For the past three decades, seaweeds contributed to many pharmaceutical applications on gall stones, renal disorders, cancer, heart disease, asthma, psoriasis, antibacterial, antifungal, antiviral, and arthritis with a wide range of metabolites like polysaccharides, phlorotannins, glycoproteins, terpenoids, alkaloids, lectins, pigments, and ketones 43. Moreover, Fucoidan has a potent antiviral activity on SARS-CoV-2 (COVID-19) viral infection 44–47. Simultaneously, laminarin also has potential antiviral activity on HIV 13, reported to possess prebiotic activity on microbial gut health 48,49, and antitumor activity 50.
Padina pavonica, Stoechospermum marginatum, Spatoglossum asperum, and Sargassum wightii are the most frequently occurring brown seaweeds in the Gulf of Mannar, Rameswaram, Tamil Nadu, India. In this present study, the four different seaweeds were collected from the Mandapam Coastal region, Rameswaram, Tamil Nadu, India followed by the extraction, purification, and evaluation of yield obtained for both Fucoidan and Laminarin. Both the polysaccharides were extracted using high polar solvent water and aqueous extraction by heat (Hot-water extraction method) was found cost-effective and the potential extraction method optimized. When treating crude extract with calcium ions (CaCl2), alginate gets precipitated and separated by centrifugation 51. Hence, 2% CaCl2 was used in this study, alginate was separated and, finally, a crude extract containing fucoidan and laminarin was precipitated by adding equal amount of ethanol.
In present study, the obtained total laminarin and fucoidan yield was higher in Padina pavonica (4.36%) and Stoechospermum marginatum (2.32%) respectively, whereas, the fucoidan yield was 9.46% in P. tetrastromatica, 5.83% in Turbinaria ornata, 3.90% in Sargassum wightii 52; 16% in Laminaria japonica. However, in another study, laminarin yield was 32% in diatoms Skeletonema costatum, 14% in Phaeodactylum tricornutum; 0.4% in Stephanodiscus meyerii and 15% in Odontella aurita 53; 3 to 6% in seaweed Laminaria hyperborea and 4 to 5% in Ascophyllum nodosum 17. The total laminarin content as storage carbohydrates in macroalgae was 1 to 25% as dry weight 54.
The estimated carbohydrate content in Laminarin was found higher in Padina pavonica with 52.15 ± 0.26 mg/60mg (86.91%), whereas for fucoidan it was 52.42 ± 1.26 mg/60mg (87.36%) in Stoechospermum marginatum. Similarly, sulphate content of fucoidan from Stoechospermum marginatum was 20.68%. Ohlsson et al. 55 reported that the seaweed biomass is rich in 90% of carbohydrate, in which 60% of were laminarin from Laminaria digitata. The total carbohydrate yield was 10–11% from the seaweed biomass 52. In another study the total fucoidan content obtained from Turbinaria decurrens and Dictyota bartayresiana were 3.89% and 8.67% respectively 23. Whereas, the range of suphate groups in fucoidan was between 5 to 40% in Sargassum swartzii 56. However, fucoidan consists of 55% of carbohydrate and 40% of sulphate in its constituent 57. Intriguingly, the carbohydrate content was greater during Summer 54. Becker et al. 58 resulted about 53% of carbohydrate from the biomass and among them, 42% were laminarin in diatom.
The monosaccharide composition of laminarin constitutes of 46.93% of fucose, 12.12% of galactose, 8.61% of arabinose, 4.23% of glucose, and 3.27% of mannose 12. However, glucose alone found reported in the laminarin 59. Concurrently, in the present investigation, laminarin derived from Padina pavonica constitutes of 79.4% of glucose, 13.2% of mannose. 1.4% of ribose, 2.5% of arabinose, and 0.7% of xylose and 0.5% of galactose. Xia et al. reported that the laminarin isolated from a marine diatom Oduntella aurita consists of about 82.23% of glucose units 60. In the case fucoidan, fucose constitutes 64.2%, galactose 14.5%, xylose 12.9%, mannose 4.3%, and rhamnose 3.2% in Stoechosermum marginatum (present study), whereas, fucoidan from Fucus vesiculosus constitutes of 59.2% of fucose, 12.6% of xylose, 2.7% of mannose, 10.4% of galactose, 1.4% of rhamnose, and 13.6% of glucose 61. The monosaccharide units of fucoidan from Turbinaria decurrens were fucose (59.3%), galactose (12.6%), mannose (9.6%), and rhamnose (6.4%) 62. Similarly, fucoidan from Fucus serratus reported 40.6% of fucose, 43.2% of glucose, 10.4% of xylose, 4.2% of galactose, and 2.7% of mannose 61. About 96.1% of fucose, and 3.9% of galactose were reported from Fucus evascens, and 38.7% of fucose, 32% of xylose, 16.2% of galactose, 5.6% of mannose, 2.3% of rhamnose, and 5% of glucose from Dictyosiphon foeniculaceus 61. Simultaneously, fucoidan purified from Laminaria digitata constitutes 67.1% of fucose, 13.7% of xylose, 14% of galactose, and 5.2% of mannose. However, fucoidan from Saccharima latissima reported with 83.8% of fucose, 7.5% of galactose, 6.7% of xylose, and 2.1% of rhamnose 61. Therefore, it has been determined that the glucose, and fucose are the major backbone monosaccharide unit of laminarin and fucoidan respectively.
The FT-IR analysis resulted that the functional groups represent the carbohydrate nature of laminarin (non-sulphated polysaccharide), and carbohydrate and sulphate nature of fucoidan (Sulphated polysaccharide) from Padina pavonica, and Stoechospermum marginatum respectivley. The presence of sulphate groups in Fucoidan was clearly seen at 1219 cm-1, and 843 cm-1 respectivley. Congruently, 843 cm-1, 842 cm-1, 842 cm-1, and 820 cm-1 representing sulphate groups of fucoidan were reported from the respective marine seaweeds, which are Padina tetrastromatica, Sargassum oligocystum, Laminaria Cichorioides, Fucus evanescens, and Laminaria japonica 52,63. In another study, 1200 cm-1, and 1201 cm-1 represents sulphonate groups of Fucoidan 23 and also in our study the same results were seen at 1219 cm-1. This similar kind of FT-IR peaks were not seen in the Laminarin of Padina pavonica and hence it is devoid of sulphate group. In laminarin, 3400 cm-1 represents the hydroxyl group (-OH) 64, whereas, hydroxyl groups were seen at 3540 cm-1, and 3385 cm-1 in the laminarin from Padina pavonica. Similarly, pure laminarin exhibited anisomeric stretching at 1620 cm-1 65, in our present study the same was obtained at 1613 cm-1 in the laminarin. Carboxylic group of laminarin was obtained at 1398 cm-1 in laminarin of Padina pavonica, whereas it was reported in the laminarin at 1420 cm-1 from Laminaria japonica 66. The presence of CC stretch (glycosidinc bonds) was determined at 1100 cm-1 in laminarin, and also the obtained result in this study as laminarin of Padina pavonica at 1118 cm-1 67. However, the fucoidan from Stoechospermum marginatum was hydrolyzed to retrieve Fucose and determined by RP-HPLC with a Standard Fucose (Sigma-Aldrich). Hence, it is confirmed that the extracted sulphated polysaccharide from Stoechospermum marginatum was Fucoidan.
The obtained mass of laminarin from Padina pavonica was 3000 to 5000 daltons, whereas, for fucoidan from Stoechospermum marginatum, it was between 2000 and 6000 daltons. About 2 to 7 kDa molecular mass of Laminarin was determined from Sargassum cichorioides, and Laminaria gurjanovae respectively 68. Graiff et al. 69 also determined the molecular mass of laminarin ranges between 2 to 7 kDa from Lamanaria digitata. The average molecular mass of laminarin was approximately 5 kDa 70–72. High molecular weight fucoidan was reported from Saragassum fusiforme at an average weight of 95 kDa 73. Similarly, high molecular weight fucoidan was obtained from Sargassum siliquosum with 107.3 kDa. However, in this present study, the obtained fucoidan was low molecular weight with 2 to 6 kDa from Stoechospermum marginatum.
The laminarin conjugated gold nanoparticles does not inhibit the Vero cells at higher concentration of 100 µg/mL 74. In another study, laminarin showed no cytotoxic effect on HDFa cell line (Normal primary dermal fibroblast) at 100 µg/mL concentration, and NHEK cell line (Primary normal human epidermal keratinocytes) at 500 µg/mL concentration 75. Simultaneously, laminarin (100 µg/mL) treated with human adipose stem cells and L929 cell line (Mouse fibroblast) exhibits no cytotoxic activity 76. However, about 2 mg/mL concentration of laminarin for 24 hours of treatment, selectively inhibited ovarian cancer (ES-2 and OV-90) and showed no cytotoxic effect on Zebrafish embryo xenograft model 9. Moreover, the depolymerized low molecular weight laminarin enhanced the proliferation rate of normal fibroblasts, and eventually suppressed the melanomas by inducing TNF-α in a wound dressing study 77. Relatively to the above studies, laminarin derived from Padina pavonica also showed no cytotoxic effects on Vero cells with an IC50 value of 123.54 ± 0.8 µg/mL.
Fucoidan from Cladosiphon okamurans had no cytotoxic effect on Vero cells upto 2 mg/mL concentration, but inhibits Canine distemper virus (CDV) replication in Vero cells 78. Concurrently, the same exhibit no cytotoxicity on Vero cells but inhibits the replication of Newcastle Disease Virus (NDV) 79. In an intriguing study, fucoidan from Turbinaria conoides potentially inhibits cancer cells (A549 – Human lung adenocarcinoma) by 25 to 75% in a dose dependent manner but doesn’t showed cytotoxicity on Vero cells 80 and human skin keratinocytes (HaCaT cell line) 81. This was again supported by another study, in which, fucoidan from Fucus evanescens possess no cytotoxic effect on Vero cells up to 2 mg/mL concentration 82. Chantree et al. evidently reported that the fucoidan isolated from Fucus vesiculosus arrests cell cycle on Cholangiocarcinoma cells (CL-6) via apoptosis, whereas, the same fucoidan doesn’t exhibits any cytotoxic activity on OUMS cells (human embryonic fibroblast cell line) 83. The present study also supports other studies by showing no cytotoxic effects on Vero cells.
Laminarin isolated from laminaria digitata enhances apoptosis in HT-29 colon cancer cells by inducing the sub-G1 and G2/M Phases 8. Whereas, laminarin induces apoptosis through Fas and IGF-IR signaling pathways and ErbB (intrinsic apoptotic) pathways 84. The four different seaweed extracts of Sargassum latifolium had shown IC50 values of 62.59 µg/mL, 110.20 µg/mL, 162.79 µg/mL and 51.15 µg/mL on HCT-116 Human colon cancer cells with promising anti-hypoxic activity 85. However, laminarin from Sargassum thunbergii inhibited human lung adenocarcinoma (A549 cell line) with IC50 values of 2.70 mg/mL and 2.85 mg/mL for 12 and 24 hours respectively 86. Intriguingly, different ranges of laminarin concentrations for 48 hours significantly suppressed the cell viability and induces apoptosis in a dose-dependent manner on liver cancer cells (Bel-7404 and HepG2) 87. In aggreement with Tian et al. 87, our study resulted that the laminarin supplementation inhibits the cell viability of HT-29 Human colon cancer cells and which is proportional to the concentration of laminarin with an IC50 value of 57 ± 1.2 µg/mL.
The fucoidan extract of Sargassum horneri 22, Chnoospora minima 88, Ecklonia cava 89, Laminaria japonica, Fucus vesiculosus, and Undaria pinnatifida 90, were found to suppress the proinflammatory cytokines such as IL-6, IL-1β, and TNF-α in vitro. Oral administration of fucoidan isolated from Laminaria japonica inhibits proinflammatory cytokines and enhanced antiinflammatory cytokines respectively 91 in vivo in cancer patients. Clinical trails on fucoidan from Fucus vesiculosus resulted that it downregulates the proinflammatory cytokines 92. High molecular weight fucoidan from Fucus vesiculosus found to induce apoptosis by the activation of caspases in MCF-7 (human breast cancer) and HeLa (cervical cancer) in vitro 93, and thus, fucoidan is a potential compound to treat melanoma including the health improvement of cancer patients in the stages of metastases 94. The fucoidan extracted from Sargassum cinereum has an IC50 value of 250 µg/mL on Caco-2 human colorectal adenocarcinoma cells in vitro 37. The IC50 values of Fucoidan isolated from the seaweed Fucus vesiculosus on DLBL (Diffuse Large B-cell Lymphoma) cell lines were 101.6 µg/mL on U2932 cell lines, 97.5 µg/mL on TMD8 cell lines, 93.7 µg/mL on NU-DUL-1 cell lines, 95.5 µg/mL on DB cell lines, 82.3 µg/mL on OCI-LY8 cell lines, and 80.0 µg/mL on SUDHL-4 cell lines 95. Whereas, in this present study, the fucoidan extracted and purified from Stoechospermum marginatum has potential anticancer activity with an IC50 value of 24.4 ± 1.5 µg/mL on human liver cancer cells (Hep G2) in vitro. Eventually, it has been resulted that, both the laminarin and fucoidan derived from PP and STM have no cytotoxic activity on Vero cells, but inhibited the proliferation cancer cells HT-29 (human colon cancer) and Hep G2 (human liver cancer).
Native laminarin induces apoptosis in LoVo (human metastatic colon carcinoma), whereas, enzyme-hydrolyzed laminarin also demonstrated potential anticancer activity over SK-MEL-28 (human melanoma), and DLD-1 (colon cancer) cell lines 96. However, laminarin sulphate hampers heparanase activity in mouse melanoma cells (B16-BL6) and inhibits the metastasis of mammary adenocarcinoma 97. Simultaneously, laminarin from Dictyota dichotoma inhibits matrix metalloproteinases MMP-2, MMP-9, and p-ERK1/2 signalling cascade and resulted in the suppression of progression and migration of human melanoma 98. Increased concentration of cytoplasmic and mitochondrial calcium levels and induced apoptosis was seen in the ovarian cancer cells treated with the laminarin isolated from Laminaria digitata 9. Similar kind of result was also reported in human colon cancer cells 18.
Regular oral administration of fucoidan isolated from Fucus evanescens significantly enhances the antitumor, and anti-metastasis activity in mice model induced with Lewis lung adenocarcinoma 99. Yang et al. recorded that the hydrolysed fucoidan had greater anticancer activity (> 75%) than the native fucoidan (37%) derived from Undaria pinnatifida 100. Comparatively, oversulfation of high molecular weight fucoidan > 30 kDa from Undaria pinnatifida had shown enhanced inhibition of cancer proliferation from 35 to 56%, whereas, oversulfation of low molecular weight fucoidan 5–30 kDa was found to inhibit from 37 to 68% 101. The molecular sulfation of fucoidan plays a vital role in the function of fucoidan, hence, a decrease in the degree of sulfation in fucoidan (< 20%) drastically minimized the anticoagulant and anticancer efficacy of fucoidan 102. Whereas, a high degree of sulfation induces anti-angiogenic activity and suppresses the growth of tumor cells 101,103. Moreover, the sulphate groups in fucoidan have higher affinity and binding with the cationic proteins in the cancer cells and inhibit cancer proliferation 103,104. Therefore, sulphate moiety is the most important factor in the function of fucoidan. In accordance with the above, the IC50 value of fucoidan on Hep G2 was found better than the same of laminarin with HT-29 due to the presence of sulphate group in fucoidan, which is advantageous for fucoidan (from STM) over laminarin (from PP) on the anticancer potential.
Fucoidan from seaweeds showed anticancer activity by enhancing the chromatin condensation, Bax, cleaved Cas-9 and poly-ADP ribose polymerase, and suppressed Bcl-2, p-PI3K, p-P38, p-Akt, and p-ERK genes in a dose-dependent manner in vitro in DU-145 prostate cancer cells 105. Similarly, fucoidan hampers PI3K, ERK, and MAPK pathway and thereby suppressing the cancer progression and also inhibits the expression of Bcl-2, Bax, and enhanced caspase-dependent apoptosis in LM3 (Murine mammary adenocarcinoma) and BEL-7402 (hepatocellular carcinoma) cell lines 106. The natural killer (NK) cells followed by IL-2 and IFN-γ were induced in mice model fed with fucoidan orally in tumour induced mice than the control mice 107–110. About 1 mg/mL concentration of fucoidan from Cladosiphon okamuranus to Huh7 liver cancer cells had increased the G0/G1 phase population, and decreased S-phase population and resulted that the fucoidan arrests cell cycle at G0/G1 phase 111.
AO/EB assay is a simple and cost-effective assay to detect apoptosis 112. When live cells are stained with acridine orange emits green fluorescence, whereas, dead cells emit orange-red by ethidium bromide due to the loss of cytoplasmic membrane of dead cells and interaction between ethidium bromide and the intercalating DNA 113. Therefore, this method is effective in identifying even mild nucleic acid damages 114. However, bright-green fluorescence illustrates early apoptotic phase when fragmented chromatin structures stained. Orange-red fluorescence resulted in the late apoptotic phase, when ethidium bromide stained with nuclei 115. Concurrently, in the present investigation, both the cancer cell lines HT-29 (colon), and Hep G2 (liver) had undergone apoptotic and necrotic cell death phase and were evidently confirmed by AO/EB staining assay under fluorescent microscopy 116,117. The fragmented DNA was visually seen as orange and red fluorescence in both the laminarin and fucoidan treated cancer cells.
Unlike programmed cell death (apoptosis), necroptosis is a passive cell death by the uncontrolled expression of inflammatory factors. Thus, necrosis deals with the immunological concept of cell cycle arrest and death 118. Moreover, cancer cells progressively develop resistant to some cancer drugs by deletion in specific genes, genetic aberration, and overexpression of anti-apoptotic proteins which is a major defect in the apoptotic death machinery 119–121. Therefore, programmed necrotic cell death is an alternate approach in anticancer treatment by inducing the immune system. However, in the current investigation, based the DNA fragmentation assay, it was evidently clear that the laminarin and fucoidan from PP and STM showed anticancer activity by forming DNA smear on both colon (HT-29) and liver (Hep G2) cancer cell lines respectively by inducing the necrotic factors based on Zhivotosky and Orrenius 122. Based on the present investigation, laminarin and fucoidan are the potential bioactive compounds for the treatment of cancer.