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. 2021 Nov 9;6(46):30998–31008. doi: 10.1021/acsomega.1c03776

Extraction of Fucoidan from Turbinaria decurrens and the Synthesis of Fucoidan-Coated AgNPs for Anticoagulant Application

Nagarajan Shanthi †, Ponnan Arumugam ‡, Marudhamuthu Murugan §, Muthiyal Prabakaran Sudhakar ∥, Kulanthaiyesu Arunkumar ⊥,*
PMCID: PMC8613821  PMID: 34841142

Abstract

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Brown seaweeds usually contain alginate as a major polymer. The second major sulfated polymer in brown seaweeds is fucoidan, which has huge potential in medicinal applications. In this study, the photosynthetic pigments from Turbinaria decurrens were first extracted using chloroform/methanol in the ratio of 1:1 (v/v), followed by fucoidan extraction with yields of 5.58% (crude) and 1.28% (purified fucoidan) from the dry weight of seaweed, whereas alginate was extracted with a yield of 14.7% DW of seaweed. The isolated fucoidan possessing anticoagulation property was identified and characterized as (1–3)-α-l-fucopyranosyl residues with sulfate groups primarily at the C4 position and to a lesser extent at the C2 position, whereas in the case of galactose, at the C3 and C6 positions. The AgNPs synthesized using isolated fucoidan exhibit strong anticoagulant activity and possess a good antibacterial property against Gram-negative clinical bacteria. Functional groups such as O–H, C–H, and S=O associated with sugar residues in sulfated fucoidan are involved in the synthesis of the nanoparticles with a spherical shape, size ranging from 10 to 60 nm, and showing polydispersity. From this study, we conclude that fucoidan-coated anionic AgNPs synthesized from T. decurrens have tremendous potential in drug development.

1. Introduction

Seaweeds (marine macroalgae) that occur in the intertidal and subtidal regions of coastal waters are categorized based on their pigmentation as red (Rhodophyceae), green (Chlorophyceae), and brown (Phaeophyceae). The cell wall of seaweeds contains commercially important polysaccharides (agar and carrageenan in red; cellulose and ulvan in green; and alginate, laminarin, and fucoidan in brown seaweeds) that exhibit various biological activities.1,2 Even though sulfated fucans are found in some marine organisms such as sea cucumber,3 sea urchin,4 and bacteria, brown algae are the major source of fucoidan.5 Isolation of fucoidan polymers from brown seaweeds reveals structural diversities in length, linear or branched, branching pattern by glycosidic linkage, the proportion of monomers (galactose, mannose, fucose, xylose, uronic acid, etc.), sulfate level, and its position in monosugar residues besides fucose (the principle sugar).6−8 These structurally varied fucoidans, which depend on species, part and age of the thallus, seasons and geography, and the extraction methods followed, are responsible for different bioactive activities.8−10 Further, being of algal origin with a wide range of bioactive properties, such as antioxidant, antiviral, antithrombic, anticancer, etc., particularly anticoagulation,11 the use of fucoidan in place of heparin as an anticoagulant certainly helps in avoiding the risk of virus contamination and its associated infections caused by animal-origin heparin.6 Anticoagulant usage in medical applications is tremendous. Anticoagulants are nothing but blood thinners or antiplatelet drugs. They prevent thromboembolic diseases,12 atrial fibrillation in stroke prevention, etc.13 However, fucoidan with promising anticoagulation property on par with heparin is yet to be isolated from brown seaweeds and applied as an anticoagulant.

Having a high surface area and a high fraction of surface atoms, silver nanoparticles (AgNPs) exhibit unique physicochemical and biological characteristics.14 Due to their outstanding antimicrobial properties, AgNPs are being employed for developing various biomedical products such as nanosilver-coated wound dressing and contraceptive devices, surgical instruments, implants, etc.15,16 Even though AgNPs are synthesized by various means through chemical and photochemical reactions such as reverse micelle thermal decomposition,17 electro- and ultrasound-facilitated reactions,18,19 and microwave-assisted processes,20 green synthesis by biological means using microbes and plant extracts21,22 is considered to be cost-effective, easy to handle, readily available, and eco-friendly.23,24 But nanoparticles (NPs) synthesized using selective biomolecules as functionalizing ligands are effective for specific applications and requirements.25,26 Although polysaccharides have long been used for preparing scaffolds, fibers, and hydrogels because of their abundance, biocompatibility, hydrophilicity, and ability to modify into different forms, employing them for synthesizing NPs of a specific size with targeted bioactive properties is considered promising for desired use and applications.27,28 A few studies have characterized the NPs synthesized using seaweed extracts, but studies using specific polysaccharides (agar, carrageenan, and alginate) and compounds are limited. The NPs synthesized using sulfated fucoidan polymers have a broad scope for biomedical applications in the field of drug delivery28 because as an anionic polymer they can readily ligand with any cationic drug. Based on the view of Jang et al.,29 we can obtain effective fucoidan and its NPs for drug delivery based on the structure and bioactivity isolated from a specific algae source. In this paper, AgNPs synthesized using fucoidan extracted from T. decurrens and showing strong antibacterial activity against two Gram-negative pathogens and also exhibiting an anticoagulant property are presented.

2. Experimental Section

2.1. Seaweed Collection

Brown alga (Turbinaria decurrens Bory de Saint-Vincent) weighing about 1 kg was collected at the subtidal region of the Mandapam coast (Lat. 09°17’ N; Long 79°07’ E, Gulf of Mannar, India). The collected Turbinaria decurrens was washed with distilled water to remove salt, sand, and epiphytes and then shade-dried in a room under air for 5 days. The shade-dried specimen was chopped and powdered using a mixer grinder.

2.2. Successive Extraction of Fucoidan and Alginate

Fucoidan and alginate were extracted by adopting a successive extraction method30 with slight modifications. The powdered specimen of 100 g was soaked in 500 mL of chloroform/methanol (1:1 v/v) solvent overnight to remove the pigments. Then, the residue was sequentially extracted in 2% CaCl2 (2 × 100 mL for 3 h at 70 °C), followed by 0.01 N HCl (2 × 100 mL at 70 °C for 3 h) and 3% Na2CO3 (1 × 100 mL at 70 °C for 3 h). Extracts obtained in 2% CaCl2 and 0.01 N HCl were combined and concentrated using a freeze dryer as crude fucoidan. The final residue extracted in aqueous 3% Na2CO3 was precipitated in acetone as sodium alginate. The total sugar31 and sulfate content32 in the crude fucoidan were recorded. Fucose, galactose, and mannose were estimated from the respective monomer standard graph prepared by a phenol-sulfuric acid method31 using the absorbance measured for total sugar estimation.

2.3. Qualitative Fucose Test

The presence of fucose in the crude fucoidan was confirmed by the cysteine–sulfuric acid–methyl pentose test.33 The crude fucoidan (50 μg/mL) and standard l-fucose (100 μg/mL) were taken in separate test tubes, and 4.5 mL of H2SO4 (conc. H2SO4/H2O 6:1 v/v) was added and mixed well. The mixture was cooled in an ice-water bath at 25 °C for 3–4 min and placed in a boiling water bath for 3 min subsequently. Then, the tubes were cooled under running tap water, and 0.1 mL of 5% (v/v) cysteine hydrochloride solution was added to each tube. The appearance of a yellowish-green color indicating the presence of l-fucose in the test solutions was observed.

2.4. Fucoidan Separation Using Ion-Exchange Chromatography

The crude fucoidan weighing 200 mg was separated through the DEAE-cellulose 52 column (42 × 3 cm) using increasing concentrations of 200 mL of NaCl (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 M). Eluents of 25 mL were collected until no more sugar was detected in the eluents by the phenol-sulfuric acid method against l-fucose standard.31 Based on the sugar content, the successive eluents such as 42nd, 43rd, 44th, 45th, and 46th fractions were pooled and confirmed the presence of the l-fucose content by a qualitative test.33 Then, the combined eluents were dialyzed (cutoff 3.5 kDa) for 24 h in distilled water and lyophilized and weighed (46 mg). Using the phenol-sulfuric acid method,31 total sugar was calculated using glucose as the standard, and individual monomers such as fucose, galactose, and mannose were recorded in purified fucoidan using its standard graph. The sulfate content32 of the purified fucoidan was recorded.

2.5. In Vitro Anticoagulation Activity

In vitro anticoagulation activity was tested using the activated partial thromboplastin time (aPTT) assay (Randox kit-APT2749).34 The platelet-poor plasma was collected from blood samples of five voluntary healthy persons in a vial containing 3.8% sodium citrate (9:1 v/v). The collected blood samples were centrifuged at 4000 rpm for 20 min, and the plasma (100 μL) from the blood was mixed with purified fucoidan (5, 10, 15, 20, and 25 μg/mL) and incubated for 1 min at 37 °C. Then, 100 μL of the prewarmed aPTT reagent was added to the mixture and incubated for 5 min at 37 °C. Thereafter, clotting was initiated by adding 100 μL of 0.25 M CaCl2, and the clotting time was recorded against the control sample plus reagents without fucoidan.

2.6. Characterization of Fucoidan

The FTIR spectrum was recorded in the range between 4000 and 400 cm–1 using a Bruker Optik GmbH, Germany (model no. Tensor 27; Software, Opus version 6.5). The 1H and 13C NMR spectra of fucoidan in 0.5 mL of D2O were measured using a Bruker Biospin Avance 400 NMR spectrometer (1H frequency 1/4 400.13 MHz, 13C frequency 1/4 100.62 MHz) at 298 K (using a 5 mm broad-band inverse probe head) equipped with a shielded z-gradient and XWIN-NMR software version 3.5 and using tetramethylsilane as an internal reference. One-dimensional 1H and 13C spectra were obtained using a one-pulse sequence. One-dimensional 13C spectra using spin echo Fourier transform and quaternary carbon detection of 42 sequences were also obtained to aid the structural detection.35,36

2.7. Synthesis of Fucoidan-Coated AgNPs

Fucoidan-AgNP synthesis37 was carried out using 1 mg of fucoidan polymer dissolved in 3 mL of distilled water and 97 mL of AgNO3 (1 × 10–1 M) and stirred well at room temperature and left undisturbed. The appearance of a yellowish-brown color was observed after 1 h indicating the formation of AgNPs, which was confirmed using UV–visible spectroscopy between 200 and 800 nm. A control group was maintained without fucoidan. The synthesized mixture was then centrifuged at 5000 rpm for 20 min, and the pellet containing AgNPs was collected. To remove the uncapped fucoidan, the pellet was again dissolved in 10 mL of distilled water and centrifuged at 5000 rpm for 20 min. Centrifugation was repeated thrice to collect fucoidan-capped AgNPs. The obtained pellet was freeze-dried and stored at 4 °C until further use.

2.8. Anticoagulant and Antibacterial Assay of Fucoidan-Coated AgNPs

The in vitro test of the anticoagulant activity using 5, 10, 15, 20, and 25 μg/mL fucoidan-coated AgNPs was carried out as described in Section 2.5. The antibacterial assay38 of 10 μg of AgNPs was evaluated against four clinical pathogens (Gram-positive: Streptococcus mutans MTCC 896 and Staphylococcus aureus MTCC 96 and Gram-negative: Pseudomonas aeruginosa MTCC 2642 and Escherichia coli MTCC 40). The assay was carried out using the disc diffusion method on Mueller–Hinton (MH) agar plates. For this, a sterile 5 mm (dia) disc loaded with AgNPs (10 μg mL–1 sterile distilled water) was impregnated on MH agar Petri plates spread with the exponential test bacteria and was then incubated at 37 °C for 48 h. The antibacterial activity was recorded by measuring the clear zone around the disc as the diameter in millimeters. The discs loaded with sterile distilled H2O and 10 μg of gentamycin were maintained as the negative and the positive controls, respectively. All of the experiments were carried out in triplicate, and the mean value was calculated with deviation.

2.9. Characterization of Fucoidan-Coated AgNPs

The FTIR spectrum was recorded in the range between 4000 and 400 cm–1 using a Bruker Optik GmbH; model no., TENSOR 27; Software, OPUS version 6.5.23 The X-ray diffraction (XRD) pattern was measured using a powder X-ray diffractometer (PXRD-6000 Shimadzu) at angle 2θ and a scan axis of 2:1 sym. The formation of AgNPswas confirmed from the PXRD peak positions using Bragg’s law.39 The morphology of the AgNPs was examined using field emission scanning electron microscopy (Hitachi SU6600) on a carbon-coated copper grid equipped with an EDAX attachment. The size and the morphology of the AgNPs were examined using atomic force microscopy (AFM, diCP II Veecs) and transmission electron microscopy (TEM). For measuring AgNPs using TEM, the freeze-dried AgNPs were placed on the carbon-coated copper grids and kept overnight in a vacuum desiccator before loading them onto a specimen holder. TEM micrographs of the AgNPs were obtained using a JEOL (JSM 100 cx) TEM instrument operated at an accelerating voltage of 200 kV.

3. Results and Discussion

3.1. Quantification of Fucoidan and Alginate

The results of the yield of crude and purified fucoidans, crude alginate, and their constituents obtained from the residue of T. decurrens after removing the pigments by chloroform/methanol (1:1 v/v) are presented in Table 1. The cell wall of the brown seaweeds mainly constitutes alginate and some extend fucoidan as matrix substances. The yield, structure, and constituents of fucoidan vary depending on brown algae species,9,10 and further, the yield was influenced by the choice of the solvents and/or methods adopted for extraction.40−42 Further, a difference in the yield was recorded when the sample of species was subjected only to fucoidan extraction compared to successive extraction for fucoidan and then alginate. The fucoidan yield obtained by direct aqueous extraction in Laminaria japonica was 2.04%43 and 2.74% by direct mild acid (0.1 N HCl) in Sargassum polycystum.44 Variation in the fucoidan yield among the 11 brown algae species was recorded depending on the methods followed.41 By successive extraction in Fucus serratus, Fucus vesiculosus, and Ascophyllum nodosum, the fucoidan yield was 6.0, 9.8, and 8.0%, respectively,10 and 4.24% in Saccharina latissima.45 Previous studies demonstrated that fucoidan was extracted using 2% CaCl2, followed by 0.01 N HCl, and alginate was extracted using 3% Na2CO3 successively after removing pigments using 85% ethanol from Saccharina longicruris, A. nodosum, and F. vesiculosus.30 In another study, 3.62% yield of fucoidan (using 2% CaCl2 and 0.01 N HCl) and 11.2% yield of alginate (using 3% Na2CO3) were obtained by successive extraction from the residues of Sargassum wightii after removing the pigments using chloroform/methanol (1:1 v/v), followed by 85% ethanol.40 Whereas, in the present study, after removing the pigments by treating samples in chloroform/methanol (1:1 v/v) and considering the thallus hardness of Turbinaria decurrens, successive extraction using 200 mL of 2% CaCl2, 200 mL of 0.01 N HCl, and 100 mL of 3% Na2CO3 was performed that resulted in a high fucoidan yield of 5.58% (w/w) and a 14.7% (w/w) alginate yield.

Table 1. Yield of Fucoidan and Alginate and Their Constituents Extracted from T. decurrens on a Dry Weight Basisa.

  sulfated polysaccharides
constituents crude fucoidan purified fucoidan crude alginate
yield (%) 5.58 1.28 14.70
total sugar (μg/mg) 34.00 ± 4.54 62.50 ± 4.21 78.32 ± 3.44
fucose (μg/mg) 12.39 ± 1.38 19.00 ± 3.72 05.61 ± 4.21
mannose (μg/mg) 06.63 ± 2.29 05.73 ± 6.81 19.51 ± 4.22
galactose (μg/mg) 11.82 ± 2.71 18.51 ± 6.38 20.78 ± 7.61
sulfate (μg/mg) 05.90 ± 0.93 09.25 ± 1.71 02.22 ± 0.46
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a

All constituents in the table are calculated using crude polysaccharides extracted from T. decurrens.

3.2. Fucoidan Constituents

Sulfated polymers dominated by fucose monomer (fucoidan) have been isolated from various brown algae species.11,46 On the other hand, recently, fucoidans with equimolar fucose and galactose, so-called galactofucoidan, have also been isolated in some brown seaweed species.10 These two major types of fucoidan isolated from the brown seaweed species are implicated mainly by extraction techniques, season, harvest location, age of the thallus, and species.47 The monomer residues and the sulfate content of fucoidan extracted using the direct method48 are varied compared to those of fucoidan extracted using the successive method49 from Undaria pinnatifida. The sulfated galactofucans are reported in various brown seaweeds such as Adenocystis utricularis,50Undaria pinnatifida,51Spatoglossum schröederi,6Lobophora variegata,7Sargassum wightii,8 and Turbinaria decurrens.40 In this study, fucoidan extracted from brown seaweed (T. decurrens) through the successive extraction method30 contains a high amount of fucose followed by galactose,51,52 and the sulfate content was more in purified fucoidan than the crude (Table 1), which implies increased bioactivity,53 and also an increased sulfate content shows increasing anticoagulation property.54

3.3. Fucoidan Isolation and Purification

In this study, crude fucoidan extracted by the successive method from Turbinaria decurrens was separated through cationic DEAE column elution by increasing NaCl gradients. Fucoidan containing sulfate as an anionic functional group and thereby giving the polymer a negative charge50 was introduced into the positively charged ion-exchange DEAE column. The strong ionic solvent (NaCl) promoted the release of sulfate groups from the gel column. As the concentration of NaCl increased in the mobile phase, more sulfate groups were released.8,9 Hence, the sulfate constituents of eluents were increased proportionally on increasing the NaCl gradient (Figure S1). In this study, the fucoidan polymer eluted using 3 M NaCl contains a high sulfate content suggesting the possibility of increased biological activities.40

3.4. Anticoagulation Activity of Isolated Fucoidan

The aPTT assay results depend on the clot detection method and assay kits used. The normal blood clotting time ranges from 25 to 43 s.55 The blood maintains fluidity as long as it is inside the vasculature and clots quickly at the sites of vascular injury as soon as it is exposed to subendothelial surfaces. In normal conditions, thrombosis and hemorrhage are prevented by a balance action between coagulation and fibrinolysis. Any imbalance that occurs favors coagulation that causes thrombosis leading to platelet aggregation, fibrin formation, and trapped red blood cells in arteries or veins. There are different types of antithrombotic drugs available in the market to treat thrombosis. Antiplatelet drugs inhibit platelet activation or aggregation, whereas anticoagulants attenuate fibrin formation and the fibrinolytic agents degrade fibrin formation.56 The compounds possessing anticoagulant properties have been evaluated using assays such as activated partial thromboplastin time (aPTT), prothrombin time, and thrombin time.57 Like heparin, fucoidan also inhibits thrombin formation, but its anticoagulant efficacy is not the same as that of the latter. The anticoagulant and antithrombotic effects of fucoidan have been mediated by catalyzing thrombin inhibition by mainly inhibiting heparin cofactor II.58 Fucoidan, like heparin, inhibits thrombin formation by forming complexes with the inhibitor. Specifically, heparin possesses antithrombin property, and in the case of fucoidan, it exhibits heparin cofactor inhibition.59 Fucoidan inhibits thrombin activity or fibrin polymerization.57

In this study, the anticoagulation activity of the isolated fucoidan from T. decurrens was assayed by in vitro aPPT activity using platelet-poor plasma.34 Using the aPPT assay, heparin (0.4 IU/mL) exhibits a clotting time of 68–75 s.55 Fucoidan of Undaria pinnatifida exhibits a five times higher anticoagulation property than the control.60 The present study emphasizes that purified fucoidan of T. decurrens shows dose-dependent anticoagulation evidenced from the aPPT assay (Figure 1a) and increased anticoagulation activity on increasing the concentration up to 20 μg/mL fucoidan in 387.8 ± 10.3 s (Figure 1b). This result shows fucoidan of brown seaweed T. decurrens as a promising anticoagulant.

Figure 1a.

Figure 1a

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In vitro anticoagulation by the activated partial thromboplastin time (aPTT) assay of fucoidan-coated AgNPs (a, b: preparation of the assay; c: blood sample showing coagulation at 54.23 ± 4.8 s by 0 μg/mL; and d: blood mixed with fucoidan-coated AgNPs showing delayed coagulation at 387.8 ± 10.3 s by 20 μg/mL).

Figure 1b.

Figure 1b

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Anticoagulation activity of T. decurrens fucoidan and its AgNPs.

3.5. Fucoidan Characterization

3.5.1. FTIR Analysis

The FTIR spectra (Figure 2) of fucoidan isolated from T. decurrens were assigned to the spectral similarities of fucoidan isolated from brown seaweeds such as Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, Fucus distichus, Fucus spiralis, and Ascophyllum nodosum.61,62 A broad band at 3431 cm–1 assigned to the OH group of monosugar and the presence of aliphatic C–H at 2827 cm–1 are recorded. The C=O stretching vibration for acetate groups is noted by the peak at 1788 cm–1, and the presence of O–C–O stretching is shown by the sharp peak at 1621 cm–1. The C–O ether bond (1079 cm–1) and the C–S–O band (872 cm–1), characteristics of the fucose principle monomer of fucoidan polymer, are observed in the FTIR spectra. Besides, sharp peaks observed at 872 cm–1 (S=O) and 775 cm–1 (C–S–O) confirm the sulfate attachment at the C4 position in the fucose monomers. As reported by Singthong,63 the band at around ∼465 cm–1 is related to the presence of the sulfate group at the C6 position of the galactose unit. The signal close to ∼1466 cm–1 is attributed to the asymmetric and symmetric stretching vibrations of COO of uronic acid. Further, signals at 1466 and 1388 cm–1 could be an indication of the presence of uronic acid residue in the fucoidan polymer isolated from T. decurrens.

Figure 2.

Figure 2

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FTIR spectra of fucoidan standard, fucoidan of T. decurrens, and its AgNPs.

3.5.2. NMR Analysis

Monomer residues with sulfates in the polymer chain are formed by different linkages that produce less stable signals in the NMR spectra.14,64 The 1H NMR data (Figure 3) support the identification of the position of glycosidic linkages and sulfate in the monosugar residues in the polymer. The spin systems attributable to the anomeric protons of α-l-fucopyranose residues and β-d-galactose residues were found in the 1H NMR spectrum. Signals observed from the ring protons (H2–H5) between 3.071 and 4.25 ppm are resonance characteristics of α-l-fucopyranose. Intense signals from the methyl protons H6, a minor signal at around 1.099 ppm, and a major signal at 1.23 ppm were observed. Methyl signals appeared at around 1.816–1.969 ppm representing α-l-fucopyranose.64,65 Similar to fucoidan characterized from various brown seaweeds, the 13C NMR spectrum of isolated polymer (Figure 4) has several intense signals in the anomeric (64.77–69.88 and 75.53–87.73 ppm) and high-field (16.62 ppm) regions that are typical of the α-l-fucopyranosyl unit. The resonance of the methyl group (C6) appeared as a strong signal at 16.62 ppm. The signal at 57.27 ppm indicates six nonlinked galactose units, and the signal at around 69.80 ppm indicates six linked galactose units. The absence of signals at around 83.0–86.0 ppm suggests a lack of C2-, C3-, or C4-linked galactose units, and the galactose residues are C1- and C6-linked. The NMR data suggest that fucoidan extracted from T. decurrens consists of (1–3)-α-l-fucopyranose residues with sulfate groups primarily at the C4 position and to a lesser extent at the C2 position and C3 and C6 of galactose residues with numerous branches in the form of α-l-fucose and β-d-galactose residues linked by (1–3)-O-glycosidic linkages as reported by Wang et al.66 in Sargassum japonica and67U. pinnatifida (Figure 4).

Figure 3.

Figure 3

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1H NMR spectrum of fucoidan isolated from T. decurrens.

Figure 4.

Figure 4

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13C NMR spectrum of fucoidan isolated from T. decurrens.

3.6. Spectral Analysis of Fucoidan-Coated AgNPs

NPs synthesized by selective biomolecules are considered to be most desired for target-specific biomedical applications rather than those synthesized from crude compounds.68,69 There are studies that have reported AgNPs synthesized using crude extracts of marine brown algae such as Sargassum wightii,70Turbinaria conoides,71 and other seaweeds.23,72,73 In this study, the synthesis of AgNPs by sulfated fucoidan isolated from the marine brown alga T. decurrens was confirmed based on the colorless solution of AgNO3 turning brownish-yellow, indicating the formation of NPs, and monitored by the UV–visible spectral band at 420 nm (Figures 5a and S2). This band was identified as a “surface plasmon resonance band” attributed to the excitation of free electrons in the NPs. The shape of the band recorded was symmetrical, which indicates that the synthesized NPs are spherical and can disperse uniformly.74

Figure 5.

Figure 5

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Fucoidan-coated AgNP characterization by the UV–visible spectrum (a), SEM (scale bar 20 μm) (b), EDAX (c), XRD (d), AFM (scale bar 500 nm) (e), and TEM (scale bar 50 nm) (f) analyses and their images.

3.7. Enhanced Anticoagulant Activity of Fucoidan-Coated AgNPs and Their Antibacterial Activity

The metal NPs possessing various bioactive properties75,76 open an avenue for synthesizing the desired-size NPs using specific biomolecules for various applications to treat different diseases. Recently, the usage of biologically synthesized AgNPs using selective active molecules is found to be effective and requires only a very less quantity when compared to active crude molecules.28,77 In this study, AgNPs synthesized using fucoidan exhibit strong anticoagulant activity compared to isolated fucoidan in a dose-dependent manner (Figure 1aa,b). Antibacterial activity against clinical pathogens was also strong, and the effect was pronounced against Gram-negative bacteria over -positive bacteria (Figure S3). This observation corroborated with an earlier study78 where the bioactivity effect was superior and depended on the concentration of AgNPs and the type of pathogen tested. Because of the high surface-to-volume ratio, NPs can undergo a high level of interaction with the bacterial cell surface with an enhanced holding capacity, promoting bioavailability that ultimately exhibited high antibacterial activity against Gram-negative bacteria. A mechanism was proposed where close contact between AgNPs and the microorganisms enhances the transfer of Ag ions to the microbial cell, while the microorganisms’ metabolic activity dissociates fucoidan that promotes the release of silver ions.79 From a previous study on fucoidan-based NPs, which were found to be effective against osteosarcoma,80 fucoidan NPs prepared in this study were more effective than its polymer. This enhanced anticoagulation and antibacterial activities of fucoidan-coated AgNPs observed in this study, which were also ascertained to act as nanocarriers to promote the intracellular delivery of fucoidan29 by controlled release with stable coated NPs that resulted in increased anticoagulant activity compared to the fucoidan polymer.81

3.8. Fucoidan-Coated AgNP Characterization

3.8.1. FTIR Study

The absorption bands at 3431 cm–1 of fucoidan standard, isolated fucoidan (Figure 2), and its AgNPs show the stretching vibrations of the OH groups of carbohydrates.82,83 Bands at 1466 cm–1 of fucoidan and 1388 cm–1 of AgNPs indicate the presence of uronic acid.84 The above characteristics of FTIR spectra confirms the formation of fucoidan-coated AgNPs. The most important bands of fucoidan (Figure 2) are those found at 1388 and 700 cm–1, corresponding to ester sulfate groups. The region at around 1466 cm–1 is assigned to the scissoring vibration of CH2 (galactose, xylose) and the asymmetric bending vibration of CH3 (fucose, O-acetyls), and the absorption at around 872 cm–1 indicates the presence of sulfate groups in the C4 position of galactose.60 The peak at 1079 cm–1 in fucoidan shows the CH stretching vibration of fucose and S=O bound to the axial position of C4 (Figure 2),60,83 which disappeared after the synthesis of AgNPs (Figure 2). These observations on the sulfate and hydroxyl groups present in the galactose and CH and S=O groups of fucose are probable functional groups involved in the reduction of AgNPs (Figure 2). In addition, a broad peak at 3431 cm–1 (OH stretching) found in the AgNPs shows that the reduction of AgNO3 decreases the intensity, which implies the involvement of the OH groups in the reduction process. This is in agreement with previous studies on sulfated polysaccharides of brown alga Sargassum muticum indicating a strong capacity to synthesize NPs from Fe3O485 and ZnO.86 Similarly, Venkatpurwar reported that sulfated polysaccharides isolated from the marine red alga Porphyra vietnamensis had a strong capacity to synthesize AgNPs.87 A peak at 1140 cm–1 was observed in all samples and in the standard, and the 1079 cm–1 peak was assigned to the presence of the RO–SO3– bond of the sulfate groups. The additional peaks at 1153 and 1117 and 966, 872, and 775 cm– 1 in Figure 2 are related to AgNPs. It is observed that mostly redox-oxidation sites of reducing sugars of fucoidan are involved in silver ion reduction in the NP synthesis.

3.8.2. FESEM and EDAX Studies

Figure 5b shows the FESEM image that visualizes the size and shape of the AgNPs. A thin organic shell covering each individual particle is found to be responsible for reducing Ag+ ions supporting interparticle binding with NPs. The synthesized AgNPs appear spherical with the size ranging between 10 and 60 nm, which indicates the polydispersity nature. A few aggregations of AgNPs were also observed in some places in the FESEM images, indicating agglomeration after some time.88 The result of EDAX (Figure 5c) gives clear information about the Ag present in the NPs, and a strong signal of the Ag atoms indicates the crystalline property. Energy-dispersive X-ray analysis is the typical technique for the analysis of metallic silver nanocrystallites.89

3.8.3. XRD, AFM, and TEM Analyses

The XRD data (Figure 5d) shows AgNPs having Bragg reflection patterns at 2θ = 32.4 and 46.4 that clearly indicate the presence of 111 and 200 sets of lattice planes, and they can be indexed as the FCC structure of silver. AgNPs synthesized using fucoidan are crystalline in nature.90 Similar to the FESEM observation (Figure 5b), the AFM image (Figure 5e) shows a three-dimensional view of the spherical shape with a 10–60 nm size supporting the polydispersity nature. The tendency of agglomeration indicates an attractive interaction among the NPs. The distribution of particle size ranging from 10 to 60 nm was calculated using Gwyddion 2.9 analysis software. The broad surface area of the NPs and the magnetic forces between them could cause considerable agglomeration between the AgNPs but not aggregation.91 The TEM analysis clearly shows that the largest size of the individual NPs is 50 nm and they are well dispersed. The shape of the NPs is almost spherical to cubical (Figure 5f) as observed.92

3.9. Fucoidan Nanoparticles for Drug Delivery

Major challenges posed in drug delivery in disease treatment are poor reachability at the target site, in vivo instability, poor bioavailability, less solubility, and absorption, but the application of drugs through nanoparticle carriers overcomes these challenges.66,93 Drugs from natural compounds formulated as nanoparticles widely practiced in Indian and Chinese systems of medicine given as dose and delivery to the target site are considered to be quick and effective. Because of their miniature size of 1–100 nm, nanoparticles as effective carriers in nanoformulated drugs can also be capped with another active drug molecule of less reachability for enhancing delivery and activity. Formulating the desired-size nanoparticles as drug and/or carriers is very important in the present context of drug delivery in modern medicine and emphasizes the potential role of seaweed sulfated polysaccharides.28,94 In this study, anionic AgNPs in the range of 10–60 nm encapsulated by anticoagulant fucoidan of T. decurrens showing strong antibacterial activity against Gram-negative clinical pathogens suitable for loading cationic drugs were identified.

4. Conclusions and Perspectives

This study concludes that the residue, after removing the pigments by chloroform/methanol (1:1 v/v) extraction, through successive extraction, crude fucoidan of 5.58% algal DW was extracted by 2% CaCl2 and 0.01 N HCl, and alginate of 14.70% algal DW was extracted subsequently by 3% Na2CO3 from the biomass of hard brown alga thallus Turbinaria decurrens. The purified fucoidan polymer yield of 1.28% algal DW possesses an anticoagulation property and is characterized, consisting of (1–3)-α-l-fucopyranosyl monomers with sulfate groups primarily at the C4 position and to a lesser extent at the C2 position, and in galactose, at the C3 and C6 positions. The AgNPs synthesized using fucoidan exhibit strong anticoagulant activity and possess an antibacterial property against Gram-negative clinical bacteria. Functional groups such as OH, CH, and S=O associated with sugar residues in the sulfated fucoidan are involved in the synthesis of nanoparticles in the range of 10–60 nm with a spherical shape and showing polydispersity, which have a tendency to agglomerate. These anionic AgNPs encapsulated by anticoagulant fucoidan of T. decurrens showing strong antibacterial activity against Gram-negative clinical bacteria are suitable for loading cationic drugs.

Acknowledgments

The authors thank the Principal, Alagappa Government Arts College, Karaikudi, and the Director, Central Electrochemical Research Institute, Karaikudi, for providing necessary infrastructure and assistance in carrying out characterization studies, respectively.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03776.

  • Fucose content of fractions collected through the DEAE-cellulose column eluted by different molar concentrations of NaCl (Figure S1); aqueous white solution containing 97 mL of AgNO3 (1 × 10–1 M) mixed with 3 mL of 1 mg of fucoidan polymer (a) turning brown as a result of fucoidan-AgNP synthesis (b) (Figure S2); and the antibacterial activity of fucoidan-coated AgNPs (Figure S3) (PDF)

Author Contributions

Nagarajan Shanthi: planning, execution, and writing; Arumugam Ponnan: experimental analysis; Murugan Marudhamuthu: experimental analysis; Muthiyal Prabakaran Sudhakar: writing and English language editing, formatting, and analysis of results; and Kulanthaiyesu Arunkumar: planning, writing, language correction, and project execution.

The authors declare no competing financial interest.

Supplementary Material

ao1c03776_si_001.pdf (381.7KB, pdf)

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