RU2194525C1 - Method for obtaining biologically active substances out of laminaria for medicinal purposes - Google Patents

Abstract

FIELD: pharmaceutics. SUBSTANCE: sugary laminaria should be reduced, degreased with chloroform at weight- volumetric ratio of raw material : chloroform being 1 : 20 for 1.5 h. Then it is successively extracted with 90-96%-rthanol, hot water and 1.5%-sodium carbonate solution. Ethanolic extraction is filtrated, dialyzed through a cellophane membrane for 2 h, concentrated and mannitol is crystallized. Aqueous extract is thrice cooled at 4 C for 1 h. Then it is dialyzed through a cellophane membrane for 6 h, evaporated to deposit a total polysaccharide-protein complex with 90%- ethanol. Then comes freeze drying at -13 C for 70 min. Sodium alginate is deposited out of carbonate extraction after pretreatment with concentrated sulfuric acid with the help of sodium carbonate followed by freeze drying at -13 C for 50 min. The method favors to simplify the process and increase product activity. EFFECT: higher efficiency. 1 dwg, 3 ex, 13 tbl

Description

 The invention relates to the pharmaceutical industry, relates to a method for producing biologically active substances from kelp and can be used in the treatment of constipation and intoxication with heavy metals.

A known method of producing polysaccharides from seaweed Laminaria saccharina L. (saccharic kelp) and L. japonica Aresch. (Japanese kelp) sem. Laminariaceae (kelp) growing in the Far Eastern Basin (USSR Author's Certificate 1275810, class A 61 K 35/78, 1984 "Method for the production of polysaccharides with laxative effect" / Zinchenko V.V., Makarevich I.F., Chushenko V. N., Obolentseva G.V., Stavrova R.F., Bakay S.I., Biryuk V.A.). Polysaccharides are obtained 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, adding 96% to the hot extract ethanol in the ratio of extract: ethanol 1: 1. Then the resulting mixture is defended, the lower suspension layer is separated, centrifuged, and after separation of the mother liquor, the precipitate is washed with 96% ethanol. The washed precipitate is dried, crushed. The yield of the finished product is 5.6% of the feedstock. The resulting complex of polysaccharides with amino acids, known under the name "Laminaride", was previously produced at the Kharkov Chemical-Pharmaceutical Production Association (KhPKhFO "Workers'Health") and was used as a laxative drug.

 The disadvantages of this method are: a) obtaining only one product from algae (polysaccharides), while production wastes are valuable substances - mannitol, sodium alginate; b) low yield of polysaccharides (5.6% of raw materials); c) incomplete precipitation of polysaccharides by ethanol due to insufficient ethanol; d) loss of ethanol when added to a hot concentrated extract for precipitation of polysaccharides; e) a low degree of purity of the target products.

 A method for the simultaneous production of mannitol, a water-soluble polysaccharide complex, laminaran, fucoidan, sodium alginate with a laxative, lipid-lowering, radionuclide-binding, hepatoprotective, antiulcer effect, from the White Sea kelp (a mixture of two types of kelp L. saccharina L. (L. digitata L.) Patent of the Russian Federation 2028153, class A 61 K 35/80, 1995 "Method for the production of biologically active substances from kelp" / Makarova R.N., Samokish I.I., Kompantsev V.A., Kaysheva N. Sh. And others .). The method is carried out by grinding raw materials, sequential extraction with 90-96% ethanol, hot water and a 1.5% solution of sodium carbonate; ethanol extraction is concentrated and mannitol is crystallized from it; a water-soluble polysaccharide complex (analogue of "Laminaride") 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 mol / l cetavlon solution is added to convert the acid polysaccharides to the precipitate, the extract is treated ethanol (to precipitate laminaran), and the remaining precipitate was 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 carbonate solution, sodium alginate is obtained.

According to this method, the raw material is first dried at a temperature of 50 ^{° C.} , after which it is freed from mechanical impurities, then the raw material is ground and extraction is carried out with various solvents. The yield of the target products (% of the feedstock) is: mannitol - 7.85, polysaccharide-protein complex (Laminaride) - 12.60, laminaran - 4.53, fucoidan - 2.14, sodium alginate - 12.41. This method of obtaining biologically active substances from kelp is close to the claimed and selected as a prototype.

 The disadvantages of this method are: a) the relatively low yield of the target products, b) the duration of the drying process of the substance, c) the low degree of purity of the obtained substances, d) the complexity and inappropriateness of separate production of fractions of a type that are not of the structure (Laminaride, laminaran, fucoidan), possessing one type of biological action (laxative effect), e) high consumption of limited and scarce solvents (ethyl alcohol, cetavlon), necessary for the preparation, purification and separation of laminaran, fukoi ana.

 The aim of the invention is the integrated processing of the White Sea algae of the Northern Basin (SB) - saccharide laminaria for the simultaneous production of mannitol, polysaccharide-protein complex (SB Laminaride) and sodium alginate, increasing the yield, purity and solubility of the target products, simplifying the production method, increasing complexing and laxative activity of the target products.

This goal is achieved in that the method for the simultaneous production of mannitol, Laminaride SB, sodium alginate, which have a laxative and detoxifying effect, from sugar kelp is carried out by grinding the raw materials, degreasing them with chloroform in a mass-volume ratio of raw materials: chloroform 1:20 for 1 , 5 hours (to remove lipophilic pigments), sequential extraction with 90-96% ethanol, hot water and a 1.5% sodium carbonate solution; ethanol extraction is filtered, dialyzed through a cellophane membrane for 2 hours, concentrated and mannitol is crystallized from it; an aqueous extract containing predominantly polysaccharides (Laminaride SB, laminaran, fucoidan) is cooled three times at a temperature of 4 ^° C for 1 h, dealized through a cellophane film for 6 h, evaporated, precipitated from it using 90% ethanol total polysaccharide-protein preparation "Laminaride SB"; dried by freeze drying at a temperature of -13 ^o C for 70 min; from carbonate extraction after pretreatment with concentrated sulfuric acid, sodium carbonate solution, three times cooling at 4 ^° C for 1 h, subsequent dialysis through a cellophane film for 6 h and treatment with 96% ethanol, sodium alginate is precipitated, which is then dried by the method freeze-drying at a temperature of -13 ^o C for 50 minutes

 According to the inventive method, the raw materials — saccharides of the kelp of kelp — are degreased with chloroform in order to remove various lipophilic substances — phospholipids, glycolipids, lipoproteins. Lipids form the basis of the extracellular membrane covering the outer walls of the epidermis of the thallus of the plant. The permeability of cells and their organoids for various solvents depends on the amount of these lipids (DA Muravyova, Pharmacognosy. M: Medicine, 1991, p. 22, 124-125). Therefore, the removal of lipophilic substances facilitates the access of extractants to the active substances contained in the cells. In addition, the degreasing of raw materials leads to the production of substances of lighter tones. So, for example, if the "Laminaride" obtained by the prototype method is a grayish-yellow powder, then "Laminaride SB" obtained by the present method, the powder is light pink in color, sodium alginate - thin plates of light orange and white colors respectively.

 Repeated cooling of solutions of the polysaccharide complex reduces their viscosity, i.e. cryolysis of polysaccharides is observed (Influence of low temperatures on the technological characteristics of solutions of a plant polysaccharide complex / N.E. Chernov, I.P. Gorodetsky, D.V. Rybachuk and others // Abstracts of the Republican Scientific Conference "Actual Issues of Search and Technology of Medicines" Kharkov, 1981, pp. 83-84). At the same time, the drying rate of plant substances increases compared with drying without preliminary freezing. Long-term drying of polysaccharide preparations is impractical because leads to a significant deterioration in the solubility of the dry preparation.

 Dialysis of polysaccharide solutions through a cellophane film allows you to clean polysaccharides from ballast substances that are larger than the target products. So, for example, if you record the UV absorption spectrum of sodium alginate in solution before and after dialysis (see drawing), you can see that after dialysis (curve 2), the spectrum has a more pronounced absorption maximum with a higher optical density than before dialysis (curve 1).

 To assess the effectiveness of binding of metal ions by polysaccharides, a complex-forming ability (CS) indicator is used, which characterizes the number of mg of metal ions bound to 1 g of polysaccharide (Davidyuk L.P., Vshivkova G.F. Comparative study of the complexing ability of pectins of different origin / Bulletin of state Nikitsky Botanical Garden ", - Yalta: GNBS, 1981. - Issue 3 (46). - P. 98-101). The content of metal ions associated with polysaccharides was determined by the method of reverse complexometric titration (Kompantsev V.A., Kaysheva N.Sh., Gokzhaeva L.P., Sizova N.M. Determination of the complexing ability of pectins and pectin-containing preparations / Environmental protection. - M .: NIITEKHIM, 1991. - P. 25-27).

 The resulting target products were subjected to qualitative and quantitative analysis.

1. Mannitol
The obtained mannitol was identified by paper chromatography (Table 1).

The authenticity of mannitol was confirmed by the following qualitative reactions:
1) 0.5 g of the drug is dissolved in 1 ml of water. 5 ml of the reagent is added to the solution (preparation of the reagent: 10% ammonia solution is added to 2 ml of a 10% copper (II) sulfate solution until a clear dark blue solution is obtained, then the solution is diluted with water to 20 ml). Shake the solution well, precipitate (mannitol).

2) 0.5 g of the drug is placed in a flask with a capacity of 50 ml, 3 ml of acetic anhydride, 1 ml of pyridine are added and heated in a boiling water bath for 15 minutes, often shaking. The reaction mixture is cooled, 29 ml of chilled water are added, stirred and allowed to stand for 5 minutes; a precipitate of mannitol-hexaacetate precipitates, which is collected on a Schott glass filter, recrystallized from ether and dried at a temperature of 60 ^o C for 1 h. The melting point of the residue is from 119 to 123 ^o C.

 The results of the analysis of the physico-chemical parameters of mannitol are shown in table 2.

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 of the prototype method was 99.56%; by the claimed method - 99.94%,
Thus, according to the above methods of identification and quantification of mannitol, the preparation obtained by the claimed method has a high degree of purity and can be used as a medicine.

II. "Laminaride SB"
The identification of the components of "Laminaride SB" was carried out using the following reactions:
2 g of the drug is placed in a conical flask with a capacity of 100 ml, 100 ml of water are added, stirred (solution A).

1) 5 ml of alcohol 95% are added to 5 ml of solution A, mixed; light brown flocculent clumps appear that precipitate (polysaccharides);
2) To 10 ml of solution A, add 0.5 g of sodium citrate, mix. 5 ml of diluted hydrochloric acid is added to the resulting solution, a gel-like precipitate (alginic acid) is formed;
3) 5 ml of solution A give a characteristic reaction to calcium with ammonium oxalate; a white precipitate of calcium oxalate is formed.

4) 8 ml of the solution give a characteristic reaction to sodium with zinc-uranyl acetate in an acetic acid medium; a yellow crystalline precipitate forms;
5) 15 ml of solution A is heated in a water bath with 8 mg of ninhydrin for 8 min, a blue-violet staining of the solution (amino acid) is formed;
6) remove the absorption spectrum in the visible region of 4 ml of solution B obtained by quantification. Only I absorption maximum is observed at a wavelength of 460 nm (reducing sugars);
7) to identify laminaran and fucoidan, the preparation was hydrolyzed with 4 mol / L sulfuric acid solution at a temperature of 100 ^o C for 6 hours, followed by neutralization of the hydrolyzate with barium carbonate. The hydrolyzate was analyzed by paper chromatography (top-down method) in a solvent system: n-butanol-pyridine-water (6: 4: 3). The developer is acid aniline phthalate. On the chromatogram, 2 spots with R _f = 0.15 (fucose) and R _f = 0.16 (glucose) were detected. As witnesses used 1% solutions of fucose and glucose;
8) 1 ml of concentrated hydrochloric acid and 5 ml of acetone are added to 5 ml of solution A, a raspberry staining of the solution appears; absorption maximum at 550 nm (fucoidan);
9) to 5 ml of solution A add 1 ml of concentrated sulfuric acid and 5 ml of a 1% solution of cysteine; yellow staining of the solution appears; There are two absorption maxima at 400 and 427 nm (fucoidan).

 10) To 5 ml of solution A add 1 ml of concentrated sulfuric acid and 5 ml of a 1% solution of thioglycolic acid; a yellow-green staining of the solution (fucoidan) appears.

 To determine the quantitative content of reducing sugars in “Laminaride SB”, 15 ml of a 10% sulfuric acid solution are added to 0.3 g of the preparation and boiled in a flask with a thin section connected to a reflux condenser for 3 hours. The contents of the flask are cooled and quantitatively transferred to a volumetric flask 50 ml, bring the volume of the solution with water to the mark and mix. 10 ml of the resulting solution is placed in a glass and neutralized sequentially with universal indicator paper: first with 1 mol / L sodium hydroxide solution, then with 10% sulfuric acid solution and, finally, 1 mol / L sodium hydroxide solution. The solution is filtered, transferred to a 25 ml volumetric flask and the volume of the solution is adjusted to the mark with water. 1 ml of a 1% solution of picric acid, 3 ml of a 20% solution of sodium carbonate and 1 ml of the resulting solution are placed in a 25 ml volumetric flask. The flask with the contents was immersed in a boiling water bath for 10 minutes, then cooled to room temperature and the volume of the solution was adjusted to the mark with water (solution B). The absorbance of solution B was measured at 460 nm.

 The content of reducing sugars in "Laminaride" (prototype) was 76.55%, in "Laminaride SB" (the inventive method) 92.05%, i.e. in the latter case, the content of reducing saccharides exceeds 1.2 times.

 Thus, as a result of the analysis of Laminaride SB, polysaccharides, amino acids, inorganic ions were identified, a high content of reducing sugars was found, and the correspondence of the obtained Laminaride SB to the Laminaride preparation was shown.

III. Sodium Alginate
The identification of sodium alginate was carried out by the following reactions:
1) 1 ml of diluted hydrochloric acid is poured into 5 ml of a 0.5% solution of the drug, a gel-like precipitate (alginic acid) is formed;
2) 1 ml of a 2% solution of calcium chloride is poured into 5 ml of a 0.5% solution of sodium alginate, a gel-like precipitate is formed (unlike gums, cellulose derivatives);
3) 2.5 ml of a saturated solution of ammonium sulfate are added to 5 ml of a 0.5% sodium alginate solution; no precipitate is formed (unlike agar, starch, cellulose derivatives, gelatin);
4) 5 ml of a 10% solution of sodium citrate and 3 ml of concentrated hydrochloric acid are added to 5 ml of a 0.5% sodium alginate solution, a gelatinous precipitate is formed (alginic acid);
5) 5 ml of a 2% solution of sodium alginate give a characteristic reaction to sodium with zinc-uranyl acetate in an acetic acid medium; a yellow crystalline precipitate forms.

 For the quantitative determination of sodium alginate, a sample of the drug is dissolved in 0.1 N. caustic soda solution, adjust the volume of the solution to 0.1 N. caustic soda solution up to 100 ml. 0.5 ml of the resulting solution is measured in a test tube. 0.5 ml of the resulting solution, 0.25 ml of a 0.5% alcohol solution of carbazole, 5.5 ml of a sulfate solution of urea and boric acid are measured in a test tube; the test tube with the solution is boiled in a water bath for 15 minutes, cooled, and the optical density of the solution is measured at 520 nm.

 The content of sodium alginate obtained by the prototype method is 91.15%, and by the present method 96.75%.

 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 (Table 3).

 As follows from table 3, the best extractants for degreasing the thalli of kelp are chloroform and diethyl ether, but the latter, due to its high explosion and fire resistance, is not practical.

 As can be seen from table 4, the highest quantitative content of active substances is provided when the mass-volume ratio of thalli kelp and chloroform 1: 20.

 As follows from table 5, the maximum content of active substances in the selected fractions is achieved by processing the raw material with chloroform for 1.5 hours

Результаты приведены в таблице 6. В этой же таблице приведены значения комплексообразующей способности (КС) очищенных путем диализа веществ.We studied the influence of the nature of the membrane on the degree of dialysis according to the optical density of the dialyzed solution (D ₁ ) and dialysate (D ₂ ). The degree of dialysis (d) is calculated by the formula

The results are shown in table 6. In the same table are the values of complexing ability (COP) purified by dialysis substances.

 As the data in table 6 show, the highest dialysis rate is characteristic of substances through cellulose acetate films, with the lowest CS; Apparently, this is due to the passage through the films of not only biologically active substances, but also of impurities that reduce CS. During dialysis through polyvinyl chloride films, despite a large CS (compared to a cellulose film), the degree of dialysis is relatively low; it is possible that the film retains not only impurities, but also active substances. The most optimal material for the dialysis membrane is cellophane: at high CS, substances exhibit a relatively high degree of dialysis.

 As the results shown in table 7 show, the optimal duration of dialysis is for all target products 6 hours, except mannitol (for it 2 hours).

As follows from table 8, the optimal cooling temperature of the extracts is 4 ^o C; at this temperature, maximum yields and solubility of the target products are achieved, and the drying time of the preparations is reduced. At 4 ^o With the viscosity of the extracts decreases, which facilitates the processes of dialysis, filtration. The viscosity of the solutions was determined by the time of expiration on a viscometer VPZH-1 at 20 ^o C.

 The results shown in table 9 show that the optimal cooling time is achieved within 1 hour

 As can be seen from table 10, the optimal cooling repetition is 3.

 Freeze drying of the preparations was carried out in the Dolina apparatus, consisting of a chamber and a condenser. The camera operates under a deep vacuum. To defrost condensate and heat the chamber, electric coils are mounted. The substances are laid out on stainless steel baking sheets and set to dry in a metal shelf. Before loading baking sheets with substances in the condenser, the temperature is brought to negative values using a compressor-condenser apparatus. The drying process of the preparations begins at a residual pressure in the vacuum drying chamber of 200-250 μm, and after drying, the pressure is 50 μm. Drying the drugs by freeze drying allows you to speed up the drying process and avoid the destruction of active substances. We have selected the optimal conditions for lyophilization of drugs.

As follows from table 11, the optimum drying temperature for the target products is -13 ^o With: this achieves a minimum drying time and the maximum content of active substances.

 In the prototype method, it was confirmed that the optimal concentration of ethanol for the precipitation of laminaran and fucoidan is a concentration in the range of 80-96% (80% alcohol was used to save alcohol), 90-96% ethanol is used to precipitate "Laminaride". Therefore, when ethanol is used in a concentration of 90%, both Laminaride SB and neutral sugars — laminaran and fucoidan — are precipitated, which, along with other reasons, explains the higher yield of the Laminaride SB fraction and lower alcohol consumption. In addition, there is no need to separate these substances, because all of them (Laminaride SB, laminaran, fucoidan) have a laxative effect, while laminaran and fucoidan are not of interest as metal detoxifiers.

 Due to the degreasing of the initial thalli of kelp with chloroform, dialysis of extracts through cellophane membranes, as well as freeze-drying of preparations to ensure the preservation of the target products, the latter have a more pronounced CS than products obtained by the prototype method. The results of determining the COP are shown in table 12.

 As follows from table 12, “Laminaride SB" and sodium alginate obtained by the present method have a more pronounced detoxifying ability than the prototype method, 1.4 times. Apparently, this is due to the high degree of purification and high yield of the target products in the present method due to the degreasing of the feedstock, dialysis of the extracts, cooling of the extracts and freeze drying, which prevents the decomposition of polysaccharides.

 The inventive method for producing mannitol, a polysaccharide-protein complex "Laminaride SB", sodium alginate is illustrated by the following examples of specific performance.

Example 1
The White Sea algae of the Northern basin (Laminaria saccharina L.) are dried at 50 ^o С in an oven, then they are freed from mechanical impurities, mollusks. Then 100 g of algae are weighed with an accuracy of ± 0.02 g, crushed on a laboratory grinder to a particle size of 3 mm, placed in a flask with a thin section with a capacity of 3 l, pour 2 l of chloroform, the flask is closed with a stopper and the raw material with chloroform is kept for 1.5 h. Then the chloroform extraction of dark green color is separated from the raw material by filtering through gauze (5 layers). The flask with raw materials is placed in a fume hood to remove residual chloroform. Then, the feed is transferred using 600 ml of 90% ethanol to the Soxhlet apparatus and extraction is carried out at the boiling point of ethanol for 3 hours. The resulting alcohol extract is filtered on a Buchner funnel under vacuum, and then dialyzed through a cellophane membrane for 2 hours. they use a dialysis device in which there are two compartments separated by a cellophane membrane, the edges of which are fixed to the inside of the case using some kind of waterproof glue. In one compartment, through the opening of the device, a dialyzable alcohol extract is placed on top, which is dialyzed through a membrane into another compartment (dialyzer). Then the resulting dialysate is evaporated to form a condensed extract (about 30 ml), after which the concentrate is crystallized at a temperature of 4 ^o C in the refrigerator. The precipitated mannitol crystals are twice purified by recrystallization from an alcohol 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 9.54 g or 9.54% of the feed.

The target product, mannitol, is a white, odorless crystal, readily soluble in water and boiling alcohol, sparingly soluble in cold alcohol, insoluble in ether. The CS of the resulting mannitol is 7.85 mg of Pb ²⁺ / g.

Then, the algae remaining after mannitol extraction is “dried” under a draft to remove alcohol residues, after which they are loaded into a 2-liter flask and 1 liter of hot (temperature 80 ^o С) water is poured. The mixture is heated on an electric stove at a temperature of 80 ^o C. Extraction is carried out for 0.5 h twice. The obtained hot water extracts are separated from the pulp by filtering through 5 layers of gauze, combined, filtered under vacuum on a Buchner funnel through a double nylon cloth. The combined filtered extract was placed in a refrigerator and kept in it at 4 ^° C for 1 h, after which the extract was taken out of the refrigerator and kept until the temperature of the extract reached room temperature. Then, the extract is cooled twice more under the same conditions. The extract is then dialysed through a cellophane film for 6 hours. The resulting dialysate 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 from the original volume). The resulting aqueous concentrate is treated with a double volume of 90% ethanol, which makes it possible to precipitate SB laminaride together with laminaran and fucoidan. The precipitate of the polysaccharide-protein complex is 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 by freeze drying in the apparatus of the Valley at a temperature in the pressure chamber -13 ^o C for 70 minutes The output of the polysaccharide-protein complex "Laminaride SB" is 19.52 g or 19.52% of the raw material.

The target product polysaccharide-protein complex "Laminaride SB" is an amorphous powder of light pink color, odorless. Virtually insoluble in alcohol. It is sparingly soluble in water with the formation of a turbid mucous solution (1 g of the drug is dissolved in 45 ml of water at 20 ^o C). CS "Laminaride SB" is 1920 mg Rv ²⁺ / g.

The remaining algae pulp is placed in a flask with a capacity of 3 L, poured 2 L of a 1.5% sodium carbonate solution and extracted in a water bath at 50 ^° C for 1 hour. The alkaline water extract is then decanted from the pulp and filtered under vacuum on a Buchner funnel. . The aqueous alkaline extract is then treated with approximately 100 ml of concentrated (99.95%) sulfuric acid by slowly adding small portions of the acid (approximately 5 ml each) and 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. The sodium alginate solution is cooled three times in the refrigerator at 4 ^{° C.} for 1 hour (after each cooling, the sodium alginate solution is kept in the refrigerator until it reaches room temperature). Next, the sodium alginate solution is dialyzed through a cellophane membrane for 6 hours. Sodium alginate is precipitated from the obtained dialysate by adding 1.5 l of 96% ethanol to it. The precipitate formed is filtered on a Buchner funnel under vacuum, and then dried by freeze drying in a Dolina apparatus at a temperature in the pressure chamber of -13 ^o C for 50 minutes.

 The yield of sodium alginate is 21.83 g or 21.83% of the feed.

The target product - sodium alginate - is a thin plate of white color, odorless, mucous taste, soluble in water (at 20 ^o With 1 g of the drug is dissolved in 15 ml of water), insoluble in alcohol. The sodium alginate CS is 2435 mg of Pb ²⁺ / g.

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

Example 2
White sea algae are prepared for extraction as described in the example. 100 g of algae are weighed, crushed to 3 mm in a laboratory grinder, placed in a flask with a thin section with a capacity of 3 l, poured 2.5 l of chloroform, the flask is closed with a stopper and the raw material is kept with chloroform for 2.0 hours. Separation of chloroform extraction, extraction of mannitol 90% ethanol is carried out analogously to example 1. The alcohol extract is dialyzed through a cellophane membrane for 3 hours. Dialysis, isolation, purification and drying of mannitol crystals are analogous to example 1. The yield of mannitol is 9.52 g or 9.52% of the raw material. The mannitol CS is 7.81 mg Rv ²⁺ / g.

Next, the extraction of pulp with water, filtering the extract is carried out as described in example 1. The obtained extract after filtration is cooled four times at a temperature of 2 ^o C for 1.5 hours (after each cooling in the refrigerator, the extract is kept until its temperature reaches room temperature) . The extract is then dialyzed through a cellophane membrane for 7 hours. Isolation from the Laminaride SB dialysate is carried out with 90% ethanol as described in Example 1. The preparation is dried by freeze drying in a Dolina apparatus at a temperature in the pressure chamber of -20 ^° C for 70 minutes. . The output of "Laminaride SB" amounted to 19.32 g or 19.32% of the raw material. CS "Laminaride SB" is 1900 mg Rv ²⁺ / g; solubility in water at 20 ^o C: 1 g of the drug is soluble in 45 ml of water.

The extraction of sodium alginate is carried out analogously to example 1, but in contrast to it, the solution of sodium alginate (after treatment with sulfuric acid and sodium carbonate) is cooled four times at a temperature of 2 ^o C for 1.5 hours; dialysis is carried out through a cellophane membrane for 7 hours; drying of sodium alginate is carried out at a temperature of -20 ^o C for 70 minutes The yield of sodium alginate is 21.63 g or 21.63% of the feed. KS sodium alginate 2430 mg Rv ²⁺ / g. Solubility in water at 20 ^o C: 1 g of the drug is soluble in 15 ml of water.

Example 3
The preparation of raw materials - thallus kelp - for extraction, the extraction of mannitol from it, is carried out analogously to example 1, but, in contrast to it, 1.5 l of chloroform is added to 100 g of algae for degreasing and incubated for 1 hour; dialysis of the alcoholic extract through a cellophane membrane is carried out for 1 hour. The yield of mannitol is 8.31 g or 8.31% of the feed. The mannitol CS is 5.63 mg Rv ²⁺ / g.

The extraction of the remaining pulp "Laminaride SB" is carried out analogously to example 1, but in contrast to it, the aqueous extract is cooled twice at a temperature of 10 ^o C for 0.5 h; the extract is dialyzed through a cellophane membrane for 5 hours; dried the preparation by freeze drying at a temperature of -11 ^o C for 80 minutes The yield of "Laminaride SB" amounted to 18.15 g or 118.15% of raw materials. CS "Laminaride SB" is 1832 mg Rv ²⁺ / g; solubility in water at 20 ^o C: 1 g of the drug is soluble in 70 ml of water.

The extraction of sodium alginate is carried out analogously to example 1, but in contrast to it, the cooling of the sodium alginate solution is carried out twice at a temperature of 10 ^o C for 0.5 h; dialysis is carried out through a cellophane membrane for 5 hours; drying of sodium alginate is carried out at a temperature of -11 ^o C for 65 minutes The yield of sodium alginate 19.71 g or 19.71% of raw materials. KS sodium alginate 2386 mg Rv ²⁺ / g. Solubility in water at 20 ^o C: 1 g of the drug is soluble in 20 ml of water.

The experimental biological part
The study of the laxative effect of the target products was carried out by recording the daily mass of feces after oral administration of a substance at a dose of 200 mg / kg in a volume of 3 ml of an aqueous solution in experiments on 42 white rats weighing 200-220 g of the Wistar line. The results of biological tests are shown in table 13. The presented results of a study of the laxative effect showed the identity of the biological effect of mannitol and sodium alginate obtained by both methods, with a slight predominance of the laxative effect of the target products according to the claimed method (the predominance of activity for mannitol is 4.2% for sodium alginate 4.9%). "Laminaride SB" obtained by the claimed method, 73% exceeds the laxative effect caused by "Laminaride" obtained by the prototype method. It is possible that this is due to the presence in the Laminaride SB fraction of additional polysaccharides (laminaran, fucoidan) and a high quantitative content of active substances.

Thus, the claimed method provides the following positive effect:
1. Complex processing of saccharine kelp growing in the White Sea of the Northern basin is provided, in which 3 products are obtained simultaneously: mannitol, polysaccharide-protein complex "Laminaride SB", sodium alginate. At the same time, the obtained substances can be used independently: a) mannitol as an osmotic diuretic to lower intracranial pressure and reduce cerebral edema, in acute renal hepatic insufficiency (MD Mashkovsky. Medicines: In 2 volumes. T.1 10- e ed. ster. - M.: Medicine, 1986. - P.493-494), as well as laxatives; b) "Laminaride SB" as a pronounced detoxifier of heavy metals and as a laxative; c) sodium alginate is similar to "Laminaride SB", and also as a stimulator of regeneration of wounds of various etiologies and as a plasma substitute. But the most widely used sodium alginate is in the technology of dosage forms as a gelling agent, a solubilizer, a binder, in the manufacture of tablets, a stabilizer of suspensions and emulsions, a film former, a solvent, etc. (Properties of alginates (by RH Mc Dowell). - Alginate Industries Limited, London).

 Laminaran and fucoidan are inappropriate to isolate as independent substances, since they, like Laminaride SB, have a laxative effect and are not of interest for other purposes.

 2. Increasing the yield of target products. So, if in the prototype method the yield of mannitol, Laminaride and sodium alginate is, respectively (% of raw materials): 7.85; 12.60; 12.41, then in the claimed method, these values reach the following values (respectively): 9.54; 19.52; 21.83. Thus, the increase in the yield of the target products by the present method is: for mannitol - 1.2 times, for "Laminaride SB" - 1.55 times, for sodium alginate - 1.76 times. The increase in the yield of the target products can be explained by repeated cooling of the polysaccharide solutions and freeze-drying of the preparations, which excludes the destruction of polymer macromolecules. In addition, the increase in the yield of Laminaride SB is apparently due to the coprecipitation of laminaran and fucoidan.

 The degreasing of thalli of kelp with chloroform promotes the destruction and extraction of various lipids that form cell membranes; this increases the access of various solvents - extractants to the active substances inside the cell, which also helps to increase the yield of active substances.

3. Increasing the purity of the target products. This is confirmed by the high quantitative content of active substances: for example, if in the target products obtained by the prototype method, the content of active substances was respectively (%) in: mannitol - 99.56; Laminaride - 76.55; sodium alginate - 91.15, then in the claimed method, these values were 99.94, respectively; 92.05; 96.75. In addition, the purification of the preparations increased the CS (mg of Pb ²⁺ / g): the CS of the target products obtained by the prototype method was 5.71 for mannitol, 1380 Laminaride, 1725 sodium alginate, and for those obtained by the present method, respectively 7.85; 1920; 2435. The excess of COP was 1.4 times for all products. The obtained "Laminaride SB" is distinguished by lighter tones of color - light pink (according to the prototype - grayish-yellow), sodium alginate - white (according to the prototype - light orange).

 An increase in the degree of purity is due to several reasons: a) the degreasing of raw materials helps release extracts from lipophilic pigments, which can bind to polysaccharides, reducing their CS; b) dialysis of extracts through cellophane membranes allows extracts to be freed of various impurities. The absorption spectrum of sodium alginate, shown in the drawing, confirms the effect of dialysis on the purification of the substance: the dialysate curve has a clear absorption maximum with a higher optical density than before dialysis; c) the use of low temperatures during the lyophilization of drugs avoids the destruction of the target products and the formation of low molecular weight fragments that "pollute" the drugs.

 Having high purity, all three target products can be used as medications, and even for injection use (mannitol, sodium alginate).

 4. Simplification of the method of complex processing of kelp due to: a) elimination of the laborious separate preparation of laminar and fucoidan, b) reduction of the drying time of the preparations: for Laminaride and sodium alginate obtained by the prototype method, the drying time is 180 and 135 min, respectively and for substances obtained by the present method, respectively 70 and 50 minutes, i.e. drying time is reduced by 2.6 and 2.7 times; c) reduction and exclusion of the consumption of limited and deficient extractants (standard, cetavlon, hydrochloric acid) due to the exclusion of the separate pressure of laminaran and fucoidan.

 5. Increasing the solubility of Laminaride SB and sodium alginate due to the use of multiple cooling of the extracts. As the data in table 8 show, the solubility of Laminaride SB and sodium alginate increases 1.9 times.

 6. Increasing the COP of the target products by 1.4 times. This allows us to recommend Laminaride SB and sodium alginate as detoxicants for heavy metal intoxication. This effect of the drugs is especially valuable if you take into account their main purpose (laxative and plasma substituting effects, respectively). "Laminaride SB", causing the gastrointestinal tract to be emptied, and sodium alginate, having a plasma-changing effect, can quickly remove heavy metal ions from the gastrointestinal tract and blood (respectively).

 7. Increased laxative effect. "Laminaride SB", mannitol and sodium alginate obtained by the present method, superior to the laxative effect of similar drugs isolated by the prototype method, respectively 73; 4.2 and 4.9%. It is possible that this advantage is associated with a high quantitative content of active substances.

 8. Adequate raw material base. Sugar laminaria forms annual large-tonnage raw material reserves in the White Sea of the Northern Basin. The State Pharmacopoeia of the USSR XI (GF USSR. XI ed. Issue 2. General methods of analysis. Medicinal plant materials. - M .: Medicine, 1990. - St. 83) recommends two types of kelp as producing plants - L. sugar and l. Japanese as the richest sources of polysaccharides, and palm-dissected kelp (L. digitata), used by the prototype method, is not an official type of raw material due to the lower content of active substances. Therefore, in the inventive method L. digitata is not used.

 The inventive method will allow, in relation to the conditions of the Murmansk scientific and technical center "Ecobiotek-Murmansk" to process the White Sea kelp. This will exclude the costs associated with the transportation and storage of raw materials, because raw materials will be mined and processed in Murmansk.

 9. Seaweed has long been used as a food product, so its processed products are completely non-toxic and biocompatible with the human body.

Claims (1)

A method for producing biologically active substances from kelp for medical purposes, characterized in that the feed is degreased with chloroform in a mass-volume ratio of feed: chloroform 1: 20 for 1.5 hours, then it is sequentially extracted with ethanol, water and an alkaline solution, the obtained extracts are dialyzed cellophane membrane for 2, 6 and 6 hours, respectively, and wherein the aqueous alkaline extracts to dialysis step trisubstituted kept at 4 ^o C for one hour with intermediate spontaneous adjusting the temperature to the room, odny dialysate was treated with 90% ethyl alcohol, precipitating the protein-polysaccharide complex comprising reducing sugars with fucoidan and laminaranom and precipitated from an alkaline dialysate sodium alginate, precipitation was dried by freeze-drying at -13 ^o C for 70 and 50 min respectively.

Images