RU2028153C1 - Method of preparing biologically active substance from laminaria - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves the sequence extraction of minced thallus of Laminaria saccharina and L. digitata for preparing of mannitol, water-soluble polysaccharide complex, laminarane, fucoidane and sodium alginate by common technology. Method ensures to prepare some products simultaneously showing strong purgative effect, and sodium alginate showing radionuclide-binding property also, and water-soluble polysaccharide complex with increased yield (up to 12.6%). EFFECT: intensified process, increased yield of polysaccharide complex. 36 tbl

Description

 The invention relates to methods for the simultaneous production of mannitol, a water-soluble polysaccharide complex, laminaran, fucoidan, sodium alginate from the White Sea algae of the genus Laminaria and can be used in medicine, pharmacy, microbiology, in industries associated with radioactive metals.

A known method of producing polysaccharides from seaweed Laminaria saccharina L.) by grinding, washing thalli of seaweed, extraction with hot water at 80 ^o C by infusion or countercurrent percolation, separating the extract from raw materials, evaporating the extract to a ratio of 1: 0.8-1: 1.2, addition of 96% ethyl alcohol to the hot extract in the ratio of extract: ethanol 1: 1. Then the resulting mixture was defended, the lower suspension layer was separated, centrifuged, and after separation of the mother liquor, the precipitate was washed with 96% ethyl alcohol. The washed precipitate is dried, crushed. The finished product yield is 5.6% of the loaded raw materials. The resulting complex of polysaccharides has a laxative effect and is known as the drug "Laminaride". This method of obtaining close to the claimed and selected for the prototype.

 The disadvantages of this method are: a) obtaining only one product from algae (polysaccharides), while production wastes are valuable biologically active substances - mannitol, laminaran, fucoidan, sodium alginate; b) low yield of polysaccharides (5.6% of raw materials); c) incomplete precipitation of polysaccharides with ethanol due to an insufficient amount of ethanol; d) loss of ethanol when added to a hot concentrated extract to precipitate polysaccharides.

 The aim of the invention is the integrated processing of White Sea algae in order to simultaneously obtain mannitol, a water-soluble polysaccharide complex, laminar, fucoidan, sodium alginate, increasing the yield of a water-soluble polysaccharide complex, expanding the raw material base, increasing the biological activity of the target product, expanding the spectrum of biological action.

 The goal is achieved in that a method for the simultaneous production of mannitol, a water-soluble polysaccharide complex, laminar, fucoidan, sodium alginate with laxative, lipid-lowering, radionuclide-binding, hepatoprotective, anti-ulcer action, from the White Sea laminaria (a natural mixture of two types of kelp - Laminaria sacchina L.) is carried out by grinding raw materials, sequential extraction with 90-96% ethanol, hot water and a 1.5% sodium carbonate solution; ethanol extraction is concentrated and mannitol is crystallized from it; a water-soluble polysaccharide complex is precipitated from an aqueous extract with 96% ethanol; while the remaining alcohol mother liquor is evaporated and laminaran and fucoidan are precipitated with 80% ethanol, the precipitate obtained is treated with a 0.4% hydrochloric acid solution and a 0.5 M solution of cetavlon is added to convert the acid polysaccharides to the precipitate, the extract is treated with ethanol ( to precipitate laminaran), and the remaining precipitate is redissolved in a 0.4% hydrochloric acid solution and treated with 70% ethanol (fucoidan); from carbonate extraction after pretreatment with concentrated sulfuric acid and sodium bicarbonate solution, sodium alginate is obtained.

According to the claimed method, the raw materials are first dried at 50 ^° C, after which they are freed from mechanical impurities. Then the raw material is ground to a particle size of 3 mm and extracted with 90-96% ethanol in the ratio of raw material: ethanol 1: 6 in a Soxhlet apparatus at the boiling point of the extractant for 3 hours. The filtered alcohol extract is evaporated to a condensed residue, crystallized, precipitated mannitol crystals purified by recrystallization. Further, after removing the residual alcohol from the algae, the latter are extracted twice with water in a ratio of 1:10 at a temperature of 80 ^o C for 30 minutes, the aqueous extracts are combined, filtered, evaporated under vacuum at a residual pressure of 0.3 atm and a temperature of 70 ^o C to density of 1.05-1.06 g / cm ³ ; the water-soluble polysaccharide complex is precipitated with 96% ethanol from the cooled extract in the ratio of extract: ethanol 1: 2, the complex is filtered, three times purified with 96% ethanol, dried. The alcohol mother liquor remaining after precipitation of the water-soluble polysaccharide complex is evaporated to 1/10 of the initial volume, after which the condensed extract is treated with 80% ethanol in the ratio of extract: ethanol 1: 3. The resulting precipitate (laminaran and fucoidan) is centrifuged. To separate laminaran and fucoidan, a centrifuged precipitate is treated with a 0.4% hydrochloric acid solution at a precipitate: hydrochloric acid ratio of 1: 5 at room temperature for 2.0 hours; then, a 0.5 M solution of cetavlon is added to the resulting solution to convert the acid polysaccharides to a precipitate. The precipitate was separated, and the supernatant was treated with 80% ethanol (to precipitate laminaran) in the ratio of supernatant: ethanol 1: 3. The resulting laminaran is filtered, purified with 96% ethanol, and dried. The precipitate formed after the addition of cetavlon is dissolved in a 0.4% hydrochloric acid solution in the ratio of precipitate: hydrochloric acid solution 1:10 and treated with 70% ethanol in a solution: ethanol ratio of 1: 3, a precipitate of fucoidan precipitates, which is filtered, purified, dried . After that, the remaining pulp of algae is extracted with a 1.5% solution of sodium carbonate in a ratio of 1:20 at a temperature of 50 ^o C for 1 h; the aqueous-alkaline extract is filtered, the filtrate is treated with concentrated sulfuric acid, the precipitate formed is separated, treated with a 1.5% sodium carbonate solution and sodium alginate is obtained by precipitation with 96% ethanol in a 1: 5 solution: ethanol ratio. Sodium alginate is filtered, dried.

 The advantages of the proposed method are the integrated processing of kelp: the simultaneous receipt of five products from the White Sea kelp: mannitol, a water-soluble polysaccharide complex, laminaran, fucoidan, sodium alginate. All target products have a biological effect. The laxative effect is described only for the polysaccharide complex from kelp. The technological scheme developed by us allows us to obtain target products with a pronounced laxative effect that exceeds the action of the official Laminaride preparation: mannitol has a laxative effect that exceeds Laminaride by 41.1%, the water-soluble polysaccharide complex - by 5.2-60.0% ( depending on the used test methods and animal species), laminaran and fucoidan - by 20.5%, sodium alginate - by 141.1%. In addition, the water-soluble polysaccharide complex has a pronounced radionuclide-binding, lipid-lowering, hepatoprotective and antiulcer effect, superior to or repeating the similar action of official drugs. Sodium alginate also exhibits a pronounced radionuclide-binding and hypocholesterolemic effect. All types of biological action for all target products, except for the laxative effect of the polysaccharide complex, we have established for the first time. The combination of laxative and radioprotective effects for sodium alginate and a water-soluble polysaccharide complex contributes to the rapid excretion of radionuclides from the body by these products.

 In addition, the inventive method provides the target products with a high yield (in terms of dry feedstock): mannitol - 7.85%; water-soluble polysaccharide complex - 12.60%; laminaran - 4.53%; fucoidan - 2.14%; sodium alginate - 12.41%. The proposed method can be used in industrial processing of kelp. This method uses a natural mixture of two types of raw materials growing in the marine basin of the White Sea: Laminaria saccharina L. and Laminaria digitata L.

 The obtained target products were subjected to qualitative and quantitative analysis (tab. 1,2,3,4,5,6,7,8,9).

 I. Mannitol.

 The obtained mannitol was identified by paper chromatography.

 The quantitative content of mannitol was determined by the method of reverse iodometry in the presence of a starch indicator. Titration was carried out until the green color of the solution. The mannitol content was 99.56%.

Thus, the distribution coefficient (R _f ), melting point, specific rotation, ash content of mannitol obtained, corresponding to those of standard mannitol, as well as a high quantitative content of mannitol indicate a high purity of the obtained product.

 II. Water soluble polysaccharide complex.

 The identification of polysaccharides was carried out using color reactions, chromatographic.

 The results of the analysis of the water-soluble polysaccharide complex shown in Table 3.4 are similar for the Laminarid preparation, which indicates the correspondence of the obtained polysaccharide complex to the Laminarid preparation.

 The quantitative content of polysaccharides was determined by the sum of reducing sugars by reaction with picric acid, spectrophotometrically. The content of reducing sugars was 76.55% in the water-soluble polysaccharide complex. In the Laminarid preparation, the content of reducing sugars reached 25.64%, i.e. the content of the amount of reducing sugars in the sample obtained by the claimed method is 3 times higher than this indicator compared with the method adopted for the prototype.

 Thus, as a result of the analysis of the water-soluble polysaccharide complex, polysaccharides were identified, a high content of reducing sugars was established, and the correspondence of the obtained water-soluble polysaccharide complex to the official Laminaride preparation was shown.

 III. Lamaran.

 The obtained indicators (table 5) are consistent with known literature data.

The monosaccharide composition of the obtained laminaran was determined by hydrolysis of 2 N. sulfuric acid at 100 ^o C for 6 hours, followed by neutralization of the hydrolyzate with barium carbonate by paper chromatography. The results are presented in table.6.

 The resulting laminaran was enzymatically digested with 1,3-β-glucanase. In this case, laminaran was broken down to glucose. The latter was determined by the colorimetric method. The laminaran content in the obtained sample was 81.22%.

 So, the analysis results confirm the identity and high content of laminaran.

 IV. Fucoidan.

The resulting fucoidan was hydrolyzed with 2 N sulfuric acid at 100 ^° C for 6 hours, followed by neutralization of the hydrolyzate with barium carbonate. The hydrolyzate was analyzed by paper chromatography. The results are presented in table.7.

 Quantification of fucoidan was carried out spectrophotometrically by the reaction of fucoidan with thioglycolic acid in a sulfuric acid medium. The fucoidan content was 72.25%.

 The results confirm the identity of fucoidan.

 V. Sodium alginate.

 Quantitative determination of sodium alginate was carried out by the method of back titration with a solution of sodium hydroxide of excess sulfuric acid remaining after its interaction with sodium alginate. The content of sodium alginate was 91.15%.

 Thus, the obtained analysis results confirm the authenticity and high quantitative content of sodium alginate.

 We have studied the dependence of the yield of target products on a number of technological parameters (tab. 10-31).

 As can be seen from table 10, the highest yield of mannitol is provided at an ethanol concentration of 90-96%.

 As follows from table 11, the maximum yield of mannitol is provided when using the ratio of raw materials: extractant 1: 6.

 From table 12 it is seen that the greatest yield of mannitol is achieved with a duration of extraction of 3 hours

As follows from table.13, the highest yield of water-soluble polysaccharide complex is provided at an extractant temperature of 80 ^o C.

 The results presented in table 14 indicate the optimal ratio of raw materials: extractant 1:10, as this provides the highest yield of the polysaccharide complex.

 As follows from table 15, the optimal duration of extraction is 30 minutes, since this achieves the maximum yield of water-soluble polysaccharide complex.

 From table 16 it follows that the optimal ratio of extract: ethanol during precipitation of the water-soluble polysaccharide complex is achieved in the case of 1: 2. This is due to the highest yield of the polysaccharide complex.

 As can be seen from table 17, the highest yield of laminaran and fucoidan is provided when using 80% ethanol.

 From table 18 it is seen that the greatest yield of the sum of laminaran and fucoidan is achieved when using the extractant in the ratio of extract: ethanol 1: 3.

 The highest solubility of the sum of laminaran and fucoidan (1.21 g in 100 ml of acid) is provided at a concentration of hydrochloric acid of 0.4%.

 From table 20 it follows that the best solubility of the precipitate is achieved by processing it with a 0.4% hydrochloric acid solution in the ratio of precipitate: acid solution 1: 5.

 From table 21 it follows that the most complete dissolution of the sediment laminaran and fucoidan is achieved with a duration of treatment with acid of 2 hours

 From the table. 22 it follows that the maximum yield of laminaran is achieved when the concentration of the precipitant is 80%.

 From table 23 it follows that the most complete deposition of laminaran is provided when the ratio of extract: ethanol 1: 3.

 As can be seen from the table. 24, the highest yield of fucoidan is provided by using ethanol at a concentration of 70%.

 From the table. 25 it follows that the maximum yield of fucoidan is achieved by treating a hydrochloric acid solution with ethanol in a ratio of 1: 3.

 From table 26 it follows that the maximum yield of sodium alginate is provided when using an extractant - sodium carbonate - in a concentration of 1.5%.

 From table 27 it is seen that the highest yield of sodium alginate is achieved when the ratio of raw materials: extractant 1:20.

From the table. 28 it follows that the optimum temperature for the extraction of sodium alginate is 50 ^o C, since this temperature provides the highest yield of sodium alginate.

 From table 29 it follows that the optimal duration of extraction of raw materials is 1.0 h, at which the yield of sodium alginate is maximum (12.41%).

 From table 30 it is seen that the most complete precipitation of sodium alginate occurs when using ethanol at a concentration of 96%.

 From table 31 it is seen that the most complete precipitation of sodium alginate occurs when the alkaline solution is treated with ethanol in a ratio of 1: 5.

 The inventive method for the simultaneous production of mannitol, a water-soluble polysaccharide complex, laminaran, fucoidan, sodium alginate is illustrated by the following specific examples.

PRI me R 1. White Sea algae (L. saccharina L. and L. digitata L.) are dried at 50 ^o C in an oven, then free them from mechanical impurities, mollusks. Then 100 g of algae are weighed to an accuracy of ± 0.0002 g, ground on a laboratory grinder to a particle size of 3 mm and extracted with 600 ml of 90% ethanol in a Soxhlet apparatus at the boiling temperature of ethanol for 3 hours. The resulting alcohol extract is filtered on a funnel Buchner under vacuum, evaporated to form a condensed extract (about 30 ml), add about 10 ml of water, the resulting pigment film is separated, then the solution is crystallized at a temperature of + 4 ^o C in the refrigerator. The precipitated mannitol crystals are twice purified by recrystallization from an alcohol-water solution. Pure mannitol crystals are filtered through a paper filter and dried at a temperature of 60 ^° C. in a vacuum oven. The yield of mannitol is 7.85 g or 7.85% of the feed.

 The target product, mannitol, is white crystals, odorless, soluble in water and boiling alcohol, slightly soluble in cold alcohol, insoluble in ether.

Further, the algae remaining after mannitol extraction is dried under draft to remove alcohol residues, after which they are loaded into a 2 L flask and filled with 1000 ml of hot (temperature 80 ^o C) distilled water. The mixture is heated on an electric stove at a temperature of 80 ^o C (temperature control was carried out by a thermometer). The extraction is carried out for 30 minutes twice. The resulting hot water extracts are poured from the pulp, combined, filtered under vacuum on a Buchner funnel through a double nylon cloth. The combined filtered extract is evaporated in a Wurz flask under vacuum at a residual pressure of 0.3 atm and a temperature of 70 ^o C to a density of 1.05-1.06 g / cm ³ (about 1/7 of the original volume). The resulting aqueous concentrate is treated with a double volume of 96% ethanol. The precipitate of the water soluble polysaccharide complex is decanted and filtered on a Buchner funnel under vacuum through a double nylon cloth. The precipitate is washed three times on a funnel with 96% ethanol, dried at a temperature of 60 ^o C in a vacuum drying oven.

 The yield of a water-soluble polysaccharide complex of the Laminaride type is 12.60 g or 12.60% of the feed.

 The target product is a water-soluble polysaccharide complex of the type "Laminaride" is an odorless, grayish-yellow amorphous powder. Virtually insoluble in alcohol. In water it dissolves sparingly with the formation of a turbid mucous solution.

The alcohol mother liquor (approximately 300 ml) remaining after precipitation of the water-soluble polysaccharide complex of the “Laminaride” type is evaporated under vacuum to 1/10 volume. Then the resulting concentrate is treated with 90 ml of 80% ethanol. The precipitate was centrifuged in a laboratory centrifuge at 2000 rpm. 40 ml of a 0.4% hydrochloric acid solution are added to the distilled precipitate in a beaker, the beaker with the mixture is closed and left for 2 hours at room temperature. After 2 hours, a 0.5 M solution of cetavlon is added to the resulting solution. At the same time, acidic polysaccharides precipitate. The addition of a 0.5 M solution of cetavlon is carried out until the formation of a precipitate of acidic polysaccharides, i.e. in the supernatant, the addition of cetavlon does not precipitate. 10 minutes after settling, the precipitate was separated by filtration through a paper filter. The supernatant (35 ml) was treated with 105 ml of 80% ethanol. A lamarana precipitate precipitates, it is filtered under vacuum on a Buchner funnel, washed twice with 96% ethanol, dried in a vacuum oven at 60 ^° C. The output of laminaran is 4.53 g or 4.53% of the feed.

 The target product - laminaran - is a powder of light beige color, odorless, soluble in water, in acids, insoluble in ethanol.

Further, the precipitate of acidic polysaccharides formed after the addition of cetavlon is dissolved in 20 ml of a 0.4% hydrochloric acid solution by stirring the mixture on a magnetic stirrer for 15 minutes. The resulting fucoidan solution is treated with 60 ml of 70% ethanol. In this case, fucoidan precipitates, the precipitate is filtered under vacuum on a Buchner funnel, washed twice with 70% ethanol, and dried in a vacuum oven at a temperature of 60 ^o C. The output of fucoidan is 2.14 g or 2.14% of the raw material.

 The target product - fucoidan - is a powder of light pink color, odorless, soluble in water, in acids, insoluble in ethanol.

The algae pulp remaining after extraction of the four products described above is placed in a 3 L flask, pour 2 L of a 1.5% sodium carbonate solution and extracted in a water bath at a temperature of 50 ^o C for 1 h. The alkaline aqueous extract is further decanted with bagasse and filtered under vacuum on a Buchner funnel. The aqueous alkaline extract is then treated with approximately 100 ml of concentrated sulfuric acid (99.95%) by slowly adding small portions of acid (approximately 5 ml) to the extract. In this case, a precipitate of alginic acid is released, which is separated by filtration on a Buchner funnel. This precipitate is transferred quantitatively to a flask with a capacity of 3 L, 300 ml of a 1.5% solution of sodium carbonate are added, stirred, the precipitate of alginic acid passes into a solution of sodium alginate. Sodium alginate is precipitated from the solution by adding 1.5 l of 96% ethanol to it. The precipitate formed is filtered on a Buchner funnel, dried in a vacuum oven at a temperature of 60 ^o C. The yield of sodium alginate is 12.41 g or 12.41% of the raw material.

 The target product - sodium alginate - is a thin plate of light orange color, odorless, mucous taste, soluble in water, insoluble in alcohol.

 Royal alcohol solutions are processed in order to regenerate alcohol from them.

PRI me R 2. Belomorsky algae are dried at 50 ^o C, free from mechanical impurities, weigh 100 g for analysis. A portion of the algae is ground to 3 mm particles and extracted with 700 ml of 96% ethanol in a Soxhlet apparatus at the boiling temperature of ethanol for 4 hours. The resulting alcoholic extract was filtered on a Buchner funnel under vacuum, evaporated to form a condensed residue (about 30 ml), about 10 ml of water was added and crystallized at + 4 ^° C in a refrigerator. The precipitated mannitol crystals are twice purified by recrystallization. Mannitol crystals are filtered and dried at 60 ^° C. The yield of mannitol is 7.80 g or 7.80% of the feed.

Then, the algae remaining after mannitol extraction is “dried” under a draft to remove alcohol residues, after which they are loaded into a 2 L flask and filled with 1500 ml of hot (temperature 90 ^o С) distilled water. The mixture is heated on an electric stove at a temperature of 90 ^o C. Extraction is carried out for 45 minutes twice. The resulting hot water extracts are poured from the pulp, combined, filtered under vacuum. The combined filtered extract is evaporated in a Wurz flask under vacuum at a residual pressure of 0.3 atm and a temperature of 70 ^o C to a density of 1.05-1.06 g / cm ³ . The resulting aqueous concentrate is treated with a three-fold volume of 96% ethanol. The precipitate of the water-soluble polysaccharide complex is decanted and filtered on a Buchner funnel under vacuum. The precipitate is washed three times on a funnel with 96% ethanol, dried at a temperature of 60 ^o C in a vacuum drying oven.

 The output of the water-soluble polysaccharide complex of the Laminaride type is 12.48 g or 12.48% of the feed.

The alcohol mother liquor (approximately 400 ml) remaining after precipitation of the water-soluble polysaccharide complex of the Laminaride type is evaporated under vacuum to 1/10 volume. Then the resulting concentrate is treated with 160 ml of 90% ethanol. The precipitate was centrifuged in a laboratory centrifuge at 2000 rpm. 45 ml of a 0.5% hydrochloric acid solution are added to the distilled precipitate in a beaker, the beaker with the mixture is closed and left for 2.5 hours at room temperature. After 2.5 hours, a 0.5 M solution of cetavlon was added to the resulting solution to convert the acid polysaccharides to the precipitate. The addition of a 0.5 M solution of cetavlon is carried out until the formation of a precipitate of acidic polysaccharides ceases. 10 minutes after settling, the precipitate was separated through a filter. The non-stop liquid (45 ml) is treated with 180 ml of 90% ethanol. The precipitate of laminaran is filtered under vacuum, washed twice with 96% ethanol, dried in a vacuum oven at 60 ^o C. The output of laminaran 4.50 g or 4.50% of the raw material.

Next, the precipitate of acidic polysaccharides formed after the addition of cetavlon is dissolved in 20 ml of a 0.5% hydrochloric acid solution by stirring the mixture on a magnetic stirrer for 15 minutes. The resulting fucoidan solution is treated with 80 ml of 80% ethanol. This precipitates fucoidan, which is filtered under vacuum, washed twice with 80% ethanol, dried at 60 ^o C. The output of fucoidan is 2.10 g or 2.10% of the raw material.

The algae pulp remaining after extraction of the 4 products described above is placed in a flask with a capacity of 3 L, pour 2.5 L of a 2.0% sodium carbonate solution and extracted in a water bath at a temperature of 60 ^o C for 1.5 hours. The alkaline extract is then decanted from the pulp and filtered under vacuum on a Buchner funnel. The aqueous alkaline extract is then treated with approximately 150 ml of concentrated sulfuric acid (99.95%) by slowly adding small portions of acid (5 ml) to the extract. In this case, the precipitated alginic acid precipitate is filtered, transferred quantitatively to a flask with a capacity of 3 L, 300 ml of a 1.5% sodium carbonate solution are added, stirred, the alginic acid precipitate passes into a sodium alginate solution. Sodium alginate is precipitated from the solution by adding 1.8 l of 96% ethanol to it. The resulting precipitate is filtered and dried at a temperature of 60 ^o C. The yield of sodium alginate is 12.35 g or 12.35% of the feed.

PRI me R 3. Belomorsky algae are dried at 50 ^o C, free from mechanical impurities, weigh 100 g for analysis. A portion of the algae is crushed to 3 mm particles and extracted with 500 ml of 85% ethanol in a Soxhlet apparatus at the boiling temperature of ethanol for 2 hours. The resulting alcohol extract is filtered on a Buchner funnel under vacuum, evaporated to form a condensed residue (about 30 ml), add about 10 ml of water and crystallize at 4 ^o C in the refrigerator. The precipitated mannitol crystals are twice purified by recrystallization. Mannitol crystals are filtered and dried at 60 ^° C. The mannitol yield is 7.13 g or 7.13% of the feed.

Further, the algae remaining after mannitol extraction is dried under draft to remove alcohol residues, after which they are loaded into a 2 L flask and filled with 500 ml of hot (temperature 70 ^o C) distilled water. The mixture is heated on an electric stove at a temperature of 70 ^o C. Extraction is carried out for 15 minutes twice. The resulting hot water extracts are poured from the pulp, combined, filtered under vacuum. The combined filtered extract is evaporated in a Wurz flask under vacuum at a residual pressure of 0.3 atm and a temperature of 70 ^o C to a density of 1.05-1.06 g / cm ³ . The resulting aqueous concentrate is treated with a single volume of 96% ethanol. The precipitate of the water-soluble polysaccharide complex is decanted and filtered on a Buchner funnel under vacuum. The precipitate is washed three times on a funnel with 96% ethanol, dried at a temperature of 60 ^o C in a vacuum drying oven. The output of the water-soluble polysaccharide complex of the Laminaride type is 11.38 g or 11.38% of the feed.

The alcoholic mother liquor (approximately 200 ml) remaining after precipitation of the water-soluble polysaccharide complex of the Laminaride type is evaporated under vacuum to 1/10 volume. Then the resulting concentrate is treated with 40 ml of 70% ethanol. The precipitate was centrifuged in a laboratory centrifuge at 2000 rpm. 30 ml of a 0.3% hydrochloric acid solution are added to the distilled precipitate in a beaker, the beaker with the mixture is closed and left for 1.5 hours at room temperature. After 1.5 hours, a 0.5 M solution of cetavlon was added to the resulting solution to convert the acid polysaccharides to a precipitate. The addition of a 0.5 M solution of cetavlon is carried out until the formation of a precipitate of acidic polysaccharides ceases. 10 minutes after settling, the precipitate was separated through a filter. The non-stop liquid (25 ml) is treated with 50 ml of 70% ethanol. The precipitate of laminaran was filtered under vacuum, washed twice with 96% ethanol, and dried in a vacuum oven at 60 ^° C. The output of laminaran was 4.11 g or 4.11% of the feed.

Further, the precipitate of acidic polysaccharides formed after the addition of cetavlon is dissolved in 20 ml of a 0.3% hydrochloric acid solution by stirring the mixture on a magnetic stirrer for 15 minutes. The resulting fucoidan solution is treated with 40 ml of 60% ethanol. This precipitates fucoidan, which is filtered under vacuum, washed twice with 60% ethanol, dried at 60 ^o C. The output of fucoidan is 1.95 g or 1.95% of the raw material.

The algae pulp remaining after extraction of the 4 products described above is placed in a flask with a capacity of 2 L, pour 1.5 L of a 1.0% sodium carbonate solution and extracted in a water bath at a temperature of 40 ^o C for 0.5 h. Water The alkaline extract is then decanted from the pulp and filtered under vacuum on a Buchner funnel. The aqueous alkaline extract is then treated with approximately 50 ml of concentrated sulfuric acid (99.95%) by slowly adding small portions of acid (5 ml) to the extract. In this case, the precipitated alginic acid precipitate is filtered, transferred quantitatively to a flask with a capacity of 2 L, 300 ml of a 1.5% sodium carbonate solution are added, stirred, the alginic acid precipitate passes into a sodium alginate solution. Sodium alginate is precipitated from the solution by adding 1.2 l of 90% ethanol to it. The resulting precipitate is filtered and dried at a temperature of 60 ^o C. The yield of sodium alginate is 12.01 g or 12.01% of the raw material.

 Target products have been tested for biological activity.

EXPERIMENTAL BIOLOGICAL PART
1. CHARACTERISTICS OF PHARMACOLOGICAL ACTIVITY OF MANNITIS
a) ACUTE TOXICITY
In experiments on 36 white rats weighing 200-230 g by the method of Kerber with oral administration it was found for mannitol LD ₅₀ > 5000 mg / kg According to the classification of toxicity of substances, mannitol should be attributed to the group of practically non-toxic substances.

b) LACK OF ACTION
The laxative effect of mannitol was studied in the manner described in the literature, for which fecal mass was recorded over a 24-hour period of time, after oral administration of mannitol at a dose of 200 mg / kg in the form of an aqueous solution in a volume of 5 ml. The experiments were performed on 36 white rats weighing 200-220 g. An official laxative Laminaride in the same dose was used as a comparison drug. It was found that the daily mass of feces after administration of mannitol was 2.4 ± 0.18 g versus 0.7 ± 0.04 g in control experiments with the introduction of physiological saline (P <0.05) and 1.7 ± 0.18 g in experiments with the introduction of laminaride (P <0.05).

 Thus, the laxative effect of mannitol exceeded the level of control experiments by 24.28%, and the official laxative preparation of laminaride - by 41.1%, which allows us to recommend the studied mannitol as an effective laxative.

2. CHARACTERISTICS OF PHARMACOLOGICAL ACTIVITY OF LAMINARAN AND FUKOIDAN
a) ACUTE TOXICITY
In experiments on 36 white rats weighing 190-230 g, the Kerber method for oral administration established for laminaran and fucoidan LD ₅₀ > 5000 mg / kg, which allows us to attribute this substance to the group of practically non-toxic substances.

b) LIABLE ACTION
The study of the laxative effect of laminaran and fucoidan by recording the daily mass of feces after its oral administration at a dose of 200 mg / kg in a volume of 3 ml of an aqueous solution in experiments on 36 white rats showed that the fecal mass in experiments with laminaran and fucoidan was 2.05 ± 0 , 32 g against 0.63 ± 0.11 g in control experiments with the introduction of physiological saline (P <0.05) and 1.7 ± 0.18 g in experiments with the introduction of the official laxative laminaride (P> 0.05) .

 Thus, the laxative effect of laminaran and fucoidan exceeded the level of control experiments by 225.3%, and laminaride - the official laxative drug - by 20.5%, which allows us to recommend the studied substances as effective laxatives.

3. CHARACTERISTICS OF PHARMACOLOGICAL ACTIVITY OF SODIUM ALGINATE
a) ACUTE TOXICITY
In experiments on 36 white rats weighing 210-230 g, the Kerber method for oral administration established for sodium alginate LD ₅₀ > 5010 mg / kg, which allows us to attribute sodium alginate to the group of practically non-toxic substances.

b) LIABLE ACTION
The study of the laxative effect by determining the daily mass of feces after oral administration of sodium alginate at a dose of 200 mg / kg in experiments on 36 white rats showed that the mass of feces in experiments with sodium alginate was 4.1 ± 0.3 g against 0.7 ± 0 , 09 g in control experiments with the introduction of physiological saline (P <0.05) and 1.7 ± 0.18 g in experiments with the introduction of the official laxative preparation of laminaride (P <0.05). Thus, the laxative effect of sodium alginate exceeded the similar effect of control experiments by 485.7%, the official drug of laminaride - by 141.1%.

c) HYPOCHOLESTERINEMIC ACTION
The hypocholesterolemic effect of sodium alginate was studied in 30 white rats with hypercholesterolemia caused by a single intraperitoneal administration of triton at a dose of 50 mg / kg. It was found that oral administration of sodium alginate at a dose of 200 mg / kg to animals with hypercholesterolemia led to a decrease in serum total cholesterol to 1.96 ± 0.13 mmol / L against 3.64 ± 0.26 mmol / L in animals with hypercholesterolemia ( control), i.e. 46.3% (P <0.001). In intact animals, the level of total serum cholesterol was 1.29 ± 0.26 mmol / L. Thus, sodium alginate causes a significant decrease in total blood cholesterol, essentially ensuring normalization of its content in animals with hypercholesterolemia (the difference in cholesterol levels in healthy animals and animals treated with sodium alginate was little significant P> 0.05).

d) RADIONUCLIDE BINDING ACTION
The study of radionuclide-binding activity (RNCA) of sodium alginate was carried out according to the method developed by the Kiev State Institute for Advanced Medical Studies, which is protected by auth. N 274598. As an indicator used strontium-85. The reference standard was citrus pectin. The test results are presented in table 32.

 The results obtained allow us to conclude that sodium alginate has a radionuclide-binding activity exceeding that of etalano-citrus pectin by 45.36%.

 The results of a study of the laxative, hypocholesterolemic, radionuclide-binding action of sodium alginate, as well as its low toxicity, make it possible to evaluate it as a promising therapeutic agent.

4. CHARACTERISTICS OF PHARMACOLOGICAL ACTIVITY OF A WATER-SOLUBLE POLYSACCHARIDE COMPLEX
a) ACUTE TOXICITY
In experiments on 36 white rats weighing 180-240 g by the method of Kerber when administered orally for a water-soluble polysaccharide complex LD ₅₀ > 3500 mg / kg In experiments on 36 white mice weighing 20-40 g by the Kerber method with intraperitoneal administration, LD ₅₀ > 1000 mg / kg was established for this complex. According to the classification of toxic substances, the investigated substance should be attributed to the group of low toxic substances.

b) LIABLE ACTION
The laxative effect of the studied substance was studied on white rats and dogs by the described methods. In one case (experiments on 18 rats), the laxative activity of substances was judged by the size of the passage (in mm) through the intestine using a label. The label was made by injecting rats with a probe into the stomach 5% cock activated carbon. 20 minutes before oral administration of the test substances in a dose of 200 mg / kg in a volume of 2 ml of an aqueous solution. Animals were sacrificed after 2 hours by decapitation and the degree of advancement of activated carbon (label) was measured. In other experiments (experiments on 18 rats and 12 dogs), the laxative effect of the test substances was judged by the amount of defecation of animals (the amount and consistency of feces) placed in special cells for these purposes. In this case, the test substance was orally administered to animals at a dose of 200 mg / kg in a volume of 2 ml to rats or 300 ml to dogs of an aqueous solution, followed by (24 g) weighing of feces (in g) and a description of their consistency. In control experiments, physiological saline was injected in appropriate amounts. It was found that with the introduction of a water-soluble complex in rats, the intestinal passage reached 42.5 ± 1.3 mm versus 6.8 ± 0.9 mm in control experiments (P> 0.05) and 33.2 ± 1.6 mm in experiments with the official laxative, laminaride, administered in the same amounts as the test substance (P <0.05).

 Defecation in rats treated with a water-soluble complex reached 10.0 ± 0.7 g per day versus 3.8 ± 0.5 g in control experiments (P <0.05) and 9.5 ± 0.7 g in experiments with an official laxative laminaride (P> 0.05), and defecation in dogs, respectively - 182.5 ± 8.4 g versus 85.0 ± 3.4 g (P <0.001) and 114.0 ± 3.9 g (P < 0.001). Thus, under the influence of a water-soluble polysaccharide complex, the laxative effect was enhanced, depending on the experimental method and animal species, by 114.7 - 523.5% compared with the control experiments, exceeding the action of the official laxative laminaride by 5.2-60.0%.

 By its chemical composition, the water-soluble polysaccharide complex obtained from the White Sea algae is closest to the official laxative laminar derived from Far Eastern algae.

 A comprehensive study of the pharmacological activity of the water-soluble polysaccharide complex revealed, along with the laxative effect, a number of additional properties: hepatoprotective, lipid-lowering, antiulcer.

c) HEPATIC ACTION
The hepatoprotective effect of the water-soluble complex was studied in experiments on white rats with dystrophic liver damage caused by three-day oral administration of 0.3 ml of carbon tetrachloride per 100 g of body weight in a 50% oil solution. The studied complex at a dose of 200 mg / kg was administered orally in 2 ml of an aqueous solution for 12 days, of which carbon tetrachloride was simultaneously administered in the last 6 days. As the reference substances, the official drug Laminaride (at a dose of 200 mg / kg) was used — a prototype in the structure and method of preparation and the official hepatoprotective drug Silibor (at a therapeutic dose of 10 mg / kg) —a prototype in action.

 Alanine aminotransferase (ALT) activity was determined in blood serum using the Bachringer Mannheim reagent kit (Germany) and alkaline phosphatase (ALP) using the Lahema reagent kit (Czechoslovakia), and total bilirubin according to Yendrashka in the modification of L.T. Lukyanova. In addition, the content of triglycerides according to Gottfritz and Rosenberg in the modification of N. A. Sentebova and N. B. Maletskaya.

 As can be seen from Table 33, the hepatoprotective effect of the studied complex was superior to the effect of laminaride and did not differ much in effectiveness from the official hepatoprotective protector of silibor, which normalized indices of alanine aminotransferase, alkaline phosphatase, triglycerides and total bilirubin in the blood and triglycerides in the tissue . These results indicate that the water-soluble polysaccharide complex isolated from the White Sea algae has distinct hepatoprotective properties, approaching in their severity to the action of the official drug silibor.

d) HYPOLIPIDEMIC ACTION
The lipid-lowering effect of the water-soluble polysaccharide complex was studied in experiments on white rats with a model of hyperlipidemia caused by daily, for 4 days, oral administration of 2 ml of a mixture of alcohol and oil solutions of vytamine D ₂ at the rate of 80,000 units per 100 g of body weight and cholesterol 200 mg / kg

 In parallel with the animals, the studied complex was injected into the stomach using a probe at the rate of 200 mg / kg in 2 ml of an aqueous solution. As reference substances, we used the official drug laminaride (at a dose of 200 mg / kg) - a prototype in the structure and method of preparation and the official anti-atherosclerotic drug polysponin (at a therapeutic dose of 10 mg / kg) - a prototype in action. In the blood serum of decapitated rats, total Ilk cholesterol and triglycerides were determined using a set of reagents from Lahema (Czechoslovakia). In the tissues of the aorta of animals, the cholesterol content was determined by T.N. Lovyagina and E.B. Bankovsky.

 The results of the experiments, presented in Table 34, indicate that the water-soluble polysaccharide complex most noticeably lowers the level of triglycerides in the blood, weaker - reduces the level of cholesterol in the blood and does not significantly affect the cholesterol content in the aortic tissues. A similar effect, but weaker in relation to triglycerides, is inherent in laminaride. The hypocholesterolemic activity of the water-soluble polysaccharide complex was slightly higher than the official anti-atherosclerotic drug polysponin. Unlike the latter, the hypocholesterolemic effect of the complex was combined with a significant hypotriglyceridemic effect. This suggests that the studied complex of White Sea algae in its hypolipidemic effectiveness exceeds the action of the official drug polysponin.

d) ANTI-ADVERSE ACTION
The study of the antiulcer action of the water-soluble polysaccharide complex in comparison with the action of the Laminarid preparation was carried out on 30 white rats weighing 185-210 g, in which gastric ulcers were caused by a combination of fasting and histamine administration according to the recommendations of the management. For this, on the first day the animals were completely starving, receiving only water. On the second and third days, complete starvation, combined with subcutaneous administration of histamine in the morning and evening of 0.4 ml of 0.1% solution (at the rate of 0.2 mg per 100 g of mass). All these animals were divided into three groups of 10 rats each. One group of rats received 0.5 g of a water-soluble polysaccharide complex with 5 ml of water orally once a day during the above three days. The second group of rats under similar conditions, 0.5 g of the drug laminarid with 5 ml of water. The third group of rats was a control. On the fourth day, all animals were killed by decapitation. After opening, a microscopic examination of the gastric mucosa was carried out using a magnifier. In animals, hemorrhagic erosions and ulcers were found mainly on the lesser curvature of the stomach and in the pylorus, the number of which was different in different groups of animals. It is characteristic that in rats treated with a water-soluble complex and the drug laminaride, a large amount of mucus was found in the stomach cavity. The results of the experiments are presented in table 35.

 As can be seen from the data presented, the number of ulcers and erosion was significantly lower in animals treated with the test substance, compared with control animals. Moreover, the effectiveness of the water-soluble complex of algae and the drug laminarid was the same.

 Thus, it should be concluded that the water-soluble complex from the White Sea algae, as well as the preparation laminarid, has a pronounced anti-ulcer activity, which, apparently, is associated with their enveloping and adsorbing properties.

e) RADIONUCLIDE-BINDING ACTION
The study of radionuclide-binding activity (RNCA) of a water-soluble complex (VK) of the Laminarid type from White Sea algae was carried out according to the method developed by the Kiev State Institute for Advanced Medical Studies, which is protected by Auth. N 274598. As an indicator used strontium-85. The reference standard was citrus pectin. The test results are presented in table 36.

 The results obtained allow us to conclude that a Laminaride type VK sample isolated from White Sea algae has a high radionuclide-binding activity superior to such activity of the reference — citrus pectin — by 280.78%.

 An analysis of the results of studies of the pharmacological activity of a water-soluble polysaccharide complex from the White Sea algae allows us to conclude that this object is multifunctional and highly effective: it has a laxative, radionuclide-binding, hepatoprotective, lipid-lowering and antiulcer effect, usually superior in strength to the corresponding lamina prototypes in structure ( ) and in action (silibor, polysponin, citrus pectin).

Thus, the claimed method provides the following positive effect:
1. A complex processing of the White Sea kelp mixture is provided, in which 5 products are obtained simultaneously: mannitol, a water-soluble polysaccharide complex, laminar, fucoidan, sodium alginate. While the known method allows to obtain only the polysaccharide complex "Laminaride", and other biologically active substances remain in the waste;
2. Increasing the yield of water-soluble polysaccharide complex: in the present method, the yield is 12.60%, in the known method - 5.6%, ie the output of the polysaccharide complex in the present method is 2.25 times higher than in the known.

 In addition, the inventive method provides high yields of other substances: mannitol 7.85%, laminaran 4.53%, fucoidan 2.14%, sodium alginate 12.41%.

 3. The expansion of the raw material base. For the first time, a natural mixture of two types of kelp (Laminaria saccharinа and Lam. Digitata L.), forming thickets in the White Sea basin, is used as a source of raw materials. In addition, the inventive method will allow, in relation to the conditions of the Arkhangelsk experimental algae plant, to process the White Sea kelp, which has annual multi-tonnage raw materials.

 4. Increase in biological activity. The water-soluble polysaccharide complex of the "Laminaride" type obtained by the present method has a laxative effect that exceeds the action of the standard official drug "Laminaride" by 5.2-60.0% (depending on the experimental technique and type of animals).

5. The expansion of the spectrum of biological action. First established:
a) The laxative effect of mannitol, which exceeds the effect of "Laminaride"41.1%;
the laxative effect of laminaran and fucoidan, superior to the action of the official laxative drug "Laminarid" - by 20.5%;
the laxative effect of sodium alginate, superior to the action of "Laminaride" - by 141.1%;
b) the radionuclide-binding action of sodium alginate, which exceeds that of the standard — citrus pectin — by 45.36%;
the radionuclide-binding effect of the water-soluble polysaccharide complex, which exceeds the similar effect of the standard - citrus pectin - by 280.78%;
c) the hypocholesterolemic effect of sodium alginate, which causes a significant decrease in total cholesterol in the blood, essentially ensuring the normalization of its content in animals with hypercholesterolemia;
the lipid-lowering effect of the water-soluble polysaccharide complex, which most significantly lowers the level of triglycerides in the blood, weaker - reduces the level of cholesterol in the blood. Moreover, the hypocholesterolemic and anti-atherosclerotic activity of the water-soluble polysaccharide complex were higher than the similar action of the drug “Laminarid” and the official anti-atherosclerotic drug polysponin;
d) the hepatoprotective effect of the water-soluble polysaccharide complex, exceeding the similar effect of "Laminaride" and approaching the severity of the action of the official hepatoprotective silibor protector;
e) antiulcer action of a water-soluble polysaccharide complex.

 In the presence of a wide range of biological effects, the studied target products turned out to be practically non-toxic, which allowed them to be recommended as effective multifunctional agents. In this case, a combination of a laxative and a radionuclide-binding action, which promotes the rapid binding and elimination of radionuclides, is especially favorable.

Claims (1)

METHOD FOR PRODUCING BIOLOGICALLY ACTIVE SUBSTANCES FROM LAMINARIA by extracting crushed raw materials, separating the extract, evaporating it and isolating the target product, characterized in that, in order to intensify the processing of raw materials and simultaneously produce several biologically active substances and increase the yield of a water-soluble polysaccharide complex, thalli are used. Laminaria saccharina and L. digitata, the crushed mixture is extracted with 90 - 96% ethanol in the ratio of raw materials: ethanol 1: 6 for 3 hours, ethanol extraction mannitol with a laxative effect is introduced and crystallized, the raw material after extraction of mannitol is treated with hot water in a ratio of 1: 10 for 30 minutes, the doubly and water-soluble polysaccharide complex has a laxative, anti-ulcer and radionuclide-binding action, precipitated with a two-fold volume of ethanol, the mother liquor is precipitated, and the mother liquor is precipitated polysaccharide complex, treated with a three-fold volume of 80% ethanol, the precipitate was separated by centrifugation, treated with a five-fold volume 0.4% hydrochloric acid solution for 2 hours and precipitated with a 0.5 M solution of cetavlon, the precipitate formed is dissolved in hydrochloric acid and fucoidan having a laxative effect, precipitated with a three-fold volume of 70% ethanol, laminaran having a laxative effect, obtained from the supernatant after precipitation of fucoidan by treating it with a three-fold volume of 80% alcohol, sodium alginate having a laxative and radionuclide-binding action is obtained by treating the remaining thallus pulp with a 1.5% solution of carbonate n atria in a ratio of 1: 20 at 50 ^o C for 1 h, then separating the extract and precipitating alginic acid by treating the extract with sulfuric acid, converting it to sodium alginate, treating the precipitate with sodium carbonate solution and precipitating the target product with ethanol.

Images