RU2417088C1 - Method for producing low-molecular chitosan - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention refers to producing chitin and its derivatives, namely to methods for producing low-molecular chitosan. Low-molecular chitosan is produced by dissolving high-molecular chitosan in 1% aqua acetic acid. It is added with hydrogen peroxide in amount 0.5-1.5 wt % to total solution in the presence of catalytic amounts of manganese oxide (IV). The solution is kept at temperature 18-50C for 30 min. Upon completing reaction time, it is added with an aqueous solution of ammonia, with reducing pH of the reaction mixture to 6.9-7.0. After setting of sediment, acetone is added, and the solution is kept for 2 hours. The sediment is filtered, washed with distilled water and acetone, dried. ^ EFFECT: use of the method provides producing the end-product with a smaller degree of polydispersity and composition heterogeneity. ^ 2 dwg, 2 tbl, 5 ex

Description

The invention relates to the field of production of chitin and its derivatives, and in particular to methods for producing low molecular weight chitosan.

In order to obtain homologues with a given degree of polymerization, various methods of physical or chemical destruction of the starting high molecular weight glycans are used. The first group of methods includes destruction under the influence of various types of high energy radiation. The kinetics of chitosan fragmentation under the influence of laser radiation pulses was studied; applied methods of viscometry and HPLC. The yield of polymer chain breaks increases with the growth of MM of chitosan (fragmentation of large molecules). The polydispersity of the resulting polymer material increases with increasing exposure time. Saturation of solutions with oxygen has little effect on the yield of breaks. Chitosan fragmentation is determined by photoabsorption processes with minimal oxidation effects (RF Patent No. 2263681, IPC ⁷ C08B 37/08, 2005).

The main disadvantage of these methods is the occurrence of side reactions of oxidation, decarboxylation, accompanied by deamination, destruction of the pyranose cycles of the structure of the polymer units. The destruction of glycosidic centers in an acidic medium also has a number of disadvantages, such as the difficulty of controlling the process and purification of the resulting low molecular weight polymer homologues.

A method has been developed for the preparation of water-soluble chitosan homologs, which involves the treatment of chitosan in acidic media. As an acidic medium, a water-acid vapor dispersion is used. A pair of an aqueous solution of monobasic acid is used, the last used is HCl, HCOOH, CH ₃ COOH (Gizatulina G.A. Depolymerization of chitin and chitosan during acid hydrolysis / G.A. Gizatulin, V.Yu. Novikov, I.N. / Current Prospects in the Study of Chitin and Chitosan: Materials of the Eighth International Conference. // Kazan, June 12-17, 2006 - P.27).

Acid depolymerization depends on the initial degree of chitin deacetylation. An increase in the degree of deacetylation leads to an increase in the number of deacetylated oligomers significantly more stable in an acidic environment than acetylated products; the monomer yield decreases (HPLC of the products of chitin and chitosan hydrolysis). The concentration of acid has a strong effect on the rate of hydrolysis. A sharp drop in MM was observed at pH 5 in comparison with pH 7 (hydrolysis duration 30 min) (Isolation and purification of highly active fractions of low molecular weight chitosan / P.A. Kuznetsov et al. // Scientific fundamentals of the production of veterinary biological preparations: Materials of the International Scientific and Practical Conference young scientists. // Schelkovo, October 5-6, 2004 - P.124-128).

The method of enzymatic hydrolysis of chitosans using specific chitinolytic enzymes, the action of which is selectively directed to the destruction of β-1,4 interblock bonds, has also gained sufficient popularity. This method is characterized by the complexity of the process control, in addition, the resulting homologs intended as raw materials of pharmacochemical technology require special techniques for purification of used biocatalysts (Separation of chitooligosaccharides by high performance liquid chromatography and thin layer chromatography / Fang Zi ao // Period. Ocean Univ . - 2005. - V.35, N 1. - P.113-115; Xia Wen-Shui. Advances in the Research and Production of Chitooligosaccharides / Xia Wen-Shui, Wei Xin-Lja // J. Appl. Chem. - 2004. - V. 21, N 4. - P.332-337; Tsaih Min Lamg. Effect of the removal of small fragments using ultrafiltration during ultrasonic processing on the kinetics of chitosan / Tsaih Min Lamg, Tseng Lan Zang, Chen Rong Huei // Polim. Degrad. and Stab. - 2004. - V.86, N 1. -P. 25-32; RF patent No. 2073016, IPC С08В 37/08, 1997).

A known method of producing low molecular weight chitosan, soluble in alkaline conditions, according to which chitosan is dissolved in an aqueous acid solution, followed by the addition of an alkali solution to the resulting chitosan solution, pH 7.0-8.0, after which precipitated chitosan is removed from the solution, and low molecular weight chitosan remains in solution or freeze-dried (RF application No. 2008113410, СВВ 37/08, 2009).

A known method of producing low molecular weight chitosan by acid hydrolysis of chitosan using nitrous acid (Allan G.G., Ryan C.A. // Carbohydr. Res. 1995. V.277. N2. - P.257-272).

However, in this case, it is difficult to obtain the target product with a molecular weight below 20 kDa and the process is accompanied by partial deamination of the polysaccharide and, accordingly, leads to a change in its properties.

A known method of producing low molecular weight chitosan by treating an aqueous suspension of 3 wt.% Freshly precipitated chitosan at pH 6.9-7.5 with hydrogen peroxide (hydrogen peroxide), 0.1 wt.%, At 85 ° C for 32 min, after filtration and triple washing is activated in a reactor with a disk stirrer (RF patent No. 2144040, IPC С08В 37/08, А61К 31/722, 1998).

A known method of producing chitosan with an 80-85% DM and the desired MM value by treating an aqueous suspension of chitosan with an initial MM of 300-600 kDa with hydrogen peroxide and a method for producing a degraded chitosan MM of 80 kDa, 68-72% DM from freshly precipitated chitosan (RF patent No. 2215749 , IPC С08В 37/08, А61К 31/722, 2003). To 5.5 wt.% The suspension, loaded into an enameled or glass reactor and diluted with demineralized water to a concentration of 3 wt.%, Heated to a temperature not exceeding 80 ° C, add hydrogen peroxide in the calculation of 0.15 wt.% Of the total weight of the suspension and with vigorous stirring, incubated for 25 minutes, then filtered, rinsing with cold demineralized water, and squeezed.

These methods of producing low molecular weight chitosan are implemented under heterogeneous conditions, i.e. in the presence of suspended particles in solution.

The technical result consists in carrying out the process of producing low molecular weight chitosan under homogeneous conditions, i.e. in the absence of suspended particles in solution, which provides a lower degree of polydispersity and compositional heterogeneity of the final product.

The technical result is achieved in that the method of producing low molecular weight chitosan involves preparing a solution of high molecular weight chitosan in 1% aqueous acetic acid, adding a solution of hydrogen peroxide in an amount of 0.5-1.5 wt.% To the total weight of the solution in the presence of catalytic amounts of manganese oxide (IV), keeping at a temperature of 18-50 ° C for 30 minutes, adding at the end of the reaction time an aqueous solution of ammonia, adjusting the pH of the reaction mixture to 6.9-7.0, and after precipitation, a cetone and incubated for 2 hours, the precipitate is filtered off, washed with distilled water and acetone, dried.

The destruction of high molecular weight chitosan H ₂ O _{2 was} studied in order to obtain polymer homologs with a degree of polymerization from 6 to 1000 aminoglucose units and preserving diabetes more than 90%.

Figure 1 shows the dependence of the molecular weight of chitosan on the concentration of H ₂ O ₂ (polymer concentration 2 wt.%, 50 ° C, reaction time 30 min).

Figure 2 shows the temperature dependence of the molecular weight of chitosan (polymer concentration of 2 wt.%, Reaction time 30 min, concentration of H ₂ O ₂ 1.5 wt.%).

Table 1 shows the dependence of the molecular weight and yield of low molecular weight chitosan on the concentration of H ₂ O ₂ (70 ° C).

Table 2 shows the temperature dependence of the molecular weight of chitosan (polymer concentration 2 wt.%, Reaction time 30 min).

The most important parameter determining the degree of destruction of high molecular weight chitosan, respectively, and the molecular weight of the final product, is the concentration of H ₂ O ₂ in the reaction medium. Studies were carried out at a constant temperature of 50 ° C. From figure 1 it is seen that with increasing concentration of hydrogen peroxide, the molecular weight decreases. However, as the concentration of H ₂ O ₂ increases, the degree of dispersion of the target product increases, as evidenced by the expansion of the molecular weights of the polymers in a series of experiments carried out simultaneously under the same conditions.

The study of the effect of temperature on the depth of the destruction was investigated in the range of 20-70 ° C. It has been established that temperature is a more promising parameter that allows more control over the molecular weight of the resulting polymer homologues. As can be seen from FIG. 2, with increasing temperature with the constancy of the remaining reaction parameters, the molecular weight decreases. Moreover, the molecular weight of the polymers obtained in a series of parallel experiments varies between 5-10%, which corresponds to the experimental error.

The use of catalytic amounts of manganese (IV) oxide can significantly accelerate the process of oxidative destruction of β-1-4 glycosidic bonds of chitosan macromolecules due to the fact that in its presence there is an active decomposition of hydrogen peroxide and, as a result, atomic oxygen released in the process provides more deep degradation at lower temperatures. It was established that in the presence of catalytic amounts of manganese (IV) oxide, it is possible at a temperature of 18 ° C to obtain low molecular weight homologs of chitosan of a given molecular weight, all other things being equal, without using heating to higher temperatures.

The most reliable and convenient method of stopping the process of chitosan depolymerization is the physical removal of H ₂ O ₂ : phase separation, extraction. The phase state of the hydrolysis products may be different (suspension or solution). This method is implemented when obtaining homologues of chitosan with a degree of polymerization of more than 30.

A distinctive feature of the proposed method for the destruction of chitosan is the use of an aqueous solution of ammonia, which, when oxidized with hydrogen peroxide residues, turns into environmentally friendly molecular nitrogen, and the peroxide decomposes to water and molecular oxygen.

Destruction of high molecular weight chitosan.

Example 1

Synthesis of chitosan with a molecular weight of 30 kDa

In a 5-liter three-necked flask equipped with a mechanical stirrer, reflux condenser, and dropping funnel, 3 liters of a 1.5 wt.% Solution of chitosan in a 1% aqueous solution of acetic acid are placed. Stirring is turned on and incubated for 10 minutes at ambient temperature (50 ° C). Then, using a dropping funnel, add 46 ml of 36.4% (0.5 wt.% To the mass of the reaction mixture) hydrogen peroxide solution. The reaction mixture was incubated for 30 minutes at a constant temperature (18 ° C). At the end, 120 ml of an aqueous solution of ammonia is added to the reaction mixture, followed by precipitation of the product. To completely isolate the product, 300 ml of acetone are added to the reaction mixture with stirring and incubated for 2 hours. The precipitate is filtered through cheesecloth on a Buchner funnel using a water jet pump, the precipitate is washed with 500 ml of distilled water, then 200 ml of acetone. The precipitate is transferred to the crystallizer in a thin layer and dried in a vacuum oven at a temperature of 30 ° C for two days. The resulting product is a brown sintered solid.

Example 2

Synthesis of chitosan with a molecular weight of 30 kDa

A 3 L three-necked flask equipped with a mechanical stirrer, reflux condenser, and dropping funnel is used to place 3 L of a 1.5 wt.% Solution of chitosan in a 1% aqueous solution of acetic acid. Mixing is turned on and incubated for 10 minutes at ambient temperature (18 ° C). Then, using a dropping funnel, add 46 ml of 36.4% (0.5 wt.% To the mass of the reaction mixture) hydrogen peroxide solution. Then add 0.0005 g (1.6 · 10 ^-5 wt.% In relation to the total weight of the solution) manganese (IV) oxide. The reaction mixture was incubated for 30 minutes at a constant temperature (18-20 ° C). At the end, 120 ml of an aqueous solution of ammonia is added to the reaction mixture, followed by precipitation of the product. To completely isolate the product, 300 ml of acetone are added to the reaction mixture with stirring and incubated for 2 hours. The precipitate is filtered through cheesecloth on a Buchner funnel using a water jet pump, the precipitate is washed with 500 ml of distilled water, then 200 ml of acetone. The precipitate is transferred to the crystallizer in a thin layer and dried in a vacuum oven at a temperature of 30 ° C for two days. The resulting product is a brown sintered solid.

Example 3

Synthesis of 9 kDa Chitosan

In a 5-liter three-necked flask equipped with a mechanical stirrer, reflux condenser, and dropping funnel, 3 liters of a 1.5 wt.% Solution of chitosan in a 1% aqueous solution of acetic acid are placed. Stirring is turned on and incubated for 10 minutes at ambient temperature (50 ° C). Then, using a dropping funnel, add 94 ml of 36.4% (1.0 wt.% To the mass of the reaction mixture) hydrogen peroxide solution. The reaction mixture was incubated for 30 minutes at a constant temperature (18 ° C). At the end, 135 ml of an aqueous solution of ammonia is added to the reaction mixture, followed by precipitation of the product. To completely isolate the product, 300 ml of acetone are added to the reaction mixture with stirring and incubated for 2 hours. The precipitate is filtered through cheesecloth on a Buchner funnel using a water jet pump, the precipitate is washed with 500 ml of distilled water, then 200 ml of acetone. The precipitate is transferred to the crystallizer in a thin layer and dried in a vacuum oven at a temperature of 30 ° C for two days. The resulting product is a brown sintered solid.

Example 4

Synthesis of 9 kDa Chitosan

A 3 L three-necked flask equipped with a mechanical stirrer, reflux condenser, and dropping funnel is used to place 3 L of a 1.5 wt.% Solution of chitosan in a 1% aqueous solution of acetic acid. Mixing is turned on and incubated for 10 minutes at ambient temperature (18 ° C). Then, using a dropping funnel, add 94 ml of 36.4% (1.0 wt.% To the mass of the reaction mixture) hydrogen peroxide solution. Then add 0.0005 g (1.6 · 10 ^-5 wt.% In relation to the total weight of the solution) manganese (IV) oxide. The reaction mixture was incubated for 30 minutes at a constant temperature (18 ° C). At the end, 135 ml of an aqueous solution of ammonia is added to the reaction mixture, followed by precipitation of the product. To completely isolate the product, 300 ml of acetone are added to the reaction mixture with stirring and incubated for 2 hours. The precipitate is filtered through cheesecloth on a Buchner funnel using a water jet pump, the precipitate is washed with 500 ml of distilled water, then 200 ml of acetone. The precipitate is transferred to the crystallizer in a thin layer and dried in a vacuum oven at a temperature of 30 ° C for two days. The resulting product is a brown sintered solid.

Example 5

Synthesis of 3 kDa Chitosan

In a 5-liter four-necked flask equipped with a mechanical stirrer, reflux condenser, dropping funnel, thermometer, 3 L of a 1.5 wt.% Solution of chitosan in a 1% aqueous solution of acetic acid is placed. Mixing is turned on and the mixture is heated to a temperature of 50 ° C. After that, 130 ml of 36.4% (1.5 wt.% To the mass of the reaction mixture) hydrogen peroxide solution are added using a dropping funnel. The reaction mixture was incubated for 30 minutes at a temperature of 50 ° C. At the end, 150 ml of an aqueous solution of ammonia is added to the reaction mixture, followed by precipitation of the product. To completely isolate the product, 300 ml of acetone are added to the reaction mixture with stirring and incubated for 2 hours. The resulting reaction mass is left for a day to settle, the upper liquid layer is decanted. 200 ml of acetone are poured into the obtained gel-like mass and, after stirring, is allowed to stand for 5 hours. After separation of the solid mass, the liquid phase is separated by decantation. The resulting product is a thick gel-like mass of white.

Table 1 The method of obtaining low molecular weight chitosan No. Chitosan diabetes,% Conc. H ₂ O ₂ in the system,% Exit, % MM, kDa one 90 5.7 72 1.0 2 90 5.1 73 1.0 3 90 4.7 78 1.0 four 90 4.2 78 1.0 5 90 4.1 75 1.2 6 90 2.0 80 1.4 7 90 1.5 81 1.6 8 90 1.3 84 1.7 9 90 1.1 82 1.8 10 90 1.0 79 2.0 eleven 90 0.9 84 1.8 12 90 0.8 85 2.0 13 90 0.7 81 1.9 fourteen 90 0.6 86 2.3 fifteen 90 0.5 85 2.5 16 90 0.4 77 3.0 17 90 0.3 83 3.6 eighteen 90 0.25 85 5.0 19 90 0.2 87 6.0 twenty 90 0.15 87 18.0 21 90 0.1 85 20.0 22 90 0.05 83 27.0

table 2 The method of obtaining low molecular weight chitosan No. Chitosan diabetes,% Conc. H ₂ O ₂ in the system,% Exit, % MM, kDa one 90 5.7 72 1.0 2 90 5.1 73 1.0 3 90 4.7 78 1.0 four 90 4.2 78 1.0 5 90 4.1 75 1.2 6 90 2.0 80 1.4 7 90 1.5 81 1.6 8 90 1.3 84 1.7 9 90 1.1 82 1.8 10 90 1.0 79 2.0 eleven 90 0.9 84 1.8 12 90 0.8 85 2.0 13 90 0.7 81 1.9 fourteen 90 0.6 86 2.3 fifteen 90 0.5 85 2.5 16 90 0.4 77 3.0 17 90 0.3 83 3.6 eighteen 90 0.25 85 5.0 19 90 0.2 87 6.0 twenty 90 0.15 87 18.0 21 90 0.1 85 20.0 22 90 0.05 83 27.0

Claims (1)

A method of producing low molecular weight chitosan, comprising preparing a solution of high molecular weight chitosan in 1% aqueous acetic acid, adding to it a solution of hydrogen peroxide in an amount of 0.5-1.5 wt.% To the total weight of the solution in the presence of catalytic amounts of manganese (IV) oxide keeping at a temperature of 18-50 ° C for 30 minutes, adding at the end of the reaction time an aqueous solution of ammonia, bringing the pH of the reaction mixture to 6.9-7.0, after precipitation, acetone is added to the reaction mass and kept for 2 hours , os docking filtered, washed with distilled water and acetone and dried.

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