Semesi, Mshigeni: Contribiitions on Halymenia ventata Phycocolloid 233 Bö tan ca Marina Vol. XX, pp. 233-237, 1977 Contributions on the Content and Nature of the Phycocolloid from Halymenia venusta Boergesen (Rhodophyta, Cryptonemiales) A. K. Semesi and K. E. Mshigeni Department ofßotany, University ofDar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania, East Africa (Received July 8, 1976) Abstract The phycocolloid from Halymenia venusta Boergesen has been studied and found to be a carrageenan which is more closcly related to - than -carrageenan. A total phycocolloid yield of 59.0 ± 2.5 % on dry weight basis was obtained. The presence of a cold water-soluble phycocolloid in the thaüi of H. venusta was also discovered and its IR spcctrum determined. Introduction The red algal genus Halymenia C. Agardh is a member of the family Grateloupiaceae, order Cryptonemiales. About 25 species of the genus have been reported in the literature (Kylin 1956). Generally members of this genus are plants of moderate to considerable size and of gelatinous consistency (Taylor 1960, Zaneveld 1959). The most widespread species on the shores of East Africa is Halymenia venusta Boergesen. Brief floristic reports have been published on this seaweed from Kenya (Isaac 1967) and from Tanzania (Jaasund 1976). In terms of frond size (Fig. 1), this is one of the largest red algae on the shores of Tanzania, sometimes growing up to 50 cm height. The gelatinous nature of the fronds of Halymenia venusta suggests the seaweed to contain appreciable quantities of phycocolloid. However, thene are no published reports on the phycocolloid content of this and, indeed, of any other species of Halymenia', nor is there any published account on the nature of its phycocolloid. Yet, from the point of view of its size and abundance, this seems to be one of the red seaweeds with greatest potential in the commercial production of seaweed colloids in Tanzania. precipitates methylene blue whereas agar does not (Stoloff and Silva 1957), that carrageenan causes an agglomeration of the milk protein, casein, whereas agar does not (Stoloff and Silva 1959, Andersen 1964). The infrared spectra of carrageenan s also show characteristic absorption peaks (Rees 1961, Andersen et al. 1968, Stancioff and Stanley 1969, Santos and Doty 1975) which are not exhibited by agar. These were among the tests conducted in the present study to reveal the phycocolloid content of Halymenia venusta and to characterize whether it is an agar or a carrageenan. Materials and Methods Plant and habitat description The specimens of Halymenia venusta ßoergesen used in this study (Fig. 1) were harvested during low tides from the shores of Oyster Bay and Msasani Bay, Dar es Salaam, Tanzania. The plants, often found growing attached to rocks along the sublittoral fringe or upper sublittoral zone by small discoid holdfasts, were picked by hand. Appreciable quantities of H. venusta torn off by wave action from sublittoral habitats are usually found äs There are t wo main groups of red algal polysaccharides of drift material along the Dar es Salaam shoreline. The commerce: agar and carrageenan. Amongst the tests which plants are of scarlet-red colour with flattened fronds, much proliferated margins and very slippery conseem to be important in the characterisation of agar and sistency. They are usually found clean and devoid of carrageenan are: total sulphate content determination, infrared spectroscopy tests, methylene blue tests, mono- noticeable epiphytes. saccharide component determination, optical rotation determination and milk reactivity tests. For example it Reparation of the seaweed for phycocolloid extraction is known that the optical rotation of agar is negative After harvesting, the fronds were carefully sorted out in whereas that of carrageenan is positive (Clingman and the laboratory to remove rock debris and other impurities. Nunn 1959, Lawson et al 1973). It is also known that the total sulphate content of carrageenan is significantly This was followed by drying on plastic trays in the sun higher than that of agar (Stoloff 1962), that carrageenan (which also facilitated the bleaching of the seaweed) and Botanica Marina / Vol. XX Brought / 1977to/you Fase. 4 by | University of Queensland - UQ Library Authenticated Download Date | 6/23/15 3:58 AM 234 Semesi, Mshigeni: Contributions on Halymenia venusta Phycocolloid Extraction by lime treatment method In the lime-modification method, the extraction procedure was basically similar to that referred to above except that 4 g of calcium hydroxide were added instead of &2% sodium hydroxide solution. Extraction with cold distüled water Freshly harvested fronds were soaked in distilled water overnight and then stirred for one hour at room temperature (26-2S °C). Because of the fresh water-soluble red pigments in the seaweed, the water in the Container turned pink. In an attempt to show whether there were any cold water-soluble phycocolloids, the coloured leachate was concentrated by boiling and then poured into 85% isopropanol. This gave a precipitate similar to that from boiled seaweeds. The precipitate was washed and dried in an oven at 60 °C. Sulphate content determination The phycocolloid (0.5 g) was oxidized using magnesium Fig. l. Growth habit of Halymenia venusta. nitrate solution (5 ml) in a crucible in a muffle furnace at 400 °C. At the end of l hr the crucible was removed and allowed to cool. The ignited mass was then checked subsequently in an oven at 60 °C. Drying was a big for specks of unoxidized material. If necessary the crucible problem because when spread on the trays, the seaweed was returned to the furnace and reheated until completely strongly adhered there, apparently due to the formation oxidized. Distilled water (4 ml) was then added to of bonds with the trays. The seaweed could be removed from the trays only by sprinkling water. This was possibly the crucible, followed by 2 ml concentrated hydrodue to the weakening of the bonds when the phycocolloid chloric acid. The solution was well mixed with a glass rod and then a drop of methyl orange solution was added absorbs water and swells. to ascertain that an excess acid was present. The mixture In the preliminary investigations which involved the was heated till everything was in solution and then washing of the freshly-harvested seaweed with fresh water checked whether still acidic with methyl orange. Then it to remove adhering salt and then drying on trays, it was was filtered and the filter paper was washed seven times learnt that a substantial proportion of the phycocolloid with distüled water till the final volume was 250 ml. The of Halymenia venusta is soluble in cold fresh water. sulphate content was then determined by precipitation äs barium sulphate with 10 ml of 0.25 M barium Chloride. Extraction procedures The phycocolloids were extracted using techniques similar to those recently described by Santos and Doty (1975) in their study of the phycocolloid homAhnfeltia concinna in Hawaii. The dried phycocolloid was weighed and the percentage yield subsequently calculated. The phycocolloid was ground into a powder, labelled and stored until required for other studies. In order to enhance an understanding of the nature of Halymenia venusta phycocolloid, phycocolloids from Eucheuma. spinosum and£". striatum known to have L- and -carrageenan, respectively, were also extracted using a similar procedure. These were also collected from Msasani Bay and Oyster Bay in Dar es Salaam, Tanzania. Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 6/23/15 3:58 AM Infrared spectroscopy determination The powdered phycocolloid (30 mg) was dissolved in 15 ml hot distilled water and then centrifuged at 2000 cycles per sec for 15 min. Small (5 ml) quantities of the solution were poured over 5 ml mercury in a 15 ml porcelain crucible. The solution was subsequently dried to a thin film cast over mercury in a vacuum desiccator at 70 °C. Upon pouring off the mercury from the crucible, the phycocolloid film was left behind. This was placed between two 12 X 5 cm pieces of cardboard with the film mounted at a l .5 X 3 cm window cut through the central portion of the cardboard-pieces. The IR spectra of the films were determined with an SP.200 Infrared Spectrophotometer, Unicam CD.0082. Botanica Marina / Vol. XX / 1977 / Fase. 4 Semesi, Mshigeni: Contributions on Halymenia venusta Phycocolloid Solubility in potassium Chloride The powdered phycocolloid (4.0 g) was added to 2.5% potassium Chloride solution with continuous stirring for l hr at room temperature (26-28 °C) and then left to stand for 2 more hours. After this time the set up was re-examined to find out whether or not the phycocolloid had dissolved in the potassium Chloride, and if so whether it could be re-precipitated with isopropanol. 235 Tab. 1. Properties of the phycocoUoid from Halymenia venusta. Aspect Tested Result 1. Phycocolloid yield: unmodified gel 59.0 t 2.5% (on dry wt. basisj 2. Phycocolloid yield: modified with lime 58.6 ± 1.5% (on dry wt. basis) 3. Total sulphate content 26.2 ß 0.4% (based on dry wt. of phycocoUoid) Methylens blue test This was done using l % of the extracted phycocolloid dissolved in hot distilled water. The solution (10 ml) was poured into a test tube and then four drops of l % aqueous solution of methylene blue were added, the test tube being held to the light against a white background. Then observations were made to detect whether a characteristic fibrous-looking agglomerate would be noticed on the Interface. Milk reactivity test A 5% aqueous solution of the extracted phycocolloid was added to fresh milk in a test tube and then shaken for a few minutes at room temperature. Observations were then made to detect whether or not flocculation appeared. Optical rotation test Optical rotation was determined with a D-polarimeter (Bellingham and Stanley Ltd. No. 593926, London), using 10 cm cells and Sodium D line (589 and 589.6 nm wavelength). To ensure dust-free samples, the Solutions were filtered bot through Whatman No. l filter paper. The concentration of phycocolloid (0.15 g per 100 ml distilled water) was determined before filtration. Results and Discussion The results of this study are summarised in Table l, and the IR spectra are shown in Figure 2 A, B, C. in addition to the IR spectra of Halymenia venusta phycocolloid, IR curves for Eucheuma spinosum and E. striatum are included (Fig. 2 D, E) to enhance comparison. 4. Solubility in potassium chloridc Soluble: the precipitate was rocovered by treatment with isopropanol 5. Methylene blue test Positive: the phycocolloid precipitated methylene btuc. 6. Milk reactivity test Positive: the phycocolloid causcd a flocculation reaction with milk 7. Optical rotation Positive: a) hot water-soluble extract b) cold water-solublc extract |Q|^5 + 1 1 à . 8, IR peaks of hot water-soluble extract a) showed broad absorption band at 1240 cm l b) showed another band at 940 cm c) showed a broad band at 860-800 cm 9, IR peaks of cold water-soluble extract a) showed a broad absorption band at 1240 cm"1 b) showed an additional weak band at l 340-1 310 cnf 1 c) Broad band at 860-800 cnf l slightly resolved to give peaks at 840 and 810 cm'1 10. IR peaks of limemodified extract a) weakened absorption at 770 cm"1 b) increased that at 940 cm"1 cause different IR absorption peaks, Do the IR absorption peaks of Halymenia venusta phycocolloid conform with the known IR spectral peaks characteristic for any of the known types of carrageenan? As shown in Figure 2 A, B, C, the IR spectra of Halymenia venusta phycocolloid show a broad absorption band at 1240 cm"1 which (Rees 1961, Anderson et al 1968, Stancioff and Stanley 1969) is characteristic of all sulphated polysaccharides. The phycocoUoid also The results strongly suggest that the phycocolloid from 1 shows an absorption peak at 940-930 cm" which is Halymenia venusta is a carrageenan. The positive optical rotation, the positive methylene blue and milk reactivity believed to be due to 3,6-anhydrogalactose (Rees 1961, tests, and the high degree of sulphation (Tab. 1), seem to Anderson et al 1968).1 The phycocolloid also has a broad band at 860-800 cm" (Fig. 2 A, B) which is believed support this view. But what kind of carrageenan is it? to be due to the possession of more than one type of Three main types of carrageenan have been reported in the ester sulphate (Stancioff and Stanley 1969). literature: t-, ê- and ë-carrageenan, differing mainly in Alkaline modification (Fig. 2A) weakened the absorption their 3,6-anhydrogalactose content and in their content peak at 770 cm"1 and apparently increased the amount and position of ester sulphate groups (Stancioff and Stanley 1969, Santos and Doty 1975). These differences of 3,6-anhydrogalactose s indicated by a slightly Botanica Marina / Vol. XX / 1977 / Fase. 4 Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 6/23/15 3:58 AM Semesi, Mshigeni: Contributions on Halymenia venusta Phycocolloid 236 i—1 l ¥ Ì Ô indicated (Fig. 2C) by the presence of a broad absorption band at 1240 cm"1 (Rees 1961, Stancioff and Stanley 1969). However, it differed from the hot water extract in its weaker band at 1340 cm"1. The broad band at 860—800 cm*1 of the cold water-soluble extract was also slightly resolved to give a peak at 840 and 810 cm"1. The presence of a single broad absorption band at 860—800 cm"1 in the IR spectra of Halymenia venusta phycocolloid suggests that the phycocolloid is not an t-carrageenan which (Rees 1961, Andersen et al. 1968, Stancioff and Stanley 1969, Penman and Rees 1973, San tos and Doty 1975) shows single sharp peak at 805 cm"1 (Fig. 2 D). The peak at 805 cm"1 has been suggested to be due to the presence of 2-sulphate on the 3,6-anhydrogalactose. As shown in Figure 2, the IR band at 930 cm"1 was rather weak, suggesting a low level of 3,6-anhydrogalactose. The phycocolloid of Halymenia venusta (Tab. 1) also showed solubility in potassium chloride. This suggests that it is more closely related to ë- than ê-carrageenan. Fig. 2. IR spectra of Halymenia venusta phycocolloid compared with IR spectra of Euchema phycocolloids: A = Halymenia venusta hot water-soluble extract: modified. B = H. venusta hot water-soluble extract: unmodified. C = H. venusta cold water-soluble extract: unmodified. D = Eucheuma spinosum phycocolloid (é-carrageenan): unmodified. E = E. striatum phycocolloid (ê-carrageenan): unmodified. The results of the present study thus seem to suggest that the phycocolloid of Halymenia venusta is akin to that ofPachymenia hymantophora (also a member of the Grateloupiaceae) which (Lawson et al 1973, Penman and Rees 1973) was reported to be a carrageenan of the ë-type. Grateloupiaelliptica, another member of the Grateloupiaceae, was also reported (Hirase et al 1967) to have a phycocolloid soluble in potassium chloride and classified s an intermediary between ê- and ë-carrageenan. Indeed when Allsobrook et al (1969) studied the phycocolloids from several members of the Grateloupiaceae (viz., species of Aedoes, Pachymenia, Grateloupia and Phyllymenia), they all found sulphated polysaccharides containing methylated monosaccharide residues in high concentration and unusual diversity, but falling within the basic pattern of carrageenan. The slightly resolved peak of the cold water-soluble extract of Halymenia venusta at 840 and 810 cm'1 (which was not present the hot water-extract) may be attributable to such diversity. Further studies therefore need to be conducted to clarify the chemical nature of Halymenia venusta phycocolloid and especially of the cold water-soluble extract which is not well documented in the literature. The finding of the present research that Halymenia venusta contains a carrageenan which occurs in a relatively high proportion (59.0 ± 2.5 % of the dry weight), seems to be of great economic significance in the tropics where this seaweed occurs in rieh abundance. stronger peak at 940 cm"1 (Rees 1961, Anderson et al. 1968). Acknowtedgement The phycocolloid which was extracted with cold water also had an IR typical of a sulphated polysaccharide s We wish to express our deep appreciation to D. A. Rees, G. A. Santos and M. S. Doty, whose reprints of their Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 6/23/15 3:58 AM Botanica Marina / Vol. XX / 1977 / Fase. 4 Semesi, Mshigeni: Contributions on /fefymeaw vtnurta PhycocoUoid publications in this topic were very valuable in our discussion; to the Chemutry Department, Univenity of Dar es Salaam for the use of their IR and optical 237 rotation Instruments; to H. K, Kabunga of the Chemistry Department for technical help in IR- and optical rotation determinations, and J. F. Haule for typing the manuscript. References Allsobrook, A. J. R», J« R. Nunnand H. Parolis. 1969. Some Grateloupiaceae polysaccharides. Proc. Intl. Seaweed Symp. 0:417-420. Andersen, H, 1964. Interactions between carrageenam and milk proteins. Proc, Intl. Seaweed Symp, 4: 377-384, Anderson, N, SM T. C S. Dolan, A* Penman, D. A. Rees, G. P. Mueller» D. J. Standoff and N. F. Stanley. 1968, Carrageenans* Part IV. Variations in the structure and gel properties of ê-carrageenan» and the chaiacterisation of sulphate esters by Infrared spectroscopy- / Chem, Soc, (C): 602-606. CHngman, A. and J. R. Nunn. 1959. Red seaweed polysaccharides. Part 3- Polysaccharides from Hypnea spictfera. / Chem. Soc.: 493-498. Hirase, S„ C. Araki and K. Watanabe, 1967, Component sugars of the polysacchaiide of the red seaweed Grateloupia elliptica. Bull Chem. Soc. Japan, 40: 1445-1448. Isaac, W. E. 1967. Marine botany of the Kenya coast. 1. A first Ust of Kenya marine algae. J. E. A fr. Íáé. Hist, Soc. 26: Penman, A., and D, A. Rees. 1973, Carrageenans. Part XI. Mild oxidative hydrolysis of ê- and ë-carrageenans and the characterisation of oligosaccharide sulphates. J. Chem. Soc. Perkin, 1: 1291-1296, Penman, A, and D. A. Rees. 1973. Carrageenans. Part IX, Methylation anatysis of galactan sulphates from Furceflaria fastigiata, Gigartina canaliculata, Giganina chamissoi, Gigartina atropurpurea, Ahnfeltia durvi aei, Gymnogongrus furcellatus, Eucheuma isiforme. E. uncinatum, Agardhiella tenerat Pachymenia hymantophora, and Gloiopeltis cervicornis. Structure of î-carrageenan. / Chem. Soc, Perkin, l : 2182-2187. Rees, S* A, 1961. Estimation of the relative amounts of isomcric sulphate esters in some sulphated polysaccharides/ Chem. Soc.: 5168-5171. Santos, G. A. and M. S. Doty, 1975, IR studics on carrageenan of Ahnfeltia concinna, a marine red alga. J. Pharm. Sei. 64 (10): 1704-1706. Stancioff, D. J. and N. F. Stanley, 1969, Infrared and chemica) 75-83, studies on algal polysaccharides. Proc, Intl. Seaweed Symp. 6: 595-609. Jaasund, E. 1976. Intertidal seaweeds in Tanzania. A field Guide. University of Troms^ 160 pp. Stoloff, L, 1962* Algal cla&sification - an aid to improved industriai utilization, Econ. ÂïÀ. 1 6 ( 1 ) : 86-94. Kylin, H. 1956. Die Gattungen der Rhodophyceen. Lund,, 672 pp. Stoloff, L. and R Silva. 1957. An attempt to deteirmne possible Lawson, C J., D. A. Rees, D. J. Stancioff and N, F. Stanley, taxonomic significance of the properties of water-cxtractable 1973. Carrageenans, Part VIIL Repeating structures of polysaccharides in red algae. Econ. Bot., Ð (4): 327-330, galactan sulphates from Furce aria fastigiata, Gigartina canaliTaylor, W, R. 1960. Marine Algae ofthe easiern tropicat and culata, Gigartina atwpurpurea, Aknfeltia durvilfaei, Gymnowbtropical coasts of the A mericas. The University of Michigan gongrus furcellatus, Euchema cottonii, E. isiforme, E. undPress, Ann. Arbor. 416 pp* natum, Agardhietla tenera, Pachymenia hymenthophora Zaneveld, L S. 1959. The utilization of marine algae in tropical and Gloiopeltis cervicornis. J. Chem. Soc. Perkin, l: south and east Asia. Econ. Bot. 13 (2): 89-131. 1277-1282. Botanica Maf ia / VoL XX / 1977 / Fase, 4 Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 6/23/15 3:58 AM Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 6/23/15 3:58 AM