Abstract
Culture media of 24 commonly occurring microalgal and cyanobacteria species were subjected to a screening for total alkaloids, saponins, and phenol compounds, especially flavonoids and tannins. Both Wagner's and Mayer's reagents were employed in the alkaloid screening. Ferric chloride solution was used to detect phenolic compounds. Tannins were identified by the gelatine-salt block test. Shinoda's color reaction was used for flavonoids. Saponins were detected by the froth method. The species investigated were isolated from different marine and brackish water habitats. Media of five species gave positive alkaloid reactions with both reagents, whereas those of seven others were reactive only in one test. For the remaining 23 species, the presence of alkaloids in the culture medium can be excluded. In the media of 16 species, phenolic compounds were present. Five species produced ferric chloride–positive phenolic compounds. In six cases, positive gelatine-salt block tests were recorded. Four media gave positive reactions to the flavonoid test. It can therefore be assumed that at least two species excreted phenolic compounds other than flavonoids or tannins. Two media gave a positive saponin test.
Introduction
Over the past decades, considerable scientific and commercial interest has focused on natural products from aquatic organisms, especially invertebrates such as porifera or bryozoa. These might constitute new sources for, among others, pharmaceuticals, enzymes, or food additives (Borowitzka, Citation1995; Sano et al., Citation1998; Apt & Behrens, Citation1999; Burja et al., Citation2001). Microalgal metabolites are currently also attracting attention both from academia and industry (e.g., Apt & Behrens, Citation1999; Luesch et al., Citation2002). There are several reasons behind the surge of interest in microalgal metabolites. One is that traditional sources for drugs of natural origin such as terrestrial higher plants or actinomycetes have already been examined extensively and, hence, screening results are suffering from a high rate of redundancy in both structural type and mechanism of pharmacological action. Here, the microalgal phyla have been recognized to provide chemical and pharmacological novelty and diversity (Shimizu, Citation2000). In the eukaryotic algae, various dinoflagellate metabolites have been found as toxins in shellfish and other invertebrates. Well-known examples are paralytic shellfish toxins, brevetoxins, ciguatoxins, and dinophysistoxins (Hall & Strichartz, Citation1990; Konishi et al., Citation2004). These compounds exhibit highly specific modes of action, (e.g., on ion channels), which are of particular interest in medical and pharmaceutical research.
Reports of potent toxicity associated with cyanobacteria growing in nature suggested that these organisms might also serve as a source of useful compounds. Screening programs, started in the 1970s to collect and examine material from the field and established that cyanobacteria indeed produce novel compounds with antineoplastic, antimicrobial, and antiviral effects (Gleason & Holmgren, Citation1981; Patterson et al., Citation1991; Piccardi et al., Citation2000; Skulberg, Citation2000).
A vast number of organisms belonging to such phyla as Chrysophyta (Chrysophyceae, Xanthophyceae, Prymnesiophyceae), Euglenophyta, Chryptophyta, and Bacillariophyta remain, however, virtually unexplored. In many respects, they are close to the very productive dinoflagellates, and there is a good chance of finding new types of compounds and bioactivity in these algae (Shimizu, Citation2000).
The active excretion of dissolved organic compounds by marine phytoplankton into their environment was a subject of debate in the 1970s and 1980s (Anderson & Zeutschel, Citation1970; Thomas, Citation1971; Williams & Yentsch, Citation1976; Wiebe & Smith, Citation1977; Sharp, Citation1977; Jensen & Solndergaard, Citation1985). These investigations and discussions were, however, largely concerned with the ecological role and quantity of excreted photosynthetically derived organic carbon rather than with the molecular characterization of individual compounds isolated from growth media. Later, attempts have been made to define the excreted compounds or at least their activity. Laane et al. (Citation1986) identified amino acids while Hama and Yanagi (Citation2001) found carbohydrates in natural phytoplankton dominated by Skeletonema costatum. and Chaetoceros. species. Intra- and extracellular lipids have been identified by Parrish et al. (Citation1994) in cultures of the dinoflagellate Gymnodinium. cf. nagasakiense.. Twiner et al. (Citation2004) describe the activity of extracellular organic compounds excreted by Heterosigma. akashiwo. (Raphidophyceae) on mammalian cells.
In addition to carbon compounds, Pujo-Pay et al. (Citation1997) noted the excretion of dissolved organic nitrogen compounds, while Lemasson and Pagès (Citation1981) reported on organic phosphorus excretion.
The current work constitutes part of a systematic investigation aiming at obtaining phytochemical information about different microalgal and cyanobacteria species, isolated from marine and brackish water habitats. Here, we concentrate on compounds released by the cultured species into the culture media rather than investigating cell-bound ones. This approach is based on the consideration that conditioning of the immediate environment of algal cells (e.g., prevention of bacterial colonisation) would require active exudation of chemicals rather than cellular storage.
The current report is part of this phytochemical investigation and presents information on alkaloids, saponins, tannins, flavonoids, and other phenolic compounds excreted into the culture media. The assays used in this work were selected because they are known to give reliable results within a reasonable time. Reports about the biological activities of the excreted compounds will follow.
Materials and Methods
Microalgae and cyanobacteria
Marine microalgae were obtained from a culture collection of the institute that is used as stock for aquaculture purposes (). Organisms from a brackish water habitat were isolated employing standard procedures from a local lake (Banter see; ). The methods used for isolation, preservation, identification, and cultivation of the organisms have been described in detail by Moore et al. (Citation1988) and Patterson et al. (Citation1991).
Table 1.. Species investigated with sources and culture conditions.
In brief, microalgae and cyanobacteria that were selected for screening from brackish and freshwater habitats were initially cultivated in 50-mL Erlenmeyer flasks at 26°C, the organisms from marine habitats at 20°C, both with continuous overhead fluorescent illumination at a light intensity of 50 µE m−2s−1. Cultures of approximately 500-mL volume were then used at a final biomass of 1.3 g/L. The media were tested for activity; portions were archived at − 18°C to establish a “library” of these active solutions for future testing.
Methods of chemical screening
The chemical screening followed Lellau and Liebezeit (Citation2001). For the analyses, 1 mL of each culture medium was used without any extraction or other enrichment procedures. The respective tests were repeated twice.
Alkaloid test
Aliquots of the media were heated on a boiling water bath with 5 mL 2 M HCl for 5 min. The acidic solutions were then filtered through premoistened filter paper to give a clear particle-free solution. The filtrates were divided into two portions. One of these was tested with Mayer's reagent [1,358 g HgCl2 in 60 mL doubly distilled water (DDW)], 5.0 KI in 10 mL DDW, bined and the total volume adjusted to 100 mL. The second portion was tested using Wagner's reagent (2.0 g KI and 1.27 g I2 were dissolved in DDW, and the final volume adjusted to 100 mL). Both reagents precipitate alkaloids. The presence of compounds of this class can be established by the occurrence of turbidity or precipitation. Only samples that gave positive alkaloid reactions with both reagents are assumed to contain alkaloids.
Saponin test
Saponins were detected by their ability to develop a honeycomb-like froth that is stable for a period of 30 min and longer. Aliquots of the media (1 mL) were added to 10 mL of DDW and shaken for about 1 min. Only samples with froth stable for 30 min are assumed to contain saponins.
Flavonoid test (Shinoda's reaction)
The group of phenol compounds determined includes flavonoids, tannins, and other phenols. Due to the presence of vicinal oxygenated functions, many of these compounds can act as ligands for metal ions. The presence of flavonoids is indicated by the development of a color ranging from orange to red or magenta within 1 or 2 min. For the preparation, a small piece of magnesium ribbon was added to the alcohol solution of an aliquot of the medium, followed by the dropwise addition of concentrated HCl.
Tannin test
Tannins were detected by the gelatine-salt block test. To an aliquot of the medium (about 5 mL), 1 mL methanol was added. Sixty microliters of an aqueous 10% NaCl solution were added to the solution to salt out nontannin compounds, thereby eliminating any false-positive results. The resulting solution was filtered through premoistened filter paper, and 1 mL each of this filtrate was placed into four glass wells. Well 1 served as a control and no reagent was added. Well 2 contained 50 µL of a 1% gelatine solution, and the mixture was observed for the formation of precipitate. Well 3 comprised 50 µL of an aqueous solution containing 1% gelatine and 10% NaCl. The mixture was also observed for the formation of precipitate. Well 4 contained, as part of the tannin test, 40 µL of an aqueous 10% ferric chloride (FeCl3) solution. Color production and precipitate formation was noted.
A greenish-blue or greenish-black color and a positive gelatine-salt block test indicate the presence of tannins of the condensed type. A bluish-black color indicates the presence of tannins of the galloyl type. A color change after the addition of ferric chloride and a negative gelatine-salt block test is induced by other phenolic compounds (Lellau & Liebezeit, Citation2001).
Results and Discussion
Tables and summarize the results obtained. Five of the 24 microalgal and cyanobacteria species screened produced positive reactions to both Mayer's and Wagner's reagents. Seven species produced positive reactions to only one of them, probably as a result of a false-positive reaction ().
Table 2.. Alkaloids in microalgae and cyanobacteria media.
Table 3.. Phenol compounds in microalgae and cyanobacteria media.
Nine species showed a color reaction with ferric chloride indicating the presence of phenolic compounds (). Three out of these produced a positive reaction for flavonoids and six gave a positive gelatine-salt block test with either gelatine solution or gelatine/salt solution indicative for tannins. The two species with a positive color reaction toward ferric chloride but without flavonoids and tannins contain other phenolic compounds. These were Prymnesium parvum. (prymnesiophytes) and Phaeodactylum tricornutum. (bacillariophytes). However, the presence of phenolic compounds other than flavonoids or tannins cannot be established when the latter two compound classes are found to be present.
Saponins were detected in only two species, Coscinodiscus wailesii. and Phaeodactylum tricornutum. (bacillariophytes, ).
One important aspect to be considered in this examination is the fact that none of the investigated organisms were in pure culture. There is evidence that symbiotic or surface-associated bacteria are the producers of compounds extractable from seaweeds and invertebrates. For a number of pharmacologically active compounds in marine invertebrates, the actual source has been identified as bacteria (Liebezeit, Citation2005). In this context, it seems to be essential to isolate all organisms involved and to verify the results obtained, on mixed assemblages on pure cultures also. However, it is possible that the exchange of nutrients, biochemical stimuli, or metabolic intermediates between these organisms is essential for the production of the allelopathic compounds found in mixed communities. Thus, after successful culture of the bacteria, microalgae, and cyanobacteria, there is the possibility that the same organisms may not produce the secondary metabolites when grown in individual culture.
Conclusions
The data presented above indicate that most of the tested microalgae and cyanobacteria excrete a considerable number and variety of chemical compounds into the growth medium. This merits further and more detailed investigations. A comparison with the little information that is available in the literature indicates that, at least for some species, a high variability in chemical composition exists probably due to control by environmental conditions.
Related Research Data
References
- Admiraal W, Peletier H, Laane RWPM (1986): Nitrogen metabolism of marine planktonic diatoms; excretion, assimilation and cellular pools of free amino acids in seven species with different cell size. J Exp Marine Biol Ecol 98: 241–263.
- Anderson GC, Zeutschel RP (1970): Release of dissolved organic matter by marine phytoplankton in coastal and offshore areas of the northeast Pacific Ocean. Limnol Oceanog 15: 402–407.
- Apt KE, Behrens PW (1999): Commercial developments in microalgal biotechnology. J Phycol 35: 215–226.
- Burja AM, Banaigs B, Abou-Mansour E, Burgess GJ, Wright PC (2001): Marine cyanobacteria – a prolific source of natural products. Tetrahedron 57: 9347–9377.
- Borowitzka AM (1995): Microalgae as sources of pharmaceuticals and other biologically active compounds. J Appl Phycol 7: 3–15.
- Gleason F, Holmgren A (1981): Isolation and characterization of thioredoxin from the cyanobacterium, Anabaena. spec. J Biol Chem 256: 8306–8309.
- Hama T, Yanagi K (2001): Production and neutral aldose compositions of dissolved carbohydrates excreted by natural marine phytoplankton populations. Limnol Oceanog 46: 1945–1955.
- Hall S, Strichartz G, Moczydlowski E, Ravindran A, Reichardt PB (1990): The saxitoxins: Sources, chemistry, and pharmacology. In: Hall S, Strichartz G, eds., Marine Toxins: Origin, Structure and Molecular Pharmacology. Washington, DC, American Chemical Society Symposium Series No. 418, pp. 29–65.
- Jensen LM, Solndergaard M (1985): Comparison of two methods to measure algal release of dissolved organic carbon and the subsequent uptake by bacteria. J Plankton Res 7: 41–56.
- Konishi M, Yang X, Li B, Fairchild RC, Shimizu Y (2004): Highly cytotoxic metabolites from the culture supernatant of the temperate dinoflagellate Protoceratium. cf. reticulatum.. J Nat Prod 67: 1309–1313.
- Lellau T, Liebezeit G (2001): Alkaloids, saponins and phenolic compounds in salt marsh plants from the Lower Saxonian Wadden Sea. Senckenbergia Maritima 31: 1–9.
- Lemasson L, Pagès J, (1981): Excretion of dissolved organic phosphorus in tropical brackish waters. Estuarine, Coastal Shelf Science 12: 511–523.
- Liebezeit G (2005): Aquaculture of “non-food organisms” for natural substances production. In: Le Gal Y, Ulber R, eds., Marine Biotechnology II, Advances in Biochemical Engineering/Biotechnology, Vol. 97. Heidelberg, Springer, pp. 1–28.
- Luesch H, Harrigan GG, Goetz GH, Horgen FD (2002): The cyanobacterial origin of potent anticancer agents originally isolated from sa hares. Curr Med Chem 9(20): 1791–1806.
- Moore RE, Patterson GML, Carmichael WW (1988): New pharmaceuticals from cultured blue-green algae. In: Fauntin DG, ed., Biomedical Importance of Marine Organisms—Memoirs of the Californian Academy of Science, Vol. 13. San Francisco, Californian Academy of Science, pp. 143–150.
- Parrish CC, Bodennec G, Gentien P (1994): Time courses of intracellular and extracellular lipid classes in batch cultures of the toxic dinoflagellate, Gymnodinium cf. nagasakiense.. Marine Chem 48: 71–82.
- Patterson GML, Baldwin CL, Bolis CM, Caplan FR, Karuso H, Larsen LK, Levine IA, Moore RE, Nelson CS, Tschappat KD, Tuang GD (1991): Antineoplastic activity of cultured blue-green algae (CYANOPHYTA). J Phycol 27: 530–536.
- Piccardi R, Frosini A, Tredici MR, Margheri MC (2000): Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc.. J Appl Phycol 12: 543–547.
- Pujo-Pay M, Conan P, Raimbault P (1997): Excretion of dissolved organic nitrogen by phytoplankton assessed by wet oxidation and super(15)N tracer procedures. Marine Ecology-Progress Series 153: 99–111.
- Sano T, He J, Liu Y, Kaya K (1998): Isolation of bioactive compounds in cyanobacteria from Chinese fresh water. Phycolog Res 46: 13–17.
- Sharp JH (1977): Excretion of organic matter by phytoplankton: Do healty cells do it? Limnol Oceanog 22: 381–399.
- Shimizu Y (2000): Microalgae as a drug source. In: Fusetani N, ed., Drugs from the Sea. Basel, Karger, pp. 30–45.
- Skulberg OM (2000): Microalgae as a source of bioactive molecules-experience from cyanophyte research. J Appl Phycol 12: 341–348.
- Thomas JP (1971): Release of dissolved organic matter from natural populations of marine phytoplankton. Marine Biol 11: 311–323.
- Twiner MJ, Dixon SJ, Trick CG (2004): Extracellular organics from specific cultures of Heterosigma akashiwo. (Raphidophyceae) irreversibly alter respiratory activity in mammalian cells. Harmful Algae 3: 173–182.
- Wiebe WJ, Smith DF (1977): Direct measurement of dissolved organic carbon release by phytoplankton and incorporation by mocroheterotrophs. Marine Biol 42: 213–223.
- Williams PJL, Yentsch CS (1976): An examiniation of photosynthetic production, excretion of photosynthetic products, and hetrotrophic utilization of dissolved organic compounds with reference to results from a subtropical sea. Marine Biol 35: 31–40.