The method of obtaining low molecular weight chitosan for radioprotective drugs

 

(57) Abstract:

Describes how to obtain low molecular weight chitosan. The invention relates to medical biotechnology and can be used to create based on natural polysaccharides new radioprotectors. The essence of the proposed method lies in the fact that the enzymatic cleavage of a solution of chitosan spend soluble complex microbial chitinolytic enzymes at pH 4.5-5.5 temperature 40-50oC for 30-60 min at a ratio of Chitinskaya activity-chitosan from 0.5 to 2.0 units of activity per 1 g of chitosan, then the reaction to withstand the weight of 3-7 min in a boiling water bath, cooled, concentrated, filtered, fractionary, allocating a fraction with srednevozrastnoe molecular weight of from 8 to 12 kDa, the resulting solution cialiswhat against water, the product is dried. The resulting substance vodorostvorima (50 mg in 1 ml) and easily sterilized by boiling, non-toxic. Survival of animals protected the obtained chitosan is 75-90%, with almost total loss of control after irradiation with a dose of 8.0 Gy. table 1.

The invention relates to medical biotechnology and can be used to create on the basis of PRIRODNYKh hydrolysis of chitosan with nitrous acid [1]. However, in this case it is difficult to obtain a product with srednevozrastnoe molecular weight below 20000 Yes, also happens chemical deamination terminal sugar residue with a change in the structure of the polysaccharide.

The closest in technical essence and the achieved result is a method of obtaining a low-molecular water-soluble chitosan by enzymatic hydrolysis of chitosan using immobilized chitinase complex [2]. The main advantage of this method is the possibility of obtaining high yield of low molecular weight water-soluble chitosan with a high degree of purity. However, the process is carried out in two stages, first at pH 4.5 to 5.0, and then at pH 6.0 to 6.5. The total time of the process ranges from 24 to 28 hours, and the resulting chitosan with a wide variation in srednevozrastnoe molecular weight of from 5 to 20 kDa.

To overcome these shortcomings provides a simplified method of obtaining low molecular weight water-soluble chitosan by enzymatic hydrolysis of chitosan solution followed by separation of the fractions characterized by radioprotective properties.

The choice of the optimal srednevozrastnoe molecular Inoi evaluate acute toxicity and radioprotective efficiency of chitosans with 30 MM, 10 and 5 kDa. Acute toxicity of samples of chitosan was determined by the mortality in experiments on nonlinear mice-albinos. Observation of animals was performed within 5 days after intravenous injection of samples in different doses. The results were processed probit method and set the values of sub-lethal doses (DM 50), and SD 84 and DM 16. The results shown in the table, indicate that with decreasing molecular weight of chitosan and decreases its toxicity.

Radioprotective protective efficacy of chitosan was investigated for nonlinear white mice and F1 hybrids(CBAC57B1). Samples with 30 MM, 10 and 5 kDa was injected intravenously before irradiation (10-20 minutes). Mice were irradiated with gamma rays Cs137 in doses of 8.0 to 8.25 G with a capacity of 1.25 G/min found that chitosan with 30 MM and 10 kDa, introduced respectively at the dose of 20 and 50 mg/kg, promotes survival 89-100% of animals with almost total loss of control. Increasing or decreasing doses of these samples leads to the reduction or absence of protective action. When using chitosan MM with 5 kDa protective effect either significantly reduced or not observed at all.

Thus, based on all properties, i.e. the manifestation of the lowest toxic The essence of the proposed method is that 1-2% solution of chitosan at pH 4.5-5.5 incubated at 40-50oWith the presence of the complex of chitinolytic enzymes in 20-60 minutes. Then the reaction mass was kept 3-7 minutes in a boiling water bath, cooled, filtered and fractionally either by chromatography or by using membrane technology, while highlighting the fraction with MM from 8 to 12 kDa. The product were dialyzed against water, and dried. The resulting substance vodorostvorima (50 mg in 1 ml) and easily sterilized by boiling, non-toxic.

Example 1.

Dissolve 1 g of crab chitosan with deacetylation (SD) of 85% and a molecular weight of about 400 kDa in 100 ml of 0.2 M sodium acetate at pH 5.0. The solution is heated to 45oTo add the filtrate of the culture fluid of Streptomyces kurssanovii (1 unit of activity) and incubated for 30 minutes at the same temperature. Then the hydrolysate stand 5 minutes in a boiling water bath, cooled, concentrated on a rotary evaporator, filtered and applied to a chromatographic column with sorbent Bio-Gel P-4, equilibrated with 0.1 M sodium acetate at pH 6.0. Spend the elution of the same solution, separating a fraction with MM 8-12 kDa, which unite, dialist against water and freeze-dried.

the industry of low molecular weight chitosan with MM 8-12 kDa was established in experiments on irradiated mice. Chitosan was administered to mice F1(CBAC57B1) intravenously at a dose of 50 mg/kg over 10-30 min before irradiation at a dose of 8.25 G: 7 protected mice 30 days after irradiation survived 7 at the end of all control animals.

Example 2.

Dissolve 1 g of chitosan Antarctic shrimp (krill) with led 90% and a molecular mass of 150 kDa 100 ml of 0.1 M sodium acetate at pH 5.5. The solution is heated to 50oTo add the filtrate of the culture fluid (0.5 units of activity) and incubated for 60 minutes at the same temperature. Then the hydrolysate stand for 3 minutes in a boiling water bath, cooled, concentrated by ultrafiltration to a volume of 10 ml, filtered and applied on the column with sorbent Sephadex G-25, equilibrated 0.15 M sodium acetate at pH 5.0. Spend the elution of the same solution, separating the fraction with molecular weight of 8-12 kDa, which unite, dialist against water and dried in the spray dryer. Obtain 0.6 g of low molecular weight chitosan in the form of a white water-soluble powder.

Efficiency study carried out analogously to example 1 on nonlinear mice albino animals irradiated at a dose of 8.0 Gy: by the end of the observation period (30 days) from 11-protected chitosan mice survived 10, in the control of ten to one.

oTo add the filtrate of the culture fluid of Streptomyces kurssanovii (2 units of activity) and incubated 45 minutes at the same temperature. Then the hydrolysate stand 7 minutes in a boiling bath, cooled, concentrated on a rotary evaporator to a volume of 10 ml, filtered and applied to a chromatographic column with sorbent Fractogel HW-50, balanced 0.05 M sodium acetate at pH 5.5. Spend the elution of the same solution, separating a fraction with MM 8-12 kDa, which unite, dialist against water and freeze-dried.

Obtain 0.5 g of low molecular weight chitosan in the form of a white powder, soluble in water.

The effectiveness of the obtained chitosan was studied as in example 2. Introduction chitosan before irradiation contributed to the survival of 8 mice out of 10, while in the control fell, all 10 mice.

Example 4

Analogously to example 1, however, take chitosan with SD 95% MM and 200 kDa.

Obtain 0.6 g of low molecular weight chitosan.

Example 5

Analogously to example 1, however, take an enzyme in the amount of 0.5 units and the hydrolysis time is 50 minutes.

Obtain 0.7 g of low molecular weight chitosan.

Example 6

Analogously to example 1, except that 0.6 g of low molecular weight chitosan.

Example 7

Analogously to example 1, except that the chitosan is taken with DM 70% and the process of hydrolysis spend 45 minutes.

Obtain 0.5 g of low molecular weight chitosan.

Radioprotective activity of samples obtained in examples 4-7, studied in four consecutive series of experiments on nonlinear mice as in example 2. Survival of animals protected chitosan, was 87%, 75%, 80% and 90%, with almost total loss of control.

The proposed method is environmentally friendly, less time consuming in comparison with known methods and allows to obtain a high quality product with the release of 50-70%.

Sources of information

1. Allan G. C., Ryan, C. A.// Carbohydr. Res. 1995. V. 277. N 2. P. 257-272.

2. Varlamov B. N., Stachenko I. A., Budanov M. C. a method of obtaining a low-molecular water-soluble chitosan. RF patent N 2073016, bull. 4, 1997.

The method of obtaining low molecular weight chitosan for radioprotective drugs enzymatic cleavage of chitosan solution, characterized in that the enzymatic cleavage spend soluble complex microbial chitinolytic enzymes at pH 4.5-5.5, a temperature of 40-50oC for 30-60 min at a ratio of Chitinskaya activity-hits, Hledat, concentrated, filtered, fractionary and the resulting solution cialiswhat against water.

 

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