Dr. Kalpa W. Samarakoon

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PURIFICATION OF FUCOIDAN WITH ANTI-INFLAMMATORY PROPERTIES USING ENZYME-ASSISTANT EXTRACTION FROM SRI LANKAN MARINE BROWN ALGA: CHNOOSPORA MINIMA

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1 Purification of Fucoidan with Anti-inflammatory Properties Using Enzyme-Assistant Extraction from Sri Lankan Marine Brown Alga: Chnoospora minima Dr. Kalpa W. Samarakoon Industrial Technology Institute (CISIR), Sri Lanka

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Dr. Kalpa W. Samarakoon 1,2 B.Sc, M.Sc (Colombo), Ph.D. (JNU, Korea), M.I.Biol S.I.P. Fernando 2 , K.K.A. Sanjeewa 2 , P. Ranasinghe 1 , G.A.S Premakumara 1 , U.K.D.S.S. Gunasekara 1 and Y.J. Jeon 2 1 Herbal Technology Section, Industrial Technology Institute (CISIR), Sri Lanka 2 Department of Marine Life Science, Jeju National University, Jeju690-756, Republic of Korea . Purification of Fucoidan with Anti-inflammatory Properties Using Enzyme-Assistant Extraction from Sri Lankan Marine Brown Alga: Chnoospora minima

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Nature is an attractive prolific source of a tremendous chemical diversity established with the living organisms on the earth. More than the 70% of the earth surface is covered by oceans and conceived that lives have originated from the sea. Terrestrial organisms Marine organisms 3 Marine Natural Products Infinite Functional ingredients Specific & untapped biological activities Background

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4 Anti-aging Anti-tumor Anti- diabetes Peptide Viamin Mineral Pigment Fiber Polyphenol Anti- hypertenstion Anti-obesity Anti-inflammation

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The new generation of Algae based natural products as functional ingredients for pharmaceutical, nutraceutical and cosmeceutical industry

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6 6 ESR is the resonant absorption of microwave radiation by paramagnetic systems in the presence of an applied magnetic field. 20ul D.W 20ul Sample 20ul AAPH 20ul POBN 20ul Sample 20ul DMPO 20ul Fe 2+ 20ul H 2 O 2 After 30min, ESR After 2.5min, ESR 30ul Sample 30ul DPPH After 2min, ESR DPPH radical Hydroxyl radical Peroxyl radical Antioxidant Activity(%) ={1-(Control-sample)/Control)}x100 Approaches

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Approaches MTT assay ( Cytoprotective effect) Formazan Optical Density mitochondria 3-[4,5-dimethylthiazol-2-yl]-2,5-diphen-Yltetrazo liumbromide (MTT) MTT FTIR Spectroscopic analysis DCFDA assay (Intracellular ROS measurement) Tetrazole

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Effect In vivo experiments (Zebra fish) Laboratory of Marine Bioresource technology Fucoidan Oxidative Stress

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Collection of Marine Algae at different sampling sites Industrial Technology Institute

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Approach Industrial Technology Institute Chnoospora minima Lyophilized Algae Powder 10 % Formaldehyde in ethanol 95% ethanol washing x 3 Enzyme –assisted extraction ( Alcalase / Cellulase ) EtOH precipitation of crude polysaccharides Alginic acid precipitate by(CaCl 2 under acidic condition) Screening bioactivity Purification by DEAE-sepharose column Isolation of Fucoidan rich fractions for characterization of monosaccharide

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Results and Discussion Industrial Technology Institute

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Table 1: Proximate chemical composition of the algae Industrial Technology Institute Sample name  Moisture content (%) Ash content (%) Protein content (%) Lipid content (%) SLGS1   Chaetomorpha antennina 2.11 ± 0.08 g 39.73 ± 0.71 f 13.56 ± 0.09 e 0.59 ± 0.07 c, d SLGS2   Halimeda discoidea 0.82 ± 0.08 d 65.72 ± 0.17 h 17.42 ± 0.47 g 0.83 ± 0.02 d, e SLGS3   Halimeda gracilis 0.41 ± 0.03 b 61.21 ± 1.00 g 20.08 ± 0.33 h 0.54 ± 0.01 b, c, d SLGS4 Caulerpa racemosa var. racemosa f. remota 0.33 ± 0.02 b 60.67 ± 0.72 g 20.45 ± 1.10 h 0.64 ± 0.07 c, d SLRS5   Gracilaria corticata var. ramalinoides 1.53 ± 0.06 e 33.84 ± 0.52 b 16.74 ± 0.06 f, g 2.27 ± 0.01 g SLRS6   Gracilaria foliifera 1.64 ± 0.04 f 39.42 ± 0.38 f 10.54 ± 0.09 c 0.94 ± 0.06 e, f SLRS7   Ahnfeltiopsis pygmaea 0.56 ± 0.04 c 37.88 ± 0.14 d 16.25 ± 0.41 f 0.15± 0.06 a SLRS8   Gracilaria corticata 0.83 ± 0.06 d 34.3 ± 0.53 c 10.26 ± 0.27 c 1.16 ± 0.05 f SLRS9 Jania adhaerens 0.17 ± 0.03 a 73.45 ± 0.70 i 4.19 ± 0.18 a 0.01 ± 0.00 a SLRS10 Gracilaria edulis 2.61 ± 0.02 h 38.17 ± 0.49 e 7.56 ± 0.29 b 0.45 ± 0.07 b, c SLBS11 Chnoospora minima 3.56 ± 0.04 i 14.54 ± 0.02 a 12.30 ± 0.20 d 0.25 ± 0.05 a, b Results represents means ± standard deviation of triplicate determinations. Superscripts in each column indicates values that are significantly different at p < 0.05.

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Algae samples Total soluble carbohydrates (%) a Total soluble Proteins (%) Total polyphenol content (%) Sample No. Polysaccharide content (%) Sulfate content (%)  Chaetomorpha antennina SLGS1P 82.24 ± 1.02 9.21 ± 0.3 0.31 ± 0.28 2.60 ± 0.16  Halimeda discoidea SLGS2P 68.44 ± 0.30 5.20 ± 0.17 0.96 ± 0.21 4.04 ± 0.36  Halimeda gracilis SLGS3P 70.04 ± 0.48 5.20 ± 0.08 0.06 ± 0.07 3.93 ± 0.47 Caulerpa racemosa var. racemosa f. remota SLGS4P 56.15 ± 0.69 10.51 ± 0.37 1.21 ± 0.56 4.38 ± 0.79  Gracilaria corticata var. ramalinoides SLRS5P 74.99 ± 0.53 1.65 ± 0.29 0.41 ± 0.28 4.27 ± 0.11  Gracilaria foliifera SLRS6P 74.22 ± 0.46 4.08 ± 0.33 1.21 ± 0.42 4.60 ± 0.15  Ahnfeltiopsis pygmaea SLRS7P 83.92 ± 0.72 4.55 ± 0.25 0.36 ± 0.35 4.04 ± 0.06  Gracilaria corticata SLRS8P 57.65 ± 0.46 9.84 ± 0.75 2.26 ± 0.35 4.71 ± 0.26 Jania adhaerens SLRS9P 64.37 ± 0.78 2.82 ± 0.54 0.31 ± 0.28 4.16 ± 0.16 Gracilaria edulis SLRS10P 84.18 ± 1.07 9.65 ± 0.16 0.66 ± 0.21 3.93 ± 0.05 Chnoospora minima SLBS11P 70.09 ± 0.21 11.80 ± 0.79 3.16 ± 0.50 4.83 ± 0.16 Table 2. Chemical composition of the crude polysaccharide fraction a Results represents means ± standard deviation of triplicate determinations

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Samples IC 50 values for radical scavenging activity (µg/mL) DPPH Alkyl Hydroxyl Chaetomorpha antennina SLGS1P >2000 278.18 ± 0.75 102.68 ± 16.00 Halimeda discoidea SLGS2P >2000 110.06 ± 2.98 1008.65 ± 8.19 Halimeda gracilis SLGS3P >2000 116.60 ± 2.59 1006.90 ± 6.40 Caulerpa racemosa SLGS4P >2000 359.48 ± 20.54 200.08 ± 8.17 Gracilaria corticata SLRS5P >2000 367.43 ± 1.74 654.13 ± 9.14 Gracilaria foliifera SLRS6P 1654 ± 37.46 382.55 ± 1.23 582.465 ± 9.29 Ahnfeltiopsis pygmaea SLRS7P >2000 377.24 ± 6.10 768.92 ± 8.10 Gracilaria corticata SLRS8P 603.38 ± 40.3 332.33 ± 15.29 287.63 ± 13.68 Jania adhaerens SLRS9P >2000 114.59 ± 5.01 281.70 ± 4.96 Gracilaria edulis SLRS10P >2000 113.09 ± 7.13 602.95 ± 12.26 Chnoospora minima SLBS11P 89.51 ± 17.00 106.80 ± 0.66 193.57 ± 3.38 Table 3. IC 50 values for the radical scavenging activities of crude polysaccharide fractions. a The values of IC 50 were determined by at triplicate individual experiments. Values are mean ± SD of three determinations.

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FTIR spectra of commercial (a) Fucoidan, (b) Alginic acid, (c) ι-Carrageenan, (d) agar and (e) Laminarin. FTIR spectra of (a) commercial fucoidan, (b) crude polysaccharides from Chnoospora minima (SLGS1P), (c) crude polysaccharides from Halimeda discoidea (SLGS5P) and (d) crude polysaccharides from Halimeda gracilis (SLGS6P).

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FTIR spectra of (a) commercial agar, (b) Gracilaria corticata var. ramalinoides (SLRS2P), (c) Gracilaria foliifera (SLRS3P) and (d) Gracilaria Edulis (SLRS11P). Sample No. Sample name Major polysaccharide identified from CP fractions SLGS1 C. antennina Fucoidan SLGS2 H. discoidea Fucoidan SLGS3 H. gracilis Fucoidan SLGS4 C. racemosa var. racemosa f. remota Fucoidan SLRS5 G. corticata var. ramalinoides Agar SLRS6 G. foliifera Agar SLRS7 A. pygmaea Carrageenan SLRS8 G. corticata Agar SLRS9 J. adhaerens Alginic acid SLRS10 G. edulis Agar SLBS11 C. minima Fucoidan

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Chemical structure of fucoidans Chemical structure of kappa-carrageenan Chemical structure Agarose (Agar)

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Fraction Polysaccharide content (%) Sulfate content (%) Protein content (%) Polyphenol content (%) F1 74.09 ± 0.21 7.37 ± 0.64 2.35 ± 0.17 4.71 ± 0.22 F2 68.44 ± 0.30 28.30 ± 0.47 0.96 ± 0.21 1.02 ± 0.40 F2,1 83.04 ± 0.48 5.20 ± 0.08 0.06 ± 0.07 0.51 ± 0.13 F2,2 70.32 ± 0.74 20.48 ± 0.72 0.21 ± 0.06 0.42 ± 0.21 F2,3 65.92 ± 0.41 32.70 ± 0.28 0.26 ± 0.04 0.44 ± 0.00 F2,4 63.52 ± 0.26 34.08 ± 0.46 0.21 ± 0.10 0.40 ± 0.15 Table 2. Chemical compositions of the polysaccharide fractions Separation and purification of the CMP by anion-exchange chromatography (DEAE-Sepharose column )

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FTIR characterization and monosaccharide analysis HPAE-PAD spectroscopy

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F2,1 F2,2 F2,3 F2,4 Fucose 19.33 71.68 77.28 97.32 Rhamnose 0.89 N.D. N.D. N.D. Arabinose 3.29 1.06 N.D. N.D. Galactose 38.08 13.97 5.81 2.68 Glucose 6.95 2.44 4.13 N.D. Xylose N.D. N.D. N.D. N.D. Others 31.46 10.85 12.78 0.00 Table 3. Monosaccharide composition of the TFA hydrolyzed polysaccharides in each F2 subtractions. Monosaccharide compositions in CMP _F2 fraction

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In vitro Anti-inflammatory activity (LPS induced NO production in Raw cells)

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Effects of CMF upon the mediation of inflammatory regulators

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In vivo evaluation of anti-inflammatory effects of CMF in LPS stimulated zebrafish embryos

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Conclusion Current findings reveal an inexpensive, efficient green extraction method to obtain fucoidan with a higher purity from the under-explored brown algae C. minima harvested from Sri Lanka. A nti-inflammatory activity of the purified fraction of fucoidan (CMF) was potent in vitro and in vivo models. Industrial Technology Institute Gracilaria edulis CMF could be efficiently utilized in the manufacturing of functional ingredients for the food, pharmaceutical and cosmetic industry

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Acknowledgements Prof. You Jin Jeon Mr. I.P.S Fernando Mr. Chaminda Lakmal HH Korean Institute of Ocean Science and Technology (KIOST) has funded for this Collaborative Research Project ( Grant No. 2016-0804) Dr. G.A.S. Premakumara Dr. Pathmasiri Ranasinghe Mr. U.K.D.S.S. Gunasekara Dr. H.M.P. Kithsiri Dr. Niroshan Wickramarachchi Mr. M.S.M Fahim

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Industrial Technology Institute Thank you

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