Immunomodulatory properties of shellfish derivatives associated with human health

Abstract

Some vital components of marine shellfish are documented as an important source for both nutritional and pharmacological applications. These bioactive compounds are multifunctional in nature and they function as anti-inflammatory, antimicrobial, anticancer, antidiabetic and anti-hypertensive agents and so on. Shellfish-derived molecules such as lectins, glycoproteins, agglutinins and various endogenous free amino acids and fatty acids display potent immunomodulatory functions associated with human health. In addition, many synthetic immunomodulating agents are commercially available in the market, but they also cause some side effects to patients. However, immunomodulating substances derived from natural origins are safer and also cheaper in cost. Shellfish derivatives regulating biological activities and immune modulation are so far underexplored. Therefore, in this review, we discuss the immunomodulatory properties of shellfish-derived compounds to emphasize their therapeutic potential in human diseases.

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Sithranga Boopathy Natarajan

Dr Sithranga Boopathy Natarajan is working as a postdoctoral fellow in the Department of Biotechnology, Konkuk University, South Korea. His current area of research is to elucidate the role of bioactive compounds found in marine shellfish associated with human health. He received his master degree in Botany followed by a Ph.D. from Annamalai University, India, in understanding the importance of marine compounds to fight against oral cancer. After his Ph.D., he worked as a postdoctoral fellow in Jeju National University. He also received a prestigious postdoctoral fellowship from the University Grants Commission, India (2010–12). He has published more than 18 research papers in various reputed journals.

Yon-Suk Kim

Dr Yon-Suk Kim is working as postdoctoral fellow in the Department of Biotechnology, Konkuk University, Chungju, South Korea. Her current area of research is to elucidate the role of bioactive compounds found in bio-resources. She received her Ph.D. degree from Konkuk University, South Korea, in understanding the importance of antioxidant and anti-inflammatory effects of plant extracts and their bioactive compounds. She has published more than 42 research papers in SCI journals.

Jin-Woo Hwang

Jin-Woo Hwang is a doctoral fellow in the Department of Biotechnology, Konkuk University, Chungju, South Korea. His current area of research is to elucidate the role of bioactive compounds found in natural resources associated with human health. He received his master degree in biosynthesis followed by a Ph.D. degree, discovering the protective effect of anthocyanin that G2/M phase arrest by H2O2 in retinal cells. He has published more than 5 research papers in various reputed, peer reviewed National and International journals.

Pyo-Jam Park

Dr Pyo Jam Park is working as a professor in the Department of Biotechnology, Konkuk University, Chungju, South Korea. His field of research is molecular biochemistry associated with human health. He received his Ph.D. degree from Pukyong National University, South Korea, in understanding the importance of bioactive compounds associated with human health. After my Ph.D., He worked as post-doctoral fellow and instructor in Pukyong National University and then he joined as an assistant professor at Konkuk University South Korea. He has published more than 116 research papers in various reputed, peer reviewed journals. He also received best paper and best presentation awards.

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1. Introduction

Immunology is a branch of science that deals with the ability of an organism to recognize the components of self vs. non-self, in an antigenic challenge. The basic functions of the immune system are to destroy foreign matter that enters the host using defense mechanisms.¹ Immunomodulation is a promising approach to treat or control various life threatening diseases such as human immunodeficiency virus (HIV),² cancer,³ multiple sclerosis⁴ and aging etc. The human immune system is a complex network that protects the body against various foreign invaders such as bacteria, virus, fungi and parasites. Human body would act as a suitable niche for the growth and development of these microbes. The vertebrate immune system is basically regulated by both innate and adaptive defense system. These immune systems could recognize, eradicate and remember the different kinds of invaders.⁵ Macrophages are mononuclear leukocytes and the primary defender of host body, which can recognize and eliminate the microbial challenges and tumor cells through the productions of cytokines such as TNF-α, IL-1B, IL-6 and IL-10. Any alteration that occurs in the immune system to encounter invading pathogen or abnormal cell phenotypes is called immunomodulation. Immunopotentiation, immunosuppression and immune tolerance are the types of immunomodulatory responses that arise against pathogens or abnormal cells. Immunotherapy is a mode of treatment by modulating the host's immune system to prevent or cure a disease. Over the past decades, there have been significant treating achievements established in the field of immunology against various diseases in human being. Immunomodulators are either chemical or biological compounds derived from synthetic or natural resources. The invading microorganisms or their toxic chemicals alert the host immune system immediately and trigger the defense mechanisms in the host.⁶ Naturally derived immunomodulators have the ability to stimulate the immune response and strengthen the immune system mediated defense. Many synthetic immunomodulators that are commercially available in the market such as azathioprine, cyclophosphamide, prostaglandins, cyclosporine A, thiocarbamate, levamisole, niridazole and penicillamine are available. In contrast, the immunomodulators derived from natural resources such as microorganisms, plants and animals have no side effects, also cheaper in cost and therefore have potential impact on immunotherapy.

Marine environment contains various living organisms with peculiar adaptations and biological properties, and is probably the most underexplored biological resources yet to be studied in immune response modulations. Marine organisms are rich in bioactive compounds due to their stress induced metabolisms. It is expected that approximately 2 210 000 species exist in the ocean. However, only around 190 000 species have been recorded so far.^7,8 During fish processing, a large quantities of body parts are considered as waste and discarded. The material such as skins, tails, scales intestines and shells are exhibiting a variety of biochemical constituents. It has been documented that approximately 40 000 tons of oysters have been harvested in Korea annually. More than 90% of the oyster content and shells are being wasted as environmental pollutant.⁹ The shells contain high amount of calcium carbonate, and relatively low amount of calcium sulfate, calcium phosphate, and amino acids. Oyster shells are also used as medicine for the treatment of dermatitis. Moreover, they are being one of the most famous traditional medicines in China, Japan and Korea.¹⁰ The sauce from a decoction of Mytilus edulis, for instance, is traditionally used to treat liver and kidney dysfunctions, impotence and menoxenia.^11,12 Shellfish are exoskeleton bearing aquatic invertebrates having hard shells to cover their soft muscles. They are further classified into mollusks, crustaceans, and echinoderms. The marine water has diverse specious of shellfish followed by some fresh water species and very few in land crabs and snails. Shellfish are economically and ethno-medicinally very important animal community of both marine and fresh water ecosystem. They have been traditionally used for many important diseases such as rheumatisms, cancer, cardiac diseases, hypertension, asthma, rickets, calcium metabolisms, nervousness and giddiness.¹³ Peptides and proteins isolated from certain marine animals and some fish proteins are acting as immunostimulants. The potential immunomodulating activity of shellfish derivatives and the scientific evaluations are emerging. Therefore, the present review is discussing the immunomodulatory role of shellfish derived biomaterials in human diseases.

2. Therapeutic importance of shellfish

Based on the literature from traditional medicine, shellfishes have rich in ethno medicinal values and their derivatives are highly potential to control many diseases. These derivatives isolated from marine animals have shown diverse biological activities such as anti-inflammation, anticancer, anti-diabetic anti-HIV activity and so on (Table 1). As a traditional medicine, Mytilus coruscus comes under the family Mytilidae has been often recommended by practitioners to regulate the functions of immune system. Hot water extraction of shellfishes Meretrix lusoria and Sinonovacula constricta stimulated the increased secretion of monoclonal antibody in the HB4C5 and SI102 hybridoma cell culture medium.¹⁴ Marine shellfishes such as prawn Nematopalaemon tenuipes and gastropods Euchelus asper and Hemifusus pugilinus were dose dependently showed immunostimulatory and immunosuppressant effects in in vitro and in vivo experiments. Extract of these animals caused cytotoxic effect at higher dosage and stimulated the macrophages at lower dosage (160 mg kg⁻¹) that evidenced from phagocytic index in carbon clearance assay.¹⁵ Akerkar et al.¹⁶ assessed the immunomodulatory activity of Nematopalaemon tenuipes and Hemifusus pugilinus by delayed type hypersensitive reaction assay. They also observed immunosuppressant activity from Nematopalaemon tenuipes and Euchelus asper. Moreover, the Hemifusus pugilinus also showed anticoagulant activity due to the presence of heparin like compound.¹⁷

Table 1 Immune mediated bioactive potential of shellfishes

Sl. no.	Source	Activity	Compounds
1	Crassostrea gigas	Anti-HIV-1 and anticancer activities	Peptide; oyster hydrolysate^71,73
2	Haliotis discus	Anti-inflammatory activity	Crude extract^27
3	Meretrix lusoria	Immunomodulator IgM secretion in culture medium	Crude extract^14
4	Mytilus coruscus	Immunomodulation
5	Mytilus edulis	Anti-inflammatory	Crude extract^12,26
6	Perna viridis	Anti-HIV activity	Crude extract^28
7	Ruditapes philippinarum	Health promoter	Peptide^70
8	Sinonovacula constricta	Immunomodulating activity	Crude extract^14
9	Cerithidea obtuse	Antidiabetic activity	Crude extract^21
10	Euchelus asper	Cytotoxic & immunomodulation	Crude extract^15
11	Nematopalaemon tenuipes
12	Astropecten polyacanthus	Anti-inflammatory activity	Steroid^29
13	Certonardoa semiregularis	Anti-HIV activity	Saponin cetonardosidase^30
14	Strongylocentrotus droebachiensis	Antibacterial activity	Peptides^68

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Some immunomodulatory compounds from shellfishes are also showing effective anticancer activity. For example, chitosan showed anticancer effect on cancer cell growth inhibition by activating the immune systems and accelerating lymphokine production. Kim and Rajapakse¹⁸ reviewed that the chitosan oligosaccharide enhances the immunomodulating activity and promote antitumor activity through the increased productions of lympokines and proliferations of cytolytic T-lymphocytes. Stimulations of pinocytic activity are most distinguished way to activate the macrophages. Wu et al.¹⁹ reported that based on the molecular weight and concentration, the low molecular weighted chitosan was significantly enhanced the macrophage activations through enhanced pinocytic activity. The results also confirm that phagocytosis could play a major role in proliferations of macrophages. Many in vivo experiments revealed that chitosan also acting as anti-diabetic agent. One of the reports demonstrates that chitosan has hypoglycemic effect in Streptozotocin-induced diabetic animals.²⁰ Extraction of Cerithidea obtusa showed effective antidiabetic effect on in vitro condition. In addition, it showed high α-glucosidase inhibition activity that was attributed to the presence of alkaloids, flavonoids, tri-terpenoids and saponins of the C. obtusa.²¹ Kahalide F is a cyclic depsipeptide derived from marine mollusk Elysia rufescens is under clinical trials for its anticancer properties. Kahalide F functions to inhibit erb2 transmembrane tyrosin kinase activity and inhibits TGFα gene expression.²²

Inflammation is defined as the response of living tissue to injury by pathogens or irritants. It is a kind of self-defensing mechanism, also related to the immune response. Various pathological complications such as diabetes, cancer and cardiac diseases have been attributed with the interactions of oxidative stress and inflammation.^23,24 A polysaccharide glycogen isolated from the shellfish Perna canaliculus showed potent anti-inflammatory activity. The purified extract of this mussel, Seatone administered orally or by intraperitoneal injection, showed remarkable anti-inflammatory activity on carrageenan-induced paw edema in rats.²⁵ Further, multifunctional bioactive peptides isolated from Mytilus edulis have been showed anti-inflammatory effect on lipopolysaccharide (LPS) stimulated macrophage cells.²⁶ Similar effect was also observed from the extract of Haliotis discus on LPS stimulated RAW 264.7 macrophage cells.²⁷ Extracts of Indian green mussel Perna viridis showed an anti-HIV activity. The extracts of this mussel showed 40–50% inhibitory effects on HIV infected human monocytes.²⁸ A steroid derived from starfish Astropecten polyacanthus prevented LPS induced pro-inflammatory cytokines such as IL-12, IL6 and TNF-α in bone marrow derived dendritic cells.²⁹ Ten novel saponins (the saccharide attached steroids or triterpenes) certonardosides A–J were isolated from starfish Certonardoa semiregularis showed potent antiviral activities including anti-HIV activity.³⁰ Similarly, the potential anti-HIV linckosides A and B, and neuritogenic steroidal glycosides isolated from starfish Linckia laevigata³¹ were successfully inhibited the chemokine receptor subtype-5.³²

3. Immunomodulating compounds from shellfishes

Marine bio-products have been widely attracted the attention of many biologists and chemists for the past few decades. More than 16 000 natural products have been isolated from marine organisms. Diverse classes of compounds, such as proteins, peptides, lipopolysaccharides, glycoproteins and lipid derivatives are having the potential to modulate the immune system.³³ All of these biochemicals are highly deserved for the regulation of immunomodulation. Marine derived water soluble and low molecular weight proteinaceous molecules such as lectins, glycoproteins, agglutinins, various endogenous peptides free amino acids and fatty acids are few examples of potent immunomodulators (Fig. 1).

Fig. 1 Current status of immunomodulating components of shellfishes.

3.1. Immunomodulating proteins

Proteins are complex molecules that are essential for the proper function of all living organisms. Some animal tissues made up of about 65–75% dry weight proteins. Proteins from marine source have received much attention to their potential bioactive and functional properties. Marine organisms including shellfishes have significant quantities of high quality proteins (10–47% (w/w)) which are being a good source of raw materials for further bioactive protein mining. Proteins are complex polymers made up of amino acids and peptides and the protein content of the shellfishes may range from 7 to 23% (w/w).³⁴ The fish and shellfish proteins are classified into three major groups, such as sarcoplasmic, myofibrillar and stroma proteins. Sarcoplasmic proteins consist of enzymes related to energy production like creatinine kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase.³⁵ The myofibrillar proteins are structural proteins of fish and shellfish muscles.³⁶ In addition, a protein paramyosin present in the invertebrate muscles is not found in vertebrate myofibrils.³⁷ However, the range of paramyosin can vary significantly from one species to another. Many protein hydrolysates, hemolymph and hemocytes are playing a vital role in the innate immune system of invertebrates by conserving several immune components such as lectins, antimicrobial peptides (AMPs), coagulation factors, protease inhibitors etc. These components are involved to modulate the immune systems which are therapeutically important.

3.1.1. Taurine. Taurine (2-amino ethane sulfonic acid) is an amino acid majorly distributed in animal tissues. Marine clam has rich in taurine, the derivative of sulfur containing amino acid cysteine. Taurine has been shown the potentials of cyto-protective and immunmodulatory effects. However, higher level of taurine in the cells mediates inflammatory lesions indicating its role in innate immunity.³⁸ Taurine is enriched in mammalian cells, especially in the immune cells such as lymphocytes monocytes and neutrophils.^39,40 Taurine accumulated phagocytes are activated to kills the pathogens on the site of inflammations, by producing a variety of antimicrobial and toxic oxidants, generated by peroxidase system of the cell. In addition, taurine is acting as a scavenger to remove unwanted materials from the cells and protect them from oxidative stress. Moreover, cellular deficiency of taurine affects the functions of immune cell. Notably, prolonged deficiency of taurine in the cells also leads to abnormalities in the immune system regulations. With the evidence of increased immune cell counts, taurine and cyclophosphamide synergistically control the S₁₈₀ tumour growth and enhance the immune functions.⁴¹ Taurine modulates the immune system by activating NF-kB and several proinflammatory mediators.⁴⁰ It also activates peroxisome proliferator-activated receptor-γ (PPAR γ) which influences to reduce the diabetic retinopathy.⁴²

3.1.2. Lectins. Lectins are glycoprotein molecules found in various animals that are involved in modulating immune functions. It is widely documented that the invertebrates fail to initiate adaptive immune response however, they do develop a series of innate immune responses.⁴³ The role of several lectins from invertebrates as modulators of innate immune response against microbe are well documented.⁴⁴ Most of the lectins isolated from clam are homologous and have related immune responses. The lectins from clams have been classified into calcium dependent (C-type) and metal independent galectins. In addition, the vertebrate C-type lectins not only recognize pathogens but also recognize the endogenous ligands which mediate inter cellular interactions during immune response. The C-type lectins play a key role in carbohydrate recognition during immune response.⁴⁵ Importantly, lectins have been reported as pathogenic recognizing receptors from marine invertebrates. Tachylectins are a group of lectins identified in the hemocytes of horse shoe crab Carcinoscorpius rotundicauda which recognize the pathogen associated molecules.⁴⁶ The tachylectin 5a and 5b isolated from plasma of Tachypleus tridentatus are major agglutinating lectins.⁴⁷ The cytokine stimulating activity of lectins from Mytilus trossulus (MTL) were studied in human peripheral blood cells. The MTL has stimulates the expression of the proinflammatory cytokines TNFα and IFNγ at higher concentrations. However, it reduces the hyper expressions of anti-inflammatory cytokine IL-10 ⁴⁸ which attribute with the immunostimulating activity of MTL. Notably, a lectin isolated from the marine bivalve Crenomytilus graynus showed potent anti-HIV activity. This mussel derived lectin was exhibiting high affinity to the glycoproteins and successfully blocked the viral entry.⁴⁹ A marine bivalve Macoma birmanica derived calcium independent lectin interfered with bacteria to recognize their surface antigens.⁵⁰ Similarly, amino acid sequence analysis of specific lectin (CGL) derived from marine mussel Crenomytilus grayanus confirms that the CGL is a novel lectin that may involve in antibacterial activity.⁵¹ Chikalovets et al.⁵² reported that the CGL was also successfully challenged Pichia pastoris induced fungal infection by strictly inhibiting the spore germination and hyphal growth of the fungus. Lectins from hemolymph of crustaceans are important component for immune recognition and antimicrobial activity and these molecules are considered as active precursor of antibodies. In addition, sialic acid is a kind of lectin isolated from the horse shoe crab Carcinoscorpius rotundicauda and blue crab Callinectes sapidus which has shown potent agglutinating activity against human pathogenic bacteria.^53–55 Similarly, scyllin is another low molecular (MW4000) lectin isolated from the hemolymph of a crab Scylla serrata showed potent antibacterial activity. The antibacterial activity of the scyllin is attributed with inhibition of nucleic acid and cell wall synthesis. However, the exact mode of inhibitory action is not yet elucidated.⁵⁶

3.1.3. Hemocyanins. Hemocyanins are large multi-subunit, oxygen carrying metalloproteins derived from marine mollusk. Hemocyanins have been isolated from several marine gastropods.^57,58 They are acting as potent immunostimulant against certain cancer with tolerable side effects.^59,60 Hemocyanins have been used as innate immunostimulant to suppress the development of superficial bladder cancer.^61,62 Further, these compounds have been acting as carriers for producing antibodies against tumour associated antigens.⁶⁰ However, the hemocyanin obtained from Concholepas concholepas (CCL) has been tested for clinical studies against superficial bladder cancer. Keyhole limpet hemocyanin (KLH) isolated from hemolymph of the giant keyhole limpet. The mollusk Magathura crenulata derived KLH has been shown tremendous immunostimulatory activities. They interact with T cell monocytes, macrophages and polymorphonuclear lymphocytes to improve the host immune response.^63,64 Arancibia et al.⁶⁵ reported a novel immunostimulant hemocyanin isolated from a gastropod Fissurella latimarginata (FLH) and demonstrated that the FLH has higher immunostimulatory activities than the traditional hemocyanins such as KHL and CCL (Table 2).

Table 2 Immunomodulating proteins and peptides from shellfishes

Sl. no.	Source	Activity	Compounds
1	Tapes philippinarum	Cytoprotective & immunomodulation, antitumor and immune stimulation	Taurine^36,41
2	Crenomytilus grayanus	Immunomodulating activity; anti-HIV activity; antifungal activity	Lectin^48,50–52,160
3	Macoma birmanica	Immunomodulation	Tachylectins^43
4	Tachypleus gigas
5	Mytilus trossulus		Lectin^48
6	Concholepas concholepas		Hemocyanins, KHL, CCL and FLH^62–64
7	Magathura crenulata
8	Fissurella latimarginata	Immunomodulation and anticancer	Lectin^60,61
9	Crassostrea virginica	Antimicrobial activity	Defensin^92
10	Crassostrea gigas
11	Mytilus edulis	Antimicrobial activity	Defensin^76
12	Mytilus galloprovincialis	Anti-HIV-1 activity	Defensin and defensin like MGD1 & 2 ^90,93
13	Mytilus edulis	And antibacterial activity	Mytilin A & B^76
14	Mytilus galloprovincialis	Innate immunity and antimicrobial activity	Mytilin B, C, D and G1 ^77
15		Antibacterial activity both Gram positive and Gram negative, antifungal activity	Myticin A and B^76
16
17	Ruditapes decussatus	Antibacterial activity	Myticin 1, 2 and 3 ^98
18	Mytilus edulis chilensis	Antimicrobial activity	Mytimycin^76,102
19	Mytilus edulis
20	Mytilus galloprovincialis
21	Hyriopsis cumingii	Antibacterial activity to Gram+	Theromacin^73,106
22	Ruditapes philippinarum	Antibacterial activity	Hydramacin^108
23	Mytilus galloprovincialis	Antimicrobial activity	Mytimacin^80
24	Litopenaeus vannamei	Antimicrobial activity	Penaeidins^109,110
25	Hyas araneus	Antimicrobial activity	Arasin 1 & 2 ^116,118,119
26	Callinectes sapidus	Antimicrobial activity	Callinectin^120,121
27	Carcinus maenas	Antibacterial activity towards both Gram positive and Gram negative bacteria	Crustins^123–130
28	Pacifastacus leniusculus
29	Fenneropenaeus chinensis
30	Scylla paramamosain
31	Penaeus monodon
32	Scylla serrata	Antibacterial activity	Scygonadin^136,138,139

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3.2. Immunomodulatory peptides

Studies on functional peptides to modulate immune responses are quite emerging and promising⁶⁶ (Table 2). Enzymatic hydrolysis of shellfish wastes contain rich in proteins that are predominantly arranged in a series of bioactive peptides. Various shellfish derived peptides have been documented for their immune stimulating properties in animal studies, although relevant clinical studies are in paucity. The shellfish protein hydrolysates effectively stimulate lymphocytes proliferations, natural killer cells (NK-cells) activity and cytokine regulation however, the specific mechanism of these activities remains unclear. Oligopeptide rich hydrolysate isolated from the oyster Crassostrea gigas has been showed immune based antitumor activity against the growth of sarcoma 180 in BALB/c mice. Administration of oyster hydrolysate significantly increased the activity of NK-cells and macrophage proliferation in S-180 bearing animals.⁶⁷ Moreover, two peptides isolated the from sea urchin Strongylocentrotus droebachiensis showed potential antibacterial activity by eliciting an innate immune response.⁶⁸ Oral administration of peptides can interact with the functions of cardiovascular, digestive, immune and nervous systems in humans.⁶⁹ Therefore, the potentials of specific dietary peptide to promote human health by reducing the risk of chronic diseases through protective immune regulations are inevitable. Hence, the isolation and utilization of peptides are ideal ways to enhance the functions of the immune system. Bioactive peptides isolated from Ruditapes philippinarum have a positive impact on body functions and ultimately influence to health promotion.⁷⁰ The marine oyster Crassostrea gigas acted as potent antidote for human immunodeficiency virus 1 (HIV-1) protease inhibiting activity. C. gigas derived peptides such as LLEYSI and LLEYSL were responsible for anti HIV-1 activity. In addition, the length of amino acid sequence and the presence of C–N-terminal in these peptides are also important in contributing their inhibitory activity.^71,72 Antimicrobial peptides (AMPs) present in invertebrates are the fundamental defense molecule with the ability to enhance innate immunity.⁷³ The molecular mechanisms of bioactive peptides from marine organisms have specific cellular targets which reveal that these peptides could represent promising drug candidates.⁷⁴ Therefore, bioactive peptides from shellfishes are the important source for modulating immunological regulations. Moreover, Kubota et al.⁷⁵ first reported the antimicrobial peptides from mollusk. However, several immunomodulating antimicrobial peptides have been isolated from a few species of bivalves such as Mytilus galloprovincialis, Mytilus edulis, Crassostrea gigas, Crassostrea virginica, Argopecten irradians, Venerupis philippinarum, Ruditapes philippinarum, Haliotis discus and Hyriopsis cumingii.^73,76–86 Based on the arrangements of the primary structure, cysteine array, disulfide bond, the shellfish derived antimicrobial peptides are classified into five groups such as defensins, myticin, mytilin, mytimycin and macin.^{73,80,81,86–91} All the four groups of antimicrobial peptides such as defensins, mytilins, myticins and mytimycin are interrelated cationic and amphipathic structured peptides balanced with four intra chain disulphide bonds. Some of the host defense AMPs derived from shellfishes are described below.

3.2.1. Defensins. Hemocytes of the marine invertebrates are extremely sensitive to microbial substances. During microbial invasion, the hemocytes degranulate and release a series of antimicrobial substances. Defensins are small cysteine-rich cationic proteins acting as host defense peptides. The defensin was first isolated and identified from acidified gill extraction of Crassostrea virginica.⁹² Three kinds of defensins referred as Cg-Def-from haemocytes and Cg-defh1 and Cg-defh2 were isolated from the pacific oyster Crassostrea gigas. New antimicrobial defensin and big-defensing were isolated from blue mussel Mytilus edulis and Mediterranean mussel M. galloprovincialis respectively.^93,94 All of them function as host defensing agents by displaying antimicrobial activity. In addition to the antimicrobial activity, defensins play an important role in immunomodulation by acting as mediator of information between both innate and adaptive immune arms. In addition, the defensins may stimulate the antibody specific immune responses on their effective therapeutic applications in humans. These antibiotic defensins are activating the molecules of immunity to prevent the invasion of pathogenic microorganisms. The defensins including human β-defensins (HBDs) are also being able to act as modulators of adaptive immune response by the recruitment of immature dendritic cells to microbial infection sites in the host. Further, processing and presentation of microbial antigens to the mature dendritic cells undergo the subsequent stimulations of the antigen specific immune response.⁹⁵ The defensins consist of six cysteines subunits which are derived from Mytilus edulis.⁷⁶ Moreover, the two defensin like peptides MGD1 and 2 derived from Mytilus galloprovincialis⁹³ showed potent antimicrobial activity by activating the immune response. Big-defensin is also a diverse group of antimicrobial peptides comprising of 79–94 amino acids.⁸³ Big-defensins are containing two distinctive domains which are differing structurally and functionally from each other. The hydrophobic N-terminal specifically controls the Gram-positive bacteria and the hydrophilic C-terminal domain controls the Gram negative bacteria.⁹⁶ The big defensin family consisting of Cg-Bigdef1, Cg-Bigdef2 and Cg-Bigdef3 were expressed from the separate genomic sequences of oysters. Big defensins derived from mollusks are closely resemble to vertebrate defensins than invertebrate defensin.

3.2.2. Mytilins. Mytilins are mollusk derived cysteine-rich cationic antimicrobial cyclic peptides containing 34 amino acids. Mytilin A and B are characterized from Mytilus edulis⁷⁶ and the isoforms mytilin C, D and G1 are identified from Mytilus galloprovincialis displayed complementary antimicrobial properties.⁷⁷ Mitta et al.⁷⁷ first time reported the complete gene sequence of antimicrobial peptides mytilin B from the Mytilus galloprovincialis. These antimicrobial peptides are potentially preventing the microbial entry to the host. For example, mytilin B and C showed different antimicrobial activities against fungus Fusarium oxysporum and bacteria Vibrio splendidus.⁷⁸ One of the isoforms mytilin A showed effective antibacterial activity against Gram positive strains such as Aerococcus viridans, Bacillus megaterium and Staphylococcus aureus and Gram negative stains such as Escherichia coli and also some marine bacteria including Alteromonas carrageenovora, Pseudomonas alginovora and Cytophaga droebachiensis.⁷⁶ Mytilin is acting as systemic antimicrobial response in the circulatory system. The massive secreted peptides mytilin could be reached in the bacteria by transported through the hemocytes. The cells containing mytilin are able to act as phagocytosing bacteria, and mytilins could execute their antimicrobial activity by cell engulfed bacteria. Mitta et al.⁷⁹ observed the migration of mytilin containing hemocytes towards the injected bacteria by electron microscope and found both bacteria and mytilin in the intracellular granules.

3.2.3. Myticins. Myticin is another cysteine rich-antimicrobial peptide (AMP) was derived from Mytilus galloprovincialis. The peptide myticin has three different isoforms such as myticin A, B and C. Mitta et al.⁹⁷ reported two isoforms of myticins, such as myticin A (4.438 Da) and B (4.562 Da), which were identified from the hemocytes and plasma of M. galloprovincialis. Myticin A and B were showed effective antibacterial activity against both Gram positive and negative bacteria. Moreover, myticin B has displayed high level of anti-fungal activity against Fusarium oxysporum and also showed antibacterial activity against the Gram negative bacteria Escherichia coli.⁷⁸ Gestal et al.⁹⁸ reported that three isoforms of myticin 1, 2 and 3 were first time isolated from the carpet shell-clam Ruditapes decussatus using the suppression subtractive hybridization technique. These isoforms myticin 1, 2 and 3 have potentially controlled the bacterial infections. Clam derived myticin isoforms and mytilin resembled closely with previously reported myticin isoforms and mytilin from Mytilus galloprovicialis.⁹⁸ Recently, Balseiro et al.⁹⁹ reported that the myticin-C acted as immune system modulator in the in vivo system. The RT-qPCR analysis was confirmed the selected immune related gene expression and significantly differed from the control. Domeneghetti et al.¹⁰⁰ found a hundred of transcript variants after in vivo immunomodulation in Italian mussels. Based on the sequence expressed they computed and synthesized the myticin-C and their related AMP. The complementary DNA (cDNA) and genomic DNA (gDNA) of 10 individuals of M. galloprovincialis were analyzed. Two basic sequences were found predominantly in all individual studied which were responsible for myticin-C (Table 5).¹⁰¹

Table 3 Immunomodulating polysaccharides from shellfishes

Sl. no.	Source	Activity	Compounds
1	Ruditapes philippinarum	Anti HIV-1 activity	Polysaccharide^141
2	Scapharca broughtonii
3	Scapharca subcrenata
4	Mytilus coruscus
5	Meretrix lusoria
6	M. petechialis
7	Sinonovacula constrictas
8	Haliotis discus hannai	Immunomodulation	Sulphated polysaccharide conjugate^125,142,143
9	Shells of shrimp & crabs	Anti HIV-1 in human CD4^+ cells	N-Carboxymethyl chitosan N,O-sulfate (NCMCS), chitosan^153,154
10		Improving the mucosal immune responses
11		Increased the expression of IL1o & TGF-β at the inductive mucosal sites	Chitosan^155–157
12		Stimulation of arginine activity	Chitosan^157,187
13	Crenomytilus grayanus	Immunomodulating and anticancer activities; antimicrobial activity	Mytilan (glycoconjugated polysaccharide)^159–161
14		Stimulation of macrophage cell counts
15		Increased counts of antibody forming cells
16	Echinaster luzonicus	Immunomodulating activity and nitric oxide productions	Luzonicoside A and D (glycosides)^164,165
17	Asterina bather	Inhibition of IL-12 production	Pyrrole oligoglycosides B–D^166

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Table 4 Immunomodulating fatty acids from shellfish

Sl. no.	Source	Activity	Compounds
1	Hyas araneus	Antibacterial activity	Lysoglycerolipids/glycerides^173
2	Crab & shrimp	Anti-inflammatory activity	Eicosapentaenoic acid (EPA)^172
3			Docosahexaenoic acid^172
4		Inhibition of NF-kB expressions	n3 poly unsaturated fatty acids^166,169,172,176,178
5		Cause susceptibility on lymphocytic proliferations, cytokine secretions and alteration of natural killer cell
6		Lipid peroxidation changes in plasma membrane eicosanoid productions
7	Thalamita crenata	Anti-inflammatory activity	Omega-3-fatty acid^170,172

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Table 5 Some of the protein sequences responsible for anti-microbial activities isolated from marine shellfishes

Sl. No.	Protein	Source	Protein sequence	GenBank
1	Defensin^82	Crassostrea gigas	MKVFVLLTLAVLLMVSADMAFAGFGCPGNQSKCNNHCKSISCRAGYCDAATLWLRCTCTDCNGKK	ACQ76262.1
2	Mytilin B^77	Mytilus galloprovincialis	MKAAVILAIALVAILAVHEAEASCASRCKGHCRARRCGYYVSVLYRGRCYCKCLRCSSEHSMKFPENEGSSPSDMMPQNENENTEFGQDMPTGETEQGETGI	AAD45013.1
3	Myticin C^101	Mytilus galloprovincialis	MKATILLAVVVVVIVGVQEAQSIPCTSYYCSKFCGSAGCSLYGCYKLHPGKICYCLHCRRAESPLALSGSARNVNEQNKEMVNSPVMNEVENLDQEMDMF	AEZ79080.1
4	Mytimycin^104	Mytilus edulis	MSLSLRMTLLFVICCVVIGMANADCCHRPYYYHCWDCTAATPYCGYRPCNIFGCGCTCRTEPHGKSCYERGDRCRCYSDKRRRRSLSFEDMSANIKFAGLDINSDGLIEQFEFIKALEQMDITDNTTMFHHWSIMDEDKDGAITLEEFDKEN	AET85056.1
5	Penaeidin^115	Fenneropenaeus indicus	MRLVVCLVFLASFALVCQGYKGTYTRPFSRPTGTRPFSPSVTGCSSCHLINFNDAVACCWRLGRCCSAVKG	AFW98240.1
6	Arasin 1 ^116	Hyas araneus	MERRTLLVVLLVCSCVVAAAAEASPSRWPSPGRPRPFPGRPKPIFRPRPCNCYAPPCPCDRWRH	ABI74601.1
7	Crustin^132	Scylla serrata	MKVQILAAMVVVATVVAMTEASRVPPYLGRDCKHWCKDNNQALYCCGPPGITYPPFIRNHPGKCPSVRSTCTGVRSYRPKLCPHDGACDFRSKCCYDACVEHHVCKTVEFY	ADW11096.1
8	Scygonadin^139	Scylla serrata	MRSSLLLGLTVVVLLGVIVPPCMAGQALNKLMPKIVSAIIYMVGQPNAGVTFLGHQCLVESTRQPDGFYTAKMSCASWTHDNPIVGEGRSRVELEALKGSITNFVQTASNYKKFTIDEVEDWIASY	AAW57403.1

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3.2.4. Mytimycins. Mytimycin (MytM) is a novel antimicrobial peptide (6233.5 Da) isolated from blue mussel Mytilus edulis⁷⁶ and from Mediterranean mussel, Mytilus galloprovincialis.⁹³ This molecule consisting of twelve cysteines connecting six intra-molecular disulfide bridges.⁷⁶ These small cationic amphipathic compounds are able to defense the invading pathogenic microorganisms. The host defensive activity is highly attributed with their immunomodulating properties of marine invertebrates. In addition, the active mytimycin AMP isolated from Mytilus edulis chilensis showed effective antifungal activity against the growth of two filamentous fungi Fusarium culmorum and Neurospora crassa.⁷⁶ Moreover, it also displayed antibacterial activities against Gram negative and Gram positive bacteria.¹⁰² Mytimycin in the mature form is closely related to mytilin than any other AMPs probably due to the C-terminal domain.⁹² The gene responsible for MytM was mainly expressed in circulatory hemocytes and very least amount was observed from mantle. A sudden and strong gene expression was observed while challenging with filamentous fungus than bacteria and yeast.¹⁰³ Cantet et al.¹⁰⁴ synthesized an AMP MytM from the hemocytes of the Mediterranean mussel M. galloprovincialis which showed potent antifungal activity.

3.2.5. Macins. Macins are positively charged antimicrobial peptides consisting of disulfide array of 8 cysteines and classified as theromacin, neuromacin, hydramacin and mytimacin. Theromacin was first identified from annelid Theromyzon tessulatum.¹⁰⁵ Further, Xu et al.⁷³ also isolated theromacin from a mollusk pearl mussel Hyriopsis cumingii. Theromacin is a cationic peptide and has profound antibacterial effect. The cDNA analysis of H. cumingii indicates the homologous to the theromacin of annelids T. tessulatum and Hirudo medicinalis. It was also confirmed by the overall amino acid similarity values and 10 conserved cysteine residues.⁷³ Theromacin and neuromacin showed membrane aggregation and permeabilizing activities against Gram positive bacteria.^105,106 Hydramacin also displayed antibacterial activity against Gram negative bacteria.¹⁰⁷ Hydramacin was first isolated from the mollusk Ruditapes philippinarum. The cDNA sequence of hydramacin (Rp-hdmc) was isolated and analyzed from Manila clam Ruditapes philippinarum.¹⁰⁸ The hydramacin Rp-hdmc matches 50% similarities with other hydramacin based on the sequence and amino acid entities. The upregulated gene expression of Rp-hdmc hydramacin in various tissues of the organisms against the infection of Vibrio tapes confirms its role in host defense.¹⁰⁸ Mytimacin is another cysteine rich antimicrobial peptides isolated from mollusk Mytilus species. Gerdol et al.⁸⁰ reported that five novel transcripts of mytimacin related to macin family were identified from the Mytilus galloprovincialis using deep RNA sequencing and the N-terminal signaling peptide anticipated for conserving the anti-microbial property.

3.2.6. Penaeidins. Penaeidins are group of peptides present in the penaeid shrimps which are the key activators of host defense immune response of shrimps. The antimicrobial peptide penaeidin was first identified from the shrimp Litopenaeus vannamei.^109,110 There are three isoforms identified such as penaeidin 1, 2 and 3 in haemocytes as well as hemolymph of penaeid shrimps. Penaeidin-3 was the most abundant peptide in the shrimp P. vannamei. Penaeidins are cationic molecule comprising of proline rich N-terminal domain followed by cysteine rich C-terminal domain. The antimicrobial activity of penaeidin could be executed by the complementary effect of both proline and cysteine rich domains. Penaeidins are mainly synthesized in haemocyte and stored in the cytoplasmic granules. Despite, penaeidins would not be secreted directly in the plasma, it would be retained in the cytoplasmic storage granules as in the mature form. The electron microscopic observations also confirmed the presence of penaeidins in granulocytes. Therefore, elimination of microorganism is engulfed by granulocytes during phagocytosisis closely resembling with human neutrophil defensin.¹¹¹ Tassanakajaon et al.¹¹² reviewed several penaeidin sequences which are coming under four major classes such as PEN2, PEN3 and PEN5 identified from various penaeid shrimp. A new penaeidins AMP gene was isolated and cloned from hemocytes of Indian white shrimp Fenneropenaeus indicus. The cDNA sequences analyzations of the peptide which responsible for penaeidin 3 consist of 243 bp and encoded with 80 amino acids. This AMP closely resembles with other penaeidin derived from other penaeid shrimps such as P. monodon, Litopenaeus vannamei and L. setiferus.¹¹³ Vaseeharan et al.¹¹⁴ also identified and cloned another sequence of antimicrobial peptide Fein-Penaeidin from Fenneropenaeus indicus which consist of 77 amino acids including 19 single peptide amino acids. Another isoforms of antimicrobial penaeidin (FiPEN) was also identified from Fenneropenaeus indicus.¹¹⁵ similarly an antimicrobial penaeidin MmPEN was isolated from another penaeid shrimp Metapenaeus monoceros. Both penaeidins Fi PEN and MmPEN were 63% resemble with penaeidin-3 isoform of P. monodon (Table 5).¹¹⁵

3.2.7. Arasins. Arasin 1 and 2 are proline and arginine rich antimicrobial peptides isolated from hemocytes of the spider crab Hyas araneus.¹¹⁶ The AMP arasin-1 has four cysteine molecules comprising of 37 amino acids. The N-terminal region containing proline and arginine residues and the C-terminal region consisting four cysteine residues comprising two disulphide bands.¹¹⁶ The full length of AMP arasin-1 showed effective antibacterial activity against both Gram-positive and Gram-negative bacteria.¹¹⁶ In addition to the bacteria, it was also significantly controlled the growth of yeast and fungus. However, the antibacterial activity of arasin-1 totally dose dependent and it did not show activity at lower concentrations but show enhanced activity at higher dose (5× MIC).¹¹⁷ Arasin 2 has been considered as putative AMP due to the lack of proper evaluations so far. However, the arasin-1 has been tested for its antimicrobial activity. The antimicrobial activity of arasin 1 also has complementary effect of both proline and cysteine rich domains which are closely resemble with the mode of antibacterial activity of penaedins.¹¹⁸ In addition, arasin like two antimicrobial peptides (arasin-like Sp and GRPSp) were isolated from the hemocytes of the mud crab Scylla paramamosain and spider crab Hyas araneus. The GRPSp showed potent antimicrobial activity specifically to the Gram positive bacteria.¹¹⁹

3.2.8. Callinectin. Callinectin is an antimicrobial peptide was first isolated from the hemocytes of blue crab Callinectes sapidus.^120,121 It resembles closely with arasin 1 which has 32 amino acids and four cysteine molecules.¹²¹ Callinectin has three isoforms such as hydroxyl-N-formyl kynurenine group, N-formyl kynurenine and hydroxyl tryptophan are varying by the functional group on the tryptophan residues. The tryptophan modification of the callinectin isoforms has been recorded from the AMP of many aquatic animals. For example, hydroxylated tryptophan occurs in an arthropod defensin MGD-2 from Mediterranean mussel Mytilus galloprovincialis and bromotryptophans including strangilocin 2 and centrocins from the sea urchin Strongylocentrotus droebachiensis.¹²² Callinectin has been reported for its antibacterial activity including Gram negative bacteria. It is highly reacting with anti callinectin-like peptides (CLP) antibodies in the granulocytes emphasizing its immunomodulating potential.¹²¹

3.2.9. Crustins. Crustins are whey acidic protein (WAP) present in the crustaceans group and play an important role in the innate immune mechanisms. Crustins are cationic cysteine rich polypeptides comprising of eight cysteine residues with four disulphide bond (MW 7–14 kDa). Crustins have been reported from various member of crustacean such as Carcinus maenas, Pacifastacus leniusculus, Fenneropenaeus chinensis, Scylla paramamosain and Penaeus monodon for its antimicrobial activity of both Gram positive and negative bacteria.^123–130 However, it was first isolated from the shore crab Carcinus maenas and called as carcinin.¹³¹ Many immunomodulating proteins are released from the haemocytes of crustacean by exocytosis. A crustins isoform Sc-ALF was isolated from mud crab Scylla serrata and analyzed. The cDNA sequence of Sc-crustin consisted of 433 base pairs and predicate molecular weight was 11.17 kDa. Anti-lipopolysaccharide factors (ALF) of Sc crustin was closely resemble with ALF of S. paramamosain¹³² Another crustin isolated from the spider crab Hyas araneus has seemed to kill Gram-positive bacteria Corynebacterium glutamicum.¹³³ Similarly, Arockiaraj et al.¹³⁴ reported a new crustin from the fresh water shellfish Macrobrachium rosenbergii. A crustin from Penaeus monodon showed potent antibacterial activity to several Gram positive and negative bacteria that was cloned and expressed in E. coli SG 13009.¹³⁵

3.2.10. Scygonadin. Scygonadin is antimicrobial peptides isolated from the seminal plasma of the mud crab Scylla serrata.¹³⁶ It is an anionic peptide and has the molecular weights 10.8 kDa. The scygonadin expresses its antimicrobial activity against Gram-positive Micrococcus luteus.¹³⁷ The mode of antibacterial activity of scygonadin might have an interaction to the phospholipids of bacterial membrane.¹²⁰ Many reports have been demonstrated the male seminal tracks are the sources of antimicrobial peptides for host defense. In this series, a 20 kDa peptide was isolated from the seminal fluid of the Scylla serrata showed the potent antibacterial activity against the commonly available marine bacteria.¹³⁸ Another isoforms of scygonadin isolated from Scylla serrata. The full length of cDNA analysis revealed that the sequence consisting of 539 base pairs and have coding capacity was 126 amino acids (Table 5).¹³⁹ Peng et al.⁵⁶ reported that the purified recombinant scygonadin from S. serrata showed antibacterial activity against Staphylococcus aureus. They observed, 97% of the S. aureus cultured bacterial cells were killed within 15 min and 50% of the bacterial cells were killed within 3 min after the inoculation. They also concluded that the analysis of the antimicrobial ability of the peptides make better understanding to the innate immune system protecting the reproductive system of the organisms.

3.3. Immunomodulating polysaccharides

Polysaccharides are long chain monosaccharides banded by glyosidic bonds. Many polysaccharides have been identified as immunomodulators which play an important role in the regulations of immune functions during the progression of infections (Table 3). These biopolymers can influence both innate and adaptive immune arm by the interaction with immune cells including T lymphocytes, monocytes and macrophages. Crustacean, mollusk and echinoderms are rich in polysaccharides and often used in a wide range of biomedical applications.¹⁴⁰ Notably, the polysaccharides derived from marine shellfishes such as abalone, mussels and clams are medicinally important. Woo et al.¹⁴¹ reported that the polysaccharides obtained from marine shellfishes Ruditapes philippinarum, Scapharca broughtonii, S. subcrenata, Mytilus coruscus, Meretrix lusoria, M. petechialis and Sinonovacula constricta showed potent anti HIV-1 activity. These polysaccharides successfully inhibited the fusion of HIV virus with CD4 protein on the T lymphocytes. Among the many polysaccharide tested, M. petechialis showed highest anti HIV activity. Further, polysaccharides derived from the shellfish Abalone Haliotis discus hannai are well known for its immunotherapeutic potentials.^142,143 Moreover, the sulphated polysaccharide conjugate exhibits significantly increased proliferation of lymphocyte, phagocytosis of macrophages, activity of natural killer cell and antibody production dose dependently in the immune suppressed mice.¹²⁵

3.3.1. Chitin and its derivatives. Chitin and its by products are shellfish derived major polysaccharides usually prepared from the shells of crabs and shrimps and has been widely utilized in biomedical field due to its non-toxic properties.¹⁴⁴ These components are also obtained from the exoskeleton of some invertebrates such as bivalves, crustacean, gastropods and echinoderms. It is a long-chain polymer of N-acetylglucosamine.¹⁴⁵ N-Carboxy methyl chitosan derived N-carboxy methyl chitosan N,O-sulfate (NCMCS), a polysaccharide has been reported to inhibit the propagation of the HIV-1 in human CD4⁺ cells.¹⁴⁶ By enzymatic hydrolysis, chitosan can be converted to chitooligosaccharides (COSs).^147,148 Interestingly, the COSs and their derivatives have also been shown potent immunomodulatory activities.¹⁴⁹ Chitosan has been used for several mucosal applications in antigen delivery,¹⁵⁰ controlled drug release¹⁵¹ and food supplementation.¹⁵² In addition, they did not display any toxicity when administrated to humans by mucosal routes. Chitosan also involves in improving the mucosal immune responses to co-administrated antigens and increases the antibody levels.^153,154 Recently, the expression of anti-inflammatory cytokines such as interleukin 10 (IL-10) and transforming growth factor (TGF-β) were increased at the inductive mucosal sites after oral administration of either chitosan or chitosan with protein conjugates.^154–157

3.3.2. Mytilan. Mytilan is a glyco-conjugated polysaccharide derived from the mantle of various species of Mytilidae family (Mytilus edulis, M. galloprovincialis and Crenomytilus grayanus). The shellfish Crenomytilus grayanus, reported for its potent immunomodulating and anticancer activities. It has remarkable ability to enhance both cell mediated and humoral immune responses.^158–161 Mytilan treatment increased the level of leukocytes counts in the peripheral blood and increased phagocytic activity of polymorphonuclear leukocytes in the experimental animals. Administration of polysaccharide mytilan had stimulating effect on macrophages observed from morphological changes, metabolic and functional activities. Intraperitoneal and subcutaneous administration of mytilan decreased the activity of 5′-nucleotidase in the macrophages of the peritoneal exudate from ++ non inbred mice and cross bagg albino (CBA) mice. Furthermore, the phagocytic activity of macrophages was increased against pathogenic microorganisms such as pseudotuberculosis, Salmonella infection¹⁶¹ Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and P. vulgaris in both in vivo and in vitro conditions.¹⁶¹ Mytilan has potent antiviral activity especially it increase the immune response against influenza virus.¹⁵⁸ Mytilan also compensated the immunosuppressant activity of doxycycline in the CBA mice.

3.3.3. Glycosides. Glycosides are the compounds containing carbohydrates bound to other functional groups by the glycosidic bond. Starfish has many biologically active glycosides and their relative compounds such as asterosaponin (oligoglycosides bind with carbohydrate chain made up of five or six sugars) and glycosylated polyhydroxysteroids with carbohydrate fraction comprising of one or three sugars.^162,163 In the series of steroid glucosides, luzonicoside A possessing a cyclic carbohydrate derivative with glucuronic acid, galactose and arabinose was isolated from Echinaster luzonicus.¹⁶⁴ Kicha et al.¹⁶⁵ reported that the immunomodulating property of the luzonicoside A and Din the RAW 264.7 murine macrophage cells. The luzonicoside A was strongly reduced the lysosomal activity at a higher concentration (10 μM) however, it significantly increased the activity and nitric oxide (NO) generations at a dose of 0.1 μM in RAW 264.7 cells. Novel furan pyrrole oligoglycosides including three new compounds have been isolated from starfish Asterina bather showed inhibitory effect on the production of pro-inflammatory cytokine TNFα. These newly isolated pyrrole oligoglycosides B–D have moderately inhibited the pro-inflammatory IL-12 cytokine production in lipopolysaccharide stimulated bone marrow derived dendritic cells.¹⁶⁶

3.4. Immunomodulating fatty acids

In marine organisms, the omega 3 poly unsaturated fatty acid (PUFA) is a very essential fatty acid which is majorly synthesized by algae and transferred to marine animal through food chain. Therefore, marine animals are the richest source of PUFA which may account the range from 1.5–10.5% of the total fatty acid levels. Many studies have been demonstrated the benefits of dietary unsaturated fatty acids on immune response of both humans and animals (Table 4). An epidemiological study revealed that the fish oil rich diet consumed Greenland Eskimos have shown less incidence of inflammatory and autoimmune diseases. These results were attributed due the effects n3 PUFA on the immune systems.¹⁶⁷ Moreover, the omega-3 fatty acids also known to inhibit the pro-inflammatory transcription factor NF-kB which induces the expression of wide range of pro-inflammatory genes.^168,169 Cell membrane is very much needed the unsaturated fatty acids to maintain the fluidity, structure and functions of the cell. Among the various marine resources, crabs are fatty acid rich organisms and crab meat is valuable for essential fatty acids rich food. Especially the shore crabs contain a huge amount of fatty acid which possibly constituting up to 90% of the total lipid content of the crabs.¹⁷⁰ The shore crabs such as Scylla serrata and Portunus pelagica have high content of omega-3-fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), with the maximum ranges of 336 and 344 mg g⁻¹ respectively of each species.¹⁶⁷

3.4.1. Omega-3-PUFA. Eicosapentaenoic acid (EPA) and DHA are the two important omega-3-polyunsaturated fatty acids important in the treatment of various diseases.¹⁷¹ Less intake of ommega-3 fatty acid is associated with various metabolic disorders including elevated glucose, triglycerides in circulation and proinflammatory effects. It was significantly reduced by the administrations of omega-3 fatty acids.¹⁷² The interesting information is 325–375 mg of omega-3-fatty acids per kg of shrimp which includes about 50% EPA and 50% DHA. Many preclinical and clinical results have shown that consumption of PUFA which contain EPA and DHA exhibits an antioxidant and anti-inflammatory effects.¹⁷³ Lipid molecules lysoglycerolipids/glycerides isolated from the hemolymph of Hyas araneus showed potent antibacterial activity. Among the pathogenic bacteria tested, it showed the maximum inhibitory zone against Shigella flexneri.¹⁷⁴ However, it was also controversially reported by several in vitro and in vivo experiments on human and animals. The results revealed that the suppressive effects of dietary fatty acids including n-3 PUFA on immune system and cause susceptibility on lymphocytic proliferations.^175–178 cytokine secretions^175,178 and alteration of natural killer cell (NK cells).^177,179,180 It was attributed to several molecular mechanisms such as lipid peroxidation changes in plasma membrane, eicosanoid productions or alterations of gene expression are involved in this action.¹⁸¹

4. Mode of actions

There are two major pathways such as innate and adaptive arms are involved to activate the immune system. During pathogen invasion, the activated immune cells play a major role to manage the immune response to destroy the invading of pathogens either through phagocytosis or inflammatory cytokine secretions. The innate immune response consist of the following components such as epithelial barrier, phagocytic leucocytes (neutrophils, monocytes, macrophages, mast cells and dendritic cells), NK cells and circulating plasma proteins. The monocyte and macrophages are the core cells of innate immune system that arise from bone marrow.¹⁸² All white blood cells mediate the immune responsibility of the immune system. The macrophages are the most plastic cells maintaining various biological functions such as development, tissue repair, homeostasis and immunity.^183–185 They can change their functional phenotypes depending on the environmental signals received. All of the activated phagocytes have the receptors molecules on their surface that can detect the harmful objects like microorganisms and kill them. This process is called as chemotaxis refers to the unidirectional movement of leucocytes responsible to chemical gradients. Leucocytes can move from a low to high concentrations of a specific chemical attractant.¹⁸⁶ Adaptive immune response is always antigen specific and more complex than innate immune response. Adaptive immune arm destroy invading pathogens and their toxic molecule or substances by clonal expansion of activated T cells against specific antigenic epitopes. This response is usually normal, however, it is crucial to distinguish foreign antigens from host to avoid autoimmune response. As we discussed earlier, many biological derivatives or biomolecules are acting as immunomodulators. If those compounds have the ability to stimulate the immune response and so is expected for the treatment of various diseases including cancer and immunodeficiency syndrome. On the other hand, if those compounds have the ability to suppress the immune responses that are useful to cure autoimmune as well as some gastro-intestinal tract diseases. The in vitro and in vivo experiments performed with shellfish derived polysaccharide chitosan stimulate the arginine activity in intestinal epithelial cells (IECs).¹⁵⁷ L-Arginine is metabolized by two major pathways in enterocytes conversion by arginase to ornithine, the precursor of polyamines, and conversion by nitric oxide synthase (NOS) to nitric oxide (NO).¹⁸⁷ NO effects on the intestine are duel action and dose dependent NO at low levels protect the mucosal injury, however at a higher level it is responsible for pro-inflammatory stimuli that will lead to cell death.^187–189

5. Conclusions

Increasing incidences of various diseases and disorders indicate the use of immunomodulators to overcome the problems. Especially, shellfish derived bioactive compound are potentially defensive in nature and they are applicable for disease control. This review elaborately discussed and consolidated the immunomodulating properties of shellfish derivatives. Moreover, the present review outlines the bioactive compounds obtained from shellfish such as proteins, polysaccharide and fatty acids which play some key roles in immunomodulating activities. More than 50% of the activities tested so far are still up to the preliminary level. Therefore, based on the information provided through available literature, the immunomodulatory effects of shellfish derivatives are highly deserved for elaborate investigation.

6. Abbreviations

AMPs	Antimicrobial peptides
CBA	Cross bagg albino
CCL	Concholepas concholepas
CD4	Cluster of differentiations
cDNA	Complementary deoxy ribonucleic acid
CGL	Lectin from Crenomytilus grayanus
Da	Dalton
DHA	Docosahexaenoic acid
EPA	Eicosapentaenoic acid
FLH	Fissurella latimarginata
GRPSp	Glycine rich peptide in Scylla paramamosain
HB4C5	Human-human hybridoma cells
HBDs	Human β-defensins
HIV	Human immune virus
IECs	Intestinal epithelial cells
IL	Interleukin
KDa	Kilodalton
KHL	Keyhole limpet hemocyanin
MGD	Mytilus galloprovincialis defensing
MTL	Lectin from Mytilus trossulus
MW	Molecular weight
MytC	Myticin
MytM	Mytimycin
NCMCS	N-Carboxymethyl chitosan derived N,O-sulfate
NK cells	Natural killer cells
NO	Nitric oxide
Nos	Nitric oxide synthase
RNA	Ribonucleic acid
Rp-hdmc	Ruditapes philippinarum hydramacin
RT-qPCR	Quantitative reverse transcription polycyclic chain reaction
TGFα	Transforming growth factor alpha
TNFα	Tumor necrosis factor-α

Acknowledgements

This paper was supported by Konkuk University in 2015.

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