Marine Algae in Pharmaceutical Science: Vol. 2 9783110837506, 9783110086263

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Marine Algae in Pharmaceutical Science Volume 2

Marine Algae in Pharmaceutical Science Volume 2 Editors Heinz A. Hoppe • Tore Levring

W DE

G

Walter de Gruyter • Berlin • New York 1982

Editors

Heinz A. Hoppe Finkenstraße 5 D-2150 Buxtehude Germany Professor Tore Levring f University of Gothenburg Marine Botanical Institute S-41319-Gothenburg/Sweden

CIP-Kurztitelaufnahme der Deutschen Bibliothek Marine algae in pharmaceutical science / ed. Heinz A. Hoppe ; Tore Levring. - Berlin ; New York : de Gruyter NE: Hoppe, Heinz A. [Hrsg.] Vol. 2 (1982). ISBN 3-11-008626-3

Library of Congress Cataloging in Publication Data (Revised) Main entry under title: Marine algae in pharmaceutical science. Includes articles from a special symposium organized in connection with the 9th International Seaweed Symposium, held in Santa Barbara, Calif., 1977. Bibliography: p. Includes indexes. 1. Marine algae-Therapeutic use-Congresses. 2. Marine pharmacology-Congresses. I. Hoppe, Heinz August, 1907II. Levring, Tore, 1913III. Tanaka, Yukio, 1929IV. International Seaweed Symposium, 9th, Santa Barbara, Calif., 1977. [DNLM: 1. Algae-Congresses. 2. Plants, Medicinal-Congresses. QV 766 M338 1980] RS165.A45M37 615'.329392 79-441 ISBN 3-11-00-7375-7 Vol. 1 ISBN 3-11-00-8626-3 Vol.2

Copyright © 1982 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Karl Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany

Preface

The increasing interest in the field of "Marine Algae in Pharmaceutical Science" gave rise to the suggestion that a special session should be organised at the X. International Seaweed Symposium in Gothenburg (Sweden) in August, 1980. The contributions submitted by participants at this symposium are published in the current second volume. We are grateful to both the speakers as well as our hosts in Sweden and we would like to express our gratitude and appreciation to the publishing house Walter de Gruyter, Berlin • New York, for their assistance. We hope this second monograph will prove to be of interest to the specialised branches of science.

Heinz A. Hoppe Tore Levring

My dear and respected friend, Professor Tore Levring, passed away on the 30th of January, 1982. His death is a great loss not only to his family and friends all over the world, but also to a specialised branch of science.

Heinz A. Hoppe *

The contributions of a special symposium "Marine Algae in Pharmaceutical Science" held in Santa Barbara, California, in August 1977 were published in 1979 in monograph form as Volume 1: Marine Algae in Pharmaceutical Science. Editors: Hoppe, Levring, Tanaka. Walter de Gruyter, Berlin • New York, 1979.

VII

Part I.

A Review

Marine Algae and their Products and Constituents H. A. Hoppe

Part II.

Articles on Special Constituents of Marine Algae

Effects of the Various Types of Carrageenan on Human Fibroplasts in vitro E. Tveter-Gallagher, Th. N. Wight, A. C. Mathieson

51

Fat Production in Freshwater and Marine Algae P. Pohl, F. Zurheide

65

Extraction and Separation of Vitamin B ^

from Marine Algae

K. C. Güven, B . Güvener, S. Cirik

81

Antifungal Activity of Different Fractions of Extracts from Indian Seaweeds Sreenivasa Rao, Y. A. Shelat

93

Anti Ulcer Substances from Marine Algae Y. Sakagami, T . Watanabe, A. Hisamitsu, T . Kamibayashi, K. Honma, H. Manabe

99

Sugar Constituents of some Sulfated Polysaccharides from the Sporophylls of Wakame (Undaria pinnatifida) and their Biological Activities H. Mori, H. Kamei, E. Nishide, K. Nisizawa

109

Effect of Seaweed Extracts on Mycobacterium P. Sreenivasa Rao, K. S. Parekh, H. H. Parekh, H. M. Mody, S. B . Trivedi, Bhaskar A. D.

123

VIII

Part III.

Miscellaneous

Seaweed Resources in Tanzania: A Survey of Potential Sources for Industrial Phycocolloids and for other Uses K. E. Mshigeni

131

Freshwater Algal Resources of Tanzania: A Review and Discussion on their Potential for Agriculture, Food Production and other Uses K. E. Mshigeni

175

Cellular Biology of Acetabularia S . Bonotto, A. Liittke

203

Specific Volume Changes in Toxin Treated Unicellular Algae B . C. Hughes

247

Relationship between Dinoflagellates and Intracellular Bacteria E. Sousa Silva

269

Virus Infection of Marine Algae A. Misra, R . Sinha, V. Iha, B . Singh, B . Prasad Sharma

289

Taxonomic Index

299

Subject Index

307

IX Contributors

Bonotto, S . , Radiobiology Dept, C. E. N . / S . C . K . , B-2400 Mol, Belgium. Cirik, S . , Inst, of Marine Science and Technology, University of Ege, Izmir, Turkey. Dave, B . A . , Tuberculosis Research Centre, Shri K. J . Metha T . B . Hospital, Amargadh 364210. India. Giiven, K. C . , Dept of Pharmacy and Technology, Faculty of Pharmacy, University, Istanbul, Turkey. Giivener, B . , Dept of Pharmacy and Technology, Faculty of Pharmacy, University, Istanbul, Turkey. Hisamitsu, A . , Faculty of Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan. Honma, K . , Faculty of Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan. Hoppe, H. A . , Finkenstrasse 5, D-2150 Buxtehude, West Germany. Hughes, B . C . , Dept of Science, Bristol Polytechnic, Coldharbour Lane, Bristol, UK. J h a , S . , Botany Dept, Mithila University, CMSc. College, Darbhanga, 846 004, India. J h a , V . , Botany Dept, Mithila University, CMSc. College, Darbhanga, 846 004, India. Kamei, H . , Bristol-Banyu Res. Institute, Shimomeguro-2, Meguro-ku, Tokyo, 153 Japan. Kamibayashi, T . , Faculty of Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan. Liittke, A . , Radiobiology Dept, C. E. N . / S . C. K . , B-2400 Mol, Belgium. Manabe, H . , Faculty of Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan. Mathieson, A. C . , Jackson Estuarine Laboratory, University of New Hampshire, Durham, N. H. 03824, USA.

X

Misra, A . , Botany D e p t , Mithila U n i v e r s i t y , CMSc College, D a r b h a n g a , 846 004 I n d i a . Mody, H. M., Central Salt and Marine Chemicals R e s e a r c h I n s t i t u t e , gar 364 002, India.

Bhavna-

Mori, H . , Tokyo Kasei-Gakuin College, S a n b a n - c h o , C h i y o d a - k u , Tokyo, 102 J a p a n . Mshigeni, K. S . , P. O. Box 35060, Dar es Salam, T a n z a n i a . Nishide, E . , Dept of F i s h e r i e s , College of A g r i c u l t u r e and V e t e r i n a r y Medicine, Nihon U n i v e r s i t y , Shimouma-3, S a t a g a y a - k u , Tokyo, 154 J a p a n . Nisizawa, K . , Dept of F i s h e r i e s , College of A g r i c u l t u r e and V e t e r i n a r y Medicine, Nihon U n i v e r s i t y , Shimouma-3, S e t a g a y a - k u , Tokyo, 154 J a p a n . P a r e k h , H. H . , Central Salt and Marine Chemicals Research I n s t i t u t e , n a g a r 364 002, I n d i a .

Bhav-

P a r e k h , K. S . , Central Salt and Marine Chemicals R e s e a r c h I n s t i t u t e , B h a v nagar364 002, I n d i a . Pohl, P . , I n s t . f . Pharmazeutische Biologie, Grasweg 9, D-2300 Kiel, West Germany. Sakagami, Y . , Faculty of Science, Tokyo I n s t i t u t e of Technology, 4259 N a g a t s u t a , Midori-ku, Yokohama, J a p a n . Sharma, B. P . , Botany D e p t , Mithila U n i v e r s i t y , CMSc. College, D a r b h a n g a , 846 004 I n d i a . Shelat, Y. A . , Central Salt and Marine Chemicals Research I n s t i t u t e , n a g a r 364 002, I n d i a .

Bhav-

Silva, E. S . , Laboratorio de Micrbiologia Experimental, I n s t i t u t o Nacional de S a u d e , A v . P a d r e C r u z , 1699 Lisboa, P o r t u g a l . S i n g h , B . , Botany D e p t , Mithila U n i v e r s i t y , CMSc. College, D a r b h a n g a , 846 004 I n d i a . Sinha, R . , Botany D e p t , Mithila U n i v e r s i t y , CMSc. College, D a r b h a n g a , 846 004 I n d i a . S r e e n i v a s a Rao, P . , Central Salt and Marine Chemicals R e s e a r c h I n s t i t u t e , B h a v n a g a r 364 002, I n d i a . T r i v e d i , S. B . , T u b e r c u l o s i s R e s e a r c h C e n t r e , S h r i K. J . Metha T . B. Hospital, Amargadh 346 210, I n d i a . T v e t e r - G a l l a g h e r , E . , Jackson E s t u a r i n e L a b o r a t o r y , Durham, N. H. 03824, USA.

XI

Watanabe, T . , Faculty of Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan. Wight, T . N . , Dept of Pathology, Univ. of Washington Medical School Seattle, Wash. 98195, USA. Zurheide, F . , Inst. f . Pharmazeutische Biologie, Grasweg 9, D-2300 Kiel, West Germany

Part I.

A Review

Marine A l g a e : T h e i r P r o d u c t s and Constituents

Heinz A .

Hoppe

Finkenstraße 5 D-2150 B u x t e h u d e F . R . of Germany

Research and utilization of marine algae have increased markedly in recent years. A number of products based on algae as a raw material have been developed and applied in many fields. This has been reported in detail in the specialist literature. In addition to well-known products of red algae such as agar, carrageenan, furcellaran e t c . , alginic acid and alginates of brown algae as well as other algal constituents are increasingly attracting interest. Research into these constituents has only just begun, but clearly there are possible applications in pharmacy and other fields. However, algae are not only a modern raw material; they also form a very interesting and multifaceted group of plants. They are said to number from 20,000 to 30,000 species. They also figure among the oldest living creatures on earth. Algae have been found as fossils from the Precambrian period. They may be 1 to 3 million years old. Recent results on fossil algae were discussed in 1975 at the First International Symposium on Fossil Algae (75), (151). The use of algae has had an interesting history, beginning in the Far East and extending to modern industry (Botanica Marina III, suppl. (1962) and IX, suppl. (1966)). In folk medicine, marine algae have been used for centuries as auxilliary materials in the pharmaceutical sciences, where phycocolloids in particular have become indispensable. Scientific

research

into algae and algal constituents as medicinal drugs is a relatively recent undertaking. New methods in phyto-chemistry. pharmacology, p h y t o t h e r a py and related fields have made remarkable contributions to research in

Marine Algae in Pharmaceutical Science, Volume 2 © 1982 by Walter de Gruyter &. Co., Berlin • New York

4 vegetable raw material. This applies not only to terrestrial medicinal plants, whose number has become greater and greater as more traditional native medicines from exotic countries have been analyzed, but in increasing measure to the algae and other marine organisms as well. Constantine (62) 1977 has pointed out many interesting aspects of this s u b ject, as have the various articles collected in "Marine Algae in Pharmaceutical Science" 1979 (12). This book singled out research into "Special Constituents of Marine Algae" and "Selected Algae and Algal Products", as well as into "Indirect Medicinal Effects", "Algae as Foodstuffs for Human Nutrition" and "Seaweed Meal for Animal Nutrition". Of course, the work of several authors active in different fields in different p a r t s of the world is likely to overlap. Furthermore, works of this sort can clearly never be complete, and must be integrated into a larger framework. A number of remarkable, recently published works point out problems in comtemporary research into algae. The FAO Fisheries Technical Paper (121), "Seaweed Resources of the Ocean", by Michanek 1975 was already mentioned in "Marine Algae in Pharmaceutical Science" (12). In 1979 Michanek reported on aspecta of phytogeographic groupings of seaweed and characterized the tropical, warm and cold regions in this r e s p e c t . The results of an Oregon State University Symposium, "The Marine Plant Biomass of the Pacific Northwest Coast - A Potential Economic Resource", were published in 1977 (25). The editor Robert W. Krauss (110) wrote: "This volume is designed to address the logic of attempting to enrich the store of resources by use of the plants of the ocean, and to examine an opportunity to ease some of the p r e s s u r e s for survival." T h u s , this book gives valuable suggestions for the utilization of marine algae in human nutrition, agriculture, pharmacy, and the production of e n e r g y . Levring (116) reports on potential yields of marine algae with special r e f erence to European species. This work provides an excellent s u r v e y of the vegetation of marine algae from the European coasts, describing the potential yield and consumption of seaweeds and presenting interesting figures and tables on such topics as zonation on the European rocky coastline,

5 water temperature in different areas, the occurrence of commercially viable algal species, and the potential yield and harvest for consumption. Levring also reported on marine vegetation in a general review published in "Marine Algae in Pharmaceutical Science" 1979 (117). "Aspects of marine natural products chemistry" is the subject of a survey by Faulkner (69) concerning new constituents of marine algae and animals, with 233 proposals for specialist research. In 1978 Hellebust and Craigie , in a handbook on "Phycological Methods" (87), published papers by 46 authors on physiological and biochemical methods employed in algal studies. In his "Einführung in die Phykologie", v . d. Hoek, 1978 (89), likewise described the most important constituents of algae in its pertinent chapters. Noelle, in his "Food from the Sea", 1981, discusses the nutritional sciences, enviromental protection, marine biology, the algal constituents and the application of seaweeds to human nutrition (136). The above-mentioned works give a detailed account of further literature. Hoppe's "Drogenkunde", 8th e d . , vol.2 ( 7 ) , presents a survey of the important algae and algal products with regard to their constituents and their potential use in industry. New discoveries in marine algae research were presented at the 10th International Seaweed Symposium, 1980, in Göteborg ( 2 3 ) . In numerous plenary and ordinary

lectures, reports were given on

"Distribution, Morphology, Taxonomy" "Ecology" "Biochemistry, Physiology" "Cultures" Within the scope of this report on the products and constituents of marine algae, the following subjects are of special interest.

6 P r o d u c t s of Marine A l g a e

Agar and carrageenan are the most important polysaccharides of the Rhodophyta ( 7 ) , ( 8 ) . Improved methods of analysis have considerably clarified their structure. Some specifications in the literature of pharmaceutical biology are quite noteworthy.

Agar

Agar can be fractionated into two products: 70% agarose and approx. 30% agaropectin ( 4 4 ) , (141). It is used as a mild laxative and for the production of suppositories, emulsions, ointments e t c . . Agar serves in bacteriology as a substratum for the cultivation of microorganisms. The polygalactosides do not decompose under the action of microorganisms. In bacteriological technology, agar has the advantage that agar plates, unlike gelatine plates, return to a solid state after repeated sterilization (157), (172). The effects on antibiotics of agar concentration, agar thickness, and medium constituents have been studied. Agar concentration was found to influence the assay sensitivity of antibiotics (166). The structure and properties of agar are described in accordance with the guidelines of the European Community for stabilizers (182). Agarose is used in electrophoresis, gel filtration, and as a molecular sieve for seperating substances of a high molecular weight (141). Its use in immunology is very interesting ( 4 4 ) .

Carrageenan

Carrageenan is a mixture of polysaccharides. It has been fractionated into kappa- and lambda-carrageenan, but it appears that the composition is in fact much more complex than this (141). Today we also know of iota-, myand ypsilon-carrageenan (141). Carrageenan has been found to be an anticoagulant and antithrombotic agent. Its use in ulcer therapy and also in the treatment of gastric and duodenal ulcers has been studied.

7 Because its effects resemble those heparin, carrageenan will be used as a prophylactic agent in arteriosclerosis and in infarction therapy ( 4 4 ) ,

(141),

( 1 7 2 ) . See also Tveter-Gallager et al. in this monograph.

Products of the Phaeophyta Of the products of the Phaeophyta-alginic acid, fucoidan, ascophyllan, laminaran and mannitol-alginic acid and alginates are the most significant. Alginic acid is usually used commercially as salts of sodium, calcium, ammonium, or potassium. New applications have arisen and demand is increasing ( 7 ) , ( 8 ) ,

(141).

Free alginic acid swells in water without dissolving. Alginic acid forms an acid-binding protection film upon the contents of the stomach, preventing inflammation and indigestion ( 1 7 2 ) . Na-, K - , Mg-, and NH^-alginates form viscous solutions when placed in water; however, they do not form gels. Solid, water-insoluble colloids are produced with the addition of calcium. Alginates are used as emulsifiers in emulsions with fats and oils, as stabilizers, as a suspension or tablet swelling agent, and in ointments. Ca-alginate is used to stop bleeding. Being resorbable, alginates form the basis of dressings which can be left i n . t h e wound ( 1 7 2 ) . Calcium alginate is reputed to be a hemostatic agent which stimulates the clotting of blood in situ and is absorbed in the tissue ( 4 4 ) . Sodium alginate is a useful adjuvant in immunization against two strains of influenza virus ( 4 4 ) . Alginate-retard tablets can be manufactured using the alginate-retard process with the usual accessory ingredients, which are free from synthetic resins and polymerisates. The tablets are capable of liberating the active ingredients in such a way that an initial dose and maintenace doses are obtained ( 1 0 9 ) .

8 The influence of the type of alginic acid on remedies for indigestion has been researched on the basis of clinical observations. The alginic acid from different species of brown algae show variations in their biological availability and their biopharmaceutical properties. Their characteristic properties, such as affinity to anorganic ions, viscosity, and molecular weight, were determined (181). The structure and qualities of sodium-, potassium-, and other alginates are described according to European Community guidelines (182).

Fucoidan

This natural, non-toxic polyelectrolyte occurs in nearly all brown algae. Fucoidan is a suitable binding agent for lead and the incidental binding of calcium is an important criterion for biological and clinical application. The binding of calcium and magnesium is undesirable. Fucoidan can be used therapeutically to suppress intestinal absorption of lead and prevent poisoning. Further studies related to other essential ions in the body, such as Mn, Cu, and Zn, are in progress (140).

Laminaran

Laminaran sulfate, which is formed with two sulfate groups per glucose unit, provides maximum stability and anticoagulant activity. The lower sulfated laminaran has antilipemic activity (44).

Constituents of Marine Algae

Besides the products agar, carrageenan, alginic acid and alginates, algae have numerous constituents which are attracting increased attention in pharmacy and other fields. Algal constituents include acids, alkaloids, and amines, antibacterial, antibiotic, antifungal, and antiviral substances, c e l lulose, enzymes, floridean starch, glycosides, inorganic substances, lipids, sterols, steroids, fatty acids, phenolic compounds (tanning substances), phytohormones, pigments, proteins, peptides, amino acids, sugar alcohols,

9 vitamins, as well as volatile constituents and toxic substances. All of these constituents are described in "Marine Algae in Pharmaceutical Science" 1979 (12). Natural products and biological substances with therapeutical effect are of increasing interest. Also, research has been conducted into a number of marine algae. The pages that follow report on new knowledge in this field.

Antibacterial,

Antibiotic, Antifungal,

and A n t i v i r a l

Substances

Several papers on this topic have recently been published. Glombitza, in a 19.78 lecture, presented a survey of this subject ( 8 1 ) . Brominated phenols have been isolated from a number of red algae. The brominated phenols generally show antibiotic activity (69). Brominated compounds, especially bromophenols, are toxic ( 4 4 ) . Polyhalogenated indols have been isolated from different marine organisms. Extracts of Rhodophyllis membranacea show strong antifungal effects ( 5 3 ) . Diterpene ketols, with eleganolone as their main compound and with antimicrobial activity, have been isolated from Bifurcaria bifurcata. Cystoseira elegans, for instance, also contains these ketols, but it is not certain whether they have therapeutic value (46). The polyphenols, trifuhalol and heptafuhalol, have been identified in Halidris siliquosa. A number of other phlorotannin oligomers could be found and partially characterized (82). Screening programs to discover bacterial, antibiotic and similar substances in marine algae have been undertaken in many parts of the world; see also Sreenivasa Rao et al. and Screenivasa Rao and Shelat in this monograph. Chilean macroscopic seaweeds (Chlorophyta, Phaeophyta, and Rhodophyta) have been assayed for their antibacterial activity and the results are reported. Some degree of antibacterial activity was found to be present in 17 of 33 seaweed extracts ( 8 8 ) .

Antimicrobial activities against different classes of

10 microorganisms in more than 60 species of marine algae from Eastern Sicily were tested. A number of them, e. g. Dictyota dichotoma, Cystoseira elegans, Laurencia obtusa show activity against Escherichia coli and other test microorganisms (55). New data was presented in another paper. Lipid extracts of algae, again from Easter Sicily, were tested for antimicrobial activity against Bacillus subtilis and Phoma tracheiphila, and for antiviral activity against tobacco mosaic virus. The Phoma fungus causes one of the most destructive diseases in citrus trees. Zanardinia prototypus and Cystoseira balearica exhibit the strongest antimicrobial and antiviral activity; Lophocladia lallemandii, while inactive against the bacterium, had a highly inhibitory effect against the virus (57). 29 further species of Mediterranean algae, some of them quite rare and growing at great depths, have also been studied. Several lipid extracts have proved efficacious against tobacco mosaic virus. The authors suggest that this exploratory study should be followed up by an inter-disciplinary effort intended to identify active compounds possessing potential economic properties (56). Chemo-therapic activity was determined in polyhalogenated terpenes from Spanish algae. The study was undertaken on the cytostatic and antibacterial activities of the polyhalogenated monoterpenes of Plocamium cartila gineum and the polyhalogenated sesquiterpenes from Laurencia obtusa and L. caespitosa. The results were published at the International Research Congress on Natural Products as Medicinal Agents, Strasbourg, 1980 (83). About 90 species of seaweeds from the French Atlantic coast have been investigated for antibacterial and antifungal activity. The largest proportion of active species occurs in Cystoseiraceae, Bonnemaisoniaceae and Rhodomelaceae (45). About 80 species of marine algae from the French Atlantic coast have been investigated in vitro for potential antimitotic properties. Some of the most interesting extracts have also been examinated for cytotoxicity and for antimetabolite properties. The results are discussed in (61).

11

The antibacterial activity of some freshwater green algae was assayed for activity against growth of bacteria. The extracts of 14 algae exhibited significant inhibitory activity against either Staphylococcus aureus or Bacillus subtilis, or both (66). Extracts of 50 species of British marine algae have been tested for antiinfluenza virus activity on inhibition of influenza neuraminidase. Significant inhibition was revealed in extracts from only six species.

Enzymes

The ionic requirements of alginate-modifying enzymes in Pelvetia canaliculata were determined. The results indicate that ionic composition may be an important factor for the mode of enzymic modification of alginate (118).

Flavonoids

These substances, with their complex structure, are present in different algae.

Haemagglutinins

Some marine algae have been tested for haemagglutinins, in particular Ptilota filicina, P.. serrata, Neoptilota californica, N. densa, Diapse ptilota. The extracts are shown to have the ability to agglutinate erythrocytes, but this activity is non-specific. An additional 49 marine algae, 7 supralittoral lichens, .2 marine grasses, and 28 marine animals have been examined for the presence of haemagglutinins. The results are tabulated in (146).

Lipids

20-carbon polyunsaturated fatty acids are biological precursors in the synthesis of prostaglandins, with possible medical applications in fertility control, labor induction, abortion, and reproductive biology. "The possibilities of the presence of prostaglandins in marine algae is worthy of research." (Patterson (141)).

12 The lipid content, lipid classes and fatty acid patterns in Laminaria pallida, Ecklonia maxima, and Macrocystis angustifolia from the west coast of South Africa were determined. The lipid content was found to be low (0,4% to 0,7% fresh weight). Fatty acid composition was analysed. The fact that polar lipids are dominant in the three kelps shows that most of the fat is structural and used in cellular membranes. The neutral lipids (triglycerides and free fatty acids) are generally higher in the blades and indicate storage of lipids and their possible use in metabolic pathways (169). See also Pohl and Zurheide in this monograph. 1-Methyllisoguanosine with muscle relaxing and antiinflammation effects, was isolated from Rhodophyta (174).

Phytohormones

Augier reported in "Botanica Marina" from 1976-8 on "Les hormones des algues" (36). We will have occasion again to refer to this paper, with its more than 800 references. The presence of growth regulatory substances in seaweeds was investigated. Gibberellin activity was found in seaweed extracts. The seasonal variations in Ascophyllum nodosum, Fucus serratus, F. vesiculosus, Laminaria digitata, L. saccharina, and L. hyperborea were examined (178). From Sargassum polycystum extracts from the Philippines the auxin activity was determined by the A vena coleoptile elongation and other tests (160). Seaweed extracts are characterised by their high cytokinin activity. Foliar application of an aqueous seaweed extract to sugar beet produced significant increases in the root weight, root sugar content and in clarified juice purity (50). The effects of post-harvest treatment of vegetables and fruits were investigated (49).

13 Polysaccharides

The most important polysaccharides of algae are agar, carrageenan, and furcellaran from red algae and alginic acid from brown algae. A number of brown algae contain fucoidan (12). But many other polysaccharides are present in algae. More work is needed on chemical structure and separation of fractions (141). (See also Mori et al. in this monograph.)

Proteins

Proteins make up approx. 60-70% of the substances of marine algae which include nitrogen. They are important for the use of algae in nutrition and food. But further studies of algal proteins are necessary. The percentage of digestible dry matter (in vitro) and crude protein concentrations were also studied in 40 species of aquatic weeds. Concentrations in some weeds were comparable to those of conventional forage species (52).

Sterols

Chlorophyta contain the following sterols: chondrillasterol, poriferasterol, 28-isofucosterol, ergosterol, cholesterol (44). Phaeophyta

The dominant sterol is fucosterol. Traces of cholesterol and biosynthetic precursors of fucosterol were also found. Brown algae also contain 24methylene cholesterol or saringosterol (44), (141). Rhodophyta

contain primarily cholesterol (44), (141). Sterols from marine algae are reported to be nontoxic and have the ability to reduce blood cholesterol level. They are also reported.to reduce the tendency to accumulate liver fats and excessive fat deposition in the heart.

14 T r a c e elements

The great significance of trace elements for human nutrition has repeatedly been confirmed in recent years by the acquisition of new knowledge. The identification of all elements is important, but it is not certain whether the total number of essential elements has already been established (120). Thus there is all the more reason to conduct thorough and painstaking r e search into the minerals and trace elements of marine algae, which contain these substances to a high degree. A review was published as early as 1953 by Vinogradow in "The Elementary Chemical Composition of Marine Organisms" (171). Munda determined the concentrations of the trace elements Co, Cu, Zn, and Mn in red and brown algae from the Icelandic coast. The results are presented in a table (132). The bromo-compounds from marine algae reportedly display interesting biological activities. As part of a project on biodynamic compounds from marine flora and fauna, bromine content was examined in some seaweeds from the central west coast of India. Chondria armata with 0.4% and Codium elongatum with 0.247% showed relatively high bromine content (134). Some species of marine algae from the coast of Goa, India, were analysed for Cu, Co, Fe, Mn, Ni, Pb, and Zn. Since the algae are of major importance as primary producers, their significance as metal concentrating agents cannot be overestimated. Primary producers are consumed directly or indirectly as detritus by fish and invertebrates, transferring their metal content to the next link in the food chain.. Trace metal accumulation in the seaweeds of the coast of Goa, well-known for their luxuriant growth, is in no way alarming at present ( 3 3 ) . But in other coastal areas, in the neighborhood of large industrial plants, the problem of the pollution is considerably more difficult. Therefore further studies are necessary, especially concerning cultivation of marine algae.

15 Toxic

Substances

Research into toxic substances in marine organisms has mainly centered on unicellular forms of algae and marin animals. Reports on the fastigiatin of Pelvetia fastigiata and the caulerpicin of Caulerpa racemosa and others has been published (69). Hallucinogenic substances have not yet been found in marine algae (151).

D i f f e r e n t Constituents of C h l o r o p h y t a ,

Phaeophyta,

R h o d o p h y t a and other

Algae

Chlorophyta

Caulerpa C. brownii contains retinol, a monocyclic diterpene (69). C. lamourouxii, Philippines, also cultivated, contains caulerpicin (phen-) azine derivative), caulerpin, sterols, palmitic acid (44), (69). The upper branches are eaten as a salad, despite their peppery and adstringent taste. Toxic for some individuals. C. proliféra, Mediterranean Sea. Caulerpenyn, acetylenes containing sesquiterpen, was isolated (34). C. racemosa contains caulerpicin. C. racemosa var. clavifera contains caulerpin (44). C. racemosa, Indian coasts. Extracts have shown hypotensive activity (135). Ç. sertularioides contains caulerpin (44). C. simpliciuscula. The exogenous and endogenous amino acids have been studied (156). Chlamydomonas Ch. reinhardtii. The antibiotic fraction is considered to be a mixture of fatty acids (44). Chorella Ch. vulgaris, Ch. ellipsoidea, Ch. saccharophila contain chondrillasterol and other sterols (44).

16 Cladophora C. pinnulata, Indian coasts. Extracts are toxic (135). Codium C. elongatum, Indian coasts. Extracts have shown antiviral activity (135). Cymopolia C. barbata, a calcareus green algae, contains an unusual group of aromatic compounds, the simplest being cymopol (69). Enteromorpha E. species, Indian coasts. Extracts have shown diuretic activity (135). Ulva U. fasciata, Indian coasts (Saurashtra). Amino acid composition in protein hydrolyzates has been studied. 21 amino acids have been detected. Aspartic acid is a dominant compound (65). U. lactuca, Indian coasts (Port Okha). Contents consisting of protein, fat, carbohydrates, sodium, potassium, calcium, phospherous, nitrogen have been studied. Potential resource for food, fodder and fertilizer (158). U. lactuca was tested with various concentrations of cadmium. Ulva accumulates considerable amounts of Cd. Its potential use for monitoring Cd pollution in coastal areas is discussed (111). U. profunda, U. rigida, U. stenophylla have also been studied (65).

Phaeophyta Ascophyllum A . nodosum. Jones has shown that toxic compounds were not extracted with chloroform, ethanol, water, and 20% sodium carbonate solution, but remain in the insoluble residue.

A . nodosum is used in fodder for sheep,

cattle, pigs and poultry as an additive source of minerals and vitamins. Since no toxic effects have been recorded, it would seem that the haemorrhaging in rabbits is atypical (101). The mercury content in the thallus of A . nodosum and agricultural products of seaweeds has been investigated (37). Stahl and Schild, in their "Drogenanalyse" (163), present exact details on the analysis of the drugs Ascophyllum nodosum and Fucus vesiculosus, discussing their purity and con-

17 stituents as well as examining the pharmacopeis and indicating their effect and use. Deutschmann et al. (67) describe anatomy and morphology. Cephalocystis C. species. The crude extract contains about 30% 6-tridecylsalicylic acid. The effect corresponds to salicylic acid (144). Cystoseira C. barbata, Turkish coasts. The sterol was identified as fucosterol (84). The structure of the thallus of C. barbata from the Crimean shore has been studied (73). Research has been made into ontogenic trends in morphology and morphophysiological analysis (106). C. corniculata, Turkish coasts of the Mediterranean Sea. The pharmacological activities of protein fractions have been studied. The lipolytic and the hypoglycemic activity is shown in (84), (85). C. crinita, Aegean, Mediterranean, and Black Sea, contains laminaran, mannitol, approx. 27% alginic acid, amino acids, oxocrinol, crinitol (terpenoids), fucosterol, vitamin B ^ - Its pharmacological activities have been studied (69), (84). Dictyopteris D. australis Sulfur-containing lipids have been isolated (69). Lipids may contain interesting functional groups. D. divaricata contains dictyopterol (69). D. plagiogramma. Sulfur-containing lipids have been isolated (69). D. undulata (formerly D. zonarioides ) contains zonarol, isozonarol, zonarene (sesquiterpene derivative) (69). D. species contain dictyopterenes and related unsaturated hydrocarbons (141). Dictyota D. dichotoma. Dictyol A and B, pachydictyol and other related compounds have been isolated (69). D. acutiloba and D. flabellata have also been studied (69). Dilophus D. crenulatus contains acetoxycrenulatin, different from other diterpenes (69). D. ligulatus contains dilophol (monocyclic compound) (69). Ectocarpus E. siliculosus. The sex attractant produced by the female gametes has been isolated. Hormone-like compounds in algae may affect the animals (141).

18 Fucus F. vesiculosus. The utilization in obesity preparations is not without problems, because the drug is not harmless (31). F . vesiculosus is used in the homoeopathy (157). Halidrys H. siliquosa. In addition to the polyphenols, trifuhalol and heptafuhalol, previously identified in the alga, a number of other phlorotannin oligomers could be found and partially characterized through their acetyl derivatives (82). Heterochordaria H. abietina. Extracts contain agents with hypotensive properties. The laminine is a choline-like basic amino acid (44). Laminaria L. angustata. Extracts contain agents with hypotensive properties. Laminine, a choline-like basic amino acid, suppressed heart action and depressed blood pressure (77). Laminine monocitrate has a transitory hypotensive effect (141). The hypotensive principle of L. japonica, commercial "Kombu" preparations, and other Laminariaceae has been studied. "Kombu" preparations have been employed as a hypotensive drug in the folk medicine of Japan. The preparation "ne-Kombu", the basal parts of the blades, has been reputed to be most effective. Two of the commercial preparations contained large quantities of histamine (0.4 and 0.11%) (77). L. saccharina was tested with various concentrations of cadmium. L. saccharina produces a considerable accumulation of Cd. The potential use for monitoring Cd pollution on coastal areas is discussed (111).

L ^ species

are employed in China in the treatment of goiter and other iodine deficiencies (24). Nereocystis N. luetkeana. Pacific coast of Canada. A systematic examination was undertaken. The alga has been analyzed for solid matter, total ash, insoluble ash, potassium, sodium, magnesium, calcium, strontium, iron, aluminum, zinc, barium, mananese, chromium, copper, cadmium, lead, cobalt, and mercury. The seasonal variations and the concentration differences between the fronds and stipes are presented, also the seasonal fluctuations in the halogens nitrate, silica, sulphur, carbonate, phosphorus, and boron contents (176).

19 Padina P . tetrastomatica, Indian coasts. Extracts have shown spasmogenic and antifertility activity (135). Sargassum S. siliquastrum, Chinese coasts. Used in China for iodine deficiencies, also as a diuretic (24), (127). S. swartzii, Indian coasts (Port Okha). Contents consisting of protein, fat, carbohydrates, sodium, potassium, calcium, phospherous, and nitrogen were studied. Potential alginophyte (158). S. tenerrimum, Indian coasts. Extracts have shown CNS depressant activity (135). S. thunbergii, coasts of Japan. The antitumor activity of a polysaccharide fraction has been studied (98). S. tortile. The active fraction contains delta-tocotrienol and the corresponding epoxide (69). S. muticum has now become firmly established on the south coast of England. T h e ' l i f e history of the alga has been studied (74). Stoechospermum St. marginatum, Indian coasts. Extracts have shown spasmolytic activity (135). Stypopodium St. zonale, tropical waters, contains the substance stypoldion, a bright red ortho-chinon, which dyes the surrounding water rust-coloured and is toxic for fish. Moreover, there was isolated stypotriol (80). Taonia T . atomaria (= Dictyota atomaria) contains Taondiol, pentacyclic isomer of delt-tocotrienol (69). Taondiol is a derivative of tocopherol (44).

Rhodophyta

Acanthophora A . spicifera, coasts of Philippines (Batangas Province). An edible seaweed which is eaten in the Philippines as a salad. On the basis of the infrared spectrum, it can be concluded that the polysaccharide is a lambda-carrageenan which is structurally modified by alkali treatment (58). A . spicifera, Indian coasts. Extracts have shown antifertility activity (135).

20 Anatheca A. montanei, coasts of Senegal, West Africa ( d r i f t i n g ) . The phycoclloid content was 33.7%, mainly iota-carrageenan. The sulfate content was 25.8% (124). Asparagopsis A. taxiformis contains polyhalogenated acetones and other polyhalogenated compounds, some possibly toxic, as well as volatily constituents. A. taxiformis is eaten by the Hawaiians (69). Bonnemaisonia B. hamifera and B. nootkana contain halogenated heptane derivatives (69). Extracts of Bonnemaisoniaceae contain phloroglucin and tocotrienol ( 144). Chondria Ch. armata contains domoic acid, anthelmintic agent, effective in expelling ascaries and pinworm without observable side effects (44). Extracts contain agents with hypotensive properties. Laminine, a cholinelike basic amino acid, has been isolated (44), (135). Ch. californica contains cylic polysulfides. The major component is a sulfone, which is antibiotic (69). Chondrococcus Ch. hornemanni, Japan, Hawai, contains halogenated monoterpenes. The major monoterpene constituent from Hawaian alga was chondrocole (69). Chondrus Ch. crispus and also Gelidium cartilagineum have been shown to posses antiviral properties attributed to the galactan units against influenza B and mumps virus in embryonated eggs (44), (78). Ch. yendoi, coasts of Japan (Hakodate). The isolation and s t r u c t u r a l elucidation of palythine has been described. Shinorine, a new amino acid, has been isolated (168). Corallina Extracts of C^ species from Indian coasts have shown anthelmintic activity (44), (135). Delisea D. fimbriata contains halogenated lactones called fimbrolides which are responsible for the antibiotic activity (69).

21 Digenea D. simplex contains kainic acids and isomeres such as alpha-allokainic acid and alpha-kainic acid lactone. Alpha-kainic acid is the most active compound with anthelmintic properties, effective in the treatment of ascariasis. Digenea simplex is also employed as a laxative (44), (24). Eucheuma Investigations have been conducted on the morphology and distribution ecology of

species, coast of Tanzania.

E. spinosum (E. s e r r a ) . This species showed the highest frequency of occurrence on the shores of small islands. Rich content of the phycocolloid carrageenan. E. striata, abundant on the coasts of Tanzania. Also labeled E. cottoni by Tanzanian e x p o r t e r s . E. okamurai, coast of Tanzania. E. platycladum, coast of Tanzania. E. speciosum f . mauritianum, coast of Tanzania, restricted in its distribution. The demand for Eucheuma is greater than n a t u r e can supply. Technology has been developed to increase production through cultivation. The results of the study by Mshigeni will serve as a useful guide for locating habitats in Tanzania from which Eucheuma can be h a r v e s t e d , and for locating areas along the West Indian Ocean shoreline where Eucheuma aquaculture could be conducted successfully (129). E. spinosum and E. striatum. The metal-binding properties of the sulfated polysaccharides kappa- and iota-carrageenan from these

species have

been investigated with respect to the toxic metals lead and cadmium. Algal polysaccharides such as those reported in a paper by Veroy et al. are no exception, particularly in view of their possible marine ecological implications and their potential application in the therapy of heavy metal poisoning and the chelation of heavy metals in wastewater (170). Furcellaria F. lumbricalis. Extracts showed a similar pharmocological activity to that of histamine (39). Gelidiella G. acerosa, Indian coasts (Port Okha). Contents consisting of protein, f a t , c a r b o h y d r a t e s , sodium, potassium, calcium, phosphorus, and nitrogen were

22 examined. Important agarophyte (158). Extracts have shown antifertility activity (135). Gelidium G. cartilagineum. Extracts have been found to be active against influenza B and mumps virus (78). G. lingulatum. Chilean coasts. The agar was found to have the following composition: galactose 52.5 %, 3.6-anhydrogalactose 34 %, 6-0-methyl-D-galactose 7.6 %, sulphate 3.6 %, glucose 1.5 %, 2-0-methyl galactose 0.9 %, mannose 0.5 %. The composition of this agar resembles that of G. filicinum (179). G. species contain taurine, nitrogen compound (141). Gracilaria G. andersoniana contains cis-phytol ( 6 9 ) . G. bursapastoris, Island of Oahu (Hawaii). Yield and gel strength of the agar have been studied ( 9 2 ) . G. coronipifolia, Hawaii. Yield and gel strength of the agar have been studied (92). G. corticata, coast of Veraval (India). Potential source of agar. Growth, reproduction and seasonal variation in agar (14.5 to 22.5 %) and gel strength have been studied (139). G. lichenoides. Extracts contain up to 0.06 % prostaglandins Eg (144). The antihypertensive effect has been studied ( 7 9 ) . G. tikvahiae, Rhode Island. The properties of the phycocolloid have been examined. The anhydrogalactose content ranged from 34 to 43 %. It is important to examine the gel-regulating

factors

( i . e. sulfate and 3,6-anhy-

drogalactose) from different sources and localities. Strong gels may be obtained during spring and early summer ( 3 5 ) . (See also Sakagami et al. in this monograph.) Gracilariopsis G. sjoestedtii, British Columbia, has been recorded for agar production (177). Gymnogongrus G. flabelliformis contains the amino acids gigartinine and gongrine (141). Halosaccion H. ramentaceum contains sterols. The main sterol is desmosterol ( 4 4 ) .

23 Hypnea H. species. Abundant on Indian coasts. H. musciformis, Veraval on the West coast of India. Extracts have shown diuretic activity (135). The alga contains sterols. 22-dehydrocholesterol is the major sterol (44). H. valentiae, Mandapam on the South Indian Coast. Seasonal variation in the yield and gel s t r e n g t h of the phycocolloids of Indian H^ species have been studied (143). H. japonica contains 22-dehydrocholesterol (44). Iridaea (Iridophycus) The antibacterial activity present in the genus is ascribed to acrylic acid (44). Laurencia The genus Laurencia has yielded a variety of halogenated sesquiterpenes, diterpenes, and acetylenes. The antibiotic activity of L

species is due to

the phenolic sesquiterpenes such as laurinterol or debromolaurinteroL The phenols and isomers are also antibiotic (69), (90), (91), (107). L^ species contain acetylenes. A series of acetylenes containing both bromine and chlorine has been described. Obtusenyn, a brownish oil, has been isolated from L^ species (107). Rhodophytin is an oily halogenated vinylperoxide (70). It was difficult to elucidate the s t r u c t u r e of laureatin, isolaureatin, laurefucin and other substances in Laurencia species. However, it proved possible with X-ray analysis. Isolaureatin is the most active substance. Scientists from the Institute of Marine Pharmacology, Oklahoma, found out that the Laurencia-substances inhibit pentobarbital-metabolism and prolong sleep. These compounds are of clinical i n t e r e s t . They are of little toxicity (42), (104), (141). L. caespitosa caespitol, isocaespitol, bisabolene-based metabolites (69). L. caraibica contains diterpenes, sesquiterpenes and non-isoprenoids. Caraibical, sesquiterpenaldehyde, was isolated (99), (100). L. cocinna contains concinndiol (69). L. decidua, Baja California. The organic halogen content was determined. Extracts showed antimicrobial activity (54). L. elata contains 0.8 % sesquiterpenalcohol elatol (154).

24 L. glandulifera contains the brominated oxygen heterocycles laurencin, laureatin, isolaureatin, laurene (a sesquiterpene hydrocarbon), and spirolaurenone ( 4 4 ) , (69), (93); 3 isomers of spirolaurenone (161); and glanduliferol, a sesquiterpenalcohol, identical with 4-bromo-alpha-chamigren-8,9epoxide (262). Alpha-bromocuparene and alpha-isobromocuparene are precursors of laurene and other aromatic sesquiterpenes from L^ species ( 6 9 ) . L. intermedia contains the sesquiterpenoid bromine laurinterol, as well as dibromlaurinterol and isolaurinterol ( 4 4 ) ,

(97).

L. intricata contains preintricatol, a bisabolene-based metabolite ( 6 9 ) . L. nidifica. A green variety from Hawaii contains halogenated acetylenes (69). L. nipponica contains the brominated compounds laurencin, laureatin, isolaureatin , lauresinol, laurefucin, acetyl-laurefucin, isoprelaurefucin, cisand trans-laurediol, alpha-bromocuparene, alpha-isobromocuparene, laurellen (44), (69), (76), (94), (95), (96),

(113).

L. obtusa contains chamigrenes ( 6 9 ) . An acetylcholine-like compound has been detected in extracts (40). L. okamurai is rich in nicotinic acid, pantothenic acid, folic acid and cobalamin (167). L. pacifica contains pacifenol, halogenated chamigrene derivative ( 6 9 ) ,

(71),

(86) and paciferol, a sesquiterpene containing bromine and chlorine ( 4 4 ) . L. papillosa. The genus Laurencia seems to be of interest as a possible source of phycocolloids. Content and nature of the phycocolloid from L. papillosa of Tanzanian shores has been investigated. A yield of approx. 33 %, with peaks akin to lambda-carrageenan, has been obtained on a dry weight basis (Mshigeni). L. perforata contains unusual sesquiterpenes, including perforatone ( 6 9 ) . L. pinnatifida. The structure of galactan has been examined ( 4 1 ) . L. snyderae. Alpha- and beta-snyderol, bromo monocyclic sesquiterpenes and concinndiol have been isolated ( 6 9 ) ,

(91).

L. spectabilis. A glycoprotein has been extracted. It contained approx. 82 % carbohydrate and 18 % protein. The main sugar was galactose. The amino acids have been examined ( 6 4 ) . L. subopposita contains acetylenes, aromatic sesquiterpenes, sesquiterpene alcohols. Oppositol has been isolated ( 6 9 ) ,

(86).

25 L. yamada. Chondria oppositiclada has been reclassified as L. yamada. The alga contains chondriol and rhodophytin, one of the most interesting of the halogenated acetylenic ethers ( 6 9 ) . Liagora L. farinosa contains lipids with groups of toxic acetylenes (142). Neogardhiella N. baileyi, Rhode Island. The properties of the phycocolloid from N. baileyi, a carrageenophyte, have been examined. The anhydrogalactose content ranged from 15 to 30 %. Strong gels would be obtained in winter and possible in early summer ( 3 5 ) . Odonthalia Extracts of O^ species contain bromophenolic compounds (141). Palmaria P. palmaria ( L . ) O. Kuntze (= Rhodymenia palmata ( L . ) G r e v . ) . In 1980 Morgan et al. published a review of chemical constituents of the alga (125). Dry weight, ash, minerals and trace elements, vitamins, nitrogenous constituents, .amino acid composition, carbohydrates, lipid content and fatty acids, sterols (cholesterol and demosterol ( 4 4 ) ) , hydrcarbon content, and pigments have been determined. A comparison of vitamin, mineral contents and essential amino acids of P. palmata and other foods was undertaken (approx. 150 references). The alga has been used for centuries in coastal areas of Europe (Great Britain, Ireland, Iceland, Norway) and more recently in northeastern North America as a food source. P. palamata contains 20 - 25 % protein on a dry weight basis and significant quantities of several vitamins and minerals. For the cultivation of P. palmata, the effect of light intensity and temperature on growth and chemical composition was tested in flowing seawater in tanks in a greenhouse (126). Variations in the content of nitrogen compounds in P. palmata from the French Atlantic coast have been examined ( 5 9 ) . Phyllophora Ph. truncata, Swedish West Coast. A paper by Westlund et al. concerns the localization of bromine and iodine at the ultrastructural level in Ph. species. The distribution of halogenated compounds appears to be very similar to the distribution of the bromine found in Odonthalia dentata and Lenormandia prolifera (175).

26 Plocamium P. cartigalineum, California, contains a mixture of halogenated monoterpenes ; major constituent cartilaginal in 3 different "chemical types" (69), (144). P. costatum contains monocyclic halogenated monoterpenes (69). Costatol has a sedative effect (144). P. violaceum , California, contains monocyclic halogenated monoterpenes (69). Polysiphonia P. lanosa contains brominated phenols (44), (141). Porphyra P. p u r p u r e a and P. umbilicalis contain sterols. The main sterol is desmosterol (44). P. thulaea. A new species from East Iceland and West Greenland is described. Chemical analyses demonstrated that the protein content is highest among Islandic Porphyra species (36.93 g/100 g d r y weight). The amino acids were determined (133). P. species. The nitrogen compound taurine and several other related compounds have been isolated (144). (See also Sakagami et al in this monograph.) Ptilota P. plumosa contains a specific group B haemagglutinin. Rogers and Blunden present information on the general s t r u c t u r a l properties of the anti-Blectin. Lectins have been shown to be extremely useful reagents for the study of erythrocyte and tumor cell s u r f a c e s . It would appear that marine algae are proving to be a f r u i t f u l unexploited source of these interesting and potentially important molecules (145). P. species contain the amino acid taurine and related compounds (141). Some P^ species are mentioned for Argentina: P. a t r o p u r p u r e a , P. capensis, P. columbina, P. endiviifolium, P. pujalsii, P. sanjuanensis, P. t a s a , P• umbilicalis, P. woolhousiae. A new species P. argentinensis is described (183). Rhabdonia R. africana, shores of Tanzania. Phycocolloid yield approx. 31% with a total sulfate content of 18.6%. Possible source of iota-carrageenan (130). Rhodomela R. larix contains brominated phenols (44), (141).

27 Rhodophyllis R . membranaeea contains polyhalogenated indols with an antifungal effect (53). Rytiphlaea R . tinctoria, Mediterranean Sea, contains lanosol and its ethyl ether. The alga from the Atlantic coast of France contains dibromophloroglucinol and a tetrabromo compound (69). Sphaerococcus Sp. coronopifolius contains brominated diterpenes, sphaerococcenol and bromosphaerol ( 6 9 ) . Cyanophyceae - Euglenaceae Trichodesmium T . erythraeum, Indian coasts. Extracts have shown diuretic activity (135). Euglena E. gracilis. The sterol composition was investigated in mass culture of Euglena gracilis grown on animal wastes for the production of single-cell protein. Ergosterol was identified as the major sterol (0.09%) (165).

Some special Possibilities for the Use of Algae

Not only do marine algae constitute raw material for pharmaceutical and technical products, they are also of interest for analysing some important problems (112). The development of sensitive instruments for routine analysis of stable isotopes opens new broad fields in the application of stable isotopes in all biosiences and in chemistry. Stable labeled organic basic compounds of metabolism are prepared with the aid of autotrophic algal cultures, using selected species of green and blue algae (102). Some constituents of medicinal plants and plant products are natural mutagens. Results of experiments with furocoumarins and furochromones have been reported in the literature. These results were obtained through an algal test system using an arginin-requiring mutant of Chlamydomonas reinhardii (147), (148),

(149).

28 In 1977 Lange-Bertalot published a paper "Algen ersetzen Messgerate" (114). Some species of submicroscopic but ecologically significant diatoms form a characteristic association, especially in heavily polluted r i v e r s . Their development is blocked, however, under the influence of excessive industrial wastewater or insufficient water purification. They are replaced by even more resistent algae. A number of p a p e r s about the biologically active s u b stances have been published recently. But new methods of analysis have been developed as well. A screening procedure for biological activity in marine macroalgae: gastroped tentacle withdrawl. This study utilizes one type of behavioral assay to test extracts from species of subtropical macroalgae for biological activity. These results are conpared with the results from four nonbehavioral marine bioassays (164).

Algae as Indicators of heavy Metal Pollution

The role of different seaweeds as indicators of metal pollution in coastal areas has recently been discussed in relation to the degree of accumulation of metals. Algae contain traces of all the elements present in their environment , with detection being limited only by the sensivity of analytical p r o cedures . Algae, being accumulators of minerals, can therefore act as indicators of extent mineralization in their habitat. Algae can accumulate high levels of metals

(between 100 - 1,000 times). Euglena can be used as an indicator,

as can Chlorella (72). With increasing industrialization, the contamination of our rivers and seacoasts by toxic substances such as lead and cadmium is getting more and more dangerous. The effect of lead compounds on primary producers such as Chlorophyta has been studied. These Chlorophyta form the basic nutrition of numerous consumers and can damage them if they are enriched with toxic substances (173).

29 Thanks to the rapid pace of industrialization, the problem of pollution of the aquatic enviroment has arisen. This in turn has provoked studies on the extent of trace metal pollution, for instance Cd, Cr, Fe, Pb and Zn, in waters as well as bottom sediments where the benthic marine algae thrive, and attempts to determine the biodeposited concentrations. As a result of these investigations, it was proposed that certain algal species be employed as indicators for pollution studies (155). Quite recently it was again pointed out that lichens too have been used as bioindicators to detect enviromental pollution. To this end, their property of reacting to certain pollutants with characteristic changes of growth is exploited. With this biological method, continual, sufficiently sensitive measurements over wide areas can often be carried out more simply than with expensive, complicated technical equipment (180). Marine algae are also used as raw materials for some other applicants. Chaetomorpha linum, Turkish coasts. Cellulose content of the pulp was 36.5%. Its papermaking properties have been investigated (108). The cellulose content of Ch. melagonium is 41%. The cellulose content of Ceramium rubrum (18.5%), Hypnea musciformis (11.4%) and other seaweeds have also been investigated (108). Dunaliella bardawil, coast of the Sinai peninsula

contains approx. 40% gly-

cerine in its dry weight, approx. 8% beta-carotene. The protein of the alga can be used for food ( 3 8 ) . Cryptopleura violacea, northern California coast, is a source of natural dye for wool yarn in homecrafts and cottage industry. Iridaea cordata, Odonthalia floccosa, Pelvetiopsis limitata, Polysiphonia paniculata, Porphyra lanceolata, Prionitis lyalli, Rhodoglossum oweniae, Rhodomela larix, and Schizymenia pacifica were also examined for use as dye stuffs. Five common mordants were used: ferrous sulfate, stannous chloride, aluminum potassium sulfate, copper sulfate, potassium dichromate (137). See "Pigments" in "Marine Algae in Pharmaceutical Science" 1979 ( 1 2 ) .

30 Marine Algae as a Source of E n e r g y

The problem of energy production is not a direct part of this survey, but as it is of urgent significance in the world today it is not out of place to mention it here. International experts in the field of seaweed research discussed this problem at the Oregon State University Symposium, March 1977 (25). Valuable suggestions were made. To qoute Krauss: "Much can be said about the potential use for marine plants to solve the energy crisis. The major problem is how to generate the biomass and mannage a sustained yield from it" (11). In this context, the former kelp industry should be mentioned. Brown seaweeds on the coast of California were made to yield acetone and other solvents , such as ethyl acetate .and other ethyl compounds, during the First World War. This industry had a considerable scope. Production was later discontinued in the USA. In the USSR, however, the production of alcohol, acetone, and organic solvents has continued (See Levring-Hoppe-Schmid "Marine Algae - A Survey of Research and Utilization", 1969). The "Central Salt and Marine Chemicals Research Institute", Bhavnagar, India (159) is engaged in the utilization of seaweeds to produce biogas by the activity of specific bacterial strains isolated from them, and to provide information necessary for understanding the operational factors involved. Bernhardt et al. have reported on the prospects and limits of fuels from plants (43). The Energy Research and New Technologies Department at Volkswagenwerk AG is intensively engaged in processes for the production of alternative fuel from biomass. The alcohols ethanol and methanol are well suited in motor vehicles. Bearing in mind the conditions obtaining in individual countries and with the application of the latest biotechnology, the production of alcohol in various countries may soon become commercially viable.

31 T h e Cultivation of Marine Algae

Marine algae h a v e long been cultivated in s e v e r a l coastal a r e a s , f o r i n s t a n c e in China and J a p a n . T o d a y , h o w e v e r , t h e cultivation of algae is worldwide. This h a s led to t h e creation of a new field - a g r a r i a n g e o g r a p h y . Algae a r e highly localized. Marine algae, as c u l t i v a t e d p l a n t s , are certainly not equal in importance to t h e well-known t e r r e s t r i a l domesticated p l a n t s s u c h as r i c e , c o r n , oil f r u i t s e t c . ; b u t t h e y play an important role in n u t r i t i o n and i n d u s t r y . Numerous s t u d i e s have been made in o r d e r to t e s t t h e most a d v a n t a g e o u s n u t r i t i v e solutions, s u b s t r a t u m s , materials f o r cultivation in tanks etc. The cultivation of marine algae was one of t h e topics at t h e FAO Technical C o n f e r e n c e on A q u a c u l t u r e in Kyoto, J a p a n , 1976, where r e p o r t s were given on t h e s t a t u s and f u t u r e of seaweed c u l t u r e and algae c u l t u r e t e c h n i q u e s . The most important raw materials s u c h as Monostroma,

Chondrus,

Eucheuma, Gelidium, Gloiopeltis, Gracilaria, P o r p h y r a , Laminaria, Macroc y s t i s , Undaria a r e mentioned (68). Michanek r e p o r t e d on t r e n d s in applied p h y c o l o g y , ocean farming p r o j e c t s , g r e e n h o u s e cultivation e t c . , with i n t e r e s t i n g numerical d a t a and detailed r e f e r e n c e s (122). T h e e f f e c t s of n i t r o g e n and seawater flow r a t e on t h e growth a n d biochemical composition of Gracilaria foliifera v a r . angustissima were i n v e s t i g a t e d in outdoor c u l t u r e t a n k s . Much r e c e n t work has examined t h e e f f e c t s of n u t r i e n t concentration on t h e growth of algae. Such d a t a a r e u s e f u l f o r optimizing yields in commercial seaweed p o n d s (115). I n c r e a s i n g c o n c e n t r a t i o n s of n i t r o g e n f e r t i l i z e r h a v e led to i n c r e a s e d growth and i n t e r n a l n i t r o g e n content of Gracilaria tikvahiae and d e c r e a s e d yields of a g a r . A g a r s h a v e h i g h e r melting t e m p e r a t u r e and g r e a t e r gel s t r e n g t h s t h a n do a g a r s e x t r a c t e d from less e n r i c h e d thalli ( 4 8 ) . I n v e s t i g a t i o n s h a v e b e e n c o n d u c t e d on t h e colonization of new s u b s t r a t a a n d recolonization of d e n u d e d i n t e r t i d a l s u r f a c e s b y b e n t h i c macrophytic a l g a e .

32 Mshigeni (128) provides a summary of field observations on the growth of seaweeds. The results of his study suggest that algae such as Acanthophora spicifera and Hypnea nidifica can propagate vegetatively. These findings have interesting agronomic implications. The carrageenans are among the most important phycocolloids of Rhodophyta. As demand for them is increasing, it is necessary to supplement raw material supply by cultivation. The cultivation of Chondrus crispus is the subject of several special studies (152), (153), (47), (60). The cultivation of Eucheuma species and other agarophytes has also been described (22), (23), (119). Field and culture studies were carried out on the Porphyra species. They seem remarkable because of the increasing importance and extent of Porphyra cultivation, not only in the FarEast (138), (103). In 1973 the value of the Pophyra harvest in Japan amounted to approx. US$ 330 million from approx. 70,000 ha (25b). Porphyra or Nori cultivation combines traditional methods with modern technology. The cultivation of Iridaea cordata was studied on the west coast of North America. Iridaea cordata contains 52-66% carrasreenan ( d r y weight) (25b). Laminaria species are cultivated in particular on the coasts of Japan. Large quantities are gathered on the Chinese coasts from wild populations. But in China great efforts have been made in the cultivation of L. japonic a. Over 100,000 metric tons are produced annually. A v e r y important raw material for the production of alginic acid and alginates, but also for food and recently the generation of energy is the Giant Kelp Macrocystis. This alga forms extensive beds along the Californian coast. Of special interest is the high productivity of Macrocystis angustifolia, whose fronds elongate at a rate of up to 50 cm per day. Studies of whole plant growth have been published (63). Regrowth after harvesting is a very important problem for marine algae serving as raw materials. The regrowth of Ascophyllum nodosum and Fucus vesiculosus has been studied. A . nodosum has been harvested commercially

33 in Europe (Norway, Iceland, northern France, British Isles, Ireland), but recently also in eastern Canada and in North America (Maine) (105). Reports on the cultivation of Ascophyllum and Fucus species have been published (150). The cultivation of marine algae was also a subject of a symposium held in Oregon, USA and published in "The Marine Plant Biomass of the Pacific Northwest Coast - A Potential Economic Resource", edited by Robert W. Krauss (25). The semi-closed culture of marine algae is discussed in (25a). The cultures of edible seaweeds and others for the

SUDDIV

of phvcocolloids on artificial

substrates are described in (25b). The domestication of Macrocystis as a marine plant biomass producer is a very important problem (25c). The productivity values for cultivated seaweeds are described in (25e). Reports have been given on the farm production of Eucheuma ( 2 5 f ) . Essential considerations for establishing seaweed extraction factories are discussed in (25g). Marine plant production and utilization (25h) and engineering of structures in the ocean with a selected bibliography on marine engineering (25i) were some other topics of the Oregon symposium. At the 10th International Seaweed Symposium 1980 at

Goteborg (23), the

cultivation of marine algae was one of the most important subjects. Two plenary lectures deserve mention as examples: "Marine Phycoculture in China" by C. K. Tseng and "The Domestication and Cultivation of Californian Macroalgae" by M. Neushul. The papers cited above in this survey, dealing with cultivation of marine algae, merely provide some examples of detailed investigations being conducted in this field. The harvesting, drying, and preparation of algae, especially for pharmaceutical use, are of the same level of importance as for terrestrial medicinal plants .

34 The cultivation of marine algae opens up the utilization of wide areas of the ocean for the production of foods, phycocolloids, chemical and pharmaceutical products. These facts point to several important future oossibilities for new areas of employment and for supplying a nutritional basis in some coastal areas of the world.

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100. Izac, R. R. et a l . : Tetrahedron Letters, 22 1799 (1981); Chem. Ztg. 81 (7/8) (1981) 101. Jones. R. T . et a l . : Effects of Dietary Ascophyllum nodosum on Blood Parameters of Rats and Pigs. Botanica Marina, XXII (6) 393-394 (1979) 102. J u n g , G . ; J ü t t n e r , F . : Herstellung stabil markierter Naturstoffe mit Hilfe autotropher Algen-Kulturen und neuere Anwendungen stabiler Isotope in den Biowissenschaften. Chemiker Zeitung, 96 (11) 603-610 (1972) 103. Kapraun, D. F . , Luster, D. G . : Field and Culture Studies of Porphyra rosengurtii Coll et Cox (Rhodophyta, Bangiales) from North Carolina. Botanica Marina, XXIII (7) 449-457 (1980) 104. Kaul, P. N. et a l . : Neue Substanzen maritimen Ursprungs als Inhibitoren des Arzneimittelmetabolismus. J . Pharm, and Pharmacol. London 30 (9) 589-590 (1978); Zbl. Pharm. 118 (12) 1291 (1979) 105. Keser, M. et a l . : Regrowth of Ascophyllum nodosum and Fucus vesicuIosus under various Harvesting Regimes in Maine, USA. Botanica Marina XXIV (1) 29-38 (1981) 106. Khailov, K. M.: Ontogenetic Trends in Morphologie Parameters of Cystoseira barbata Thalli. Botanica Marina, XXII (5) 299-311 (1979) 107. King, T . J . et a l . : Obtusenyn, ein neungliedriger cyclischer Äther mit Acetylengruppierung aus Rotalgen. Tetrahedron L e t t . , 1453 (1979) Chemiker Zeitung, 103 (10) (1979) 108. Kiran, E. et a l . : Studies on Seaweeds for Paper Production. Botanica Marina, XXIII (3) 205-208 (1980)

43 109. Klaudianos, S . : Alginat-Retard-Tabletten. Deutsche Apotheker Zeitung, 118 (19) 683-684 (1978) 110. Krauss, R. W.: Marine Plants - A Renewable Resource for Mankind. In: Krauss, Robert W.: The Marine Plant Biomass. Oregon State University Press (1977) 1-7 111. Kremer, B. P . , Markham, J . W.: Meeresalgen als Kadmiumwarner. Umschau, 80 (21) 664-665 (1980) 112. Kremer, B. P . : Endosymbiontische Algen in Wirbellosen. Deutsche Apotheker Zeitung, 121 (4) (1981) 113. Kurosawa, E. et a l . : Tetrahedron Letters, 4135 (1973); Chem. Z t g . , 98 (4) (1974) 114. Lange-Bertalot, H . : Bioindikatoren: Neue Algen ersetzen Messgeräte. Umschau, 77 (19) 642-643 (1977) 115. Lapointe, B. E . , R y t h e r , J . H . : The Effects of Nitrogen and Seawater Flow Rate on the Growth and Biochemical Composition of Gracilaria foliifera v a r . angustissima in Mass Outdoor Cultures. Botanica Marina, XXII (8) 529-537 (1979) 116. Levring, T . : Potential Yields of Marine Algae with Emphasis on European Species. In: Krauss, Robert, W. ( e d ) : The Marine Plant Biomass. Oregon State University P r e s s . (1977) 251-270 117. Levring, T . : The Vegetation in the Sea. In: Hoppe-Levring-Tanaka ( e d s . ) : Marine Algae in Pharmaceutical Science. Walter de G r u y t e r , Berlin • New York (1979) 3-23 118. Madgwick, J . et a l . : Ionic Requirements of Alginate-Modifying Enzymes in the Marine Alga Pelvetia canaliculata Decene et T h ü r . , Botanica Marina, XXI. (1) 1-3 (1978) 119. Mairh, O. P . , Sreenivasa Rao, P . : Culture Studies on Gelidium pusillum ( S t a c k h . ) Le Jolis. Botanica Marina, XXI (3) 169-174 (1978) 120. Mertz, W.: Biochemie der Spurenelemente - ernährungswissenschaftliche Aspekte. Seifen-Öle-Fette-Wachse, 106 (14) 383 (1980) 121. Michanek, G.: Seaweed Resources of the Ocean. FAO Fisheries Technical Paper No. 138 (1975) 122. Michanek, G.: Trends in Applied Phycology. Botanica Marina, XXI (8) 469-475 (1978) 123. Michanek, G . : Phytogeographic Provinces and Seaweed Distribution. Botanica Marina, XXII (6) 375-391 (1979)

44 124. Mollion, J . : Infrared and Chemical Studies of the Carrageenan from Anatheca montagnei Schmitz, (Solieriaceae) from Senegal, West Africa. Botanica Marina, XXIII (3) 197-199 (1980) 125. Morgan, K. C. et a l . : Review of Chemical Constituents of the Red Alga Palmaria palmata (Dulse). Economic Botany, 34 (1) 27-50 (1980) 126. Morgan, K. C . , Simpson, F . J . : The Cultivation of Palmaria palmata. Botanica Marina, XXIV (5) 273-277 (1981) 127. Mosig, A . : Der Arzneipflanzen- und Drogenschatz Chinasund die B e deutung des Pen-Ts'ao Kang-Mu. VEB Verlag Volk und Gesundheit, Berlin (1955) 128. Mshigeni, K. E . : Field Observations on the Colonization of new Substrata and Denuded Intertidal Surfaces by Benthic Macrophytic Algae. Botanica Marina, XXI (1) 49-57 (1978) 129. Mshigeni, K. E . : The Economic Algal Genus Eucheuma (Rhodophyta, Gigartinales): Observations on the Morphology and Distribution Ecology of Tanzanian Species. Botanica Marina, XXII (7) 437-445 (1979) 130. Mshigeni, K. E. et a l . : Studies on the Phycocolloid from the Red Seaweed Rhabdonia africana Jaasund (Gigartinales, Rhabdoniaceae). Botanica Marina, XXII (7) 447-450 (1979) 131. Mumford j r . , Th. F . : Growth of Pacific Northwest Marine Algae on Artificial Substrates - Potential and Practice. In: Krauss, Robert W. ( e d . ) : The Marine Plant Biomass. Oregon State University Press (1977) 139-161 132. Munda, J . M.: Trace Metal Concentrations in some Icelandic Seaweeds. Botanica Marina, XXI (4) 261-263 (1978) 133. Munda, J . M., Pedersen, P. M.: Porphyra thulaea sp. nov. (Rhodophvceae, Bangiales) from East Iceland and West Greenland. Botanica Marina, XXI (5) 283-288 (1978) 134. Naqui, S . W. A. et a l . : Bromine Content in some Seaweeds of Goa (Central West Coast of India). Botanica Marina, XXII ( 7 ) 455-457 (1979) 135. Naqui, S . W. A. et a l . : Screening of some Marine Plants from the Indian Coast for Biological Activity. Botanica Marina, XXIV (1) 51-55 (1981) 136. Noelle, H . : Food from the Sea. Springer Verlag, Berlin-HeidelbergNew York (1981) 137. Novak, K . , Rasmussen, R . A . : Seaweed Dyes. Botanica Marina, XXIV (6) 343-346 (1981)

45 138. O o h u s a , T . : Diurnal Rhythm in t h e Rates of Cell Division, Growth a n d P h o t o s y n t h e s i s of P o r p h y r a yezoensis (Rhodophyceae) c u l t u r e d in t h e L a b o r a t o r y . Botanica Marina, XXIII (1) 1-5 (1980) 139. Oza, R. M.: Studies on Indian Gracilaria, IV. Seasonal Variation in Agar and Gel S t r e n g t h of Gracilaria c o r t i c a t a J . A g . o c c u r i n g on t h e coast of V e r a v a l . Botanica Marina, XXI (3) 165-167 (1978) 140. P a s k i n s - H u r l b u r t , A. J . et a l . : Fucoidan: I t s Binding of Lead and o t h e r Metals. Botanica Marina, XXI (1) 13-22 (1978) 141. P a t t e r s o n , G. W.: S u r v e y of Chemical Components and E n e r g y Cons i d e r a t i o n . I n : K r a u s s , Robert W. ( e d . ) : The Marine Plant Biomass. Oregon State University P r e s s (1977) 271-287 142. Paul, V. J . , . F e n i c a l , W.: Toxische A c e t y l e n g r u p p e n e n t h a l t e n d e Lipide a u s marinen Algen. T e t r a h e d r o n L e t t . , 21 3327 (1980); Chem. Z t g . , 105 (1) (1981) 143. Rama Rao, K . , K r i s h n a m u r t h y , V . : S t u d i e s on Indian H y p n a c e a e , I . Seasonal Variation in Phycocolloid Content in two Species of H y p n e a . Botanica Marina, XXI (4) 257-259 (1978) 144. Roche F o r s c h u n g s i n s t i t u t , Dee Whe, Neusüdwales: Biologisch aktive S u b s t a n z e n von Meeresorganismen. Pharmazeutische Zeitung, 125 (31) 1486-1489 u n d (32) 1543-1550 (1980) 145. R o g e r s , D. J . , B l u n d e n , G . : S t r u c t u r a l P r o p e r t i e s of t h e Anti-B Lectin from t h e Red Alga Ptilota plumosa ( H u d s . ) C. A g . . Botanica Marina, XXIII (7) 459-462 (1980) 146. R o g e r s , D. J . et a l . : A S u r v e y of some Marine Organisms f o r Haemagglutinins. Botanica Marina, XXIII (9) 569-577 (1980) 147. Schimmer, O . : Natürliche Mutagene in h ö h e r e n P f l a n z e n . D e u t s c h e A p o t h e k e r Zeitung, 118 (48) 1818-1823 (1978) 148. Schimmer, O . : U n t e r s u c h u n g e n zur mutagenen Potenz von F u r o c u marinen u n d F u r o c u m a r i n d r o g e n - ein B e i t r a g z u r Risikoabs c h ä t z u n g . D e u t s c h e Apotheker Zeitung, 120 (6) 308-309 (1980) 149. Schimmer, O. et a l . : Phototoxicity and Photomutagenicity of F u r o c o u marins and Medical Plants with Furocoumarins in Chlamydomonas r e i n h a r d i i . Planta medica, 40 (1) 68-76 (1980) 150. S c h o n b e c k , M. W., Norton, T . A . : The E f f e c t s on t h e Growth of Fucoid Algae of some S y n t h e t i c Materials u s e d in t h e C o n s t r u c t i o n of C u l t u r e A p p a r a t u s . Botanica Marina, XXIII (7) 433-434 (1980) 151. S c h u l t e s , R . E, Hofmann, A . : Pflanzen d e r G ö t t e r . Hallwag V e r l a g , B e r n - S t u t t g a r t (1980)

46 152. Simpson, F . J . et a l . : The Cultivation of Chondrus crispus. Effect of pH on Growth and Production of Carrageenan. Botanica Marina, XXI (4) 229-235 (1978) 153. Simpson, F . J . , Shacklock, P. F . : The Cultivation of Chondrus crispus. Effect of Temperature on Growth and Carrageenan Production. Botanica Marina, XXII (5) 295-298 (1979) 154. Sims, J . J . et a l . : Tetrahedron Letters, 3487 (1974); Chem. Ztg. 98 (12) (1974) 155. Sivalingam, P. M.: Biodeposited Trace Metals and Mineral Content Studies of some Tropical Marine Algae. Botanica Marina, XXI (5) 327-330 (1978) 156. Smith, M. M., Gayler, K. R . : Free Amino Acids in the Marine Green Alga Caulerpa simpliciuscula. Botanica Marina, XXII (6) 361-365 (1979) 157. Spegg, H. ( e d . ) : Der Pharmazeutisch-technische Assistent. Band III. Botanik • Drogenkunde, 2. Auflage, Deutscher Apotheker Verlag, Stuttgart (1977) 158. Sreedhara Murthy, M., Paresh Radia: Eco-Biochemical Studies on some Economically important Intertidal Algae from Port Okha (India). Botanica Marina, XXI (7) 417-422 (1978) 159. Sreenivasa Rao, P. et a l . : Seaweed as Source of Energy. I . Effect of a Specific Bacterial Strain on Biogas Production. Botanica Marina, XXIII (9) 599-601 (1980) 160. Sumera, F. C . , Cajipe, G. J . B . : Extraction of Auxin-like Substances from Sargassum polycystum. Botanica Marina, XXIV (3) 157-163 (1981) 161. Suzuki, M. et a l . : Tetrahedron Letters, 821 (1974); Chem. Z t g . , 98 (6) (1974) 162. Suzuki, M. et a l . : Tetrahedron Letters, 1807 (1974); Chem. Z t g . , 98 (9) (1974) 163. Stahl, E . , Schild, W.: Pharmazeutische Biologie - 4. Drogenanalyse I I . : Inhaltsstoffe und Isolierungen. Gustav Fischer Verlag, StuttgartNew York (1981) 164. Targett, N. M.: Gastropod Tentacle Withdrawal: A Screening Procedure for Biological Activity in Marine Macroalgae. Botanica Marina XXII (8) 543-545 (1979) 165. Templeton, J . F. et a l . : Characterization of Ergosterol as the Major Sterol from Euglena gracilis grown on Animal Waste. Pianta medica, 33 (4) 377-378 (1978)

47 166. Toama, M. A . et al.: Effect of Agar Percentage, Agar Thickness, and Medium Constituents on Antibiotics Assay by Disc Diffusion Method. Pharmazie, 33 (2/3) 100-102 (1978) 167. Tokida, J . , Hirose, H . : Advance of Phycology in Japan. VEB Gustav Fischer Verlag, Jena (1975) 168. Tsujino, J. et al.: Isolation and Structure of a new Amino Acid Shinorine, from the Red Alga Chondrus yendoi Yamada et Mikami. Botanica Marina, XXIII ( 1 ) 65-68 (1980) 169. Velimirov, B . : Fatty Acid Composition of Kelp on the West Coast of South Africa and some Ecological Implications. Botanica Marina, XXII ( 4 ) 237-240 (1979) 170. Veroy, R. L . et al.: Studies on the Binding of Heavy Metals to Algal Polysaccharides from Philippine Seaweeds, I . Carrageenan and the Binding of Lead and Cadmium. Botanica Marina, XXIII ( 1 ) 59-62 (1980) 171. Vinogradov, A . P . : „The Elementary Chemical Composition of Marine Organisms." Sears Foundation for Marine Research, Yale University, New Haven, (1953) 172. Wagner, H . : Pharmazeutische Biologie - 2. Drogen und ihre Inhaltsstoffe. Gustav Fischer Verlag, Stuttgart-New York (1980) 173. Weber, A . : Algen und Schwermetalle - Untersuchungen zur Anreicherung und Toxizität von Blei. Deutsche Apotheker Zeituner, 119 ( 7 ) (1979) 174. Wells, Robert J . : Biolostically Active Substances from Australien Marine Species. International Research Congress on Natural Products as Medicinal Agents, Strasbourg, July 6-11, 1980 175. Westlund, P. et al.: Localization and Quantification of Iodine and Bromine in the Red Alga Phyllophora truncata (Pallas) A . D. Zinova by Electron Microscopy and X-Ray Microanalysis. Botanica Marina, XXIV ( 3 ) 153-156 (1981) 176. Whyte, J. N. C . , Englar, J. R . : Seasonal Variation in the Inorganic Constituents of the Marine Alga Nereocystis luetkeana; Part I , Metallic Elements, Part I I , Non-metallic Elements. Botanica Marina, XXIII ( 1 ) Part I 13-17 (1980), Part II 19-24 (1980) 177. Whyte, J. N. C . , Englar, J. R . : Chemical Composition and Quality of Agars in the Morphotypes of Gracilaria from British Columbia. Botanica Marina, XXIII ( 5 ) 277-283 (1980) 178. Wildgoose, P. B. et al.: Seasonal Variations in Gibbellin Activity of some Species of Fucaceae and Laminariaceae. Botanica Marina, XXI ( 1 ) 63-65 (1978)

48 179. Zanlungo, A. B . : Polysaccharides from Chilean Seaweeds. P a r t IX. Composition of t h e Agar from Gelidium lingulatum. Botanica Marina, XXIII (12) 741-743 (1980) Addendum

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Part II

EFFECTS OF VARIOUS TYPES OF CARRAGEENANS ON HUMAN FIBROBLASTS IN VITRO* Eleanor Tveter-Gallagher Jackson Estuarine Laboratory, University of New Hampshire Durham, New Hampshire, U.S.A. 03824 Thomas N. Wight Department of Pathology, Univ. of Washington Medical School Seattle, Washington, U.S.A. 98195 Arthur C. Mathieson Department of Botany and Plant Pathology Jackson Estuarine Laboratory, University of New Hampshire Durham, New Hampshire, U.S.A. 03824

Introduction Carrageenans are sulfated polysaccharides extracted from Gigartinalean red algae (1). As a result of their gelling, stabilizing and textural properties they have a wide variety of industrial uses and are of considerable economic significance. All carrageenans contain repeating galactose units (sulfated or non-sulfated) joined by alternating ¿*-l-3, fb 1-4 glycosedic linkages, which resemble the sulfated glycosaminoglycans (GAG) in the connective tissue stroma of vertebrate cells (2,3). The various types of carrageenans(iota, lambda, kappa) are differentiated by their amounts of 3,6 anhydro-D-galactose (3,6 AG), as well as the number and positions of sulfated ester groups (1). At present, 70% of all carrageenan products are utilized by the food industry (1) and it is estimated that the average human consumption of carrageenan is approximately 250 mg/day •Scientific Contribution No. 122 of the Jackson Estuarine Laboratory

M a r i n e A l g a e in P h a r m a c e u t i c a l S c i e n c e , V o l u m e 2 © 1982 by W a l t e r d e G r u y t e r &. C o . , Berlin • N e w Y o r k

52 (personal communication, D. Stancioff, FMC Corp.). Early studies (4) on the effects of carrageenan suggested that most of the ingested phycocolloid material passed through the gut unabsorbed but whole body organs were not analyzed in these studies. More recent investigations (5) suggest that both native and degraded carrageenan can be absorbed in rats and guinea pigs. Furthermore, it has been shown that molecular weight and/or types of carrageenan can influence this absorption, while the absorption and retention of carrageenan within the liver is species dependent (6). Numerous reports have also demonstrated that carrageenans exert diverse physiological effects when administered to test animals. Selected examples of these effects include: (a) antiviral (7) and ulcerogenic properties (8-10); (b) promotion of inflammatory reaction (11); and (c) induction of connective tissue formation (12,13). Yet the cellular mechanism by which these phycolloids exert their physiological effects is unknown. Structurally related macromolecules, the glycosaminoglycans, are known to influence several basic cell processes such as proliferation, migration, differentiation, attachment and connective tissue synthesis (14). It therefore became of interest to determine whether the sulfated carrageenans possess similar biological properties as the glycosaminoglycans. To determine this, we have examined the influence of both low and high molecular weight carrageenans on the growth and metabolism of cultured human vertebrate cells. Specifically we have addressed the questions of: 1) whether various types of carrageenan differentially affect the growth of human skin fibroblasts in vitro and 2) whether a particular functional property of these cells, i.e., the synthesis and secretion of connective tissue of glycosaminoglycans, is affected by these phycocolloids.

53 Materials and Methods Cell culture

The human skin fibroblasts used in the experiments were kindly supplied by Dr. George Martin, Department of Pathology, Uni2

versity of Washington. The cells were cultured in 35 mm petri dishes, at 37°C and in a 5% CC>2-95% air atmosphere; an initial seeding density of 3.5 x 10^ cells/dish was employed. The control medium was a Dulbecco-Vogt modification of Eagle's minimal medium, supplemented with 10% fetal calf serum. It was exchanged every 4 8 hours throughout the course of the experiment, unless otherwise indicated. The test medium consisted of a control medium, plus one of the following supplements added at 400 jigm/ml: a) low molecular weight (11,600) iota carrageenan (Marine Colloids, FMC Corp., Rockland, ME); b) high molecular weight (178,000) iota carrageenan (Marine Colloids, FMC Corp., Rockland, ME); c) high molecular weight kappa carrageenan; d) chondroitin 6-sulfate with a molecular weight of ~ 20,000 (Miles Laboratories, Kanakee, IL); and e) heparin (Sigma Chemical Co., St. Louis, MO) with a molecular weight of 3000 to 6 000. The average molecular weights of the degraded carrageenan fractions (11,000 and 178,000) were estimated using viscosity and electrophoresis (15). Kappa carrageenan extracted from gametophytic plants of Chondrus crispus were prepared according to the methods of Craigie and Leigh (16). The test substances were added to a small volume (0.5 ml) of twice distilled sterile water and heated to 80°C to facilitate the solubilization of the carrageenans. The mixture was allowed to cool and then added to the control medium. Twice distilled sterile water (5.0 ml), without additives, was added to the medium as a control. The medium was then filtered to assure maximum sterility.

54 Measurement of cell growth and DNA synthesis Cells were initially plated in 35 mm dishes at a density of 4 3.5 x 10 cells/dish in complete control medium. Plating efficiencies were determined after one day and found to average approximately 75%. On day 1, the cultures were transferred to each of the test media and sufficient numbers of dishes were maintained to determine the growth response for at least 12 days. Every other day, cells were removed by digestion on 1 ml of a 0.05% trypsin, .005 M EDTA in phosphate buffered saline (PBS) of 37 C. The trypsin was then inactivated by adding an aliquot of 10% calf serum and a portion of the resultant cell suspension used for counting in a hemocytometer chamber. The percent cell viability for each medium was tested using the trypsin blue exclusion test (17). Control and test media were changed every 4 8-72 hours. The synthesis of DNA was mea3 sured on days 12-14 by labelling each dish with 3 jiCi Hthymidine for 24 hours and precipitation of the nucleic acids with 20% TCA after solubilization of the cell pellet with 1 N NaOH (18). The TCA precipitates were collected by filtration; the amount of activity was determined by placing the filters in Liquiflor and counting them in a Packard Tri Carb Scintillation Counter.

Measurement of glycosaminoglycan synthesis and secretion Control cultures and those treated with test substances were double labelled for 48 hours with 5 jaCi/ml 35S-sulfate 3 (carrier free) and 2.5 pCi/ml of H-glucosamine on day 12 of the growth curve. Following labelling, the medium was removed and saved for analysis, the cell layer was harvested by trypsinization. The medium, cell pellet and trypsinate supernatant were extracted for glycosaminoglycans as previously described (19). Briefly, this procedure consists of boiling the samples for 5 minutesf followed, by prona.se digestion (0.5 mg/ml in 0.2 M Tris-HCl

buffer, pH 8.0) for 18 hours at 60°C.

55

Fig. 1. Control culture. and form multiple layers. similar. sulfate appear

Celts appear elongated, overlapped} Cultures treated with Chondroitin

Fig. 2. Cells treated with various types of oarrageenan were polygonally shaped and ceased to divide when contacted by adjacent cells.

56 Ice cold trichloroacetic acid (TCA) was added to a final concentration of 10% and the precipitates were removed by centrifugation. The supernatants were exhaustively dialyzed at 4°C, initially against 0.01M Na2SOjj and finally against distilled water. Samples of isotopically labelled material were taken for liquid scintillation counting.

Results Morphology and cell growth Control fibroblasts and those treated with chondroitin sulfate were elongate and spindle shaped; they grew in orderly parallel and swirling arrays, forming multiple cell layers at confluency. Fibroblasts treated with high or low molecular weight carrageenans and heparin were polygonal in shape; they did not exhibit an orderly growth pattern and only appeared to form monolayers (Fig. 1 and 2). The addition of various types of carrageenan, as well as chondroitin sulfate and heparin, to the culture medium of human fibroblasts did not interfere with cell growth for the first 5-6 days, as the growth rates in the logarithmic phase of all treated cultures were the same as the control (Fig. 3). However, cultures treated with heparin as well as with low molecular weight, high molecular weight or kappa carrageenan reached saturation densities ( ~ 4 x 10^) at a much lower cell number than control cultures (7.5 x 10^) or cultures treated with chondroitin sulfate. Viability tests revealed that differences among tested compounds could not be attributed to cell death and that none of the additives were cytotoxic (Table I). Carrageenan and heparin-treated cells appeared to adhere more tightly to the substratum and to one another since longer periods of trypsinization (as compared to controls) were required for detachment and disaggregation of the cells.

57 Fig. 3. Growth curve of cells treated with various substances over 12 days. Chondroitin sulfate(CS), Low molecular weight iota carrageenan(LMW), High molecular weight iota carrageenan (HMW), Kappa carrageenan(Kappa).

Table I. Incorporation of 3H-thymidine into DNA(DPM cells) and cell viability after 12 days in culture. tration of test substances was 400 ngm/ml.

Final cell no.

Viability

x

106 Concen-

^H-thymidine/ 10 6 cells

Control

ZOxlO5

91%

35,470

Chondroitin Sulfate

75x10"*

90%

22,470

L M W Carrageenan

5.3xl05

89%

25,290

H M W Carrageenan

4.0xl05

89%

28,870

Heparin

3.7xl0 5

90%

13,250

Kappa Carrageenan 3.5xl05

89%

14,720

58 Table II. The incorporation of S5S-sulfate into proteoglycans of human fibroblasts in vitro. Counts are given for the medium, pericellular matrix3 and cell-bound components.* Matrix

Medium

Total

Cells

Control

408,555 (88%)

41,619 (9%)

8,989 (3%)

459,163

Chondroitin Sulfate

618,213 (80%)

136,129 (18%)

11,352 (2%)

765,694

1,194,454 (90%)

110,100 (8%)

18,967 (2%)

1,323,521

124,255 (14%)

63,150 (8%)

869,222

Heparin HMW Carrageenan

681,817

(78%)

LMW Carrageenan

716,462 (82%)

81,185 (9%)

65,842 (9%)

863,489

Kappa Carrageenan

658,371 (84%)

92,806(11%)

31,817 (5%)

782,994

* DPM/K)6 c«lli

3 Furthermore,

H-thymidine incorporation into DNA (Table I) was

depressed in the carrageenan and heparin-treated cultures at the end of the growth period, with kappa carrageenan and heparin exhibiting the most conspicuous reduction

(50-60%).

Glycosaminoglycan synthesis All cultures treated with test substances exhibited increased 35 3 incorporation of S-sulfate and H-glucosamine into TCA soluble, nondialyzable material (Table II). The majority of the labelled macromolecules appeared in the medium (80-90%) with less activity being associated with the pericellular matrix (10-20%) or cell bound components (0-10%). No obvious differences were observed in the distribution of the labelled macromolecules in each of the culture compartments among the experimental groups (Table II).

59 Discussion The effect of heparin and other polysulfated compounds on the growth of vetebrate cells has been studied by a number of investigators; their results suggest that polysulfated sugars are effective in decreasing cell saturation density (20-26) while sugars containing low sulfate content either exhibit no effect on cell growth or they are slightly stimulatory (22, 27-29). Our finding that both low and high molecular weight carrageenan were as effective as heparin in decreasing the saturation density of human skin fibroblasts growing in vitro is not surprising, as carrageenans have been shown to be both structurally and functionally similar to the heparins (30-32). Although the sugars that constitute the chains in the respective molecules are different (galactose for carrageenan vs. glucosamine: iduronic/glucoronic acid for heparin), the degree of sulfation and the optical rotary dispersion pattern of these molecules in solution are similar (30,31). Such findings suggest that the two groups of molecules are structural analogues and possess similar stereochemical configurations of anionic substitutes. Further evidence for their biological similarities is demonstrated in a recent study by Kindness et al (33) who showed that carrageenans (iota, lambda, kappa) like heparin, prolong blood clotting time and potentiate the inactivation of thrombin and Xa by antithrombin III. It is of interest to note that in their study desulfated carrageenans and desulfated heparin did not potentiate the inactivation of antithrombin III, suggesting that the degree of sulfation is of fundamental importance to the biological activity of this molecule. The mechanism by which carrageenan and other related sulfated sugars affect cell growth is unknown. For example, carrageenan may be cytotoxic to cells. Costachel and associates (21) reported that heparin was cytotoxic to a Syrian Hamster sarcoma

60 line at 100-2000 jagm/ml. However, our finding that carrageenantreated cells initially grew at the same rate in logarithmic growth as control cells argues against the cells being adversely affected. Also, at the final cell densities, carrageenantreated cells exhibited the same degree of viability(as determined by trypsin blue exclusion tests) as control cells. Such observations suggest that carrageenan's action is not one of cytotoxicity. Another possibility is that the polyanionic carrageenans combine with mitogenic components present in the serum to inactivate them or make them unavailable to the cell. Recent work by Paul and associates (34) has shown that heparin can effectively block the growth of 3T3 cells in vitro by first binding to a major mitogen in serum, the plateletderived growth factor. However, the lack of any effect on the growth rate of carrageenan-treated cells during early logarithmic growth, coupled with the fact that all cultures were changed routinely with fresh medium, argues against the test substance inactivating a mitogen in the medium. The finding that the treated cells grow logarithmically at approximately the same rate but reach much lower saturation densities suggests that cessation of growth may in some way be related to contact inhibition. A consistent observation of the carrageenan-treated cells was that they appeared more polygonal and flatter and had less overlapping than untreated cells. Furthermore, the fact that carrageenan-treated cells were more difficult to release from the dish than control cells argues that carrageenan affects some process that influences the interaction of the cell surface with its substratum. The change in shape and apparent modification of the adhesive properties of cells in vitro is often accompanied by a change in the cell surface properties (23, 35,36). Such findings suggest that the site of action for carrageenan may be at the cell surface. Carrageenan has been shown to interact with the plasma membrane of erythrocytes (37) , and recent

61 studies by Ehresmann et al (7) demonstrate that a carrageenanlike polysaccharide isolated from marine algae blocked the absorption of viruses to the surface of cultured cells. Although these studies suggest that carrageenan may act at the cell surface, specific binding studies need to be done to confirm or deny- this suggestion. The occurrence of increased glycosaminoglycan synthesis and decreased cell growth in cells treated with carrageenan resemble similar studies with heparin, which showed the same effect on WI 38 cells in vitro (25) . Inhibition of cell growth is often accompanied by increased macromolecular synthesis (38,39) and it may be that this sequence is responsible for the fact that carrageenan is effective in promoting connective tissue formation in experimental systems in vitro (12,13,40,41). However, it should be stressed that increased synthesis of glycosaiflinoglycans may not be a necessary consequence of inhibition of cell division since recent studies by Miller et al (39), using cultured chondrocytes, demonstrated that when cell proliferation was inhibited by hydroxyurea, no elevated synthesis of glycosaminoglycans was observed. On the other hand, when cells were inhibited from dividing by cyclic AMP, there was a concomittant increase in GAG synthesis. Such data indicates the potential importance of cyclic AMP in the control of GAG synthesis. Cyclic AMP has been shown to stimulate glycosaminoglycan synthesis in other systems (38) and it may be that carrageenan is in some way mediating its effect through cyclic AMP. The physiological significance of carrageenan-like molecules such as heparin is that they have been shown to be effective anticoagulant agents (42) and have been used clinically to treat inflammatory and allergenic diseases (43). Furthermore, heparin has been shown to accelerate the recovery of burn patients and promote wound healing in humans and animals (4446). Recent reports indicate that heparin may in fact retard the development of atherosclerosis by inhibiting the myointimal

62 proliferation of arterial smooth muscle cells (47) . The similar biological activities of carrageenan and heparin, as demonstrated in this study, illustrate the value of using chemically defined carrageenans as molecular models by which mammalian systems involving the glycosaminoglycans may be perturbed and explored. The possibility of using carrageenan as an effective, clinically therapeutic substitute for heparin should not be overlooked.

Acknowledgements This project was funded by the Office of Sea Grant, National Oceanic Administration, U.S. Department of Commerce, through a grant (#R/LRS-3) to the University of New Hampshire. We also wish to thank Dr. Donald Cheney for supplying us with kappa producing Chondrus plants, and Ms. V. Lloyd for her typing.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Marine Colloids Division, FMC Corporation, Carrageenan, Monog. 1, (1977). Baslow, M.: Marine Pharmacology, Krieger Press, New York, 56 (1977) . Stone, A.L.: Biopolymers 11, 2625 (1972). Hawkins, W.W., Yaphe, W.: Can. J. Biochem 43, 489 (1965). Grasso, P., Gargalli, S., Butterworth, K., Wright, M: Fd. Cosmet. Toxicol !L3, 195 (1975) . Pittman, K.A., Goldberg, L., Coulston, F.: Fd. Cosmet. Toxicol 14, 85 (1976). Ehresmann, D.W. , Deig, E., Hatch, M. : J. Phycol 1_3, 37 (1977). Benitz, K. , Goldberg, L., Coulson, F. : Fd. Cosmet Toxicol 11, 565 (1973). Crasso, P., Sharratt, M. , Carpanini, F.: Fd. Cosmet Toxicol 11, 555 (1973).

10. Watt, J.f Marcus, R.: J. Pharm. Pharmac 22, 130 (1970).

63 11. Vinegar, R. , Traux, J.F., Selph, J.L.: Fed. Proc. 35, (1976). 12. McCandless, E.I., Lehoczky-Mowa, J.: Growth 2JB, 143 (1964). 13. Jackson, D.S.: Biochem J. 6_5, 277 (1957). 14. Wight, T.: in Progress in Hemostasis and Thrombosis, Grune and Stratton, New York, 1 (1980). 15. Personal Communications, Norman Stanley, Marine Colloids Division, FMC, Rockland, Me. 16. Craigie, J.S., Leigh, C.: in Handbook of Phycological Methods, ed. Hellebust, J.A., Craigie, J.S., Cambridge University Press, New York, 109 (1978). 17. Phillips, H.J. in Tissue Culture Methods and Application, ed. Kruse, P., Patterson, M.K. Academic Press, New York, 406 (1973). 18. Vogel, A., Raines, E., Kariya, B., Rivest, M.J., Ross, R. : Proc. Nat. Acad. Sei. U.S.A. 75, 2810 (1978). 19. Wight, T.N., Ross, R. : J. Cell Biol. 6J7, 675 (1975). 20. Heilbrunn, L.V., Wilson, W.L.: Proc. Soc. Exp. Biol. Med. 70, 179 (1949). 21. Costachel, 0., Fadei, L., Nachtigal, M.: Exp. Cell Res. 34, 542 (1964). 22. Lippman, M.: in Epithelial-Mesenchymal Interactions, R. Fleischmayer, R.E. Billinham, eds., Williams and Wilkins Co., Baltimore, Md. (1968). 23. Goto, M., Kataoka, Y., Kimura, T., Goto, K., Sato, H.: Exp. Cell Res. 82, 367 (1973). 24. Ohnishi, T., Ohshima, E., Ohtsuka, M.: Exp. Cell Res. 9^, 136 (1975). 25. Wever, J., Schachtschabel, 0.0., Sluke, G., Wever, G.: Mech. Aging and Develop. L4, 89 (1980). 26. Hoover, R.L., Rosenberg, R., Haering, W., Karnovsky, M.I.: Circ. Res. £7, 578 (1980). 27. Morrison, L.M., Murata, K., Quilligan, J.J., Schjeide, O.A., Freeman, L.: Proc. Soc. Exp. Biol. Med. 118, 770 (1965). 28. Takeuchu, J.: Cancer 26, 797 (1966). 29. Yang, T.K., Jenkin, H.M.: Proc. Soc. Exp. Biol. Med. 159, 88 (1978). 30. Nemeth-Csoka, M., Kajtar, M., Kajtar, J.: Conn. Tissue Res. 5, 1 (1966). 31. Arnott, S., Scott, W.E., Rees, D.A., McNab, C.G.A.: J. Mol. Biol. 90, 253 (1974). 32. Nemeth-Csoka, M., Kajtar, J., Kajtar, M.: Conn. Tissue Res. 3, 207 (1975).

64 33. Kindness, G., Long, W.F., Williamson, F.B.: Enhancement of antithrombin III activity by carrageenans. Thrombosis Res. 15, 49 (1979). 34. Paul, D., Niewiarowski, S., Varma, K.G., Rucker, S.: Thrombosis Res. .18, 883 (1980) . 35. Schnebli, H.P., Burger, M.: Cancer Res. 33* 3306 (1973). 36. Gallagher, E., Harris, N., Morin, P., Wight, T.N.: SEM Symposium, Vol. II, 779 (1978). 37. Piletz, E.P. Goldberg, L., Coulston, F.: Life Sciences 17, 969 (1975). 38. Goggins, J.F., Johnson, G.S., Pastan, I.: J. Biol. Chem. 247, 5759 (1972). 39. Miller, R.P., Husain, M., Lohin, S.: J. Cell Physiol. 100, 63 (1979). 40. DiRosa, M.: J. Pharmacol Pharmac 2£, 89 (1972). 41. Chandrasekaran, E.V., Bachhawat, B.H.: Biochem. Biophys. Acta. 1T7, 265 (1977). 42. Jeanloz, R.W.: in The Carbohydrates, W. Pigman, D. Horton, eds., Vol. II, Academic Press, New York, 607 (1970). 43. Dougherty, T.F., Dolonitz, D.A.: Amer. J. Cardiol. }A_, 18 (1964) . 44. McCleery, R.S., Schaffarzick, W.R., Light, R.A.: Surgery 26 , 548 (1949) . 45. Fenton, H., West, G.B.: Brit. J. Pharmiacol. 20, 507 (1963). 46. Saliba, M. , Dempsey, W.C., Kruggel, J.: Amer. Med. Assoc. 225, 261 (1973). 47. Clowes, A., Karnovsky, M.: Nature 265, 625 (1977).

FAT PRODUCTION IN FRESHWATER AND MARINE ALGAE Peter Pohl and Friedrich Zurheide Institut für Pharmazeutische Biologie, Universität Kiel, Grasweg 9, 2300 Kiel, Germany

1. Introduction Fatty acids of algae: Algae produce a great variety of fatty acids and lipids. A review of these compounds has recently been published (1). In brief, the main algal fatty acids are saturated and cis-unsaturated with mostly 12 - 20 carbon atoms and 0 to 6 double bonds. Lipids of algae: The predominating unpolar and polar lipids of algae are shown in fig.1 and fig.2. UNPOLAR LIPIDS

0 H •• C-O-I C-H I C-O-C-H

i

Triglycerid«»

I •9C-O.-Ct-H H

(n)

Hydrocarbons 0 C-OH

Fr«» fatty acids

Fig.1 : Unpolar lipids of algae

M a r i n e A l g a e in P h a r m a c e u t i c a l S c i e n c e , V o l u m e 2 © 1982 by W a l t e r d e G r u y t e r &. C o . , Berlin • N e w Y o r k

66 POLAR L I P I D S

R - C

R - c ' - g - l — O-ch2 -O-CH

R ' - C ^

1

ö(-)

W

P h o s p h a t i d y l choline

Phosphatidyl

-O-CH,

R - C R

«0

I -C^-H—O-CH

-O-CH HjC-O-M-CHfCHj &-> I ,. NH/>>

| 2 N(CH3)3 (PC)

serine(PS)

CH,OH

Phosphatidyl

ethanolamine(PE}

Phosphatidyl inositol (PI)

R-C*

TT

-o-ch2 OH

I H-C-OH

-O-CH

>H

H.C-O-P-O-CH, H

P h o s p h a t i d y l glycerol ( P G )

2

C

" ° V CHjSOjH

S u l p h o q u i n o s y l diglyceride (Sulpholipid.SL)

R-C2=

-O-CH,

-O-CH,

I '

R - C -

-C-CH

H,C-0\

0

H,C-C

Monogalaclosyl

[/OH

OH

CH5

diglyceride

(MGDG)

Fig.2: Polar lipids of algae

Oigalactosyl diglycerid« (DGDG)

67 2. F a c t o r s influencing the b i o s y n t h e s i s of f a t t y acids a n d lipids i n algae So far, there appear to exist at least three m a j o r

factors

h a v i n g a n influence o n the b i o s y n t h e s i s of algal fatty acids a n d lipids . These are the light conditions, the w a t e r temperature, a n d the contents of n i t r o g e n i n the n u t r i e n t medium. a) Light: This f a c t o r has b e e n shown to enhance the formatio n of p o l y u n s a t u r a t e d C^g and C^g fatty acids a n d of p o l a r lipids (MGDG, DGDG, SL, PG) + ^ in v a r i o u s freshwater algae

(2 - 6). b) Temperature: D e c r e a s i n g temperatures a p p a r e n t l y l e a d to a n i n c r e a s i n g degree of u n s a t u r a t i o n i n the fatty acids of algae (7 - 11). c) Contents of n i t r o g e n

etc.) i n the n u t r i e n t

medium: I n a d d i t i o n to the before m e n t i o n e d two factors (light a n d temperature), w e succeeded i n f i n d i n g a third factor w h i c h is of great influence o n the f o r m a t i o n of fatty acids a n d lipids i n freshwater and marine algae. This factor is the content of n i t r o g e n (such as n i t r a t e or ammonium)

in

the n u t r i e n t medium. W e came across this factor w h e n we grew freshwater algae i n m e d i a containing y e a s t extract. These algae t e n d e d to p r o d u c e larger amounts of p o l y u n s a t u r a t e d fatty acids. We t h e n i n v e s t i g a t e d this p h e n o m e n o n a n d found out that the h i g h n i t r o g e n content of y e a s t extract (ca. 10 %) was responsible for the effect. 3• Influence of the n i t r o g e n content i n the m e d i u m o n the fatty acids a n d lipids of freshwater algae E x p e r i m e n t s w i t h b a t c h cultures of Chlorophyceae a n d Euglenophy+

^ A b b r e v i a t i o n s : See fig.2

68 fatty acids

Bracteacoccus minor

('/. of total fatty acids)

KNO3 0,0015*/.

0,003*/.

Chlorella vulgaris KNO3

0,01*/.

0,0003*/.

0,0015V.

Qor/.

12: 0

tr

tr

tr

tr

tr

tr

14: 0

o.s

0.4

0.5

0,8

14,4 A.I

1.1

1.0

15.7

28,8

20,6

13,8

3.6

0.8

1.7

2,9

6.6

7.6

0.4

3.4

5.8

0.7

0.9

6,2

8.9

16: 0 16:

1

16 : 2

15,0

1.0 4,1

16: 3

1.2

1.4

16 4 16: 0 18. 1 16 : 2

1.9

6.1

6.3

-

-

-

1.0

2.0

2.6

4,8

1.0

18: 3

44,4

13,5

8.7

30,0

8.6

18.5

22.2

20,9

14,2

17.9

tr 1,1 14,9

12.1

29.1

33.2

8.0

39,6

51,6

Fig.3: Fatty acids of Bracteacoccus minor and Chlorella vulgaris grown in an inorganic medium with varying concentrations of KNO^ ceae showed that in these organisms the biosynthesis of polyunsaturated C^G and C^Q fatty acids (16:2, 16:3, 16:4, 18:2, 18:3)+^ and of chloroplast lipids (MGDG, DGDG, SL, PG; see fig.2) was enhanced by high concentrations of KNO^ or NH^Cl (more than ca. 0.001 % or 200 yumoles, respectively) (6,12). Probably these concentrations will be still lower when the algae are grown in chemostate (continuous flow) cultures. This, however, has not yet been investigated with freshwater algae. Fig.3 shows the fatty acids of Bracteacoccus minor and Chlorella vulgaris (Chlorophyceae) grown in batch cultures at different concentrations of KNO^. The biosynthesis of lipids is also controlled by the nitrogen content of the medium, the predominant lipids at higher N-levels being MGDG, DGDG, SL, and PG. At lower N-levels (below +

^18:2, for example, indicates a straight chain fatty acid with 18 carbon atoms and 2 double bonds.

69 Front NL

s \

CD CD

'

C

>

:

CD

;

C=> A

CD DGDG SL cz> Jmoles KNO^/l) Influence of nitrogen This effect could be clearly demonstrated: Fig.10 and 11 show the lipids and fatty acids of seven samples of Enteropha linza. Three of them were kept at 1.6, 16, and 80 pmoles KNO-j/l, and at a low phosphate level (1 pmole/l). Four samples were kept at 1.6, 16, 40, and 80 jamóles KNO^/l, and at a high phosphate level (10 ^unoles/l). In both series, increasing concentrations of nitrate led to A) an increase in the formation of glycolipids (MGDG, DGDG, SL) and simultaneously to a decrease of triglycerides (fig.10) and B) to an increase of polyunsaturated fatty acids (16:4, 18:4, partly 18:3) and a decrease of fatty acids with a low degree of unsaturation (16:0, 18:1, 18:2) (fig. 11).

77

( l i m ó l e s NO;/!)

Fig.10: Influence of the nitrogen content (KNO,) of the nutrient medium on the lipids of Enteromorpha linza kept in 20 1 laboratory tanks under controlled conditions for 4 weeks (pH 8.2; 4000 lux; 10 ) 5. Conclusions The results show that in freshwater algae as well as in marine algae the biosynthesis of fatty acids and lipids depends strongly on the environmental conditions at each locality and can change rather rapidly. This must be taken into account when investigations are carried out on the lipids and fatty acids in algae.

78 Ifjmote PQ|7l

32-

Wnmoles PO'7l

£ 30o 0 1 2« 26 16:0

2422

20-

16:3

1B-

12-

10-

18:2

6-

20

40

60

Fig.11: Influence of the trient medium on linza kept in 20 conditions for 4

16:3 > (3t>-16-. «0 100 o

60

80

(pmoles NO,"A)

nitrogen content (KNO,) of the nuthe fatty acids of Enxeromorpha 1 laboratory tanks under controlled weeks (pH 8.2; 4000 lux; 10°)

79 References 1

Pohl,P.,Zurheide,F.: Fatty acids and lipids of marine algae and the control of their biosynthesis by environmental factors. In: Marine Algae in Pharmaceutical Science, Ed. H.A. Hoppe, T. Levring and Y. Tanaka, Walter de Gruyter, Berlin-New York, 1979 (p. 473-523)

2

Rosenberg,A., Pecker,M.: Lipid alterations in Euglena gracilis cells during light-induced greening. Biochemistry 3, 254-258(1964)

3

Nichols,B.W.: Light induced changes in the lipids of Chlorella vulgaris. Biochim.Biophys. Acta 106,274-279(1965)

4

Rosenberg,A., Gouaux,J.: Quantitative and compositional changes in monogalactosyl and digalactosyl diglycerides during light-induced formation of chloroplasts in Euglena gracilis. J.Lipid Res. 8,80-83(1967)

5

Constantopoulos,G., Bloch,K.: Effect of light intensity on the lipid composition of Euglena gracilis. J.biol.Chemistry 242,3538-3542(1967)

6

Pohl,P., Wagner,H.: Control of fatty acid and lipid biosynthesis in Euglena gracilis by ammonia, light and DCMU. Z.Naturforsch.

7

27b,53-61(1972)

Ackman,R.G., Tocher,C.S.: Marine phytoplankter fatty acid acids. J.Fish.Res.Bd.Canada

8

25,1603-1620(1968)

Allen,C.F., Good,P., Holton,R.E.: Lipid composition of Cyanidium. Plant Physiol. 46,748-751(1970)

9

Kleinschmidt,M.G. McMahon,V.A.: Effect of growth temperature on the lipid composition of Cyanidium caldarium. I. Class separation of lipids. Plant Physiol. 46,286-289 (1970)

80 10

Kleinschmidt,M.G., McMahon,V.A.: Effect of growth temperature on the lipid composition of Cyanidium caldarium. II. Glycolipid and phospholipid components. Plant Physiol. 46,290-293(1970)

11

Adams,B.L., McMahon,V.A., Seckbach,J.: Fatty acids in the thermophilic alga, Cyanidium caldarium. Biochem.Biophys.Res.Commun. 42,359-365(1971)

12

Pohl,P., Passig,T., Wagner,H.: Über den Einfluß von anorganischem Stickstoff-Gehalt in der Nährlösung auf die Fettsäure-Biosynthese in Grünalgen. Phytochemistry 10, 1505-1513(1971)

EXTRACTION AND SEPARATION OF VITAMIN B 1 2 FROM MARINE ALGAE K.C. G u v e n ^ , v

B. G u v e n e r ^ ,

$. C i r i k ^ * * )

'Depart, of Pharmacy and Technology, Faculty of Pharmacy, University of Istanbul

(xxl v ' I n s t , of Marine Science and Technology, University of Ege, Izmir/TURKEY Introduction Vitamin B ^ has been used extensively for therapeutic purposes. Many bacteria may be the principal source of this vitamin. I t exists in animals, vegetables and also in sea water and marine algae. Various papers have been published about the distribution of vitamin B ^ in marine and fresh water algae and sea. In 1952, four years after the isolation of vitamin B ^ f r o m natural sources, vitamin B-|2 content of Chlorella, a fresh water alga, was f i r s t l y demonstrated by COMBS (1). One year later vitamin B ^ has been determined for the f i r s t time in marine algae (0.5-1 mcg/g) by ERICSON and BANHIDI (2). Later various papers were published in t h i s f i e l d by ERICSON and LEWIS (0.07-0.35 mcg/g) (3), BLACK and WOODWARD (0.004-0.08 mcg/g) (4), TEERI and BIEBER (0.082-0.312 mcg/g) (5), KUTSEVA and BUKIN (up to 0.311 mcg/g) (6), EMANUIL0V et al. (0.08-2.16 mcg/g) (7), KANAZAWA et a l . (0.028 mcg/g) (8) and GUVEN et a l . (0.025-0.055 mcg/g) (9, 10). Most comprehensive accounts on vitamin B ^ content of algae have been written by KANAZAWA (11, 12). I t was shown that sea water also contains vitamin B ^ (0.2-25 mmcg/1) (13-20).,;.. The question related with the source of vitamin B-^ in algae has not been elucidated yet. I t i s not clear that the source of vitamin B-,o in algae i s

Marine A l g a e in Pharmaceutical Science, Volume 2 © 1982 by W a l t e r de Gruyter &. Co., Berlin • New York

82 exogeneous or endogeneous. Some algae undoubtedly produce vitamin B ^

when

grown in pure culture (21). FORD and HUTNER proved that blue-green, brown and red algae, but not green algae, synthesize vitamin B-|2 (22). Vitamin B-j2 is required by a large number of algae (23). Firstly Euglena gracilis has been shown to require vitamin B-J2 and it was used in the bioassay of vitamin B ^

(24). For the elucidation of the source of vitamin B-|2 (24).

For the elucidation of the source of vitamin B ^ studied on vitamin B ^

in algae, FRIES in 1959

and cobalt free nutrient solution of Goniotrichum

elegans (red alga) (25) and stated that only the vitamin B-|2 contained series showed good and visible growth while the addition of cobalt had no effect. CARLUCCI and BOWES demonstrated that the content required vitamin found in pure cultures of algae is variable depending on the vitamin level of the external medium (26). PEDERSEN showed that vitamin B-|2 concentration has a role on the growth of Litosiphon pusillus and Ectocarpus fasciculatus, two brown algae, cultured in artificial sea. In the concentration of 1 mcg/g vitamin B ^

stimulated the growth while 10 mcg/g inhibited (27).

According to ERICSON and LEWIS vitamin B-|2 factors were produced by bacteria and then accumulated by the algae (3). On the other hand vitamin B-|2 content of algae is variable depending on the division of algae. Vitamin B-J2 extraction techniques in natural sources are as follows: For the extraction of vitamin B-^ in sea water, gel filtration with Sephades G 2 5 (28), dilution (29), phenol (13), o-cresol and CCl^ extractions (30), dialysis with EDTA and ion-exchange resin chromatographic (14) techniques were used.

For the extraction of vitamin B^ 2 in fresh water algae BROWN et al. used hot aqueous methanol in the presence of ammonia and cyanide (21), PRATT water, isopropanol

(70 %), perchloric acid (31) and in marine algae ERICSON

and BANHIDI boiling water (2), HASHIMOTO water at 80°C (32), KANAZAWA water added KCN at pH 4.5-5 (11), GUVEN et al. phosphate buffer (9, 10) maceration techniques. In addition to the extraction, purification on charcoal and separation by ion-exchange resine and paper chromatographic techniques were applied in this laboratory.

83 For the determination of vitamin B ^ various organisms were used in bioassay as Euglena gracilis (29), Cyclotella nana (16), Monochrysis lutheri (33), Thalassiosira pseudonana, Bellerochea polymorpha (34), Lactobacillus leichmanii (20), in sea water, Euglena gracilis (31, 32), Escherichia coli (21), Ochromonas malhamensis (31) in fresh water algae and Lactobacillus leichmanii

(2, 8-10), Euglena gracilis (32) in marine algae.

In this work vitamin B-|2 content of fourteen algae was investigated and the factors influenced this determination were studied. EXPERIMENTAL I- Material The algae investigated are listed below: Chlorophyceae Enteromorpha linza

(Linn.) J. Agardh

Phaophyceae Cystoseira corni culata

Hauck

Cystoseira crinita

Bory

Dictyopteris membranacea

(Stackhouse) Batters

Halopteris scoparia

(Linn.) Sauvageau

Rodophyceae Acanthophora del ilei

Lamouroux

Digenea simplex

(Wulfen) C. Agardh

Gigartina teedii

(Roth) Lamouroux

Gracilaria confervoides

(L.) Grev.

Grateloupia dichotoma

J. Agardh

Halophytis incurvus

(Huds.) Batters

Liagora farinosa

Lamouroux

Polysiphonia subulifera

(C. Ag.) Harvey

Rytiphlaea tinctoria

(Clem.) Ag.

These algae were collected from the coasts of Aegean and Mediterranean Sea.

84 I I - Method 1- Extraction of vitamin B-|2 100 g dried alga material was macerated in 1000 ml phosphate buffer (pH 6) in dark. After centrifugation and f i l t r a t i o n the vitamin B-|2 was adsorbed on 100 g charcoal. The charcoal part was separated and extracted by shaking with 1000 ml 65 % hot ethanol or in Soxhlet with 1000 ml 6b % ethanol. The extracts were evaporated in rotary vacuum evaporator. The residues were called as crude extracts. 2- Purification and separation of vitamin B ^ from crude extract. For this purpose two techniques were used. 2.1- Ion-exchange column chromatography The crude extract was applied on ion-exchange column chromatography with Ionenaustaucher IV (E. Merck, Darmstadt) (1.5 x 10 cm). The column was eluted with 150 ml 65 % ethanol with a flow rate of 6 drops/min. The eluats were concentrated in vacuo. 2.2- Preparative paper chromatography For this purpose the crude extract was applied on S

S 2043 b paper. Sol-

vent system of sec.-butanol saturated with HCN solution (0.01 %) was used in descending paper chromatography. For location of vitamin B ^ on chromatograms, reference vitamin B-|2 (Cyanocobalamin reference standard USP) was used. After developing, the spot of reference vitamin B ^ was detected by visual inspection and the corresponding band of algal extract on chromatogram was cut out and eluted with water. The eluates were concentrated in vacuo.

85 3- Determination of vitamin B-|2 For this purpose Cyanocobalamin Reference Standard solution was prepared according to the USP XIX technique and vitamin B ^ was determined using the same technique as explained below (3b). Stock culture of Lactobacillus leichmanii ATCC 7830 was prepared and then inoculated in broth tubes. It was suspended and added in Basal Media containing Cyanocobalamin reference standard solution and or algae extract. After incubation the turbidity of standard solution and samples is determined by measuring the percent light transmission at 600 nm in a spectrophotometer (Spekol, Jena, DDR).

For the calculation, standard concentration-response curve for each test was used. From this standard curve, the vitamin B ^

content for each sample

was determined and these values were multiplied by the dilution factor. Results and Discussion The standard curves of vitamin B-|2 (Cyanocobalamin) are shown in Fig. 1 and the amounts of vitamin B ^

in marine algae are listed in Table 1.

Aqueous extracts containing vitamin B ^

isolated from natural materials

are often accompanied by vitamin B ^ analogues. The separation of these analogues is difficult, because they are closely related to vitamin B ^ The impurities may seriously enhance or depress the growth (36).

For the purification the charcoal adsorption technique was firstly used by LALAND and KIEM in 1936 during isolation of vitamin B ^

from liver (37).

Later for industrial production other methods including column chromatography on cellulose, ion-exchange resin, paper and thin-layer chromatographic techniques and electrophoresis were used (36). In this work

for the purification of vitamin B ^ charcoal adsorption

technique and for the separation, ion-exchange column and paper chromato-

86 graphic techniques which are earlier applied on algae in this laboratory were used with some modification. According to the results obtained from these techniques the purification of the crude extract decreases the vitamin B ^ content. The comparison of the findings obtained by ion-exchange and paper chromatographic techniques showed that the latter gave higher values of vitamin B ^ -

It can be con-

cluded that the elution of vitamin B ^ on ion-exchange column has not been conpletely achieved. These findings showed that, .the amount of vitamin B ^

is variable depending

on the purification techniques used. On the other hand filtration of the aqueous extract obtained from brown algae showed difficulties. This is thought to be due to the alginic acid content of algae that may disturb the results.

The other problem is the sensitivity of vitamin B ^ to light and pH. Consequently one or other of these agents would have destroyed it during the extraction procedure. For the stability the extraction was made at pH 6 and in dark. Another problem in the determination of vitamin B ^ content of algae is the assay methods used. It was shown that assay methods have a role on the findings of vitamin B ^ amount. According to KANAZAWA the values obtained with two different organisms Euglena and Ochromonas are not similar. Among the determination methods applied on vitamin B ^ » microbiological assay with Lactobacilli known to be sensitive enough. The complication with all Lactobacilli is that they respond not only to vitamin B ^ > employing paper chromatography is the way to eliminate this interference (36). For this purpose the microbiological assay was made after the separation of vitamin B ^ by paper chromatography. Among the fourteen algae investigated in this laboratory vitamin B ^ tent

con-

of only 3 had already been tested by KANAZAWA (11,12). The results

Table I. Vitamin B 1 ? content of tested algae mcg/100 g dry weight.

Crude extract

Purified crude extract by C.C.* p,.C.**

Enteromorpha linza

1,,427

0.125

-

Cystoseira corniculata

0.,090

0.886

0,.943

Cystoseira crinita

0.,241

0.441

1,.012

Algae

Dictyopteris membranacea

0,.124

0.421

0,.721

Halopteris scoparia

0.,269

0.556

0,.501

Acanthophora del ilei

0,,172

0.486

1,.510

Digenea simplex

0..162

0.047

2,.899

Gigartina teedii

0,,330

0.353

1,.295

Gracilaria confervoides

0.,315

0.245

0,.042

Grateloupia dichotoma www Halophytis incurvus

0,.259

0.970

8,.535

0,,214

0.297

1..222

Liagora farinosa

0..063

0.024

2,.436

Polysiphonia subulifera

0..370

8.659

14,.850

Rytiphlaea tinctoria

-

4.603

15,.815

*Column Chromatography on Ionenaustauscher IV uu Paper Chromatography ***Güven, K.C. and Kizil, Z.: Unpublished data

Table II. The comparison of vitamin B ^ content of algae mcg/100 mg.

Algae

Kanazawa 1961, 1963

Enteromorpha linza

9.75 xx

Present authors^***) 1.427

0.125

Gigartina teedii

5.54^)

3.49^ ^

0.330

0.353

1.295

Digenea simplex

8.36^

1.69^**)

0.162

0.047

2.899

(^Euglena method [ uu v 'Ochromonas method

v

^^Microbiological

assay with Lactobacillus leichmanii

88 of this work and those obtained by KANAZAWA are not similar. I t can be attributed to the difference between the methods (Table I I ) . Conclusion According to these results i t can be concluded that the problem concerning the extraction and determination of vitamin B ^ have not been solved yet. Present authors propose that the extraction, separation and determination techniques of vitamin B ^ must be standardized. SUMMARY In this work 1 green, 4 brown and 9 red algae were investigated in respect to their vitamin B ^ contents. Aqueous algal extract was purified by charcoal adsorption technique and the crude extract was separated by ionexchange column and preparative paper chromatography. Lactobacillus l e i c h manii was used for microbiological assay. The results showed that the amount of vitamin B-|2 in algae depends on the methods used in the extraction, purification and determination techniques. The authors propose that all techniques used for the determination of vitamin B ^ content of algae must be standardized. Acknowledgments The authors thank to the S c i e n t i f i c and Technical Research Council of Turkey for supporting this work and to Prof.Dr. E. Dizdar for his help on cellecting the algae with his research ship " P i r i Reis".

89

0,1

0.2

I 0.3

I OA

— I T — I 0,5 0,6 0,7

I 0,%

I 0,9

I 1.0

I 1.1

1,2

1.3

1.4

1,5

Concn.Q2mmc^ml Fig. 1.

The standard curves of vitamin B 1 2 plotted in the assay. These curves were d a i l y prepared for each assay.

90 REFERENCES 1- Combs, G.F. 1952. Algae (Chlorella) as a source of nutrients for the chick. Science 216. 453 - 454. 2- Ericson, L.-E. and Z.G. Banhidi. 1953. Bacterial growth factors related to vitamin B ^ and folinic acid in some brown and red seaweeds. Acta Chem. Scand. 7. 167 - 172. 3- Ericson, L.-E. and L. Lewis. 1953. Occurence of vitamin B-|2 factors in marine algae. Arkiv Kemi 6. 427 - 442; C.A. 48. 7684 (1954) 4- Black, W.A.P. and F.N. Woodward, 1957. The value of seaweeds in animal feedingstuffs as a source of minerals, trace elements, and vitamins. Empire J. Expt. Agric. 2jj. 51 - 59. 5- Teeri, A.E. and R.E. Bieber. 1958. B - complex vitamins in certain brown and red algae. Science ]2J_. 1500-1501. 6- Kutseva, L.S. and V.N. Bukin. 1957. Marine plants and saprofels as sources for vitamin B 1 2 . Dokl. Akad. Nauk. S.S.S.R. 215. 765 - 767; C.A. 52. 4107 (1958) 7- Emanuilov, I., L. Nachev, P. Velcheva and T. Daov. I960. Vitamin B ^

in

certain Black sea algae. Mezhdun. Konf. Vitamin., Dokl. Suobsht., Sofia 59 - 66; C.A. 60. 9592 (1964) 8- Kanazawa, A., A. Saito and D.R. Idler. 1966. Vitamin B in Dulse (Rhodymenia palmata). J. Fish. Res. Bd. Can. 23. 915 - 916. 9- Giiven, K.C., E. Mutluay, E. Aktin, and H. Koyuncuo^lu. 1975. Chemical and Pharmacological investigations on Corallina rubens. Planta Medica 27. 5 - 13. 10- Giiven, K.C., E. Gliler and Y. Ylicel. 1976. Vitamin B 1 2 content of Gelidium capillaceum Kiitz. Botanica Marina XIX. 395 - 396. 11- Kanazawa, A.: 1961. Studies on the vitamin B - complex in marine algae - I. On vitamin contents. Kagoshima Daigaku Suisen Gakubu Kiyo U). 38 - 69. 12- Kanazawa, A.: 1963. Vitamin in algae. Bull. Jap. Soc. Sci. Fish. 29. 713 - 731.

91 13- Cowey, C.B. 1956. A preliminary investigation of the variation of v i t a min B-|2 in oceanic and coastal waters. J. Mar. B i o l . Assoc. U.K. 35. 609 - 620; C.A. 51. 4073 (1957) 14- Kashiwada, K., D. Kakimoto, T. Morita, A. Kanazawa and K. Kawagoe. 1956 - 57. Vitamin B ^ in sea water. I I . The assay method and the d i s tribution of vitamin B ^ in the ocean. Nippon Suisan Gakkaishi 22. 637 - 40; C.A. 52. 7792 (1958) 15- Daisley, K.W. and L.R. Fisher. 1958. Vertical distribution of vitamin B 1 2 in the sea. J. Mar. Biol. Assoc. U.K. 37. 683 - 686; C.A. 53. 16620 (1959) 16- Ryther, J.H. and R.R.L. Guillard. 1962. Marine planktonic diatoms. I I . Use of Cyclotella nana for assays of vitamin B-^ in sea water. Can. J. Microbiol. 8. 437 - 445; C.A. 57. 17189 (1962) 17- Suprunov, A.T., A.G. Benzhitskii and L.N. Bugaeva. 1967. Content and distribution of vitamin B ^ in the l i t t o r a l zone of the Black sea. Din. Vop. Gidrokhim. Chern. Morya 133 - 142; C.A. 71. 94628 (1969) 18- Natarajan, K.V. 1970. Distribution and significance of vitamin B-^ and thiamine in the Subarctic Pasific Ocean. Limnol. Oceanogr. 15^. 655 - 659; C.A. 74. 45436 (1971) 19- Ohwada, K. and N. Taga. 1972. Distribution and seasonal variation of vitamin B ^ » thiamine and biotin in the sea. Mar. Chem.

61 - 73

20- Inoue, A., H. Koyama and S. Asakawa. 1973. Vitamin B-|2 contents in sea water along the coast of Fukuyama in 1970 and 1971. J. Fac. Fish. Anim. Husb. J_2. 13 - 20; Aq. Sei. & Fish. Abst. 4. 4138 (1974) 21- Brown, F., W.F.J. Cuthbertson and G.E. Fogg. 1956. Vitamin B-^ a c t i v i t y of Chlorella vulgaris Beij and Anabaena cylindrica Lemm. Nature 177. 188 22- Ford, J.E. and S.H. Hutner. 1955. Vitamins and Hormones 1J3, 101; in Smith, E.L. 1965. Vitamin B ^ » Methuen Co. & Ltd., London 23- Droop, M.R. Organic micronutrients. In Physiology and Biochemistry of Algae (Ed.) R.A. Lewin) Academic Press, New York, 1962 p. 141 - 159

92 24- Hutner, S.H., L. Provasoli, E.L.R. Stokstad, C.E. Hoffmann, M. Belt, A.L. Franklin and T.H. Jukes. 1949. Assay on antipernicious anemia factor with Euglena. Proc. Soc. Exp. Biol. Med. 70. 118 - 120 25- Fries, L. 1959. Goniotrichum elegans: a marine red alga requiring Vitamin B 1 2 . Nature. 183. 558 - 559 26- Carlucci, A.F. and P.M. Bowes. 1972. Vitamin B ^ » thiamine and biotin content of marine phytoplankton. J. Phycol. 8. 133 - 137 27- Pedersen, M. 1969. Marine brown algae requiring vitamin B ^ -

Physiol.

Plant. 22. 977 - 983; C.A. 72. 52204 (1970) 28- Daisley, K.W.: 1961. Gel filtration of sea water: Separation of free and bound forms vitamin B-^- Nature 191. 868 - 869 29- Daisley, K.W. 1958. A method for the measurement of vitamin B ^

concen-

tration in sea water. J. Mar. Biol. Assoc. U.K. 37. 673 - 681; C.A. 53. 16620 (1959) 30- Suprunov, A.T. and Z.A. Muravskaya. 1963. Method of determination of vitamin B-^ in sea water. Tr. Sevastopol'sk Biol. St., Akad. Nauk. S.S.S.R. 26. 463 - 466; C.A. 61. 6778 (1964) 31- Pratt, R. and E. Johnson. 1968. Deficiency of vitamin B ^

in Chlorella.

J. Pharm. Sei. 57. 1040 - 1041 32- Hashimoto, Y. 1954. Vitamin B ^

in marine and fresh water algae. J.

Vitaminol. (Japan) 2- 49 - 54; C.A. 49. 7651 (1955) 33- Martin, S.L.Y. 1973. Clarifying a method for the measurement of Vitamin B-|2 in sea water. Tethys b. 95 - 105 34- Swift, D.G., R.R.L. Guillard. 1977. Diatoms as tools for assay of total B ^ activity and cyanocobalamin activity in

sea water. J. Mar. Res. 35.

309 - 320 •h h 35- The United States Pharmacopeia. 1975. XIX

rev., Mack Publishing Co.,

Easton, Pa, p. 613 36- Smith, E.L. 1965. Vitamin B 1 2 , Methuen & Co, Ltd. New York 37- Laland, P., A. Klem. 1936. Experimenta to isolate the antianemic principle of the liver. Acta Med. Scand 88. 620 - 623

ANTIFUNGAL INDIAN

ACTIVITY

OF

DIFFERENT

FRACTIONS

OF

EXTRACT5

FROM

SEAWEEDS

Sreenivasa

Rao,

P.

and Y.A.

5helat

Central Salt and M a r i n e Chemicals Bhavnagar 364002, India

Research

Institute

Introduction

Rao

and

Shelat

(1979)

from

about

from

Saurashtra

observed

twentyseven

that

The

of

were

antifungal

form

as

(1979)

active

from

and

principles

fraction

Halimeda

against

extracts

Phaeophyceae

the

tuna.

A and

indica,

Gelidiella

acerosa.

and

Hypnea

musciformis.

The

following

Sabroud's ria

burnsii.

were

were

test

isolated

the

method

seaweeds

Amphiroa

used

in

India

and of

than

semipurified

strains

obtained

from on

Marine Algae in Pharmaceutical Science, Volume 2 © 1982 by Walter de Gruyter &. Co., Berlin • New York

cinctum.

on

study:

Alterna-

sp.,

Phytophthora

Rhizopus

maintained

antennina,

Helminthosporium

notatum.

and

yeast

Chemical

medium

sp.,

sp.,

oryzae.

National

e t al

corticata

maintained

present

Rao

asperum,

Sarqassum

Gracilaria

vasinfectum,

sp.

by

: Chaetomorpha

anceps,

fungal

a

Spathoqlossum

tenerrimum.

Penicillium

were

species

developed

Colletotrichum

Poona,

collected

strains

organisms

niqer.

Fusarium

Rhizoctonia

strains

more

the

Aspergillus

crassa.

Laboratory,

from

the

N eurospora

Yeast

obtained

obtained

were

fungal

during

sp.,

sp.,

of w h i c h

used

Curvularia

Pvthium

extracts

Rhodophyceae.

Sarqassum

different

agar

all

qymnospora.

Cystoseira

crude

seventeen

against

B by

following Padina

the

seaweeds,

coast,

crude

Chlorophyceae those

screened

:

94 Candida albicans. Candida tropicalis and

Saccharomvces

cervisiae. Evaluation of antifungal activity

of the two fractions of

the seaweeds was done as described

by Shelat

(1979).

Results The two different

fractions obtained

from the nine

algae when tested against seven test fungi showed activity in each case

(Table 1).

selected different

It can be observed

both the fractions of almost all the seaweeds under gation showed activity against Neurospora Rhizopus oryzae showed activity with few Fraction A of Gracilaria

corticata

crassa.

that investi-

while

extracts.

showed activity

all the organisms tested except Candida albicans.

against while

fraction B of the same alga showed activity against

Candida

albicans. Fraction A of Hypnea musciforrnis showed maximum

activity

against Candida albicans. while fraction B showed activity against this pathogen. from Chaetomorpha

no

Fraction A and B obtained

antennina showed

activity against

Candida

t ropicalis. It is necessary to take up a detailed fraction B of Gracilaria corticata

study on the effect of

and fraction A of Hypnea

musciforrnis against Candida albicans. a human

pathogen.

It was observed from the data (Table 2) that fraction A and B of Chaetomorpha

antennina. Halimeda tuna.

asperum. Cvstoseira

indica. Sargassum

tenerrimum and H ypnea musciforrnis

Spathoglossum

cinctum.

Sarqassum

showed activity

against

95 Colletotrichum musciformis substance

sp.

can

for

Of

be

use

these,

fraction

considered against

for

A and B of

developing

Colletotrichum

an

Hypnea

antifungal

sp.

Discussion Burkholder growing

et. .al ( 1 9 6 0 )

in P u e r t o

tamarensis

showed

Candida

albicans.

alcohol

and ether

representing Rhodophyta Shelat

(1979)

cinctum other

test

Khaleefa extracts

reported no

fungi.

were

Candida

in

of C a n d i d a

that

by

activity

stated

that when

against

Candida and

can

that

crude when

while

albicans,

it

of

crude extract

this,

extracts seaweed

is n o t

and

of

activity

was

it

and

in

can

traces

be

observed

of t h e

algae,

It m a y

be

extract

active

and

Rao

Sarqassum

it s h o w e d

recognised. of

Phaeophyta

tropicalis

activity

From

crude

be w e l l

a fraction

vice

Candida

the

water,

species

activity.

extracts the

algae

against

prepared

algal

Chlorophyta,

against

marine

Goniaulex

activity

( 1 975)

fractionated,

the

that

for antifungal

tropicalis.

fractionating

fungal

tropicalis

Phyla:

them

albicans.

tropical

inhibitory e,t a l

However,

cinctum

with

mentioned

of n u m e r o u s

activity

against case

major

tested

showed

Sarqassum

remarkable

three

and

working

Rico waters

is

against

anti-

further active Candida

versa.

Acknowledgement We

are

Salt the

thankful

and Marine

to

Dr.

K.S.

Chemicals

encouragement

and

the

Rao,

Acting

Research

Director,

Institute,

facilities

given.

Central

Bhavnagar

for

96 R eferences 1.

Eurkholder, P.R., Burkholder, B o t . M a r . 2, 1 4 9 - 5 6 (1960).

2.

Khaleefa, A.F., Khurbousha, M . A . M . , Metwalli, A., M o h e s e n , A.F. and Serwi, A.: Bot. Mar. 163-165 (1975).

3.

Rao, P.S. and Shelat, Y.A.: I n t e r n a t i o n a l S y m p o s i u m M a r i n e A l g a e of the I n d i a n O c e a n R e g i o n , C S M C R I , Bhavnagar, India, Abstracts 47 (1979).

4.

Rao, ibid

5.

Shelat, Rajkot,

P.S., Kalpana 47 (1979). Y.A.: India

S. P a r e k h

Ph.D. thesis, (1979).

L.M.

and

and Almodovar,

H a n s a H.

Saurashtra

Parekh

L.R.s

!

University,

on

97

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u ai H 01 ta -F c ID IH ID

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c
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10 C h a) • -p CL H < to

3 •H -C •p

VO

I-

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t-

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+>

>

JZ a.

e

3 •H h 10 (0 3

>

(H • 3 a LJ ta

•

a 10

-a- h -

in vo

I

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i-

h-

c\j ^ r

I I

l i

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I

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i l

in vo

i

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m

c o od

I-

t\j T-

• c\i

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o

sz -p

10 a u->

u

•

JZ -p c •rl E rH 0) zu

» Q. 10

N •H (01 JZ •H ir c

>

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a (0 E •rl ÍH O Q 10

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+>

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to rH rH tu •H TJ •H H tu L3 r-

I I I

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m

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Q o

a co

•

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10 >10 0.82 ( 0.32-1.2 )

0.035 ( 0.015-0.056 )

a

C o n t r o l g r o u p was given s a l i n e i n s t e a d of a t e s t compound. ^ V a l u e s in parentheses are p e r c e n t a g e s of a n i m a l showing hyperlipemia.

As the conclusion, we found that 3 kinds of the sulfated polysaccharides from sporophylls of Undaria pinnatifida are composed of fucose, galactose, glucuronic acid and ester sulfate as major components, and that the molar ratio of galactose to fucose is at a high ratio in comparison with the sulfated polysaccharides from other brown algae (1, 12, 13, 14, 15, 16), in particular the ratio was higher with C polysaccharide. Thus, the polysaccharides may be grouped under the name of fucogalactan-sulfates. From the fact that the C polysaccharide showed remarkable activities for anticoagulation as well as lipoprotein clearance, it seemed that the relative content(s) of galactose

120 and / or ester sulfate may be mainly related to the biological activities as far as these sporophyll sulfated polysaccharides are concerned.

Summary Three sulfated polysaccharides ( A, B and C ) were fractionated from an acidic aqueous extract of sporophylls of Undaria pinnatifida by DEAE-Sephadex column chromatography. They behaved each as a single component on electrophoretic test. Sugar constituents and ester sulfate of these polysaccharides were determined by GLC and PC, and it was found that Fuc, Gal and GlcUA are main components. All of them contained ester sulfate bound at an equatorial position. The molar ratios of Fuc : Gal : GlcUA : SC>42- were 1 : 0.77 : 0.33 : 0.32, 1 : 0.87 : 0.12 : 1.12 and 1 : 1.76 : 0.17 : 3.37 for A, B and C, respectively. Antithrombin activities of them were 1/27, 1/3 and 2 times that of heparin, respectively. In the lipoprotein clearance test, only C showed a remarkably high activity and it was about 1/2 3 times that of heparin.

References 1. Abdel-Fattah, A.F., Hussein, M.M.D. and Salem, H.M. : Carbohyd. Res. 33, 9-17 (1974). 2. Usui, Y. , Toriyama, T. and Mizuno, S.: The Meeting of Agric. and Biol. Chem. Soc. Jap. 1979 (oral presentation). 3. Moss, J.N. and Dajani, E.Z.: Screening Methods in Pharmacology. Vol. II (ed. by Turner, R.A. and Hebborn, P.) 121-143, Acad. Press. New York (1971). 4. Fujikawa, T., Anno, T. and Wada, M.: Nihon Nogeikagaku Kaishi 49_, 667-669 (1975) (in Japanese) . 5. Igarashi, 0., Iwaki, E. and Fukuba, H.: Agric. and Biol. Chem. 35 1836-1843 (1971). 6. Nevins, D.J., English, P.D. and Albersheim, P.: Plant Physiol. 42, 900-906 (1967).

121 7. Sawardeker, J.S., Sloneker, J.H, and Jeanes, A.R.: Anal. Chem. r7, 1602-1604 (1965). 8. Knutson, C.A. and Jeanes, A.: Anal. Biochem. 24, 470-481 (1968). 9. Dodgson, K.S. and Price, R.G.: Biochem. J. 8£, 106- 110 (1962). 10. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R. J.: J. Biol. Chem. 193, 265-275 (1951). 11. Shimada, K., Igarashi, M. and Asada, T.: J. Med. Soc. Toho, Japan 18, 939-944 (1971) (in Japanese). 12. Larsen, B., Haug, A. and Painter, T.J.: Acta Chem. Scand. 20, 219-230 (1966). 13. Percival, E. : Carbohyd. Res. 1_, 272-283 (1968). 14. Percival, E.G.V. and Ross, A.G.: J. Chem. Soc. 717-720 (1950) . 15. Abdel-Fattah, A.F., Hussein, M.M.D. and Salem, H.M.: Carbohyd. Res. 33, 19-24 (1974). 16. Abdel-Fattah, A.F., Hussein, M.M.D. and Salem, H.M.: Carbohyd. Res. 33^, 209-215 (1974).

EFFECT

Sreenivasa

OF

Rao,

SEAWEED

P.,

K.5.

EXTRACTS

Parekh,

ON

H.H.

Central Salt and Marine Chemicals Bhavnagar 364002, India Trivedi,

S.B.

and

B h a s k a r A.

MYCOBACTERIUM

Parekh

Research

and

H.M.

Mody

Institute

Dave

Tuberculosis Research Centre, Amargadh 364210, India.

Shri

K.J.

Mehta

T.B.

Hospital

Introduction Burkholder Nadal

et

effective reports

et

al

(1960),

al. ( 1 9 6 4 ) against

of the

Mycobacterium.

to

find

out the

clinical

seaweed

Hence

effect

isolates

et

that

al

(1962)

the

Mycobacterium.

Indian

of

Starr

reported

However,

extracts

present

and

seaweed

there

of s e a w e e d

are

inhibiting

investigation extracts

of M y c o b a c t e r i u m

Martinaz-

extracts

were no

the

was

against

growth

undertaken the

tuberculosis.

Results The

fraction

lipid

Caulerpa

taxifolia,

Gracilaria fractions ( 1 978) sis.

corticata - Fraction

and Rao

atypical

and

H37RV

Various addition

of

ejt a l

five was

Padina were

sp.,

of E n t e r o m o r p ha

Gelidiella

fractionated

A and B - by ( 1979)

typical

used

medicated

the e x t r a c t s

and

the

they

strains

as a c o n t r o l

acerosa obtain

method

and

two

given

were tested

of M y c o b a c t e r i u m

by

Parekh

against

two

tuberculo-

strain.

Lowenstein-Jensen

of f r a c t i o n

to

prolifera,

A a n d B in t h e

media were

prepared

concentration

Marine Algae in Pharmaceutical Science, Volume 2 © 1982 by Walter de Gruyter & Co., Berlin • N e w York

of

16,

by 32,

124 64 and 12B ng/ml of the medium.

The bacteria were isolated

from the sputum of the diagnosed case of tuberculosis admitted at Shri K.J. Mehta T.B. Hospital, Amargadh. After six weeks of incubation period, the final results revealed complete inhibition of the growth of tubercle bacilli in the cultures indicating high effectiveness of antibiotic substance isolated from seaweeds against various tubercle bacilli (Table 1). It is interesting to note that the antibiotic

substances

isolated as fraction A and B from the seaweeds under investigation were particularly effective against atypical form of tubercle bacilli.

However fraction B of the alga under

test were more effective against atypical strains than that of fraction A.

It was further observed that all the concen-

trations of both the fraction from Enteromorpha

prolifera

were completely inhibitory to the growth of atypical bacteria tested. In order tQ know the minimum inhibitory concentration

(MIC)

value for various strains of M.tuberculosis, fraction A and B of Enteromorpha prolifera were used and the results showed that MIC for both typical and atypical ranged between 50 /ug to 100 /ug per ml of the medium for fraction A and 100 /jg per ml of the medium for fraction B (Table 2). Acknowledgements The authors are thankful to Dr. D.J. Mehta, the then

Director

and Dr. K.S. Rao, Acting Director of Central Salt and Marine Chemicals Research Institute, Bhavnagar for encouragement and facilities provided.

125 References 1.

Burkholder, P.R., Hurkholder, Bot. Mar. 2, 149-156 (1960).

2.

M a r t i n a z - N a d a l , N . G . , R o d r i g u e z , L.V. and C a s i l l a s , Antimicro A g e n t s Chemother. 6B-72 (1964).

3.

Parekh,

4.

Rao, P.5., P a r e k h , K.5. and P a r e k h , H.H. s I n t e r n a t . S y m p . o n M a r i n e A l g a e of t h e I n d i a n O c e a n R e g i o n , C S M C R I , Bhavnagar, India, Abstracts 47 (1979).

5.

Starr, T . J . , D e i g , E.F., C h u r c h , K.K. and A l l e n , T e x a s Repr. Bio. Med. 20, 2 7 1 - 2 7 6 (1962).

K.5.:

Ph.D.

Thesis

L.M.

and A l m o d o v a r ,

L.M.: C.:

(1978).

M.D.:

126

in •p u

ra

H -P X ID

13 Ol m 3 (0 01 III q-

•

+> •H

>

•ri

-P U (D M

ra

H a u

i-t

ai XI D -P •H •P C •=C » *—

a) H XI 10 1-

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

i 1 1 i 1 1 1 1 1 1 i m 1 n cq 1 E 1 • u. 1 3 1 • 1 1 -H 1 M "D 1 O 1 tu 1 C 1 E 1 E 1 1 > U 1 D X I - p i ra 1 1 1 c 1 1 -H 1 o 1 *D 1 I ra rH i a . C I E 1 L-l 1 \ i 10 i 1 Dl i 1 1 3 i 1 i 1 c i 1 -ri i 1 1 CO i 1 c 1 ID •H 1 • 1 • H 1 -ri 1 M • m 1 -P 1 01 i "I a, Syringodium and Halodule.

5hese also support epiphytic growths

of many algal species. Review of the Literature on Tanzania1s Seaweed Resources: Tanzania's marine algal resources were first published upon by Sonder (63) from specimens which had been collected in Zanzibar by Dr. A. Roscher in January 1859. Sonder reported 40 species from Zanzibar, which included the blue-green alga Lynghya majuscula; the green algae Ulva reticulata, Halimeda macroloba; the brown algae Turbinaria decurrens, Oystoseira myrica; the red algae Bucheuma spinosun, Gracilaria lichenoidest Iaurencia papillosa, and Gelidium rigiduia (= Golidiella acerosa) to mention but a few (see Table 2 for entire list).

I11 all he reported 1 species represe-

ntative of the Division Cyanophyta, 10 species of the Chlorophyta, 8 of the Ehaeophyta, and 21 of the Hhodoplvta.

Thus over 50/o of the taxa

reported by Sonder (63) were Red Algae. Hauck (16, 17, 18, 19) is sometimes also cited as having published on soue taxa from Zanzibar. The taxa in oueation included Halimeda macroloba. Padina commersonii. Gracilaria corticaba, Sargassum lendi/;erum var. nonbassaense (see Table 3

for entire list).

The material published upon

by Hauck had been collected by J.LI. Hildebrandt in 3ast Africa, the Red Sea, •-"Thesis on file in the Library of the University of Dar es Salaam.

135 and Malagasy Republic.

A close scrutiny on Hauck's papers reveals,

however, that in almost all instances where the name Zanzibar is mentioned, he had referred to the collection sites ac "Mombasa, Zanzibar." This implies that the specimens had been collected at Mombasa.

Hie inclusion

of the name Zanzibar was due to the fact that Mombasa was then ruled by the Sultan of Zanzibar, and was considered to be part of Zanzibar.

Die

taxa reported by Hauck from Zanzibar should, therefore, be considered amongst the Kenyan rather than the Tanzanian algal flora.

Since, however,

the nature of the marine environment on the shores of Mombasa and those in Tanzania are very similar, it is to be expected that the algal species of the two countries are basically similar. Schmitz (56) contributed significantly to our knowledge of Tanzania's marine red algal resources.

In this work he enumerated 68 taxa occurring

in Tanzania and Kenya (see Table 4).

2ie taxa from Tanzania were based on

material that had been collected by Dr. Stuhlman in Zanzibar, by Er. bischer in Kikogwe (located just south of the Pangani River estuary), and by Holst in Dar es Salaam.

The taxa reported by Schmitz included agaro-

phytes such as Gracilaria confervoides (= G. verrucosa), G. corticata, Gelidium rigidum (= Gelidiella acerosa) and G. pannosum, and also carrageenophytes such as Eucheuma inerme, 3. striata, E. platycladum and E. spinosum, to mention but a few. Schröder (57) reported 82 marine algal taxa from East Africa (Table 5), and these were mostly from Tanzania. These included the blue-green alga lyngbya majuscula; the green alga Haliaeda opuntia; the red algae Gelidium rigidum (= Gelidiella acerosa), Gracilaria corticata, Gr. radicans, Hypnea muscifoimis, H. hamulosa, Eucheuma striata; and the brown algae Sargassum aquifolium, S. polycystum, Turbinaria conoides and T. decurrens. Additional records of Tanzania's marine algal resources are those by Post (53) , who made a contribution on a new species of Bostrychia (viz., tangatensis) collected from mangrove habitats at Kisosora, Tanga, near the mouth of the Mkulumuzi River, and also from Kibaoni, north of Mkokotoni, Zanzibar; those by Anderson (1) on Eucheuma striata from Zanzibar, and also by Dickinson (9) on E. cottonii and E. serra from Zanzibar. Gerloff (11) also made a useful contribution on the marine algae of Dar esSalaam, enumerating 48 taxa (Table 6).

Of these, 11 were representative

136 of the Division Chlorophyta, 13 were nenbers of the Braeophy ta, and 24 were representatives of the Hhodophyta. Here again the Hhodophyta comprised 500 of all the taxa. Amongst the green algal species recorded by Gerloff are Ulva fasciata, U. reticulata, Valonia macrophysa and Enteromorpha ¿.ompressa; amongst the members of the Ehaeophyta are Cystoseira myrioa, Hornophysa triquetra, Sargassum subrepandum, S. portierianum and Turbinaria oraata; and amongst the Hhodophyta are Gelidiella acerosa, Pterocladia capillacea, Gracilaria arcuata, G. corticatat G. foliifera, Hypnea musciformis and Laurencia papillosa. liore recently Taylor (64, 65) made contributions on the species of Turbinaria and Oaulerpa collected by Professors G.P. Papenfuss and H.j?. Scagel from East Africa during the International Indian Ocean Expedition in 1962.

Papenfuss (48) advanced our knowledge on the morphology and

taxonomy of Hoimophysa (ffucales:

Cystoseiraceae) of the region.

He

and Jensen (51) also made contributions on Cystoseira (Pucales: Cystoseiraceae), and with Chiang he (49) also made contributions on some species of Galaxaura from the region.

Subsequently, Papenfuss and

Edelstein (50) made a useful contribution on the morphology and taxonomy of the species of Sarconema

(Gigartinales, Solieriaceae).

She most recent and most extensive contributions on the taxa of seaweeds occurring in Tanzania are, however, those by Jaasund (22, 23, 24, 25, 26, 27, 28, 29, 30).

In his annotated l i s t of Tanzania's green, brown and red

marine algae (26), 291 taxa are included.

Of these 172, representing

about 600 of the total, are members of the Hhodophyta; 68 (i.e., 230 belong to the Chlorophyta; and 51 (i.e., 170) are members of the Ehaeophyta. The predominance of the Hhodophyta seerie; to be the general rule for tropical algal floras. Some of the species were reported by Jaasund as new to science.

ITLva pulchra, Ceramiua nulti.jugum and Turbinaria

tanzaniensis, are examples of such species. It might be pointed out that some of the taxa reported from Tanzania by Sonder (63), Schmitz (56), Schröder

(57) and Gerloff (11), etc., will

probably be shown to be but synonyms, nevertheless, it should be added also that most of the reports on the marine algae of Tanzania have been confined to the macroscopic members. The Cyanophyta and the Phytoplankton have been essentially overlooked.

Only recently have the phytoplankton

137 been investigated (e.g., by Bryceson, 1976: unpublished Hi.D. thesis*). It seems, therefore, that the total number of Tanzanian marine algal species most probably exceeds 500.

Tanzania thus has a relatively rich marine

algal flora. Potential Economic Uses of Seaweeds in general: Daring the past three to four decades there has been a world-wide interest in exploratory surveys of the seaweed resources of the world with a review to tap them for industrial use.

Biis interest was especially stimulated

by the difficulties experienced during V/orld V7ar II when the supplies of the hydrocolloid agar from Japan could no longer reach the western world. The gelling substance

agar, which is obtained from certain species of red

algae (e.g, species of Gelidiella, Gelidium, Pterocladia, Gracilaria, mentioned above) is indispensable as^solid culture medium in microbiology, and also finds innumerable applications in pharmaceutical and other industrial preparations as a gelling-, thickening-, and emulsifying agent (44, 46). Ihe production of agar from seaweeds in Japan is believed to have begun as far back as about 1660 (20, 44).

Subsequent research on this hydrocolloid

revealed it to be a polymer of galactose and anhydrogalactose sugars of D - and 1- configurations, respectively, and to exhibit negative optical rotation. A small amount of sulphate is also present in some of the galactose and anhydrogalactose units. liore recently methoxyl and pyruvate groups have also been detected in agar (2, 3). Other hydrocolloids of industrial importance also discovered from seavreeds during the past century are carra,f