6narsico-fucoidan.pmd J.T. Narsico et al. 45 SCIENCE DILIMAN (JANUARY-JUNE 2018) 30:1, 45-59 Fucoidan Content in Phil ippine Brown Seaweeds Joemark T. Narsico Hokkaido University Joyce A. Nieva Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research Alper James G. Alcaraz University of Saskatchewan, Canada Elad io G.M. Anino V University of the Philippines Manila Norchel Corcia F. Gomez Marco Nemesio E. Montaño* Marine Science Institute University of the Philippines Diliman ABSTRACT This study aims to determine which brown macroalgae in the Philippines has the highest content of par tially purif ied fucoidan. Percent fucoidan content of brown seaweeds Sargassum spp. , Pad ina sp. , Hyd roclathrus sp. , Turbinaria ornata J. Agardh, Hormophyza cuneiformis PC Silva, and Dictyota d ichotoma Lamouroux were determined in f ifty sites across 14 provinces in Northern Luzon (Cagayan, Ilocos), West Luzon (Pangasinan), the eastern seaboard of Luzon (Quezon Province, Camarines, Sorsogon), Central and Eastern Visayas (Bohol, Cebu, Negros Oriental, Negros Occidental), and N o r t h e r n M i n d a n a o ( C a m i g u i n , L a n a o d e l N o r t e , M i s a m i s O r i e n t a l , M i s a m i s O c c i d e n t a l ) . C r u d e a n d s e m i - p u r e f u c o i d a n w e r e e x t r a c t e d t h r o u g h a c i d h y d r o l y s i s a n d e t h a n o l p r e c i p i t a t i o n u s i n g 5 0 g r a m s o f dried and milled seaweed biomass. Extracts were verif ied using infrared spectroscopy with fucoidan from Fucus vesiculosus as standard. Sargassum spp. is the most widely distributed source of fucoidan found in all sites. T. ornata was found in only 11 sites. Both have signif icantly higher percent _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online Fucoidan content in Philippine brown seaweeds 46 c o n t e n t ( p ≥ 0 . 0 5 ) o f f u c o i d a n t h a n o t h e r s a m p l e d s e a w e e d s . H i g h e r percent content of semi-purif ied fucoidan were observed in D. d ichotoma from Bohol (1.53%), H. cuneiformis from Cebu (2.17%), Hydroclathrus sp. f r o m P a n g a s i n a n ( 2 . 2 3 % ) , P a d i n a s p . f r o m Q u e z o n P r o v i n c e ( 3 . 6 9 % ) , Sargassum spp. from Camiguin (4.30%), and T. ornata from Cagayan (7.03%). Keywords: Brown seaweeds, distribution, fucoidan, fucoidan yield INTRODUCTION The Philippines is known for its diverse marine flora, particularly seaweeds. The country contains 966 taxa of macroalgae, with 893 species in 82 families. Brown macroalgae (Order Ochrophyta, Phaeophyceae) are represented in 171 taxa with 153 species in 10 families (Ang et al. 2013). Brown algae contain fucoxanthin, a xanthophyll pigment responsible for its distinguishing brown color. Their cell walls are composed of cellulose, alginic acid, and other algal polysaccharides (Trono 1 9 9 7 ) . Fucoidan is a sulfated polysaccharide commonly found in brown seaweeds, as well as in marine invertebrates (Mak et al. 2013). Fucoidan is absent in green algae (Chlorophyceae) and red algae (Rhodophyceae) (Berteau and Molloy 2003). Fucoidans are structurally diverse macromolecules with a backbone of a (1,3)- and (1,4)- linked α(1,4)-bonded α-L-fucopyranose residues. The polysaccharide may be arranged in stretches of alternating α(1,3)- and α(1,4)-bonded L-fucopyranose residues or through (13)-α-fucan (Ale et al. 2011). Fucoidans have been extensively studied due to their biological activity which includes anti-inflammatory, antioxidant, anticoagulant, antitumor, antiangiogenic, antithrombotic, antiviral, and immunomodulatory properties. It is widely studied because it comes from different inexpensive sources, and has a potential for drug development or as a functional food resource (Li et al. 2008). Despite the diverse potential applications of fucoidans, information on the fucoidan content of seaweeds in the Philippines remain limited. This information is necessary to guide manufacturers and researchers on which species and sites should be utilized for the maximization of the extraction of the algal polysaccharide. In this study, we determined the fucoidan content of brown seaweeds collected from different J.T. Narsico et al. 47 Figure 1. Percent fucoidan content (semi-purif ied) of Dictyota collected in Pangasinan, Sorsogon, Negros Occidental and Bohol. provinces all over the Philippines through acid hydrolysis extraction and fractional ethanol precipitation. We further compared which among the species has the highest content of fucoidan. MATERIALS AND METHODS Sample collection Gratuitous permits were issued by the different regional off ices of the Bureau of Fisheries and Aquatic Resources (BFAR). Fresh seaweed thalli were collected from the intertidal zones across f ifty sites in fourteen provinces in the Philippines (Figure 1, Table 1): Northern Luzon (Cagayan, Ilocos); West Luzon (Pangasinan); the eastern seaboard of Luzon island (Quezon, Camarines, Sorsogon); Central and Eastern Visayas Islands (Bohol, Cebu, Negros Oriental, Negros Occidental); and Northern Mindanao Island (Camiguin, Lanao del Norte, Misamis Oriental, Misamis Occidental). One-time sampling was conducted in all the sites. Not all species were found in Fucoidan content in Philippine brown seaweeds 48 Sargassum Patar, Bolinao, Pangasinan 17-19 May 2010 16°19’59.11"N 119°47’13.95"E Patar, Bolinao, Pangasinan 17-19 May 2010 16°19’59.11"N 119°47’13.95"E Trensiera, Bolinao, Pangasinan 17-19 May 2010 16°26' 24.84"N 119°56' 45.87"E Lucero, Bolinao, Pangasinan 17-19 May 2010 16°24’10.50"N 119°54’22.91"E Villa Manzano Norte, Alabat, Quezon 25-30 Nov 2011 14°1’43.17"N 122°5’17.56"E Sabang, Alabat, Quezon 25-30 Nov 2011 14°3’28.52"N 122°9’33.78"E Gonzaga, Cagayan 25-29 July 2010 18°17’16.30"N 121°59’24.89"E Sta. Ana, Cagayan 25-29 July 2010 18°28’49.06"N 122°8’26.09"E Sta. Ana, Cagayan 25-29 July 2010 18°28’49.06"N 122°8’26.09"E Blue Lagoon, Ilocos Norte 25-29 July 2010 18°37’22.49"N 120°51’34.16"E Burgos, Ilocos Norte 25-29 July 2010 16°2’44.93"N 119°45’13.97"E Burgos, Ilocos Norte 25-29 July 2010 16°2’44.93"N 119°45’13.97"E Burgos, Ilocos Norte 25-29 July 2010 16°2’44.93"N 119°45’13.97"E Lioes, Ilocos Norte 25-29 July 2010 18°0' 59.68"N 120°29' 11.37"E Pangil, Ilocos Norte 25-29 July 2010 18°0’24.19"N 120°29’20.19"E Bgy Nailon, Bogo City, Cebu 10-16 Aug 2010 11°2’52.02"N 124°2’28.96"E Bgy Nailon, Bogo City, Cebu 10-16 Aug 2010 11°2’52.02"N 124°2’28.96"E Alcoy, Cebu 10-16 Aug 2010 9°40’25.97"N 123°30’18.65"E Alcoy, Cebu 10-16 Aug 2010 9°40’25.97"N 123°30’18.65"E Maribago, Mactan, Cebu 10-16 Aug 2010 10°17’8.17"N 124°0’26.30"E Catmon, Cebu 10-16 Aug 2010 10°43’20.72"N 124°1’4.35"E Dalaguete, Cebu 10-16 Aug 2010 9°45’54.64"N 123°32’10.47"E Dalaguete, Cebu 10-16 Aug 2010 9°45’54.64"N 123°32’10.47"E Bgy. Paypay, Daanbantayan, Cebu 10-16 Aug 2010 11°13’20.67"N 123°58’56.78"E Ubojan East, Garcia-Hernandez, Bohol 10-16 Aug 2010 9°36’32.40"N 124°18’7.14"E Larapan, Jagna, Bohol 10-16 Aug 2010 9°38’55.90"N 124°22’7.31"E Pamilacan Island, Baclayon, Bohol 10-16 Aug 2010 9°29’25.84"N 123°54’59.53"E Pamilacan Island, Baclayon, Bohol 10-16 Aug 2010 9°29’25.84"N 123°54’59.53"E Sitio Basdio, Loon, Bohol 10-16 Aug 2010 9°48' 2.22"N 123°47' 20.61"E Punta-Cruz, Maribojoc, Bohol 10-16 Aug 2010 9°43’56.69"N 123°47’54.99"E Sitio Daorong, Bgy. Danao, Panglao, Bohol 10-16 Aug 2010 9°32’41.70"N 123°46’1.16"E Bgy. Lawis, Panggangan Island, Calape, Bohol 10-16 Aug 2010 9°54’13.80"N 123°50’27.84"E Bgy. Lawis, Panggangan Island, Calape, Bohol 10-16 Aug 2010 9°53' 40.86" N 123°50' 29.37"E Pungtod Island, Panglao, Bohol 10-16 Aug 2010 9°40’41.37"N 123°51’0.69"E Sitio Hoyohoy, Bgy. Tawala, Panglao, Bohol 10-16 Aug 2010 9°33’23.51"N 123°48’32.91"E Sto. Domingo, (Bicol) 8-13 Nov 2010 13°23’42.40"N 123°11’29.20"E Sto. Domingo, (Bicol) 8-13 Nov 2010 13°23’42.40"N 123°11’29.20"E Pasacao, (Bicol) 8-13 Nov 2010 13°30’30.59"N 123°0’27.70"E Bulusan, Sorsogon 8-13 Nov 2010 12°44’52.69"N 124°8’33.59"E Bulusan, Sorsogon 8-13 Nov 2010 12°44’52.69"N 124°8’33.59"E Sangay, Sorsogon 8-13 Nov 2010 13°36’22.02"N 123°32’51.26"E Matnog, Sorsogon 8-13 Nov 2010 12°35’59.45"N 124°6’24.24"E Pinagtigasan, Sorsogon 8-13 Nov 2010 14°9’56.41"N 122°58’34.42"E Poblacion, Oroqueta City, Misamis Occidental 18-24 Oct 2010 8°28’55.05"N 123°49’37.89"E Poblacion, Oroqueta City, Misamis Occidental 18-24 Oct 2010 8°28’55.05"N 123°49’37.89"E Talisayan, Poblacion, Misamis Oriental 18-24 Oct 2010 9°0’53.98"N 124°52’15.25"E Cantaan, Guinsilaban, Camiguin 18-24 Oct 2010 9°5’36.28"N 124°47’29.84"E Tagcatong, Carmen, Misamis Oriental 18-24 Oct 2010 9°5’36.28"N 124°47’29.84"E Tagcatong, Carmen, Misamis Oriental 18-24 Oct 2010 8°31’8.56"N 124°37’48.51"E Gingoog, Misamis Oriental 18-24 Oct 2010 8°49’57.16"N 125°5’49.66"E Kauswagan, Lanao del Norte 18-24 Oct 2010 8°12’3.60"N 124°4’57.57"E Liangan, Maigo, Misamis Oriental 18-24 Oct 2010 8°9’36.72"N 123°56’20.24"E Bgy. Roque, Mantigue Island, Camiguin 18-24 Oct 2010 9°10’14.48"N 124°49’26.41"E San Jose, Negros Occidental 17-20 Sept 2010 9°25’10.65"N 123°14’36.85"E Sipalay City, Negros Occidental 17-20 Sept 2010 9°44’47.75"N 122°23’50.14"E Dumaguete, Negros Oriental 17-20 Sept 2010 9°18’17.53"N 123°18’40.40"E Genera Site Date Latitude Longitude Sample Collection Table 1. Sampl ing sites and GPS coord inates J.T. Narsico et al. 49 Genera Site Date Latitude Longitude Sample Collection Table 1. Sampl ing sites and GPS coord inates (Cont.) Hinobaan 2, (Negros) 17-20 Sept 2010 9°32’23.05"N 122°30’56.19"E San Juan, (Negros) 17-20 Sept 2010 10°36’32.86"N 122°54’36.25"E Bais City, (Negros) 17-20 Sept 2010 9°34’44.33"N 123°10’19.55"E Hinobaan 1, (Negros) 17-20 Sept 2010 9°35’57.65"N 122°27’50.81"E Sta. Catalina, (Negros) 17-20 Sept 2010 9°19’40.42"N 122°51’54.28"E Lazi, (Negros) 17-20 Sept 2010 9°19’21.00"N 123°19’33.01"E Maria 2, (Negros) 17-20 Sept 2010 10°43’52.81"N 122°56’2.08"E Turbinaria Trensiera, Bolinao, Pangasinan 17-19 May 2010 16°26' 24.84"N 119°56' 45.87"E Villa Manzano Norte, Alabat, Quezon 25-30 Nov 2011 14°1’43.17"N 122°5’17.56"E Gonzaga, Cagayan 25-29 July 2010 18°17’16.30"N 121°59’24.89"E Blue Lagoon, Ilocos Norte 25-29 July 2010 18°37’22.49"N 120°51’34.16"E Maribago, Mactan, Cebu 10-16 Aug 2010 10°17’8.17"N 124°0’26.30"E Punta-Cruz, Maribojoc, Bohol 10-16 Aug 2010 9°44' 4.20" N 123°47' 26.04" E Sitio Hoyohoy, Bgy. Tawala, Panglao, Bohol 10-16 Aug 2010 9°32' 57.91"N 123°46' 52.84"E Sangay, Sorsogon 8-13 Nov 2010 13°36’22.02"N 123°32’51.26"E Talisayan, Poblacion, Misamis Oriental 18-24 Oct 2010 9°0’53.98"N 124°52’15.25"E Bgy. Balite, Sagay, Camiguin 18-24 Oct 2010 9°6’14.35"N 124°42’48.10"E Bgy. Roque, Mantigue Island, Camiguin 18-24 Oct 2010 9°10' 16.99"N 124°49' 22.38"E Sipalay City, Negros Occidental 17-20 Sept 2010 9°44’47.75"N 122°23’50.14"E Hinobaan 2, (Negros) 17-20 Sept 2010 9°32’23.05"N 122°30’56.19"E San Juan, (Negros) 17-20 Sept 2010 10°36’32.86"N 122°54’36.25"E Padina Patar, Bolinao, Pangasinan 17-19 May 2010 16°19’59.11"N 119°47’13.95"E Perez, Alabat, Quezon 25-30 Nov 2011 14°10’48.33"N 121°55’27.49"E Gonzaga, Cagayan 25-29 July 2010 18°17’16.30"N 121°59’24.89"E Sta. Ana, Cagayan 25-29 July 2010 18°28’49.06"N 122°8’26.09"E Burgos, Ilocos Norte 25-29 July 2010 16° 2’44.93"N 119°45’13.97"E Alcoy, Cebu 10-16 Aug 2010 9°40’25.97"N 123°30’18.65"E Maribago, Mactan, Cebu 10-16 Aug 2010 10°17’8.17"N 124°0’26.30"E Bgy. Paypay, Daanbantayan, Cebu 10-16 Aug 2010 11°13’20.67"N 123°58’56.78"E Pamilacan Island, Baclayon, Bohol 10-16 Aug 2010 9°29’25.84"N 123°54’59.53"E Sitio Basdio, Loon, Bohol 10-16 Aug 2010 9°47’51.87"N 123°47’1.22"E Sto. Domingo, (Bicol) 8-13 Nov 2010 13°23’42.40"N 123°11’29.20"E Bulusan, Sorsogon 8-13 Nov 2010 12°44’52.69"N 124°8’33.59"E Sipalay City, Negros Occidental 17-20 Sept 2010 9°44’47.75"N 122°23’50.14"E Dumaguete, Negros Oriental 17-20 Sept 2010 9°18’17.53"N 123°18’40.40"E Caliling, (Negros) 17-20 Sept 2010 9°59’32.31"N 122°28’14.94"E Hinobaan 1, (Negros) 17-20 Sept 2010 9°35’57.65"N 122°27’50.81"E Hormophyza Patar, Bolinao, Pangasinan 17-19 May 2010 16°19’59.11"N 119°47’13.95"E Trensiera, Bolinao, Pangasinan 17-19 May 2010 16°26' 24.84"N 119°56'45.87"E Lucero, Bolinao, Pangasinan 17-19 May 2010 16°24’10.50"N 119°54’22.91"E Maribago, Mactan, Cebu 10-16 Aug 2010 10°17’8.17"N 124°0’26.30"E Dalaguete, Cebu 10-16 Aug 2010 9°45’54.64"N 123°32’10.47"E Sto. Domingo, (Bicol) 8-13 Nov 2010 13°23’42.40"N 123°11’29.20"E Bulusan, Sorsogon 8-13 Nov 2010 12°44’52.69"N 124° 8’33.59"E Talisayan, Poblacion, Misamis Oriental 18-24 Oct 2010 9°0’53.98"N 124°52’15.25"E Hinobaan 2, (Negros) 17-20 Sept 2010 9°32’23.05"N 122°30’56.19"E San Juan, (Negros) 17-20 Sept 2010 10°36’32.86"N 122°54’36.25"E Hydroclathrus Patar, Bolinao, Pangasinan 17-19 May 2010 16°19’59.11"N 119°47’13.95"E Patar, Bolinao, Pangasinan 17-19 May 2010 16°19’59.11"N 119°47’13.95"E Dictyota Lucero, Bolinao, Pangasinan 17-19 May 2010 16°24’10.50"N 119°54’22.91"E Ubojan East, Garcia-Hernandez, Bohol 10-16 Aug 2010 9°36’32.40"N 124°18’7.14"E Pamilacan Island, Baclayon, Bohol 10-16 Aug 2010 9°29’25.84"N 123°54’59.53"E Bulusan, Sorsogon 8-13 Nov 2010 12°44’52.69"N 124°8’33.59"E Hinobaan 2, (Negros) 17-20 Sept 2010 9°32’23.05"N 122°30’56.19"E San Juan, (Negros) 17-20 Sept 2010 10°36’32.86"N 122°54’36.25"E Maria, (Negros) 17-20 Sept 2010 10°20’58.91"N 122°50’54.02"E Fucoidan content in Philippine brown seaweeds 50 the same locations. The type of seaweeds collected varied among sampling sites. Samples were washed with distilled water to remove salt and epiphytes, air dried, and milled for extraction. Voucher specimens were dried, identif ied, labeled, and kept at the GT Velasquez Phycological Herbarium. Study collection sites were limited by geopolitical and accessibility considerations. Extraction of fucoidan Milled samples were used for extraction following the methods presented by Ale et al. (2011) with slight modif ications. Fifty grams of dried and milled seaweed thalli were briefly acid-treated using dilute hydrochloric acid, heated, allowed to cool, and centrifuged. Residues were discarded afterwards. The solution was neutralized to pH 7.0 using sodium hydroxide pellets, forming brown precipitate of crude fucoidan. Partial purif ication was performed by fractional precipitation using 30% and 60% ethanol, in order to remove alginate contamination and to precipitate semi-pure fucoidan. Semi-pure fucoidan extracts were subjected to infrared spectroscopy to verify signature peaks of functional groups of fucoidan from Fucus vesiculosus. Using FT-IR spectrophotometer, dry fucoidan was mounted to the attenuated total reflectance (ATR) accessory sample holder and scanned from 400 cm -1 to 4000 cm-1. Fucoidan from F. vesiculosus (Sigma Aldrich: F5631) was used as standard for structure elucidation. Data analyses Percent fucoidan content of the sampled seaweeds from different sites were pooled within respective provinces. Percent fucoidan content was calculated using the weight of the semi-purif ied fucoidan (in grams) divided by the dried and milled biomass (50 g) of seaweeds. The resulting value was then multiplied by 100. The average value of the percent fucoidan content from its corresponding seaweed sources were plotted on the Philippine map through QGIS, an open-source geographic information software. There were differences in the occurrence of seaweeds per sampling site. Statistical analyses on the percent content were conducted via one- way ANOVA and Tukey’s Multiple Comparison Test. RESULTS The diversity and occurrence of brown seaweeds varied signif icantly among sampling sites. Samples were grouped and pooled up to genus level. Of the major J.T. Narsico et al. 51 groups, Sargassum was consistently present and collected in all of the 14 provinces, Turbinaria in 11 provinces, Padina in 10 provinces, Hormophysa in six provinces, Dictyota in four provinces, and Hydroclathrus in only one province (Figures 1-6). Crude fucoidan content varied from 10.23% to 24.55% (Sargassum spp. , Turbinaria ornata), whereas partially-purif ied fucoidan yield were at 1.89% to 7.03% (Padina sp. , T. ornata) based on dry weight. Sargassum samples from Camiguin had the highest fucoidan content (4.3%) among the provinces while samples from Pangasinan had the lowest content (1.89%). Turbinaria from the northern Philippine provinces of Cagayan and Ilocos had the highest content (7.03% and 6.85%, respectively), Figure 2. Percent fucoidan content (semi-purif ied) of the brown seaweed Hormophysa collected in six provinces in the Philippines. Figure 3. Percent fucoidan content (semi-purif ied) of Hydroclathrus. Hydroclathrus was only observed in Pangasinan. Fucoidan content in Philippine brown seaweeds 52 Figure 4. Percent fucoidan content (semi-purif ied) of Padina collected in 10 provinces in the Philippines. Figure 5. Percent fucoidan content (semi-purif ied) of Sargassum. Samples were collected in 14 provinces in the Philippines. Highest fucoidan yield was observed in Camiguin. J.T. Narsico et al. 53 while those obtained in Cebu had the lowest fucoidan content at 0.74%. Padina from the Quezon Province had the highest content at 3.69%, while the Negros Occidental samples had the lowest content at 1.3%. Hormophysa from Cebu had the highest content at 2.17%, while the Misamis Oriental samples obtained the lowest content at 0.99%. Dictyota from Bohol had the highest content at 1.53%, while the Pangasinan samples had the lowest content at 0.19%. Hydroclathrus from Pangasinan averaged at 2.23% fucoidan content. Fucoidan percent content among the genus were also compared. There was no signif icant difference between the contents of Sargassum, and Hormophysa, Hyd roclathrus, Padina, and Turbinaria (p ≤ 0.05). However, the percent content of Sargassum compared to Dictyota was signif icantly higher (p ≥ 0.05). Percent content from Turbinaria was signif icantly higher compared to Dictyota, Hormophysa, and Padina (p ≤ 0.05). There was no signif icant difference between the yields of Sargassum and Turbinaria (p ≤ 0.05). Additionally, there was no signif icant difference between the fucoidan contents of Padina and Hormophysa, Hydroclathrus and Dictyota, and Hormophysa against Hydroclathrus and Dictyota (Figure 7). Figure 6. Percent fucoidan content (semi-purif ied) of Turbinaria collected in 11 provinces in the Philippines. Samples from Cagayan obtained the highest fucoidan yield. Fucoidan content in Philippine brown seaweeds 54 Representative data on the semi-purif ied Fucoidan from Sargassum, Turbinaria, and Padina showed peaks similar to the standard fucoidan from F. vesiculosus (Figure 8). There were broad bands at 3321–3415 cm-1 and small peaks at 2941-2945 cm-1, indicating signature vibrations of OH groups and CH of pyranoid rings, and C6 of fucose and galactose, respectively (Kim et al. 2010). A peak at 1732 cm-1 indicates the O-acetyl group (Chandia and Matsuhiro 2008; Kim et al. 2010; Synytsya et al. 2010). Sulfate stretch was observed at 1241–1242 cm-1, which are peaks unique to ester sulfates. Finally, centered peaks were observed between 830–842cm-1, corresponding to C-O-S with sulfate at equatorial and/or axial positions (Bilan et al. 2004; Kim et al. 2010). Figure 8. IR spectra of semi-purif ied fucoidan from different brown seaweed species and F. vesiculosus. Figure 7. Percent fucoidan content of brown seaweeds in the Philippines. Comparison of the fucoidan yield of samples gathered from different provinces in the country from 2010-2011. Signif icant differences between the following comparisons were observed at 95% conf idence interval: Sargassum and Dictyota, Turbinaria and Padina, Turbinaria and Hormophysa, and Turbinaria and Dictyota. J.T. Narsico et al. 55 DISCUSSION In this study, we sampled six genera of brown macroalgae from more than 50 sites within 14 provinces in the Philippines. Some seaweeds were at minimal distribution, if not absent, in sampling sites; hence, it was not feasible to extract suff icient fucoidan for analyses. All samples were not collected in the same sites at the same time during the year. Seasonal differences and varying distribution patterns could account for the absence or presence of certain species in the sampling sites. For instance, Dictyota is known to be widely distributed in the Luzon and Visayas regions (Trono 1997), but it was not found during cer tain collection periods in sampling sites in Mindanao. On the other hand, Sargassum was collected in all provinces because it is widely distributed and grows during wet and dry seasons. Trono (1997) detailed the distribution and seasonal variation of brown seaweeds in the Philippines. Thus, the differences in their distribution affect the comparison of fucoidan content among the provinces. As a consequence of this irregularity, we pooled the data collected in each genus or species per provinces. This gives us an estimate of how much content can be obtained in seaweeds from representative sites per province. At present, there is no standardized purif ication procedure for fucoidans. Classical methods of extracting fucoidan involve a multi-step aqueous extraction using an acid which is usually hydrochloric acid (Ale et al. 2012). Fucoidan extracted using HCl is similar to the fucoidan supplied by Sigma-Aldrich. It is important to note that the characteristics of fucoidan are dependent on the extraction technique. Different extraction methods and purification treatments of fucoidans have resulted to varied compositional results and structural suggestions for fucoidan and other polysaccharides (Ale and Meyer 2013). Among the seaweeds sampled, Dictyota had the lowest fucoidan content while Sargassum and Turbinaria had the highest. It is expected that Dictyota will have the lowest fucoidan content because of its fleshy and soft fronds. Fucoidan yield and monosaccharide composition are also affected by plant age or maturity (Skriptsova et al. 2010). Aside from differences in fucoidan content, seaweeds are also reported to exhibit a relatively large variation in composition and structural properties, even those belonging to the same order or family (Ale et al. 2011), resulting to an array of varied intensities of bioactivities. The amount and composition of algal metabolites are influenced by complex exogenous factors and endogenous biological and biochemical processes. Mature and reproductive stages of the macrophyte reportedly produce signif icant amounts of fucoidan compared to young thalli (Zvyagintseva et al. 2003; Skriptsova et al. 2010). Fucoidan content of reproductive Fucoidan content in Philippine brown seaweeds 56 ҁ  tissues of f ive macroalgal species were 1.3-1.5 times higher compared to their sterile counterparts. Fucoidan generally accumulates in the reproductive structures of brown seaweeds, whose reproduction cycle also affect the changes in fucoidan monosaccharide composition (Skriptsova et al. 2012). Geographical location, seaweed species, and seasonal variations may also influence the differences in polysaccharide composition and their chemical structure. In a study conducted by Sinurat et al. (2016), Sargassum polycystum from three different sampling sites in Indonesia exhibited differences in their fucoidan and ash contents. The maturation cycle of the seaweed also influences the changes in fucoidan content. This cycle is primarily influenced by the changes in season. In temperate countries, the increase in water temperature influences the growth and maturity of the seaweeds. This was observed in the brown seaweed Undaria pinnatifida collected from September-October 2011 in three different mussel farms in New Zealand, wherein seaweed samples exhibited low fucoidan content on July, an increase in the content on September, and a drastic drop on October 2011. Aside from the changes in the fucoidan content, uronic, sulphate, fucose, and protein contents of the seaweeds were also affected by the month of harvest. The study also suggested that there were variations in the crude fucoidan content and composition between the two different sampling sites (Mak et al. 2013). Sargassum and Turbinaria are potential sources of fucoidan because of their greater fucoidan content and wide distribution in the Philippines. For future studies, it is recommended that the relationships between the fucoidan content of the seaweeds, and the seasonal variations and the reproductive cycles should be investigated. It is also recommended to f ine scale the sampling sites, and to focus on locations where there is a high chance of collecting the same type of species in certain months. For example, the preliminary results of this study suggest that the provinces of Camiguin, Negros Oriental, and Pangasinan are possible locations for establishing multiple collection sites for Sargassum. For Turbinaria, possible sampling sites can be established in Cagayan, Ilocos Norte, and Quezon Province. By establishing multiple sites in these provinces, researchers and investors can be assisted in deciding which areas should be prioritized for collection and which months the seaweeds should be collected. It is recommended to further study the life cycle and physiology of these fucoidan-yielding species. It is also important to develop culture techniques for local brown seaweeds to prevent the overharvesting of these species in the wild and to create a steady supply of brown seaweeds in the J.T. Narsico et al. 57 market. For instance, culture techniques for brown seaweeds Undaria and Laminaria were already developed (Tseng and Fei 1986). Possible investors and farmers may culture the brown macroalgal species in the sites identif ied. ACKNOWLEDGEMENTS This study was funded by the Bureau of Agricultural Research of the Department of Agriculture, Republic of the Philippines. We thank Ms. Hannah Faith Dormido for making the maps presented in this study. This is contribution no. 452 of the Marine Science Institute, University of the Philippines. REFERENCES Ale MT, Meyer AS. 2013. Fucoidans from brown seaweeds: an update on structures, extraction techniques and use of enzymes as tools for structural elucidation. Royal Chemistry Society Advances. 3:8131-8141. Ale MT, Mikkelsen JD, Meyer AS. 2011. Important determinants for fucoidan bioactivity: a critical review of structure-function relations and extraction methods for fucose- containing sulphated polysaccharides from brown seaweeds. Marine Drugs. 9(10):2106- 2130. Ale MT, Mikkelsen JD, Meyer AS. 2012. Designed optimization of a single-step extraction of fucose-containing sulfated polysaccharides from Sargassum sp. Journal of Applied Phycology. 24(4):715-723 Ang PO, Leung SM, Choi MM. 2013. A verif ication of repor ts of marine algal species from the Philippines. Philippine Journal of Sciences. 142:5-49 Berteau O, Mulloy B. 2003. Sulfated fucans, fresh perspectives: structures, functions, b i o a c t i v i t i e s , a n d b i o l o g i c a l p r o p e r t i e s o f s u l p h a t e d f u c a n s a n d a n o v e r v i e w o f enzymes active toward the class of polysaccharides. Glycobiology. 13(6):29R-40R. Bilan MI, Grachev AA, Ustuzhanina NE, Shashkov AS, Nifantiev NE, Usov AI. 2004. A highly regular fraction of a fucoidan from the brown seaweed Fucus distichus L. Carbohydrate Research. 339:511–517. Chandia N, Matsuhiro B. 2008. Characterization of a fucoidan from Lessonia vadosa (Phaephyta) and its anticoagulant and elicitor properties. International Journal of Biological Macromolecules. 42:235-240. K i m E J , Park S Y, Lee J Y, Pa r k J H Y. 2 0 1 0 . F u co i d a n p r e s e n t i n b r ow n a l g a e i n d u ces apoptosis of human colon cancer cells. BMC Gastroentorology. 10:96. Fucoidan content in Philippine brown seaweeds 58 Li B, Lu F, Wei X, Zhao R. 2008. Fucoidan: Structure and bioactivity. Molecules. 13:1671- 1695. Mak W, Hamid N, Liu T, Lu J, White WL. 2013. Fucoidan from New Zealand Undaria pinnatifida: Monthly variations and determination of antioxidant activities. Carbohydrate Polymers. 95(1):606-614. Sinurat E, Saepudin E, Peranginangin R, Hudiyono S. 2016. Characterization of fucoidan e x t r a c t e d f r o m S a r g a s s u m p o l y c y s t u m d i f f e r e n t h a b i t a t s . I n t e r n a t i o n a l J o u r n a l o f Chemical, Environmental and Biological Sciences. 4(1):96-99. Skriptsova AV, Shevchenko NM, Zvyagintseva TN, Imbs TI. 2010. Monthly changes in the c o n t e n t a n d m o n o s a c c h a r i d e c o m p o s i t i o n o f f u c o i d a n f r o m U n d a r i a p i n n a t i f i d a (Laminariales, Phaeophyta). Journal of Applied Phycology. 22:79–86. Skriptsova AV, Shevchenko NM, Tarbeeva DV, Zvyagintseva TN. 2012. Comparative study o f p o l y s a cc h a r i d e s f r o m r e p r o d u c t i ve a n d s te r i l e t i s s u e s of f ive b r ow n s e a weed s . Marine Biotechnology. 14:304-311. Synytsya A , Kim W, Kim S, Pohl R, Synytsya A, Kvasnicka F, Park Y. 2010. Structure and antitumour activity of fucoidan isolated from sporophyll of Korean brown seaweed Undaria pinnatifida. Carbohydrate Polymers. 81:41-48. Trono GC. 1997. Field guide and atlas of the seaweed resources of the Philippines. Makati: Bookmark. Tseng CK, Fei XG. Macroalgal commercialization in the Orient. In: Ragan MA , Bird CJ, e d i t o r s . Pr o ce e d i n g s o f t h e Tw e l f t h I n t e r n a t i o n a l S e a w ee d S y m p o s i u m . Tw e l f t h I n t e r n a t i o n a l S e a w e e d S y m p o s i u m ; J u l y 2 7- A u g u s t 1 , 1 9 8 6 ; S a o Pa o l o , B r a z i l . Netherlands: Springer. 1986. pp 167-172. Zvyagintseva TN, Shevchenko NM, Chizhov AO, Krupnova TN, Sundukova EV, Isakov VV. 2003. Water-soluble polysaccharides of some far eastern brown seaweeds. Distribution, structure, and their dependence on the developmental conditions. Journal of Experimental Marine Biology and Ecology. 294(1):1-13. _____________ Joemark T. Narsico is a graduate of the Marine Science Institute under the Marine Biology program. He is currently a Ph.D. student at Hokkaido University in Japan. His dissertation focuses on the biochemical studies of polysaccharide lyases from a marine bacterium. His research interests include microbial biotechnology and algal polysaccharides. Joyce A. Nieva is a graduate of the Marine Science Institute under the Marine Biotechnology program. She is currently pursuing ecological chemistry for her Ph.D. in Alfred Wegener Institute in Bremerhaven, Germany. J.T. Narsico et al. 59 Alper James Alcaraz is currently a Ph.D. Research Fellow on Molecular and Environmental Toxicology at the University of Saskatchewan, Canada. He was a research associate at the Marine Science Institute working on inland macroalgal farming techniques and seaweed polysaccharide chemistry. He also lead an ecotoxicology research project that was used in the development of offshore effluent standards, partnering with government and private sectors. He graduated with a Bachelor’s degree in Chemistry and an M.S. in Environmental Science in UP where he also worked with leading scientists in the marine sciences. Elad io G.M. Anino V was one of the research assistants in Dr. Montaño’s team, working on algal polysaccharides from brown and red seaweeds during his time in the Seaweed Chemistry Laboratory of the Marine Science Institute. He is currently a fourth year medical student in the University of the Philippines College of Medicine. His research interests include biomedical applications of marine natural products and drug delivery systems. Norchel Corcia Gomez is a graduate student in the Marine Science Institute under the program Marine Biotechnology. As a research assistant in the Seaweed Chemistry Lab, she has worked with algal polysaccharides from brown and red seaweeds under the supervision of Dr. Montaño. Her research interests include marine natural products, and marine pollution. She plans to study about microplastics in the marine environment for her MS thesis. Marco Nemesio E. Montano, Ph.D. is a retired professor from the Marine Science Institute. He is a member of the National Academy of Science and Technology of the Philippines. He was awarded by the Integrated Chemists of the Philippines as the Most Outstanding Chemist Award for the year 2012 for his dedication and professional excellence in supporting the seaweed industry in the Philippines. He has published numerous papers and spearheaded projects on various research topics on algal polysaccharides, marine pollution and marine natural products.