health-promoting
Biomedicine
A B S T R A C T Extensive research on marine algae, especially on their health-promoting properties, has been conducted Various ingredients with potential biomedical applications have been discovered and extracted from marine algae Alginate oligosaccharides are low molecular weight alginate polysaccharides present in cell walls of brown algae They exhibit various health benefits such as anti-inflammatory, anti-microbial, anti-oxidant, anti-tumor and immunomodulation Their low-toxicity, non-immunogenicity, and biodegradability make them an excellent material in biomedicine Alginate oligosaccharides can be chemically or biochemically modified to enhance their biological activity and potential in pharmaceutical applications This paper provides a brief overview on alginate oligosaccharides characteristics, modification patterns and highlights their vital health promoting properties
1 Introduction
Brown algae are a large group of multicellular algae and one of the
essential and integral components of the marine ecosystem (Lavaud &
Goss, 2014) They are biologically diverse, with thousands of different
species, forming the dominant vegetation in the intertidal and subtidal
zone of rocky shores (Bartsch et al., 2008) They live from coastal-
estuarine to deep-sea regions and have many unique features, such as
fast growth, distinctive structure, strong adaptiveness, and wide
distri-bution Their ecological significance is based in part on their
contribu-tions to marine biomass and marine carbon cycling (Lavaud & Goss,
2014) Brown algae provide food and habitats for many other organisms
and promote the prosperity of the entire marine biosphere (de Mesquita
et al., 2018; Santelices, 2007) Brown algae are also economically
important for human beings as a kind of food, especially in Asian
countries In addition, they are used as natural feed or fertilizer due to
their high mineral and trace elements, and as a source of biological
products such as alginates, mannitol and iodine (Afonso et al., 2019;
Arioli et al., 2015; Overland et al., 2019; Salehi et al., 2019) Brown
algae have tremendous potential as a source of novel functional
com-ponents that are not present in terrestrial plants More recently, the
valuable ingredients extracted from brown algae, e.g alginates,
fucoi-dan and laminaran, have been explored for nutrient and drug
develop-ment (Ford et al., 2020; Garcia-Vaquero et al., 2018; Generali´c Mekini´c
et al., 2019; Gunathilaka et al., 2020; Saraswati et al., 2019; Thanh et al.,
2013; Zou et al., 2019)
Alginate is a linear acidic polysaccharide distributed widely in cell walls of brown algae (Sari-Chmayssem et al., 2015; Synytsya et al.,
2015) Alginate consists of hexuronic acid residues β-D-mannuronic acid (M) and α-L-guluronic acid (G) with exclusively 1 → 4 glycosidic link-ages Its chelation, gelation, and hydrophilic properties have led to its wide application in food, cosmetic and biomedical industries (Donati & Paoletti, 2009; Lee & Mooney, 2012) Increasing evidence indicates that, when used as a therapeutic adjuvant, drug carrier, wound healing ma-terial and biological scaffold, alginate could improve antitumor immune efficacy in ovarian cancer, melanoma, liver cancer, and breast cancer (Fan et al., 2019) However, the direct therapeutic effects of alginate in biomedical applications have been greatly limited due to its macromo-lecular structure, poor solubility and low bioavailability (Zhu et al.,
2020) Alginate can be digested chemically or enzymatically, producing alginate oligosaccharides (AOS), which have lower molecular weights and lower viscosity AOS have better solubility and bioavailability (Trincone, 2015) As a consequence, particular interest has been focused
on AOS due to their better pharmacological activities and beneficial effects in biomedicine
In this paper, we present an overview of the structure, biological activities and modification patterns of AOS We also discuss the recent developments of using AOS in treating chronic and degenerative
* Corresponding author at: Qufu Normal University, 57 Jingxuan West Road, Qufu, China
E-mail address: leichen_2018@qfnu.edu.cn (L Chen)
Contents lists available at ScienceDirect Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
https://doi.org/10.1016/j.carbpol.2021.118408
Received 7 April 2021; Received in revised form 23 June 2021; Accepted 3 July 2021
Trang 2diseases, modulating human gut microbiota and promoting curative
effect of traditional drugs as therapeutic adjuvant or drug carrier
2 Structure and modification patterns of alginate
oligosaccharides
2.1 Structure of AOS
As a degradation product of alginate, AOS are a mixture of linear
oligomers, which consist of β-D-mannuronic acid (M) and α-L-guluronic
acid (G) (Fig 1) at different ratios and degrees of polymerization (DP)
(Rioux & Turgeon, 2015) The overall composition of the two uronic
acids and their distribution along the oligomer chain vary widely
depending on the species of algae and influence the properties of AOS
As shown in Fig 1, three types of oligomer blocks (2 ≤ DP ≤ 25) are
typically obtained: M, G, and mixed MG blocks, according to different
source and degradation methods
Structurally, the monomeric units M and G are a pair of C-5 epimers
due to their different orientation of carboxyl group at the C-5 position
(Grasdalen, 1983) The molecular formula of M or G units is C6H10O7
and the conversion relationships of AOS relative molecular weight (Mr)
and the degree of polymerization can be described as shown in Eq (1):
Mr=DP⋅Mr(C6H10O7) − (DP − 1)⋅Mr(H2O) (1)
When the atomic weights of carbon, hydrogen and oxygen, and the
molecular weights of H2O are plugged into the formula, the DP can be
calculated using Eq (2):
Finally, the epimerization at C-5 results in significant differences in
spatial structure and physicochemical properties of their oligomers,
respectively The equatorial configuration of the β-1,4-glycosidic bond
predestines a stretched chain conformation of poly M while the axial
linkage of the α-1,4-glycosidic bond implicates the tendency to helix
formation of poly G (Mackie et al., 1983; Plazinski, 2011) Generally, the poly G involving axial linkage is more rigid than the equatorially linked poly M
2.2 Production of AOS from alignate
At present, the three main methods for AOS production are acid hydrolysis (AH), oxidative degradation (OD) and enzymatic digestion (ED) Each method has its own advantages and limitations
The main feature of acid hydrolysis of alginate is that it results in random cleavage along the polysaccharide chains and produces AOS fragments with unmodified hexuronic acid residues at both termini (Fig 2) Therefore, the AOS produced by acid hydrolysis can maintain the inherent structure of alginate Acid hydrolysis has been widely used for producing a series of AOS with different degrees of polymerization because of its low cost, ease of control, simplicity and availability However, only under high temperature and pressure conditions can AOS with molecular weight below 4000 be obtained through acid hydrolysis
In addition, a large number of inorganic salts are generated during the final neutralization stage of acid hydrolysis The application of acid hydrolysis has been limited due to equipment corrosion, high energy consumption and residue waste pollution
Another chemical degradation method is oxidative degradation, which has higher reaction efficiency and high yield compared to acid hydrolysis As an easily degradable reagent that creates only water as an oxidation by-product, hydrogen peroxide (H2O2) has been widely used
to produce functional AOS with high purity and quality As shown in
Fig 2, AOS residues are easily ring-opened at the reducing end to form carboxyl group during the oxidative degradation This additional carboxyl radical could induce novel bioactivities of oxidative AOS Zhou
et al reported that guluronate oligosaccharide (GOS) prepared by oxidative degradation (GOS-OD), but not GOS produced by acid hy-drolysis or enzymatic digestion, significantly reduced the lipopolysac-charide (LPS)-stimulated overproduction of nitric oxide (NO) in RAW
Fig 1 The structure of alginate and AOS: monomers; chain conformation and fragments of AOS product 2 ≤ n ≤ 25
Trang 3264.7 cells (Zhou et al., 2015) The existence of the additional carboxyl
group may play an important role in the NO-inhibitory and subsequent
anti-inflammation effect of GOS-OD
The enzymatic digestion may be the most promising method for
producing AOS due to its key advantages such as site-specific cleavage
reaction, mild reaction conditions, efficient reaction rate, and high
re-action yields Alginate lyases are key enzymes that specially catalyze the
degradation of alginate They break the O–C4 bond to uronic acid
residues through a β-elimination reaction that leads to formation of the
4,5-unsaturated hexuronic acid residue at the non-reducing terminus
(Fig 2) This unsaturated terminal structure with a C4–C5 double bond
can be detected at 230 nm The unsaturated AOS have various novel
bioactivities such as antioxidant, anti-tumor, neuroprotective and
immuno-stimulation (Iwamoto et al., 2003; Iwamoto et al., 2005; Tusi
et al., 2011) Compared with saturated AOS, the unsaturated AOS
showed efficient anti-obesity effects in high-fat diet (HFD)-fed mice
through activating the AMPK signaling pathway (Li et al., 2019)
Although the enzymatic digestion of alginate has various advantages
and bright application prospects, most studies are still at laboratory
level The discovery and development of novel enzymes with high yield,
activity and stability is still in high demand for the achievement of in-dustrial production of AOS
2.3 Modification of AOS
Recently, various approaches, i.e vanadylation, sulfation, selenyla-tion or oxidaselenyla-tion, have been used to modify the backbone of AOS and improve their physicochemical and biological properties For example, vanadyl AOS (VAOS) presents a higher antioxidant activity than un-modified AOS in hydroxyl and DPPH radical scavenging systems (Liu
et al., 2015) In addition, VAOS exhibited strong anti-proliferation ac-tivities against human hepatoma cell line BEL-7402 VAOS could markedly inhibit tumor progression in non-small cell lung cancer (NSCLC) (Zhou et al., 2018) It was synthesized through slowly adding vanadium (IV) oxide sulfate hydrate into the AOS solution under con-dition of constant stirring and pH 12 during whole process The vana-dium content of VAOS could reach to about 3.0% According to FT-IR spectral analysis, introduction of vanadyl groups changed the absorp-tion peak of different funcabsorp-tional groups of the oligosaccharide chain such as C–O–C, C––O and C–O A similar infrared shift has also been
Fig 2 The different structures of AOS fragments produced by three degradation methods and hypothetical structure of modified AOS
Trang 4found in FT-IR spectra of vanadyl (IV)/chondroitin sulfate A (CSA)
complex (Etcheverry et al., 1994) Although the exact structure of VAOS
remains to be determined, it is believed that VAOS is a new type of
coordination compound rather than a covalent compound based on the
available data As previously reported, the FT-IR spectra in this study
indicate the hypothetical structure of VAOS in the form of coordination
through the hydroxyl oxygen of carboxylate group and the glycosidic
oxygen of G or M moieties (Etcheverry et al., 1994; Etcheverry et al.,
1997) As shown in Fig 2, both the C–O–C and the O––C–O occupy
two coordination positions This speculated coordination structure may
play a key role in improving the bioavailability of vanadium and
bioactivity of AOS The definite structure-function relationship of VAOS
requires further investigation
A recent study of selenium-containing alginate oligosaccharides (Bi
et al., 2020) showed that the modification of AOS was driven by two
reaction steps: Poly M and SO3-Py were each suspended in dimethyl
methanamide, and then the two solutions were mixed and reacted to
obtain the sulfonated Poly M (S-PM) in the sulfonation step; while in the
selenylation step, the sulfur in S-PM was partially replaced by Se
through reacting with Na2SeO3 in the presence of excess barium chloride
(BaCl2) in 5% HNO3 at 60 ◦C for 8 h According to the FT-IR spectra of
PM, S-PM, and Se-PM, the specific absorption of S––O and C–O–S
detected only in S-PM and Se-PM, confirming that the sulfation
substi-tution was successfully prepared Furthermore, the spectrum of Se-PM is
similar to that of S-PM, which suggests that their carbon-skeleton
structure was the same In addition, the measured S content in Se-PM
decreased to about 60% of that in S-PM, implying that some S bound
is replaced by Se Therefore, they speculated that Se might be in the form
of -SeO3 covalently bound to sites that were originally occupied by S
(Fig 2) As a new covalent compound, Se-PM combines the advantages
of Se and AOS and exhibits various bioactivities including anti-
oxidation, anti-inflammation and neuroprotection that are superior to
those of Se itself or Se-free oligosaccharides (Bi et al., 2019; Bi, Lai, Cai,
et al., 2018; Bi, Lai, Han, et al., 2018) The resultant low molecular
weight Se-PM exhibited enhanced neuroimmunoregulatory activity in
LPS-induced BV2 microglia probably due to the covalent structure
formed during the replacement process that could attenuate the nitric
oxide (NO) and prostaglandin E2 (PGE2) secretion, as well as the
inducible NO synthase-20 (iNOS) and cyclooxygenase-2 (COX-2)
expression, depending on the treatment dose
A different kind of sulfated mannuronate oligosaccharides (S-MOS)
were developed by reacting mannuronate oligomers with ClSO3H in
formamide (Liu et al., 2005) 13C-nuclear magnetic resonance (NMR)
analysis suggested that sulfate modification of mannuronate occurs at
the hydroxyl groups of C-2 and partial C-3 with different degrees of
substitution
2.4 Structure-function relatioship of AOS
The size, composition and structure of AOS vary due to algal species
(Guo et al., 2016; Li, Jiang, Guan, & Wang, 2011), degradation patterns
(Li et al., 2019; Wang et al., 2019) and modification methods (Bi et al.,
2020; Liu et al., 2015) The structural features of AOS, including degrees
of polymerization, the G/M ratio, residue structure and spatial
confor-mation, are responsible for biological functions of AOS The following
are some examples that reflect structure-function relationship of AOS
2.4.1 DP
AOS DP5 (pentamer composed of randomly arranged G and M)
ob-tained by enzymatic digestion rather than AOS DP2, DP3 or DP4,
exhibited significant inhibitory functions on the growth of osteosarcoma
cells in vitro (Chen et al., 2017) Unsaturated guluronate oligomers
(DP3–DP6) significantly enhanced the bacterial phagocytosis of
mac-rophages, especially GOS DP5 (the guluronate pentamer) showed the
maximum enhancement among all oligomers measured (Xu, Bi, et al.,
2014) These studies suggested that AOS with different DP, even derived
by the same degradation patterns, still exhibited distinct biological ac-tivities Interestingly, AOS with anti-obesity effects tend to have a lower average DP, usually no more than 4 in many reports (Guo et al., 2016,
2017; Li et al., 2019; Nakazono et al., 2016; Wang et al., 2020)
2.4.2 G/M ratio
Xu et al reported that the unsaturated guluronate oligosaccharides (GOS) prepared by enzymatic digestion exhibited macrophages- activating effect in mouse immune response while GOS prepared by other methods or mannuronate oligosaccharides (MOS) showed very low or no such effects (Xu, Wu, et al., 2014) A series of studies have shown the potential of the guluronate rich alginate OligoG CF-5/20
(containing >85% G residues) as an effective treatment in chronic
res-piratory disease (Nordgård & Draget, 2011; Pritchard et al., 2016;
Pritchard et al., 2019; Sletmoen et al., 2012) The OligoG could directly interact with mucin and reduce its linearization and flexibility, leading
to effective detachment of cystic fibrosis (CF) mucus (Ermund et al.,
2017; Pritchard et al., 2016)
MOS on the other hand, plays important roles in the treatment of human melanoma and Alzheimer's disease (AD) MOS produced by M- specific alginate lyase strongly inhibits anchorage-independent colony formation of human melanoma cells compared to polymannuronate and GOS (Belik et al., 2020) It is suggested that MOS might be a potential drug candidate for synergistic tumor therapy More recently, two groups demonstrated that MOS (DP2-DP11) could significantly inhibit the ag-gregation of amyloid-β (Aβ) oligomer and Aβ fibril formation, although their respective MOS samples were prepared by different methods (Bi
et al., 2021; Wang et al., 2019) Their results suggested the mannuronate component and proper DP may play pivotal roles in alleviating AD
In addition, G/M ratio may determine spatial conformation of AOS and affect their gelation, mechanical properties and biological activity
As mentioned in 2.1, G block has a tendency to form a helical confor-mation while M block has a relatively straight chain-like conforconfor-mation due to their different linkage at C-1 and C-4 (Mackie et al., 1983; Pla-zinski, 2011) Alginate with higher M residues was characterized by higher tensile strength and percent of elongation than alginate with dominated G bocks (Costa et al., 2018) It is well-known that alginates containing G-blocks can form strong hydrogels in the presence of diva-lent cations such as calcium in the so-called egg-box model (Grant et al.,
1973) However, several reports also indicate that such cooperative ionotropic gelation only occurs when the length of the G blocks involved
in the dimerization exceeds a certain length (Skjåk-Bræk et al., 1986;
Smidsrød & Haug, 1972) For example, 3 and 8 ± 2 contiguous G resi-dues are required to form stable junction zones for Sr2+- and Ca2+- induced gelation respectively (Stokke et al., 1991; Stokke et al., 1993) Thus, AOS with low G/M ratio and DP might not form stable crosslinking conformation even in the presence of divalent cations and maintain high water solubility which is conducive to their delivery in vivo while AOS with higher DP and GM ratio may maintain gel-forming property similar
to alginate, which makes them have potential application as drug car-riers G blocks normally form stiffer, brittle, and mechanically more stable gels On the contrary, M blocks form a softer and more elastic gel (Ashikin et al., 2010) As a potential drug carrier candidate, MOS were added in an alginate-based drug delivery system, which improved me-chanical properties and antifungal activity of the whole delivery system (Szekalska et al., 2019) The flexible conformation of MOS may facilitate penetration into bacterial cells and makes them show better antimi-crobial effect than GOS, which usually forms stiff chains (Hu et al.,
2005)
2.4.3 Special terminal structure
AOS derived by enzymatic digestion usually have unsaturated structure at non-reducing end while AOS obtained by oxidative degra-dation are always ring-opened at reducing end to form carboxyl groups (Fig 2) The unsaturated and oxidative terminal structure are key fac-tors determining biological function of AOS
Trang 5The unsaturated mannuronate oligosaccharides (UMOS)
predomi-nantly contains mannuronic acids (M/G ratio = 2.12) and exhibits
su-perior anti-obesity effect via suppressing the accumulation of
triglycerides and improving the intestinal microflora (Kim, 2018) The
unsaturated alginate oligosaccharides (UAOS) also exhibited significant
anti-obesity effect (Li et al., 2019) Moreover, this anti-obesity effect was
only related to the unsaturated structure but independent of the G or M
composition
MOS derived from enzymatic digestion and oxidative degradation
exhibited a similar inhibition to Aβ oligomer aggregation However, the
underlying molecular mechanisms of these two kind MOS in the
treat-ment of AD were different, although their DP and G/M ratio were very
similar The unsaturated MOS enhanced autophagy to promote
clear-ance of amyloid precursor protein (APP) and Aβ in AD cell models (Bi
et al., 2021) while the oxidative MOS reconstituted gut microbiota and
improved anti-neuroinflammation responses to inhibit AD progression
(Wang et al., 2019) These studies suggest that the molecular mecha-nisms of AOS are greatly affected by different terminal structures
It also suggests that there is a direct relationship between structural characteristics of AOS and their biological activities The molecular size, G/M ratio and terminal structure play important roles in determining the biological functions and action modes of AOS
In order to obtain AOS products with stable structure and unique function, several important aspects need to be considered: 1) The source
of alginate; The G/M ratio, content and order of alginate are always different due to algal species and their respective environments
Algi-nate derived from Laminaria japonica with an M/G ratio of 1.86 (Guo
et al., 2016) was degraded by alginate lyase from Pseudomonas sp HZJ
216 for 6 h at 30 ◦C, producing AOS with DP 2 to 6 However, when the
alginate source changed to another brown seaweed, Laminaria sp with
M/G ratio of 2.28, AOS products were mainly oligomers of DP 2 and 3 under exactly the same degradation conditions (Li, Jiang, Guan, & Wang, 2011) Therefore, the uniform alginate source is a prerequisite for obtaining stable AOS 2) Production technology; Production techniques greatly affect the structural properties and biological functions of AOS products For example, the type of alginate lyase is the most essential factor affecting the final product in enzymatic technique An alginate lyase that could specially produce AOS with DP 5 to 7 was reported (Huang et al., 2013) Recently, two mannuronate-specific alginate lyase
were found in a marine bacterium Formosa algae and the human gut microbe Bacteroides cellulosilyticus (Belik et al., 2020; Stender et al.,
2019) These alginate lyases can be applied to produce special AOS products The development and standardization of efficient and stable production technology is the key to ensure the quality, yield and variety
of AOS products 3) Purification and characterization methods; In most cases, AOS are mixtures of oligomers with varied DP, different G/M ratios and sequences Thus, it is very important to develop purification and analytical methods for qualification and quantification of AOS Nowadays, FT-IR and NMR spectroscopy are widely used to obtain
Table 1
Anti-tumor activities of AOS
Mechanism of anti-tumor Cancer style and Cell line Reference
Inhibition of cell growth and
colony formation Human bone cancer cell MG-63 Chen et al (2017)
Human melanoma cells SK-MEL-5, SK-MEL-28, and RPMI-7951 Belik et al (2020)
Human prostate cancer cells DU145 and PC-3 Han et al (2019)
Human aneurysm Yang et al
(2017)
Human myeloma RPMI-8226 cells Arlov et al
(2015)
Induction of cell apoptosis Human non-small cell lung cancer
cells A549 and LTEP-a-2 Zhou et al (2018)
Reduction of angiogenesis
induced by HGF Human myeloma RPMI-8226 cells Arlov et al (2015)
Fig 3 The proposed mechanism of AOS for suppressing the tumorigenicity of prostate cancer cells
Adapted according to Han et al (2019)
Trang 6precise and key information for AOS structure (Bi et al., 2020; Liu et al.,
2005; Liu et al., 2015; Lundqvist et al., 2012) High performance liquid
chromatography (HPLC), mass spectrometry (MS) and related
tech-niques are used to purify and characterize AOS (Fu et al., 2013;
Jona-than et al., 2013; Zhang et al., 2006)
3 Potential applications of AOS for biomedicine
3.1 Cancer therapies
Cancer is a major public health problem worldwide In the last
de-cades, numerous active substances with anti-cancer effects have been
reported As a natural product from marine algae, AOS and their
de-rivatives have various anti-cancer effect (Table 1)
Han and colleagues reported on AOS's capacity to inhibit prostate
cancer cell growth via the Hippo (Ste20-like protein kinase, mutations in
this gene lead to tissue overgrowth, or a “hippopotamus”-like
pheno-type) /YAP (Yes-associated protein) pathway (Han et al., 2019) AOS
trigger the activation or suppression of regulatory processes in prostate
cancer cells involving various transcription factors and effectors (Fig 3)
First, AOS could activate the Hippo signaling pathway, which negatively
regulates YAP activity through a cascade of phosphorylation Reduced
expression of the oncogene YAP and an increase in phosphorylated YAP
protein were observed in prostate cancer cells in response to AOS
treatment The recruitment of both the coactivator YAP and c-Jun (a
component of the transcription factor activator protein-1, belongs to the
Jun family containing c-Jun, JunB, and JunD) into the upstream
response region of the sialyltransferase ST6Gal-1 promoter was largely
blocked due to the absence of YAP As a result, a decrease of ST6Gal-1,
which plays a fundamental role in growth, migration, and invasion of
prostate cancer cells was detected at different levels of transcription,
translation and sialylation both in cultured cells and in a xenograft
mouse model Finally, proliferation of human prostate cancer cells was
attenuated at a non-cytotoxic concentration of AOS (0.5 mg/ml for cell
lines; 2.5 mg/kg for mouse models) via repression of the Hippo/YAP/c-
Jun pathway and sialylation caused by downregulation of ST6Gal-1
gene expression (Han et al., 2019)
In a clinical trial, AOS could significantly suppress aneurysm
recur-rence caused by endovascular aortic repair (EVAR) (Yang et al., 2017)
Compared with the control, the size of residual aneurysms was
signifi-cantly reduced after 2-year treatment with AOS (oral administration, 10
mg/day) The incidence of EVAR-related adverse effects, such as back
and chest pain, pulse-less legs, persistent cough and wound infection were also reduced The clinical trial demonstrated that AOS with DP 3 to
6 effectively reduced aneurysm recurrence after EVAR As a bioactive small molecule, AOS could cause changes of related signal pathway such
as the expression of miR-29b (a small noncoding RNA) and toll-like re-ceptor signaling According to their results, the expression levels of miR- 29b in aortic aneurysm patients were significantly reduced after AOS treatment Consequently, it affected the toll-like receptor (TLR) signaling pathway involving variety of downstream related factors such
as mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB), interleukin 1 (IL-1) β, and interleukin 6 (IL-6) They concluded that AOS can prevent the regeneration of aneurysms by reducing the level of TLR4, NF-κB, IL-1β, and IL-6 via the inhibition of miR-29b Myeloma, also known as plasma cell tumor, is a malignant tumor originating from the plasma cells in the bone marrow The major char-acteristic of myeloma cells is the high expression of the cell surface heparin sulfate proteoglycan syndecan-1 (Sdc-1) (Gambella et al.,
2015) Human growth factor (HGF) interacts with Sdc-1 and increases downstream HGF signaling, promoting angiogenesis, cell migration and tumor growth (Aref et al., 2003) Blocking the interaction between HGF and Sdc-1 can effectively inhibit the proliferation of tumor cells Arlov
et al found that sulfated AOS could directly bind to HGF, preventing HGF from interacting with Sdc-1 (Arlov et al., 2015) The sulfated AOS- bound HGF was released from the surface of myeloma cells when appropriate sizes of sulfated AOS were used In contrast, no HGF release was observed upon treatment with non-sulfated AOS
A recent study showed that VAOS, a novel modified coordination compound, have an effective inhibitory effect on NSCLC in both cultured cells and mouse models with transplanted tumor cells (Zhou et al.,
2018) It was further confirmed that VAOS (12.5, 25, 50 μM) could induce apoptosis of NSCLC cells by activating protein kinase B (AKT) to increase intracellular reactive oxygen species (ROS) levels Phosphorus colorimetric analysis showed that VAOS significantly inhibited the dephosphorylation activity of phosphatase and tensin homolog on chromosome 10 (PTEN), another member of the protein tyrosine phos-phatases (PTPases)-upstream factor of AKT In addition, ectopic PTEN overexpression decreased VAOS-induced apoptosis In vivo, VAOS treatment (intraperitoneal injection, 30 mg/kg for 2 weeks) triggered the hyperactivation of AKT through significant reduction of phosphatase activity of PTEN, leading to ROS accumulation and apoptosis of NSCLC cells
Fig 4 The proposed mechanism of Se-PM for anti-inflammation effect
Adapted according to Bi, Lai, Cai, et al., 2018 and Bi, Lai, Han, et al., 2018)
Trang 73.2 Anti-inflammation
Frequent uses of anticancer drugs cause a series of side effects,
especially gastrointestinal inflammation A variety of agents, such as
probiotics, selenium, volatile oils and prebiotics have been used to
reduce pharmacotherapy-induced intestinal disruption (Araújo et al.,
2015; Justino et al., 2014; Lee et al., 2017; Ting et al., 2017; Yao et al.,
2013 However, little success has been reported in these efforts Novel
bioactive substances are urgently needed to assist the recovery of
inflammation after cancer therapy
It has been shown that AOS could effectively alleviate the
inflam-mation caused by busulfan, a drug used for patients with chronic
myeloid leukemia (Zhao, Feng et al., 2020) AOS also enhances integrity
and migration ability of IPEC-J2 cells (porcine small intestinal cell line)
(Xiong et al., 2020) The underlying molecular mechanisms are
multi-faceted and wide-ranging, including the regulation of AOS on cellular
transcriptome, and the subsequent responses of different types of
in-testinal cells SiRNA trials in IPEC-J2 cells confirmed that AOS
per-formed their function by interacting with mannose receptors on the cell
surface Consequently, the extensive remodeling of transcriptomes in
different type small intestine cells was observed The researchers
iden-tified 184 active and differentially expressed genes, including multiple
transcription factors and associated cofactors Finally, AOS (oral gavage,
10 mg/kg for 2 weeks) rescued the developmental time line of different
types of small intestinal cells (enterocytes, goblet, Paneth, tuft cells)
disrupted by busulfan, and their respective functions involving
micro-villi organization, cell junction, antibacterial humoral response, and
others In addition, the improved plasma metabolome further verified
that AOS could recover small intestinal function in patients undergoing
anticancer chemotherapy Moreover, fecal microbiota transplantation
(FMT) from AOS-treated mice was an effective measure for alleviating
small intestine mucositis through reconditioning gut microbiota and
improving blood metabolome on a multi-omics scale (Zhang et al.,
2020) In another study, it showed that AOS alleviated TNF-α-induced
inflammatory injury by decreasing pro-inflammatory cytokine (IL-6 and
TNF-α) concentrations and TNF receptor 1 (TNFR1)-mediated apoptosis
rate in TNF-α-treated IPEC-J2 cells (Wan et al., 2020)
Se-PM, a modified AOS derivate, significantly attenuated the in-flammatory response in LPS-activated murine macrophage RAW264.7 cells, primary microglia and astrocytes by suppressing the activation of NF-κB and MAPK signaling pathways (Bi, Lai, Cai, et al., 2018; Bi, Lai, Han, et al., 2018) The cytotoxicity of Se-PM was measured by CCK-8 assay It showed that Se-PM did not exert any cytotoxic effects on pri-mary glial cells when the concentration of Se-PM reached 0.8 mg/ml As shown in Fig 4, Se-PM treatment significantly decreased phosphoryla-tion of NF-κB inhibitor (IκB-α), Akt p38 kinases (p38), extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) It lead to reduced phosphorylation and nuclear translocation of tran-scription factors, especially p65 Therefore, the expressions of down-stream target genes iNOS and COX-2 were dramatically reduced As a result, the production of related pro-inflammatory mediators, including ROS, NO, PGE2 and the secretion of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6 also decreased in cells incubated with Se-PM Finally, Se-PM inhibited the inflammatory response in different types of LPS-triggered cells, respectively Moreover, Se-PM remarkably suppressed carrageenan-induced pro-inflammatory cytokine production and LPS-triggered microglial and astrocytic activation in different mouse models Se-PM might block the interaction between LPS and re-ceptors on the surface of LPS-induced in RAW264.7 cells These studies might contribute to the comprehensive understanding of the potential health benefits of Se-PM for alleviating the prolonged and excessive inflammation induced by human degenerative diseases, including can-cer, cardiovascular diseases, type 2 diabetes, and arthritis
3.3 Anti-microbial activities
Microbial infections represent one of the major causes of human diseases The biofilm-forming abilities and the resulting increased drug resistance of pathogens make them a formidable challenge and remain difficult to overcome (Ceri et al., 1999; Moskowitz et al., 2004) AOS have exhibited great potential in the treatment of microbial infections
caused by the most common opportunistic pathogens including
Pseu-domonas aeruginosa, Acinetobacter baumannii, and Candida species
(Pritchard, Jack, Powell, et al., 2017; Pritchard, Powell, Jack, et al.,
Fig 5 The proposed mechanism of AOS for anti-oxidation effect
Adapted according to Jiang et al (2021) and Zhao, Han, et al (2020)
Trang 82017; Stokniene et al., 2020; Tøndervik et al., 2014)
Powell et al reported that the OligoG CF-5/20 could significantly
reduce the biomass, thickness and density of biofilm of Pseudomonas
aeruginosa during early biofilm formation or even after biofilm
estab-lishment (Powell et al., 2018) They revealed that the OligoG CF-5/20
not only inhibited bacterial biofilm growth but also destroyed the
well-defined structure of established biofilm They further demonstrated
that OligoG CF-5/20 could rapidly diffuse into the whole biofilm and
make the biofilm collapse from the inside, due to disrupting the
struc-ture of extracellular polysaccharides matrix and DNA-Ca2+-DNA
bridges, both of which are essential for biofilm formation and
matura-tion (Bales et al., 2013; Flemming & Wingender, 2010) In addition, the
synergistic use of OligoG CF-5/20 with antibiotics enhanced the efficacy
of antibiotics Most recently, Stokniene et al has reported the artificial
bi-functional compounds of OligoG CF-5/20–polymyxins exhibited
prolonged antimicrobial and antibiofilm activities against multidrug-
resistant Gram-negative bacterial pathogens compared to parent
anti-biotic whilst reducing antianti-biotic toxicity to human (Stokniene et al.,
2020)
OligoG CF-5/20 also showed an effective inhibitory effect on
in-fections caused by Candida albicans through reducing the candidal
growth, mycelium formation and invasion (Pritchard, Powell, Khan,
et al., 2017) Moreover, the gene expression and protein production of
key phospholipase as the major virulence factor was significantly
decreased in OligoG pre-treated C albicans ATCC 90028 cells
3.4 Anti-oxidation
Oxidative stress reflects an imbalance between the systemic
mani-festation of ROS and the innate antioxidant defense systems
Distur-bances in the normal redox state of cells can cause toxic effects through
the production of peroxides and free radicals that damage proteins,
lipids, and DNA Oxidative stress leads to cell dysfunction and apoptosis
via driving disruptions in normal mechanisms of cellular signaling In
humans, oxidative stress is thought to be associated with the occurrence
of the development of various degenerative diseases, especially atherosclerosis
Studies have shown that AOS treatment can significantly enhance the activity of antioxidant enzymes and the accumulation of free radical scavengers, including superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in human umbilical vein endothelial cells (HUVECs) (Jiang et al., 2021; Zhao, Han, et al., 2020) AOS treatment also reduced
H2O2-induced ROS accumulation, the production of malondialdehyde (MDA), one of the final products of lipid peroxidation, and the secretion
of endothelin-1 (ET-1), a signaling molecule to stimulate ROS and su-peroxide generation In addition, AOS protected HUVECs against oxidative stress-induced apoptosis by regulating expression of genes involving in the caspase-mediated apoptosis pathway and integrin-
α/FAK/PI3K pathway (Fig 5) These studies suggested that AOS can protect endothelial cells, due to their effective antioxidant and anti-apoptotic activities, providing a promising therapeutic strategy for preventing and treating atherosclerosis
3.5 Anti-obesity
Obesity is a metabolic disorder characterized by excessive accumu-lation of body fat, leading to a variety of diseases, such as hypertension, hyperlipidemia, diabetes and even cancer It has become a major problem threatening human health In addition to general recommen-dations with regard to proper diet and exercise, several different ap-proaches to treat obesity have been proposed, such as the use of functional food supplements that have anti-obesity effects As a naturally-derived food additive with various beneficial effects, AOS might be a candidate for the treatment of obesity (Guo et al., 2016,
2017; Li et al., 2019; Nakazono et al., 2016)
Li et al investigated the anti-obesity effects of the unsaturated AOS
(UAOS) from the enzymatic degradation of Laminaria japonicais, in a
high-fat diet (HFD) mouse model (Li et al., 2019) They found that UAOS showed stronger anti-obesity effects than acid hydrolyzed saturated AOS (SAOS), judged by the greater reduction in body and liver weights,
Fig 6 The proposed mechanism of AOS for anti-obesity effect
Adapted according to Li et al (2019) and Wang et al (2020)
Trang 9adipose tissue mass, serum and liver lipid contents Similar studies have
also shown that the UAOS provides greater biological activity (Guo
et al., 2016, 2017; Nakazono et al., 2016) UAOS causes significant
in-crease on both AMPKα and acetyl-CoA carboxylase (ACC)
phosphory-lation in adipocytes, suggesting that UAOS acts as an anti-obesity agent
through AMPK signaling
In another study, it is observed that AOS treatment induces reduction
in size of adipocytes and lipid metabolism improvement, such as
decrease of TG and low-density lipoprotein cholesterol (LDL-C) levels
and inhibition of lipogenesis genes expression (Wang et al., 2020) AOS
alleviated HFD-induced metabolic disorders and inflammation via
modulating gut microbial communities, and the consequent release of
microbiota-dependent short-chain fatty acids (SCFAs) and reduction of
endotoxin levels AOS treatment recovered the HFD-disturbed gut
microbiota by increasing the abundance of specific and beneficial gut
microbiota, including Akkermansia muciniphila, Lactobacillus reuteri and
Lactobacillus gasseri (Fig 6) These results showed that the inhibitory
effect of AOS on obesity was closely related to their regulation of
in-testinal microorganism
3.6 Alzheimer's disease therapies
Alzheimer's disease (AD) is a chronic neurodegenerative disease AD
is characterized by the deposition of amyloid-β (Aβ) in the brain forming
plaques (plaques); and the hyperphosphorylation of tau protein causing
neurofibrillary tangles (NFTs) and loss of neurons, accompanied by glial
cell proliferation (Jana & Pahan, 2010) The Aβ peptide is generated
from amyloid precursor protein (APP) by β-secretase (BACE) and
γ-secretases cutting orderly (Lazarov & Demars, 2012; Mattson, 2004)
Recently, several studies have shown that AOS plays an important role with different action modes in the pathogenesis of AD
The effects of two different forms of AOS, MOS (derived from enzymatic digestion) and Se-PM (modified with Se) on the treatment of
AD were evaluated by Bi et al (2020) and Bi et al (2021) Both MOS and Se-PM significantly inhibited the aggregation of Aβ1–42 oligomer, which
is suggested to be the most neurotoxic form, and suppressed Aβ1–42, APP and BACE1 expression in N2a-sw cells In addition, ROS production and oxidative stress levels were decreased by MOS and Se-PM These results suggested that the Se and the unsaturated double bonds in the AOS might be a vital factor in exerting their biological function Subse-quently, they found that MOS could activate autophagy via suppressing mTOR signaling pathway, promoting clearance of intracellular accu-mulation of APP and Aβ in AD cell models; while Se-PM could attenuate cell apoptosis and improve cell survival of N2a-sw cells via reducing the
expression of cytochrome c and enhancing the mitochondrial membrane
potential
Studies revealed that GV-971, a kind of MOS derived from oxidative degradation, exerts its role in treating AD by targeting the gut-brain axis (Wang et al., 2019) GV-971 could improve cognitive functions by remodeling gut microbiota, reducing the production of abnormal me-tabolites, especially phenylalanine and isoleucine, preventing the inva-sion of peripheral immune cells to the brain, inhibiting neuroinflammation, and reducing brain Aβ deposition and tau hyper-phosphorylation (Fig 7) A phase 3 clinical trial showed that GV-971 significantly reversed cognitive impairment in patients with mild to moderate AD In addition, GV-971 had been demonstrated to cross the blood-brain barrier, and directly bind to multiple subregions of Aβ to inhibit the formation of Aβ fibril and destabilize the Aβ aggregates into
Fig 7 The proposed mechanism of AOS for AD therapies effect
Adapted according to Wang et al., 2019)
Trang 10non-toxic monomers The elaboration of the action mechanism of GV-
971 undoubtedly provides an important scientific basis for the in-
depth understanding of targeting gut microbiota as a novel treatment
strategy for AD, and a detailed experimental basis for the research and
development of similar drugs as a novel treatment path for AD At
present, the GV-971 has already been marketed in China and approved
by the FDA in proceeding international Phase 3 clinical trials
3.7 Drug carrier
Considering that alginate hydrogels are difficult to degrade in vivo,
Park et al proposed AOS as a drug matrix in oral sustained release
formulations (Park et al., 2021) They simulated the passage of an oral
drug through the stomach into intestine via controlling pH of the
reac-tion solureac-tion in vitro AOS obtained by enzyme digesreac-tion could form
spherical gel containing lysosome through liquid dropping test and
protect the lysosome from degradation or hydrolysis under acidic
con-ditions (pH 1.2) Then, the gel was dissolved and the embedded
lyso-some was released at near-neutral pH (pH 6.8) The morphological
integrity and antimicrobial activity of lysosomes were largely preserved
Therefore, they suggested that AOS have potential as oral delivery
sys-tem of drugs, proteins, and lysosomes for the treatment of metabolic
diseases In addition, AOS could alleviate enterotoxigenic E coli-induced
intestinal mucosal damage in weaned pigs, and improve intestinal
morphology and growth (Wan et al., 2016, 2018) The above studies
indicate that AOS may not only serve as carriers to deliver special drugs,
but also act as stimulators on intestinal microecology
In another study, AOS with high M content were used as matrices for
drug delivery and enhancing the antifungal activity of posaconazole
(POS) (Szekalska et al., 2019) They used the freeze-thaw technique to
prepare AOS gels This method involves solvent crystallization during
freezing, which led to the compression of the space of AOS chains,
increased forces between AOS chains, allowing their connection and
formation of the hydrogels after thawing In this way, the conventional
antifungal drugs were embedded in AOS with appropriate
concentra-tions and proporconcentra-tions in order to obtain optimal mucoadhesive and
prolonged drug release Finally, it was shown that POS-containing
mucoadhesive films, based on AOS for buccal delivery, possessed
larger zones of inhibition and reduced the growth of all tested Candida
spp
In general, AOS have improved water solubility and bioavailability
due to their short chain length Although AOS are different from alginate
in molecular size, they still maintain the properties of forming effective
gel similar to alginate by adjusting the AOS size, proportion and
con-centration and using proper preparation technique These studies
indi-cated that AOS related gels also had good mucoadhesive and swelling
properties and prolonged drug release effect, all of which are important
and necessary for a drug carrier (Park et al., 2021; Szekalska et al.,
2019) AOS have additional properties such as antimicrobial effect,
enhancing drug efficacy and inducing physiologic and pathophysiologic
stress response (Liu et al., 2019; Stokniene et al., 2020; Tusi et al., 2011)
Therefore, AOS, as a kind of natural and emerging drug carrier with dual
curative effect, have a broad application prospect and are worth further
research and development
3.8 Other medical effects
In addition to the above multiple pharmacological benefits, AOS
have other medical effects AOS can alleviate pulmonary hypertension
in mice via restoration of the TGFβ1/p-Smad2 signaling pathway and
restraining the activation of P-selectin/p38MAPK/NF-κB (Feng, Hu,
et al., 2020; Hu et al., 2019) AOS could prevent D-galactose-mediated
cataracts in C57BL/6 J mice through inhibiting oxidative stress and up-
regulating genes related to antioxidant system (Feng, Yang, et al., 2020)
Two studies showed that AOS exhibited protection effect on senescent
cardiomyocytes and could alleviate myocardial reperfusion injury (Guo
et al., 2017; Mou & Miao, 2019) AOS were also applied in aquaculture industry due to their beneficial effects on growth performance,
immu-nity, and disease resistance of tilapia (Oreochromis niloticus) (Van Doan
et al., 2016)
3.9 Safety and commercialization of AOS
Biosafety assessment of AOS is important for their potential appli-cations in biomedical field None of AOS (-ED, -AH, and -OD) exhibited cytotoxic effects on RAW264.7 cells at a concentration of 1 mg/ml in WST-8 assay (Xu, Wu, et al., 2014) AOS with concentration of 1 mg/ml did not cause toxicity to endothelial cells, isolated from human aneu-rysm (Yang et al., 2017) Zhao et al found that AOS treatment at different doses (0.05, 0.1, 0.2, 0.4, and 0.8 mg/ml) showed no cyto-toxicity on HUVECs (Zhao, Han, et al., 2020) No obvious cell toxicity and mutagenicity of AOS were observed in mice after 31 days of oral administration at dosage of 600 mg/mouse/day (Ogawa et al., 2001) A double-blind, randomized, placebo controlled, 3 days, dose-escalation phase I study (NCT00970346) to test the in vivo safety and tolera-bility of inhaled OligoG CF-5/20 in healthy volunteers have been completed, as well as a multicenter, randomized, placebo controlled, crossover phase II study (NCT01465529) with 28 days treatment periods
to evaluate the safety, tolerability and preliminary efficacy of OligoG CF-
5/20 in subjects with CF chronically colonized with Pseudomonas
aeru-ginosa At present, OligoG CF-5/20 is in phase IIb/III clinical trials in CF
patients In the recently completed phase III trial (NCT02293915), GV-
971 (900 mg twice a day for 24 weeks) was demonstrated to meet the
primary endpoint, with statistical significance (p < 0.001) No serious
adverse events were observed, with similar incidence rate between GV-
971 and placebo group (Wang et al., 2019) Nowadays, GV-971, a low- molecular-weight mannuronic acid oligomer, has been manufactured into sodium oligomannate capsules and successfully marketed in China
In general, all these studies demonstrated that the doses of AOS had no obvious toxicity and side-effects for different cell lines, mouse models and human patients, which indicated safety of AOS for utilization as food supplements, drug carriers or pharmaceutical ingredients How-ever, the future applications of AOS in biomedical field still require a large number of perspective efforts on the metabolism and safety of all AOS products, due to their diverse composition, variable structure and multiple modification styles
Commercialization is a good way to realize large-scale application of AOS products At present, there are only a few commercially available AOS-based biomedical products The GV-971 related products have been marketed in China after more than 20 years of research and development GV-971 is the first new Alzheimer drug to receive regu-latory approval globally since 2003 This typical and successful case will provide great inspiration and a wealth of practical experience for the development of AOS-based drugs in future In addition, the drug candidate Oligo G CF-5/20 has proven to be safe Its serial products are being developed by a pharmaceutical company named AlgiPharma, which has successfully completed five clinical trials (NCT00970346; NCT01465529; NCT01991028; NCT02157922; NCT02453789) including a drug deposition study in CF patients Multiple formulations for inhalation, oral, and topical administration for the treatment of respiratory diseases and microbial infections are expected to be ready in the near future
4 Conclusion and perspective
In summary, the physicochemical and biochemical properties of AOS, such as small molecular size, low viscosity, high water solubility, and intestinal absorption, make them suitable for preparation in a va-riety of dosage forms for different administration modes, including inhalation, intraperitoneal injection (i.p.) and oral gavage (Pritchard
et al., 2016; Wang et al., 2019; Zhou et al., 2018) AOS could perform their unique biological functions in three modes, as extracellular