Evaluation of Various Methodologies Used in Purification of Biologically Active Carbohydrates Derived from Marine Life Evaluation of Various Methodologies Used in Purification of Biologically Active Carbohydrates Derived from Marine Life.

Although various biologically active drugs derived from natural products, especially marine life such as antiproliferation and antioxidant drugs have been launched...


Introduction
Carbohydrates are molecules consisting mainly of three basic elements of carbon, oxygen and hydrogen with the empirical formula (CH 2 O). Carbohydrates can be classified according to its length into monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides comprise the basic building blocks of carbohydrates. This type of carbohydrates is known as simple sugars; therefore, it cannot be hydrolysed into simpler forms of carbohydrates. Monosaccharides structure is based on the number of carbon atoms, for instance, three carbons monosaccharide is known as triose, four carbons (tetrose), five carbons (pentose) and six carbons (hexose). Monosaccharides can be classified into aldehydes or ketones, according to the carbonyl group. In addition, carbohydrates have a chiral carbon that can give monosaccharides their structural configuration. Chiral carbon is represented via dextro-rotary(D) or levo-rotary (L) isomers [1,2].
Oligosaccharides are shorter sugar polymers, typically consisting of two to about hundred monosaccharides, attached together via glyosidic linkages. Oligosaccharides can be found freely in the blood or it can be attached to proteins or lipids forming a special type of carbohydrates [2].

Glycoconjugate
Glycoconjugate is a carbohydrate moiety that is covalently attached to a protein or lipid molecule. Carbohydrate which linked protein residues known as glycoproteins. There are subtype of glycoproteins known as proteoglycans (PG), which is composed of a particular amino-sugars known as glycosaminoglycan (GAG) attached to core protein residues [3]. GAGs are a group of long, Finally, HA is considered as simple GAG-type, which is unbranched and unaltered GAG. Its basic repeating disaccharides is composed of GlcA (1→3) GlcNAc [4]. GAGs biosynthesis starts from the rough endoplasmic reticulum (RER), where core protein in synthesised, afterwards, thus core protein is transferred to Golgi, where protein glycosylation occurs. In Golgi, several monosaccharides catalysed from their precursor and attached to the core protein residues in a process known as protein glycosylation, resulting in N-linked or O-linked glycan. N-linked glycans occur when GAG monosaccharides attached to asparagine (Asn) residue withing core protein residues. On the other hand, O-linked glycans occur when GAGs monosaccharides attach to serine (Ser)or threonine (Thr) residues of the core protein [1,[5][6][7]. GAGs structures are very diverse, and their synthesis is not template driven, like that of other biomolecules. All these structural modifications give GAGs their structural heterogeneity and wide range of biological functions. GAGs structural heterogeneity can lead to several cellular and extracellular functions, which depend on GAG types and the cell type that would vary from simple cellular function to cellular morphogenesis [8].

GAGs Derived from Marine Life as A Potential Pharmaceutical Agent
Recently, there has been increase demand for development of new drugs from natural products such as bacteria, animals including marine life. Marine life has derived several pharmaceutical utilities as therapeutic agent [9]. One of the largest phylain the animal kingdom known as molluscs, including snails, bivalves, squid and others. Various molluscs were used for dietary intake, as it has excellent dietary benefits. Over the past several years, there has been an increased demand for developing new anti-cancer agents derived from natural products such as plants, bacteria and animals, including marine life. The main advantage of natural product-derived anticancer drugs that it would have acceptable side-effects than of other chemically synthetic drugs [10]. There are several drugs either approved by the United State Food and Drug Administration (US-FDA), European Medicines Agency (EMA) orin clinical trial that have been derived from marine sources. For instance, Cytarabin that was the first marine-derived chemotherapeutic drug isolated from a sponge (Tethya crypta) [11]. Dolastatin which is a polypeptide product from marine mollusc (Dolabellaauricularia) can inhibit microtubule assembly as well astubulin polymerization, resulting in cell apoptosis. Although it was not successfully approved, this drug progressed to advanced phases in clinical trials, and its derivatives are still being considered as anticancer candidate [12,13].
Halichondrin is a tubulin inhibitor agent, which was isolated from Japanese sponge (Halichondria okadai), is synthetic macrocyclic ketone with enhanced antitumor activities against breast cancer. The US-FDA has approved its synthetic analogue since 2010 [14]. Cyclic-peptide drug Kahalalide-F was isolated from the mollusc (Elysiarubefescens) has anti-lysosomal properties.
Ithasentered phase-I clinicalt rials against prostate cancer and phase-II against hepatocellular carcinoma, non-small cell lung cancer and melanoma [15,16]. Didemnin-B isolated from (Carribean tunicate) shows inhibition of DNA-synthase that leads to cell cycle arrest [17]. Many molluscs have been used for dietary intake, as they have great nutritional benefits. Traditionally, molluscs have been used as natural treatments in Chinese traditional medicine; therefore, there has been great interest in investigating their valuable elements to be used as pharmaceutical resources [18].
The high content of uncommon polysaccharides structures in marine organisms make them a favourable candidate of natural products with various pharmaceutical benefits, including in cancer therapy. Generally, mollusc soft-body structural investigations have revealed high amounts of carbohydrates with some proteins and lipids, which makes carbohydrates a major area of interest from a pharmaceutical perspective [19].
Polysaccharides from vertebrates and invertebrates have emerged as powerful platforms for medical utilities, as they have various biological activities such as immunostimulatory, antioxidant, and antitumor activities [9,20,21]. Polysaccharides isolated from invertebrates have shown very different structures to common mammalian polysaccharides in terms of monosaccharide composition, molecular weight, degree of sulphation, type of glycosidic linkages, and branching points. For instance, a novel polysaccharide isolated from the head of ashrimph as a novel combination structure between heparin and HS known as a hybrid hep/HS structure [22]. This structural diversity in polysaccharides has generated additional features for the senovel molecules that could make them potent tool singlecobiology, especially as pharmaceutical agents [9]. Although there are several studies of marine life as source of promising biologically active substances that can be used as pharmaceutical target for various disorders such as cancer, however, various purification techniques were used to purify biologically active GAG and using different structural analysis techniques to characterize this active compound made this field of carbohydrates challenging. These major issues in biologically active GAGs from marine life including, extraction procedure, purification step and characterisation technique, which has created a wide gap on the final structure for GAGs to be used as pharmaceutical agent.
There is no standard method to extract GAGs from in marine life to purified active compounds. GAGs extraction and purification methods from marine source mainly follow three consecutive steps, namely, de-shell (shell removal), de-fat (lipid removal) and proteolysis. However, there are inconsistency in GAG extraction techniques. Indeed, the nature of the GAG polysaccharide derived from marine source, which has an unknown structure and lacks any chromophore that affects adopting an appropriate purification step. For instance, size-exclusion chromatography requires a well-known molecular weight sample, and lectin affinity chromatography requires a particular ligand to allow the complex to interact. This lack of standardization has assigned as major issues in GAGs purification from marine life. Therefore, GAGs with potent biological activity, for instance, antiproliferative or anticoagulant activity were not assigned as pure polysaccharide, but it could be identified as proteoglycan, as other biomolecules could play a role in the biological function. The aim of this review is to evaluate different methodologies used to extract and purify GAG-derived from marine life with potent biological activities.

Purification Methods of GAG-Derived from Marine Life
The literature has demonstrated several purification techniques to obtain purified GAGs from different marine life species. Firstly, de-fat technique, which considered as the first step in GAGs purification. Next, proteolysis technique which is used to ensure removing on N-linked or O-linked proteins that is attached to the polysaccharides chain. Finally, using various chromatographic methods in order to obtain GAG purified chain.

GAG-Defat Techniques
The Defat process is the first step of the purification, which

Species
Defat Process Using Acetone Reference

Sea Cucumber (Stichopus Japonicus)
The soft body was homogenized with chloroform/methanol, then, filtered to ensure fat removal [39] De-Fat Method Using Acetone and Ethanol: According to this method, lipid-soluble molecules were removed by the action of acetone and ethanol. Acetone will remove lipid-soluble molecules; while ethanol was used to enhance the dehydration process (Table 4) [40].

Seagrass (Phanerogam Zostera marina)
The sample was submerged in acetone overnight, then, washed with acetone and ethanol, until the supernatant become clear. [40] De-fat Method Using Ethanol (

GAG-Proteolysis Techniques
This is the second step in GAG purification process, after defat, which lead to cleave GAG chain from the attached protein chain.
This process mainly concerns about cleaving the polysaccharide chain, thaerefore, protein residues were hydrolysed either via enzymatic or chemical techniques. Enzymatic extraction, which mean removing of proteins by using enzymes, for instance, alcalase, papain or pronase [42]. On the other hand, chemical extraction by using different chemical such as Aluminium sulfate [36]. The following literature distinguished different techniques to remove protein residues using different concentrations, centrifugation, incubation period and temperatures.

Proteolysis Technique Using Papain Enzyme: Papain is a
proteolytic enzyme derived from Papaya (Carica papaya) It was used to remove protein residues. Different proteolysis techniques were applied using papain as proteolytic enzyme (Table 6) [43,44].

Bivalve Mollusc (Scapharca inaequivalvis)
The dried body was dissolved in (1g/20 mL) 0.1 M sodium acetate buffer pH 5.5, then, 1 mg papain/1 g of the dried materials were mixed and incubated for 24 h at 60 °C [28] Bivalve Mollusc (Nodipecten nodosus) 25 g of dried materials were dissolved in 500 mL (0.1 M sodium acetate) buffer (pH 5.5) with 5 g of papain in 5 mM EDTA, and 5 mM cysteine. Mixture was incubated overnight at 60°C. After incubation period, the mixture was centrifuged 2000 × g for 10 min. This proteolysis method was been repeated three times [25] Chordata Chondrichthyes (Raja clavate) Proteolysis method were dependent on dissolving the 5 g of Raja clavata on alcohol, then added 250 mL of (0.1M sodium acetate buffer, EDTA 5 mM, cystein 5 mM pH 6). Afterwards, immediately added 510 mg of papain. The mixture was incubated for 24 hours at 60 °C, then filtered with 138 mL of distilled water. [44] Bivalve Mollusc (Amussium pleuronectus) 5 g of defatted tissue in 100 ml (100 mM sodium acetate, 5 mM EDTA and cysteine pH 5.5) and 100 mg of papain/1g of defatted tissues was added and the buffer. The mixture was incubated at 60°C for 24 h. [26] Chordata Chondrichthyes (Aetobatus narinari) [27] Fish (Salmo salar) [31] Chordata Tunicata (Styela plicata) [24] Fish (Monkfish, codfish, spiny dogfish, salmon and tuna) 1 g of defatted tissue was dissolved in 10 mL (100 mM sodium acetate containing 5 mM EDTA and 5 mM cysteine buffer pH 5.5). Then, added 60 mg of papain to the mixture. The mixture was incubated at 60°C for 24 h [30] Proteolysis Technique Using Alcalase Enzyme: Is a serine endopeptidase that is suitable for the hydrolysis of proteins. It has previously been used to produce soluble hydrolysates that digest the protein in the defatted tissues (Table 7) [45,46,47].

Species Proteolysis Techniques Using Actinase-E Reference
Bivalve Mollusc (Mactra chinensis) 30 g of dried tissue was dissolved in 120 mL of Tris-acetatebuffer (pH 8.0) containing actinase-E, then, the mixture was incubated for 18 h at 45°C. Afterwards, 600 mL of NaOH buffer was added and the mixture was cooled at 4°C for 18 h. [35] Fish maw 2 g of dried fish was homogenized in 100 mL distilled water,then, 10 mg/mL actinase-E was added. The mixture was incubated at 55°C for 48 h, then, the mixture was centrifuged at 4000 x g for 30 min. [48] Bivalve Mollusc (Ruditapes philippinarum, Scapharca broughtonii, Mizuhopecten yessoensis, Turbo cornutus, Crassostrea nippona, Corbicula japonica, Mytilus galloprovincialis, Neptunea intersculpta, Pseudocardium sachalinense and Crassostrea gigas) 10 mg of actinase-E g of dry powder was dissolved in 50 mM Trisacetate (pH 8.0), then, the mixture was incubated for 48h at 45°C. Afterwards, the sample was further treated with a solution containing 0.5 M NaOH and 0.3M sodium borohydride (20 mL/g of dried powder), the mixture was incubated at 4 °C for overnight. Finally, proteins were removed by centrifugation at 1000 x g for 30 min. [34] Proteolysis Technique Using Pectinase Enzyme: Pectinase enzyme derived from Aspergillus niger, which is used to purify pectins molecules from the sample (Table 9). Table 9: Proactinase enzyme was used to cleave pectin polysaccharides chain from residual proteins.

GAG Purification Using Various Chromatographic Techniques
The aim of this process is to purify the biologically active components that is derived from polysaccharides, especially GAGs.
The purification techniques were mostly used chromatographic techniques to purify GAGs according to their charge or size structure.

GAG Purification Using Anion-Exchange Chromatography:
Anion-exchange chromatography was one of the most common methods for GAGs purification and fractionation. As GAGs known to carry highly negatively charged molecules such as heparin Penta saccharide structure [49]. Therefore, various purification techniques were particularly based on this feature. Anion-exchange resins were used with various solvents such as high molar NaCl solution in order to exchange the biologically active molecules.

Quaternary Aminoethyl (QAE)-Sephadex: QAE functional
group is used as strong anion-exchanger. In this method, QAE functional group is attached to the sephadex resin, which is composed of cross-linked dextran with epichlorohydrin, to allow separation of negatively charged molecules (Table 12) [50]. Table 12: Shows different chromatographic techniques using QAE as anion-exchange resin, which purify GAGs according to their negatively charge.

Mactra chinensis
Crude GAG sample was dissolved in distilled water, then applied to DEAE-sepharose fast flow column. GAGs were eluted using gradient elution of two buffers, which are 50 mM sodium phosphate (pH 6) as buffer (A), and 2.0 M NaCl in 50 mM sodium phosphate (pH 6) as buffer (B) at flow rate 2 mL/min over 180 min. [35] Marine mollusc (Cerastoderma edule) Crude GAG sample was dissolved in distilled water. Then applied to anion exchange column (16 X 200 mm), which packed with 10 mL of DEAE-Sepharose fast flow. GAGs were eluted using a linear gradient (0-1.5 M) NaCl over 70 min. Elution was monitored using UV 280 nm [9] DEAE-Cellulose: In this methods, DEAE anion exchanger is attached to the cellulose resin (Table 14) [51]. Table 14: Shows different chromatographic techniques using DEAE-cellulose as anion-exchange resin.

Size-exclusion Chromatography (SEC)
This method depends on molecules size, as SEC purifies molecules according to their molecular size. Sephadex is a gel that contains dextran with epichlorohydrin, where G-100 refer to a particular property of Sephadex as G-100 has fractionation range 4000-15000 kDa (Table 15).

Species Chromatographic Techniques Using SEC Column Reference
Raja radula Raja clavata GAGs sample was applied to sephadex G-100 column (2.5 X 70 cm) using (0.05 M) sodium acetate as elution buffer at flow rate 10 mL/h.

Conclusion
In conclusion, GAGs have wide range of structural diversity as well as biological activity, therefore, setting up a standard method in GAG purification would be result in highly purified biologically active compound from GAGs, which can be used as pharmaceutical agent. As conducted in this review, there are three main steps are used in purification of GAG-derived from marine life, which are de-fat, proteolysisand polysaccharide chain purification, however, various techniques were used in each method that resulted in lack of standard method in GAG purification. Hence, it is highly recommended to adapt versatile standard method to allow better purification of biologically active molecules derived from GAGs. Thus, it would be possible by evaluating the current purification techniques used in GAGs analysis and assign a gold standard method on purification techniques.