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Water Soluble Polysaccharides of Marine Algal Species of Ulva (Ulvales, Chlorophyta) of Indian Waters

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Water Soluble Polysaccharides of Marine Algal Species of Ulva (Ulvales, Chlorophyta) of Indian Waters Indian Journal of Marine Sciences Vol. 30, September, 2001, pp. 166-172 Water soluble polysaccharides of marine algal species of Ulva (Ulvales, Chlorophyta) of Indian waters A. K. Siddhanta*, A.M. Goswami, B. K. Ramavat, K.H. Mody & O.P. Mairh Marine Algae & Marine Environment Discipline, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, Gujarat, India Received 13 November 2000, revised 3 May 2001 Cold and hot water extracts of four different species of Ulva viz. U. reticulata, U. lactuca, U. rigida and U. fasciata were studied for their polysaccharide (PS) contents. In both the cold (CWE) and hot water (HWE) extracts relatively higher yield of polysaccharides were obtained in Ulva fasciata (6.5 and 16% respectively). Ulva lactuca was found to contain higher amounts of protein (33.1% in CWE), uronic acid (35.7% in HWE) and sulfate (23.8% in HWE). Cold water extracts were found to be enriched with hexose sugars, comprising a part of structural polysaccharide, whereas the hot water extracts were rich in rhamnose, xylose as well as glucose. The average molecular weight of these polymers were found to be in the range 1.14 to >2.0×106 Da. Seasonal variation of PS of U. fasciata were also studied alongside. For this, cold and hot water soluble polysaccharides (PS) were isolated separately from the samples of Ulva fasciata Delile, collected monthly from a single location during the season of algal growth (September-March) of the year 1995-96 from the west coast of India. Yield (17-21%) and viscosity (203 247 cps) of HWE were high during the active period of growth (October-February) of algae. Given the abundance of Ulva species in Indian waters coupled with the potential utilities of their polysaccharides, the results obtained in this investigation would be useful in product development and bioprospecting strategies. Species of the marine algal genus Ulva Linnaeus the Indian coasts. Though there are few reports on the (Ulvales, Chlorophyta) are known for their various cultivation, laboratory culture and growth studies of uses like feed, manure, folk medicines, dietary fiber Ulva of Indian waters16,17, there are no reports on the contents as well as for the rheological and gelling chemical nature of the polysaccharide (PS) contents properties of their sulfated polysaccharides (SPS)1-4. of these species. Therefore, the studies on the PS of Polysaccharides isolated from Ulva lactuca Linnaeus four different taxa of Ulva viz. U. reticulata Forssk., have also shown antiviral activity against influenza U. lactuca, U. rigida C. Ag. and U. fasciata, collected virus and the same from Ulva conglobata Kjellman from the Indian coasts, were undertaken for the first are reported to have antiretroviral properties5-7. time. Ulva fasciata being one of the most proliferating Intensive studies were done, since 1941 by various as well as poorly utilized algae, study on the seasonal groups of researchers on the chemical nature, changes in the biosynthetic process and structure and rheological properties of these sulfated physicochemical properties of its PS was undertaken polysaccharides and potential industrial utilisation of in view of the potential utility of these phycocolloids. these mucilagenous polysaccharides were suggested8-12. Cell-wall polysaccharides, isolated from different Materials and Methods species of Ulva have been identified as sulfated Collection and extraction ⎯ Different species of polymers of rhamnose, xylose, and glucuronic acid Ulva collected were - U. reticulata Forssk. from (glucuronoxylorhamnan) along with small amounts of Ervadi (lat. 9o 25′ N, long.79o 08′ E), in January 1997; 13,14 arabinose, galactose and mannose . Various groups U. lactuca Linnaeus from Porbandar (lat. 21o 38′ N, of workers have also reported glucan, a polymer of o 2,11,15 long. 69 37′ E), in February 1997; U. rigida C. Ag. glucose units (1,3 linked) from Ulva . Recently, o o 10 from Gopnath (lat. 21 18′ N, long. 72 14′ E), in Lahaye et al. have also reported SPS containing o January 1998; U. fasciata Delile from Diu (lat. 20 45′ iduronic acid residue from Ulva. N, long. 70o 58′ E), in September 1996. For the The species of the family Ulvaceae, particularly seasonal variation studies, U. fasciata was collected U. fasciata Delile and U. lactuca, occur abundantly on monthly during its season of growth i.e. September to _________ *Corresponding author March (1995-1996) from along the coast of Diu at the E-mail : [email protected] time of low tide. All the algal specimens were SIDDHANTA et al. : POLYSACCHARIDES OF ULVA 167 deposited with CSMCRI Herbarium, Bhavnagar, UV-VIS 160A spectrophotometer using glucuronic India. The algae were washed with tap water to get rid acid as standard21. Neutral sugars were determined as of mud, dirt and sand and then the same was dried in their alditol acetates by GC-MS analysis on a BP-225 the shade and powdered in a rotating ball mill. After column22 using a Hewlett-Packard 5890 Series II GC depigmentation of the dry algal powder by methanol machine connected to 5971 MSD. in a Soxhlet apparatus, 50 g of alga was extracted in Viscosity ⎯ Viscosity of polysaccharide samples o 20 volumes (w/v) of distilled water (DW) at 4-5 C. was measured by using a Brookfield viscometer at The cold water extract (CWE) was first filtered, 30oC in 1.6% and 5% solutions of SPS using #1and concentrated and precipitated with acetone (4 volume, #2 spindle. Measurements for each sample were done v/v). The precipitate was dissolved in minimum DW, in duplicate and an average value was considered. dialysed (MWCO 12,000 Da) against DW and lyophilized. The algal residue after cold water Results and Discussion extraction was extracted in DW at 80oC for 3 hr. From Chemical analyses data of the sulfated poly- the filtrate, called hot water extract (HWE) was saccharides obtained from cold and hot water extracts obtained following the same procedure used for the of four different species of Ulva are presented in CWE. The crude PS products obtained from cold and Tables 1 and 2 respectively. Analytical data of SPS hot water extracts have been designated as REC/REH fractions obtained from the DEAE cellulose column (U. reticulata), LAC/LAH (U. lactuca), RIC / RIH chromatography of crude polysaccharide samples are (U. rigida) and FAC/FAH (U. fasciata). presented in the Table 3. Table 4 describes the Fractionation and purification ⎯ Fractionation monthly variations of physicochemical properties of hot water extracts (HWE) of four different of the cold and hot water extracted product from species of Ulva were made on a DEAE cellulose U. fasciata. The constituent sugar ratio and viscosity anion exchange column (Cl- form, 45 × 3 cm) by a (in cps) of hot water extracts are presented in Table 5. stepwise gradient elution of NaCl (0.0, 0.5, 1.0, Percent yield has been calculated based on the dry 1.5 and 2.0 M) at a flow rate of 25 ml/hr. Fractions weight of the alga taken for extraction. Other were dialyzed and freeze-dried to obtain the physicochemical parameters have been presented as products (A1, A2, A3 for U. reticulata; B1, B2, the percentage of extracted polysaccharide. B3, B4 for U. lactuca; C1, C2, C3, for U. rigida; Polysaccharides of the species of Ulva D1, D2, D3 for U. fasciata). Repeated ion- In cold water extracts, the yields of exchange chromatography of major fractions polysaccharides are in the range of 1.2-6.5%. Low (product of 0.5 M NaCl eluent) were made by a yields of these products precluded further stepwise gradient elution of NaCl (0.15, 0.30, purification and hence characterization of these 0.45, 1.5 and 2.0 M). Products obtained were CWEs were done using the crude products. The designated as: (A2-1, A2-2, A2-3 for U. reticulata, yield as well as the sugar contents are higher in B2-1, B2-2, B2-3 for U. lactuca C2-1, C2-2, C2-3, FAC (cold water extract of U. fasciata). High for U. rigida; D2-1, D2-2, D2-3 for U. fasciata. protein content (17-33%), which is a characteristic Molecular weight of major fractions obtained from of Ulva polysaccharides23, was found in all the the second DEAE cellulose column were polysaccharide samples except in U. fasciata determined by gel filtration on Seralose CL-4B containing low protein (9%). Uronic acid content column and standard dextrans as molecular weight was found to be high in LAC (cold water extract markers (580,000-19,500 Da). Blue dextran (2 × of U. lactuca). Ash content of RIC (cold water 6 10 Da) was used to determine the bed volume of extract of U. rigida) was high in comparison to the the column. others, which appears to be due to the higher Chemical analyses ⎯ Moisture and ash contents sulfate and uronic acid contents that are associated were determined after oven-drying (120oC for 2 hr) with metal ions. Galactose, mannose and glucose and igniting (550oC for 6 hr) the polysaccharide contents were found to be relatively higher than samples, respectively. Total sugar and protein were xylose content in CWE samples which indicate estimated by the methods of Dubois et al.18 and that the presence of neutral polysaccharides as Lowry et al.19 respectively. Sulphate content was well as ‘glucan’ in these products (Table 1). determined20 after hydrolysis with 1N HCl. Uronic In all the hot water extracts, yields of acid was analysed colorimetrically on a Shimadzu polysaccharide products were higher, compared to 168 INDIAN J. MAR. SCI, VOL. 30, SEPTEMBER 2001 their cold water counterparts. Uronic acid, sulfate and phosphate buffers of pH 7.0, 7.6 and 8.0.
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