The method of obtaining water-soluble polysaccharides of brown algae

 

(57) Abstract:

The invention relates to carbohydrate chemistry and relates to a complex of biologically active water-soluble polysaccharides, in particular of laminarans and fucoidans. The method provides for processing fresh or frozen, or dried seaweed solvents in order to remove low molecular weight substances, extraction of polysaccharides of 0.1 N hydrochloric acid at room temperature and with water at 50 to 60C. the Separation of laminarans and fucoidans and their subsequent fractionation is carried out using hydrophobic chromatography. Consistently received extracts of polysaccharides applied on the column separately. As hydrophobic sorbent used polychrome-1. The technical result is to simplify the process and increase the yield of the target product. 2 C.p. f-crystals, 2 tab., 2 Il.

The invention relates to carbohydrate chemistry and relates to a method of obtaining biologically active water-soluble polysaccharides of vegetable origin, in particular, of brown algae. These polysaccharides can be used in medicine, including in the chemical-pharmaceutical industry, fisheries, Microbiology, etc.

Bureij on the structure and biological activity of water-soluble polysaccharides: laminaran, fucoidans [1-13]. For example, laminaran are cryoprotectants [6], anticancer drugs [7] , activates the immune system of fish [2], fucoidans have anticancer, anticoagulant, antidromically, antiviral activities, including HIV, hepatitis and herpes etc., [3-5, 7, 14].

The chemical structure of water-soluble polysaccharides of brown algae is very diverse [1-13]. Laminaran - low molecular weight 1 3; 1 6-D - glucan, are heterogeneous in molecular mass, location, and content -1--->6-linked glucose residues. The fucoidans are a family of sulfated Homo - and heteropolysaccharides, where the main monosaccharide residue is L-fucose, and in L-fucose in their composition found galactose, mannose, xylose and glucuronic acid. The fucoidans are heterogeneous in molecular mass, monosaccharide composition, degree of sulfation. Content and structural characteristics of these polysaccharides vary considerably depending on the source and within the same source depending on the season and age of the algae [1, 8].

In connection with the necessity of communication structure - biological activity, obtaining medicinal fractionation.

In the literature described complex methods of obtaining water-soluble polysaccharides: allocation of laminarans and fucoidans from the same extract. This may be due to the fact that to obtain, for example, fucoidans prefer to use algae that do not contain laminaran. So, a brown seaweed Fucus vesiculosus, fucoidan, which currently is a commercial product, and that its biological activity and structural characteristics of the most widely studied, practically does not contain laminaran [14] . If necessary, obtain laminaran the fucoidans contained in significant amounts in all investigated brown algae are removed from the solution in the form of insoluble salts with heavy metals or insoluble complexes with cetavlon (cetyltrimethylammonium bromide) [12, 16]. Extracting fucoidan from these complexes is a time-consuming process associated with large losses of the target substance.

Analysis of the currently known methods of separation and fractionation of water-soluble polysaccharides of brown algae indicates that the processes of making them consist of several General stages[12, 14, 16, 17]:

1. Remove neskovic the allocation of polysaccharides using dried seaweed.

2. Extraction at elevated temperature (sometimes at different temperatures - 20-25oand 60 - 90oC) water-soluble polysaccharides from the crushed algae or deionized and free of grease algae water or water with the addition of CaCl2or weak acids with the addition of the formaldehyde removal of polyphenols, or without it[12, 14, 16, 17].

3. The separation of laminaran and fucoidan or deposition of a substance in the form of complexes with cetavlon or using anion exchange resins, which fucoidan sorbed. Laminaran, if it is contained in the algae, and in the first and in the second case remains in solution [12, 16].

The closest proposed solution method, allowing to isolate and fractionate water-soluble polysaccharides, designed for brown seaweed Laminaria cichorioides [16]. This seaweed contains approximately the same number of laminarans and fucoidans and is the best object to develop comprehensive way of obtaining laminarans and fucoidans. Prototype method includes: fractional extraction of algae L. cichorioides different solvents (methanol, chloroform, ethanol) at different temperatures in order to remove discomolo N HCl at 70oC. Separation of laminarans and fucoidans catalona, laminaran remains in solution, from which it after dialysis precipitated aqueous ethanol (80%). The destruction complex fucoidans with cetavlon hold 3 M CaCl2at 60oC. Fucoidans after dialysis fractionary using anion exchange resin DEAE-Sephadex A-25 (Cl-form). The fucoidans elute from the ion exchanger and simultaneously fractionary content of sulphates increasing gradient of sodium chloride in water (1.25, 1.5, 1.75 and 3 M NaCl solutions). The method allows to divide laminaran and fucoidans and to fractionate the fucoidans on the degree of sulfation.

The disadvantages of this method include the multistage and the complexity of the process associated with the use of cetavlon and anion exchange resins, namely, the destruction ataulaevich complexes, the need for dialysis solutions. This all leads to the loss of fucoidans, in addition nicosulfuron fraction of fucoidans not visadelta catalona and also lost. As shown in [14], where a method for separation and fractionation of water-soluble polysaccharides from F. vesiculosus, using ionoobmennoi chromatography, part of fucoidans from F. vesiculosus not cerastium against HIV infection. In the method prototype, there is no fractionation of laminaran. Our proposed method is shown in Fig.1 and includes:

1. Sequential processing of dry (50% aqueous ethanol) or, preferably, fresh or frozen brown seaweed ethanol (1:1, by weight), acetone and chloroform, leave salt, partially polyphenols, low-molecular substances (including mannitol, mono-, oligosaccharides, amino acids, peptides, pigments, lipids, iodine-containing substances, vitamins a, b and so on). In the solvent-extracted about 30-40% of the chemicals from the dry weight of the algae. Next alcoholic extract of brown algae can be directly used in the cosmetic industry and medicine as iodine-containing drug, a treatment for sore throat, diseases of the mucous membranes. Substances extractable with acetone and chloroform, can also be used in the cosmetic industry [16].

2. Sequential extraction of polysaccharides from the rest of algae 0.1 N HCl with added (1 ml/l) 40% formaldehyde at room temperature, and water at elevated temperature (60 - 70oC). Sequential extraction at different temperatures allows already at the stage of selection to refractionist axle composition (table. 1, 2). So, fraction F-1 from L. cichorioides consisted of fucose (65%), galactose (25%), mannose (5%), ramnose (3%) and xylose (2%), fraction F-2 from L. cichorioides consisted of fucose (92%) and galactose (5%).

3. The extract is separately applied on the column with a hydrophobic carrier (polytetrafluoroethylene). Laminaran adsorbed hydrophobic media. Vysokoerudirovannye the fucoidans easily elute with water (PL.1, 2, Fig. 2, A). A stepped gradient of ethanol in water elute with media laminaran mixed with nicosulfuron puktana (5% aqueous ethanol, PL. 1.2 content of sulphates not more than 5%) and the fraction of laminaran (10-, 15 -% aqueous ethanol, PL. 1, 2, Fig.2).

4. The fucoidans from solution wikidot fully 80% aqueous ethanol, or sequentially 60 - and 80% aqueous ethanol. Faction, eluruume with Polychrome-1 5-, 10 -, or 15% aqueous ethanol, freeze-dried. The yield of water-soluble polysaccharides is depending on the type of algae from 10 to 25% of dry weight of algae (table. 1).

5. The hydrophobic carrier recovered by ethanol, which column suiryudan more hydrophobic than laminaran, substances that make up about 20-30% of the dry weight of the algae. If necessary (for example, in the case of strong pig and and/or hydrochloric acid, depending on the nature of the pigment.

Identification of the obtained fractions was performed using the following methods. Monosaccharide composition of the fractions was determined after hydrolysis with 2 N HCl (100oC, 1 hour) carbohydrate analyzer "Biotronik, Germany. Polysaccharides in the fractions were also identified using the13C-NMR spectroscopy. NMR spectra-13C can include polysaccharides to a certain type [4, 13, 16]. So, for laminaran are characteristic signals C1 (103-104 M. D.), C3 ( - 1,3, 85-87 M. D.) and C6 atoms (61-62 memorial plaques). In NMR spectra-13C fucoidans are characteristic signals C1 atoms (96-100 M. D.), intense signals about 68 and 72 M. D. and 73-78 memorial plaques relating to the ring C-atoms desulfation and deglycosylation and sulfated and glycosylated, respectively, and signals C6 atoms (17,9-18,3 m D.). For alginic acid characteristic signals C1 (100-102 M. D.) C6 (175-178 memorial plaques). NMR spectra-13C is received on the device Bruker WM-250 in D2O at 40oC. as an internal standard was used methanol (50, 15). In Fig.2 shows the C13NMR spectra characteristic of fractions F (fucoidans), nicosulfuron fractions FL (mixture of fucoidan and laminaran) and L (laminaran). As can be seen from the table. 2, a new way for the first time gives the possibility of obtaining a natural cm is further investigation.

Previously we have proposed a method of extracting laminarans from extracts of dried seaweed with hydrophobic media - Polychrome-1 (polytetrafluoroethylene) [17], but the method does not involve the allocation of fucoidans, as well as stepwise fractionation of fucoidans and laminaran order to obtain standard fractions of these biologically active, heterogeneous polysaccharides. The method also provides for the processing of algae solvents that may upon receipt of laminaran, but it is undesirable in the case of hydrophobic media to create a technology of complex processing of algae, i.e. not only to obtain laminaran, but also for other water-soluble polysaccharides.

Our proposed method was applied for the separation and fractionation of water-soluble polysaccharides from brown seaweed L. cichorioides (example 1), L. japonica (example 2) and F. evanescens (example 3). These algae are widespread in the far East and, as you know, are significantly different from each other, the content and the structures they are composed of polysaccharides. The highest content (12%) of laminaran characteristic of L. cichorioides, the smallest (less than 1%) for L. japonica, F. evanescens SODERZhANIYa structural characteristics of fractions, obtained by the method prototype, as well as from each other depending on the type of algae and the type of extraction (table. 2). In the IR spectra of the fractions of fucoidans derived from L. cichorioides the proposed method has an absorption band at 840 cm-1indicating the axial position of the sulfate group (ie, C4). For fractions isolated from F. evanescens characteristic band at 820 cm-1indicating the presence of sulfate groups in the Equatorial position (i.e., when C2 or C3). This difference is significant, as the manifestation of various biological effects of fucoidans depends on the presence and position of sulfate in these polysaccharides.

Example 1 (Fig. 1, 2, PL. 1, 2).

5 kg of chopped fresh seaweed L. cichorioides fill of 5 kg of ethanol and incubated at room temperature for at least one day, the algae is separated and poured sequentially with acetone (5 kg) and chloroform (5 kg), in each case, maintaining the mixture at room temperature for at least one day with stirring.

After treatment with solvents spend sequential extraction of polysaccharides from the rest of algae 0.1 N HCl with added (1 ml/l) of 40% aqueous formaldehyde (5L; 4-5hours) under stirring at room temperatrure extracts separately applied to the column (h cm) with a hydrophobic carrier, Polychrome-1 (polytetrafluoroethylene). Fucoidan elute with water (10 l) to negative reactions to sugar (phenol-sulfuric acid method) [18]. A stepped gradient of ethanol in water elute fractions of laminaran mixed with fucoidan (5% aqueous ethanol; about 3 l) and laminaran (10-, 15% aqueous ethanol, approximately 2.5 l), each time washing the carrier to negative reactions to sugar. Fucoidan from solution wikidot consistently 60 - and 80% aqueous ethanol. Precipitation fucoidans washed with alcohol and acetone and dried in air. Solutions of laminaran, eluruume with Polychrome-1 5-, 10 -, or 15% aqueous ethanol freeze-dried. Media regenerate ethanol (5 l), which column suiryudan more hydrophobic than laminaran, substances that make up about 20-30% of the dry weight of the algae, and water (7-10 l). If necessary (for example, in the case of strong pigmentation of the medium after passing extracts) conduct additional regeneration 0.5-1.0 M solutions of alkali and/or hydrochloric acid. The outputs of laminaran and fucoidan are, respectively, 10.1 and 13.7% of the dry weight of algae L. cichorioides. The outputs of the individual fractions of polysaccharides from the dry weight of the algae are shown in table 1.

Example 2 (Fig. 1, PL. 1, 2).

5 kg of crushed fresh water and pour sequentially with acetone (5 kg) and chloroform (5 kg), in each case, maintaining the mixture at room temperature for at least one day with stirring.

After treatment with solvents spend sequential extraction of polysaccharides from the rest of algae 0.1 N HCl with added (1 ml/l) of 40% aqueous formaldehyde (5 l; 4-5 hours) with stirring at room temperature (cold extraction) and water (5 l; 4-5 hours) at elevated temperature (50 - 60oC; hot extraction). The extract is separately applied to the column (h cm) with Polychrome-1. Fucoidan elute with water (about 10 l) to negative reactions to sugar (phenol-sulfuric acid method [18]). A stepped gradient of ethanol in water elute fractions of laminaran mixed with fucoidan (5% aqueous ethanol; about 3 l) and laminaran (10-, 15% aqueous ethanol, approximately 2.5 l), each time washing the carrier to negative reactions to sugar. Fucoidan from solution wikidot fully 80% aqueous ethanol. Precipitation fucoidans washed with alcohol and acetone and dried in air. Solutions of laminaran, eluruume with Polychrome-1 5-, 10 -, or 15% aqueous ethanol, freeze-dried. Media regenerate ethanol (5 l), which column suiryudan more hydrophobic than laminaran, substances, comprising about 20-30 the following bandwidth extracts) conduct additional regeneration 0.5-1.0 M solutions of alkali and/or hydrochloric acid. The outputs of laminaran and fucoidan are, respectively, 0.5 and 5.3% of the dry weight of algae L. japonica. The outputs of the individual fractions of polysaccharides from the dry weight of the algae are shown in table 1.

Example 3 (Fig. 1, PL. 1,2).

5 kg of crushed frozen algae F. evanescens fill of 5 kg of ethanol and incubated at room temperature for at least one day, the algae is separated and poured sequentially with acetone (5 kg) and chloroform (5 kg), in each case, maintaining the mixture at room temperature for at least one day with stirring.

After treatment with solvents spend sequential extraction residue algae 0.1 N HCl with added (1 ml/l) of 40% aqueous formaldehyde (5 l; 4-5 hours) with stirring at room temperature (cold extraction) and water (5 l; 4-5 hours) at elevated temperature (50 - 60oC; hot extraction). The extract is separately applied to the column (h cm) with Polychrome-1. Fucoidan elute with water (10 l) to negative reactions to sugar (phenol-sulfuric acid method [18]). A stepped gradient of ethanol in water elute fractions of laminaran mixed with fucoidan (5% aqueous ethanol; about 3 l) and laminaran (10-, 15% aqueous ethanol, approximately 2.5 l), kao 60 - and 80% aqueous ethanol. Precipitation fucoidans washed with alcohol and acetone and dried in air. Solutions of laminaran, eluruume with Polychrome-1 5-, 10 -, or 15% aqueous ethanol, freeze-dried. Media regenerate ethanol (5 l), with which the column is eluated with more hydrophobic than laminaran, substances that make up about 20-30% of the dry weight of the algae and water (7-10l). If necessary (for example, in the case of strong pigmentation of the medium after passing extracts) conduct additional regeneration 0.5-1.0 M solutions of alkali and/or hydrochloric acid. The outputs of laminaran and fucoidan are, respectively, 3.9 and 18.5% of the dry weight of algae F. evanescens. The outputs of the individual fractions of polysaccharides from the dry weight of the algae are shown in table 1.

Example 4 (Fig. 1, PL. 1, 2).

500 g of dry algae L. cichorioides fill of 5 kg of 50% aqueous ethanol and incubated at room temperature for at least one day, the algae is separated and poured sequentially with acetone (5kg) and chloroform (5 kg), in each case, maintaining the mixture at room temperature for at least one day with stirring.

After treatment with solvents spend sequential extraction residue algae 0.1 N HCl with added (1 ml/l) of 40% aqueous formaldehyde is elevated temperature (50 - 60oC; hot extraction). The extract is separately applied to the column (9 x 70 cm) with Polychrome-1. Fucoidan elute with water (10 l) to negative reactions to sugar (phenol-sulfuric acid method [18]). A stepped gradient of ethanol in water elute fractions of laminaran mixed with fucoidan (5% aqueous ethanol; about 3 l) and laminaran (10-, 15% aqueous ethanol, approximately 2.5 l), each time washing the carrier to negative reactions to sugar. Fucoidan from solution wikidot consistently 60 - and 80% aqueous ethanol. Precipitation fucoidans washed with alcohol and acetone and dried in air. Solutions of laminaran, eluruume with Polychrome-1 5-, 10 -, or 15% aqueous ethanol, freeze-dried. Media regenerate ethanol (5 l), with which the column is eluated with more hydrophobic than laminaran, substances that make up about 20-30% of the dry weight of the algae and water (7-10 l). If necessary (for example, in the case of strong pigmentation of the medium after passing extracts) conduct additional regeneration 0.5-1.0 M solutions of alkali and/or hydrochloric acid. The outputs of laminaran and fucoidan are, respectively, 11 and 12.5% of the dry weight of algae L. cichorioides.

Captions to drawings

Fig. 1. The allocation of the hypoxia fractions (A - F-2, IN - FL5-2, - L15-2) obtained from L. cichorioides offered by way of

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17. T. N. Zvyagintseva)350.

1. The method of obtaining water-soluble polysaccharides of brown algae, characterized in that conduct processing of brown algae solvents (ethanol, acetone, chloroform), sequential extraction of polysaccharides from the remainder of the algae of 0.1 N hydrochloric acid at room temperature and water at 50-60oWith the purpose of preliminary fractionation, separation of laminarans and fucoidans and their subsequent fractionation using hydrophobic chromatography, using as a media Polychrome-1 (polytetrafluoroethylene).

2. The method according to p. 1, characterized in that the processing is subjected to fresh, or frozen, or dried seaweed.

3. The method according to p. 1, characterized in that the extract of polysaccharides applied on the column separately.

 

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