Fucus vesiculosus. These algal species are rich in polysac- charides (algic acid and its salts, fucoidan) and contain mannitol, lipids, iodine, and trace elements ...
Pharmaceutical Chemistry Journal
Vol. 38, No. 6, 2004
DEVELOPMENT OF EXTRACTION TECHNOLOGY AND CHARACTERIZATION OF EXTRACT FROM WRACK ALGAE GRIST E. D. Obluchinskaya1 and S. A. Minina2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 38, No. 6, pp. 36 – 39, June, 2004. Original article submitted June 09, 2003.
molecular weight of various fucoidan fractions ranges in order of magnitude from 103 to 105, which influences the solubility of this polysaccharide and determines its extraction properties. The proposed complex technology of wrack algae includes the stage of separating a fucoidan-containing extract from the initial grist. Since the technical yield of fucoidan in this stage was relatively low (66.65 ± 1.17%), it was necessary to develop more effective methods for obtaining a rich fucoidan-containing extract from wrack algae grist. The aim of this study was to solve this task and to characterize the products obtained in various stages of the proposed technology. Wrack algae grist was prepared and processed according to the scheme depicted in Fig. 1. The initial dry material was comminuted and sequentially extracted with chloroform and acetone in order to obtain a lipid – pigment concentrate. After removal of the extractant, the residue was treated with 96% ethanol in order to extract mannitol. Mannitol was purified by repeated recrystallization from ethanol. After removal of ethanol vapor, the grist was extracted with a 10% aqueous ethanol solution. The extract was separated by filtration, concentrated in vacuum, and treated with 96% ethanol in order to precipitate polysaccharides. This precipitate was separated on a centrifuge and dried to obtain fraction F1. Then, the algal residue obtained after extraction was treated with purified water at 40 – 70°C for 1.5 h on a water bath. The tincture was cooled, filtered, and treated with 96% ethanol in order to precipitate polysaccharides. This polysaccharide fraction was also separated on a centrifuge and dried to obtain fraction F2. Fractions F1 and F2 were combined, dissolved in purified water, and filtered. The combined extract was concentrated by ultrafiltration using a column filled with hollow fibers and simultaneously dialyzed by adding purified water. The obtained fucoidan-containing solution was lyophilized.
Wrack algae are a promising material for obtaining various biologically active substances. The most widespread species of these algae in the Russian Barents and White Seas are Fucus vesiculosus. These algal species are rich in polysaccharides (algic acid and its salts, fucoidan) and contain mannitol, lipids, iodine, and trace elements (Co, Ni, Mo, Mn, Ca, etc.) [1]. All components of algae possess pronounced pharmacological activity and find use in medicine [2 – 8]. The existing technologies used for processing brown algae allow only some of the biologically active substances contained in this material to be isolated, including mannitol, sodium alginate, iodine, and lipid concentrate [2, 9 – 12]. Various methods of purification used in the technology of these products leave many biologically active substances in wastes. For example, in the existing sodium alginate technology, the raw material is preliminarily treated with aqueous acid solutions in order to remove water-soluble components [2], which include polysaccharides, proteins, amino acids, and other valuable substances. Therefore, it is necessary to develop a complex technology for the processing of algae that would provide for obtaining the maximum number of useful biologically active substances from these macrophytes [4]. We have developed a new technology for such a complex processing of wrack algae, which not only yields the traditional final products, such as mannitol and sodium alginate, but also retains the lipid complex and gives an extract containing fucoidan [13]. Fucoidan is a complex sulfated polysaccharide, usually isolated from brown algae, in which the main monosaccharide unit is L-fucosa [1]. Fucoidans isolated from various algal species have different structures, compositions of monosaccharide residues, and degrees of sulfation. For most algal species, the structure of fucoidans is not yet studied [14]. The 1 2
Murmansk Institute of Marine Biology, Kola Scientific Center, Russian Academy of Sciences, Murmansk, Russia. St. Petersburg State Chemico-Pharmaceutical Academy, St. Petersburg, Russia.
323 0091-150X/04/3806-0323 © 2004 Springer Science+Business Media, Inc.
324
E. D. Obluchinskaya and S. A. Minina
Comminuted dry material Sequential extraction with chloroform and acetone, filtration, purification, and drying
Grist
Lipid – pigment concetrate
Extraction with 96% ethanol, filtration, purification, and drying
Mannitol Grist 1) Extraction with 10% aqueous ethanol (20°C); 2) Extraction with purified water; 3) Precipitation with 96% ethanol; 4) Combined extracts (F1 + F2); 5) Purification and filtration.
Fucoidal-containing extract
Sodium alginate
Fig. 1. Scheme of Complex Processing of Wrack Algae.
The residual grist was treated further in order to obtain sodium alginate. To this end, the grist was first extracted with water at 45 – 50°C and then doubly extracted with a 2% aqueous sodium carbonate solution. The extracts were combined, filtered, and treated with sulfuric acid (concentrated sulfuric acid was added until obtaining a total solution concentration of 0.4%). The precipitate was separated by filtration and dissolved in 1.5% aqueous sodium carbonate solution. Sodium alginate was purified by recrystallization from ethanol and dialysis, filtered through a Nutsch filter, and dried in a thermal box. The physicochemical characteristics of various components isolated from wrack algae are presented in Tables 1 – 4. MATERIALS AND METHODS The experiments were performed with dried and comminuted seaweeds (Fucus vesiculosus) from the Barents
TABLE 1. Physicochemical Characteristics of Lipid – Pigment Concentrate Obtained by Complex Processing of Wrack Algae Grist Characteristic
Qualitative TLC analysis
Description
TLC pattern shows three dark-blue spots with Rf = 0.023, 0.115, and 0.805 (lipid fractions) Optical absorption spectrum Spectrum of 0.5% solution of lipid concentrate in ethanol has maxima at 410 and 645 nm and minimum at 525 nm (due to chlorophyll contained in lipid – pigment complex) Weight loss upon drying 8.12 %
Sea, which is one of the most widely occurring shrub-forming wrack species in the Russian northern seas. Based on the published data [2 – 4, 7, 11] and the results of preliminary investigations [13], we selected the following solvents for extraction: acetone and chloroform (in the stage of obtaining lipid – pigment concentrate); 96% ethanol (isolation of mannitol); 10% aqueous ethanol solution and purified water (isolation of fucoidan-containing complex); 2% aqueous sodium hydrocarbonate (NaHCO3) solution (isolation of sodium alginate). The extraction with 10% aqueous ethanol solution was performed using either the percolation technique, which ensures obtaining high-quality extracts with a high yield of biologically active substances (in the optimum regime), or the traditional double maceration technique [15, 16]. Aqueous tinctures were obtained by the method of stepwise maceration with stirring. The lipid – pigment concentrate was qualitatively analyzed by TLC in a benzene – chloroform (50 : 50) system. The spots were revealed with a 5% phosphomolybdic acid solution in methanol. The lipid fractions are manifested by three dark-blue spots [17]. Mannitol was identified using a qualitative reaction, whereby it precipitates when the extract is treated with a 10% aqueous copper sulfate solution and 10% aqueous ammonia solution [18]. The qualitative analysis of a fucoidan-containing extract was performed as follows. Treatment of a 2% aqueous solution of this extract with an equal volume of 96% ethanol results in the formation of a flocky precipitate of polysaccharides. Adding concentrated sulfuric acid and a 1% aqueous solution of L-cysteine leads to the appearance of a yellow color and optical absorption peaks at 396 and 430 nm [19]. The content of fucoidan in the initial (dried and comminuted) material and in the extract was determined spectrophotometrically upon reaction with L-cysteine and sulfuric acid [19]. According to the results of such analyses, the content of fucoidan in the initial material was 14.70 ± 1.70%. The quality of sodium alginate was assessed according to the pharmacopoeial article (FS 42-3383–97. Sodium alginate for medicine). The total amount of extracted substances was determined as described in the State Pharmacopoeia (RSP-XI) [20].
TABLE 2. Physicochemical Characteristics of Mannitol Obtained by Complex Processing of Wrack Algae Grist Sample of mannitol Characteristic
Melting point, °C Rotatory
power [a ]20 D
reference
product of proposed technology
166 – 169
167.6 ± 1
+ 23…+24 £ 0.05
+23 0.04
£ 0.1
0.09
(c, 0.1;
Na2B4O7 solution) Ash content, % Weight loss upon drying, %
Development of Extraction Technology
325 Fucoidan yield, %
Yield, % 60 40
1 1
2
2
20 0 Maceration
60 50 40 30 20 10 0 0
Percolation
Fig. 2. Histograms of the content of (1 ) fucoidan and (2 ) total extracted substances in extracts obtained by methods of percolation and double maceration.
RESULTS AND DISCUSSION We have studied the peculiarities of the technology of obtaining a fucoidan-containing extract from wrack algae grist. First, we determined the influence of the method of extraction with a 10% aqueous ethanol solution on the yield of fucoidan and on the total yield of extracted substances. For the comparison, we used the percolation technique and the method of stepwise maceration with stirring (Fig. 2). It was established that the two-step maceration provided for a 21.36 ± 1.11% yield of fucoidan into the tincture. The percolation technique led to an increase both in the fucoidan yield and in the total amount of extracted substances. Subsequently, we used this method in the stage of extraction with a 10% aqueous ethanol solution. In this case, a dynamic equilibrium in the grist – extractant system for a 10% aqueous ethanol solution is attained within 12 h and further increase in the extraction time is inexpedient (Fig. 3). As a result, the technical yield of fucoidan in this extraction stage reached 51.86 ± 1.34%. The extraction with purified water was performed in the regime of stepwise maceration with stirring and heating.
2
4
Characteristic
Solubility Identity polysaccharides fucoidan Weight loss upon drying
4.51 ± 0.36 %
Sulfate ash and heavy metals
28.76 ± 0.64 % £ 0.001 %
Quantitative content of fucoidan
95.58 ± 0.71 %
10 12 14 16 18 20 22 24 Time, h
We have also studied the temperature dependence of the content of fucoidan in the aqueous extract. It was found that the optimum temperature interval for the aqueous extraction of the residual grist is 40 – 80°C (Fig. 4). At 40 – 50°C, the yield of fucoidan in the grist – extractant system is stabilized within 3 – 3.5 h. Further increase in the temperature does not lead to any significant increase in the target product yield, while high temperatures (90 – 100°C) even lead to a de-
TABLE 4. Physicochemical Characteristics of Sodium Alginate Obtained by Complex Processing of Wrack Algae Grist Characteristic
Description
Solubility
Identity
Product of proposed technology
Fine powder of light brown or creamy color Soluble in water, insoluble in organic solvents
8
Fig. 3. Kinetics of fucoidan extraction from wrack algae grist with 10% aqueous ethanol solution.
TABLE 3. Physicochemical Characteristics of Fucoidan-Containing Extract Obtained by Complex Processing of Wrack Algae Grist Description
6
Insoluble residue in boiling water Weight loss upon drying Sulfate ash and heavy metals
Requirements (FS 42–3383–97)
Product of proposed technology
Amorphous odorless powder of cream or light-gray (with creamy tint) color Sparingly soluble in water, forms turbid colloidal solution; practically insoluble in 95% ethanol, chloroform, and ether [20, p.175] Qualitative reaction with 2% CaCl2 solution (jelly precipitate formation) Qualitative reaction with dilute HCl solution (jelly precipitate formation) Qualitative reaction with saturated (NH4)2SO4 solution (no precipitation) Characteristic reaction B for sodium [20, p.159]
Amorphous odorless powder of creamy color Meets pharmacopoeial requirements
£ 0.2 %
0.14 %
£ 20.0 %
11.3 % 12.7 % £ 0.001 %
£ 35.0 % £ 0.001 %
Arsenic £ 0.0002 % Quantitative content 90.8% < [algin] < 106% of algin
Positive reaction (jelly precipitate) Positive reaction (jelly precipitate) No precipitate formation Positive reaction
£ 0.0002 % 92.17%
326 Fucoidan yield, % 30 20 10 0 0 10 20
E. D. Obluchinskaya and S. A. Minina Fucoidan yield, % 30
1
20 30
40
50 60 70 Temperature, °Ñ
80
90
100
Fig. 4. Temperature dependence of fucoidan yield from wrack algae grist extracted with purified water.
crease in the content of fucoidan in the extract, which is explained by the thermal lability of this polysaccharide. Extraction of the grist with purified water at a temperature of 45 ± 5°C with stirring at a rate of n = 80 – 90 rpm reaches equilibrium within 3 h in the first step and within 1 – 1.5 h (Fig. 5) in the second step (due to a decrease in the content of fucoidan in the grist). The total yield of fucoidan in this stage reached 36.29 ± 0.97%. In conclusion, this investigation showed that a fucoidan-containing extract should be prepared from wrack algae grist by sequential extraction of the residual grist by a 10% aqueous ethanol solution and purified water. The total yield of fucoidan into the combined extract amounted to 88.15 ± 1.11%. REFERENCES 1. G. K. Barashkov, Comparative Biochemistry of Algae [in Russian], Pishchevaya Prom-st, Moscow (1972), pp. 6 – 34. 2. A. I. Arazashvili, Biologically Active Substances and Other Natural Compounds in Marine Algae [in Russian], Tbilisi (1980). 3. I. S. Gurin and I. S. Azhgikhin, Biologically Active Substances of Hydrobiota: A Source of Components for New Drugs and Preparations [in Russian], Moscow (1981). 4. V. P. Zaitsev, I. S. Azhgikhin, and V. G. Gandel’, Complex Use of Marine Organisms [in Russian], Moscow (1980). 5. M. Ya. Rozkin, M. N. Levina, V. S. Efimov, and A. I. Usov, Farmakol. Toksikol., 54(5), 40 – 42 (1991). 6. G. Lewis, N. Sanley, and G. Guist, in: Commercial Production and Applications of Algal Hydrocolloids, C. Lembi (ed.), Washington University, Seattle (1988).
2
10 0
0
1
2
3
4
5
6
Time, h
Fig. 5. Kinetics of fucoidan extraction from wrack algae grist with hot water during (1 ) first and (2 ) second maceration.
7. V. J. Chapman and D. J. Chapman, Seaweeds and Their Uses, Chapman and Hall, London (1980). 8. S. Colliec, C. Boisson-Vidaal, and J. Jozefonvich, Phytochemistry, 35(3), 697 – 700 (1994). 9. R. N. Makarova, I. I. Samokish, V. A. Kompantsev, et al., RF Patent 2028153; Byull. Izobret., No. 4 (1995). 10. A. V. Podkorytova, N. M. Aminina, L. S. Zimina, and O. A. Kusheva, RF Patent 2070808; Byull. Izobret., No. 36 (1996). 11. H. A. Hoppe, T. Levring, and Y. Tanaka, Marine Algae in Pharmaceutical Science, Berlin (1979). 12. J. B. Larripa, M. Mudry-de-Pargament, M. Labal-de-Vinuesa, and A. M. S. Mayer, The 12th International Seaweed Symposium (1987), pp. 151 – 151, 491 – 496. 13. E. D. Obluchinskaya, The All-Russia Seminar “Recent Advances in the Chemistry and Technology of Plant Materials” [in Russian], Barnaul (2002), pp. 252 – 254. 14. A. I. Usov and O. S. Chizhov, Chemical Investigations of Algae [in Russian], Khimiya, Moscow (1988). 15. I. A. Murav’ev, Drug Technology [in Russian], Meditsina, Moscow (1975), Vol. 1. 16. V. D. Ponomarev, Extraction of Medicinal Raw Materials [in Russian], Moscow (1976). 17. L. A. Sirenko, A. I. Sakevich, L. F. Osipov, et al., Physicochemical Methods for Investigation of Algae in Hydrobiological Practice [in Russian], Naukova Dumka, Kiev (1980). 18. State standard GOST 8321–74. Mannitol. 19. A. I. Usov, G. P. Smirnova, and N. G. Klochkova, Bioorg. Khim., 27(6), 444 – 448 (2001). 20. USSR State Pharmacopoeia, XIth Ed. [in Russian], Meditsina, Moscow (1987), Vol. 1., p. 295.
