RU2269542C1 - Modified chitosan - Google Patents

Abstract

FIELD: organic chemistry of natural compounds, chemical technology, medicine.

SUBSTANCE: invention relates to the group of chitosan-containing compounds. Invention relates to synthesis of modified chitosan of the following structure:

wherein n = 150-1400. The modified chitosan possesses the bactericidal activity, in particular, antituberculosis activity.

EFFECT: valuable medicinal properties of modified chitosan.

1 tbl, 1 dwg, 3 ex

Description

The invention relates to the field of chemical technology, namely to a group of compounds containing chitosan. The resulting modified chitosan can be used in medicine, cosmetology, agriculture, veterinary medicine, food and light industry.

Chitosan is a physiologically active compound of natural origin, widely used in cosmetics and medicines. It is obtained by hydrolysis of chitin - the main component of the external skeleton of arthropods:

n = 150-1400

Chitin (from the French "chitine" and Greek "chitone", which means "skin", "shell") is widespread in nature, it is part of the cell wall of fungi and bacteria, where it performs a supporting and protective function.

By its chemical structure, chitosan is a high molecular weight polymer (MM ~ 30000-260000 cu) from the group of polysaccharides built from glucosamine residues (acetylglucosamine) linked by β (1 → 4) glycosidic bonds. In its structure, physicochemical properties and biological role, chitosan is similar to plant cellulose.

Recently, in various fields of medicine and cosmetology there has been an increased interest in the use of chitosan as a mild therapeutic agent with a weak antiseptic effect. It is especially valuable that chitosan, being a product of the vital activity of living organisms, is harmless to humans.

An effective antibacterial agent is known - the antibiotic streptomycin, the molecule of which is a tricyclic carbohydrate containing two guanidine groups in one of the cycles [Encyclopedia of drugs. 8th ed., Ed. Yu.F. Krylova, M., "Radar", 2001, p. 813].

The structural formula of streptomycin has the following form:

Streptomycin by its chemical nature, like chitosan, belongs to aminoglycosides, is an effective broad-spectrum antibiotic that is active against tuberculosis mycobacteria, plague pathogens, tularemia, brucellosis, dysentery, strepto-, gono- and meningococci, produced by a radiant fungus (of the genus Streptomyces )

However, streptomycin is quite toxic and with prolonged use affects the auditory nerve.

Closest to the proposed solution is the carboxymethylchitosan preparation obtained by the reaction of chitosan with monochloracetic acid (Pat. RF No. 2185387, class 08 08 37/08, 2000).

The disadvantages of the known drug include insufficient bactericidal activity and a complex method of obtaining.

The technical task of this invention is to increase the bactericidal activity of the drug.

To solve the technical problem, a modified chitosan of the following structure was synthesized:

where n = 150-1400

Since the polymeric forms of drugs are usually preferred due to the enhancement of the effect due to the presence of many active groups located at molecular distances, lower toxicity, and also the deposition effect (longer therapeutic effect) of the drug, it seemed advisable to guanidize the amine groups of chitosan by converting them from low-active amine groups into highly effective guanidine.

Modified chitosan is prepared as follows. Due to the low thermal stability of chitosan, carbodiimide (NH = C = NH), the tautomer of monomeric cyanamide (NH ₂ CN), was chosen as a soft guanidizing agent.

Two commercial samples of chitosan succinate from Bioprogress and Himmed, dried by sublimation, were used as the starting chitosan feed. According to ultrafiltration (osmosis on membranes), their molecular weights are 30,000 and 26,000; degree of polymerization, respectively, n = 150 and n = 1400. The reaction was carried out with swollen chitosan succinate in an acetonitrile solution containing a calculated amount of 50% aqueous carbodiimide:

where n = 150-1400

To complete the process, the reaction mixture after 24 hours under normal conditions was heated for another 10 hours at 40 ° C. Modified chitosan differs from the original in a looser structure.

и

при ~3350 и 1600-1650 см^-1. Для сравнения на чертеже приведен также спектр гуанидированного полиэтиленимина, содержащий гуанидиновые полосы (кривая 2). ИК-спектр исходного хитозана полос при 3350 и 1600 см^-1 не содержит [хитин и хитозан]. Поскольку мы не располагали методом осмометрического определения молекулярных масс для определения молекулярной массы модифицированного хитозана был использован следующий прием. Навески исходного модифицированного хитозана растворяли в 2% уксусной кислоте и определяли их характеристические вязкости. Равенство полученных значений (η_исх=0,41 дл/л; η_мод=0,43 дл/л) свидетельствует о том, что в ходе реакции деструкция полимерной цепи хитозана не имела места. В свою очередь, небольшое возрастание молекулярной массы вследствие модификации, составляющее около 20%, также не на столько значительно, чтобы повлиять на характеристическую вязкость.An infrared spectroscopic study of the obtained polymer (see drawing) indicates the appearance of a significant number of guanidine groups in the structure of modified chitosan (curve 1): intense bands

and

at ~ 3350 and 1600-1650 cm ^-1 . For comparison, the figure also shows the spectrum of guanidized polyethylenimine containing guanidine bands (curve 2). The IR spectrum of the original chitosan bands at 3350 and 1600 cm ^-1 does not contain [chitin and chitosan]. Since we did not have the method of osmometric determination of molecular masses, the following technique was used to determine the molecular mass of the modified chitosan. Samples of the initial modified chitosan were dissolved in 2% acetic acid and their intrinsic viscosities were determined. The equality of the obtained values (η _ref = 0.41 dl / l; η _mode = 0.43 dl / l) indicates that during the reaction the destruction of the polymer chain of chitosan did not take place. In turn, a slight increase in molecular weight due to modification, amounting to about 20%, is also not so significant as to affect the intrinsic viscosity.

The appearance of guanidine groups in chitosan is also recorded by chemical analysis: colorimetry with eosin.

The antimicrobial activity of guanidized chitosan was studied by serial dilution. Bacteriostatic activity on the strain of Pseudomonas aeruginosa was 0.075%, bactericidal 0.15%.

Due to the proximity of guanidized chitosan to streptomycin, the drug was also tested for anti-tuberculosis activity. It also noted increased effectiveness due to the polymer nature of the drug (compared to streptomycin) and compared to unmodified chitosan.

The examples given illustrate the practical implementation of the synthesis of guanidized chitosan.

Example 1

A portion of 25 g (0.1 mol) of chitosan succinate (n = 150) was ground in a coffee mill into a fine yellowish powder and mixed in a flask with a 100 ml solution of acetonitrile containing 4.5 g (0.1 mol) of carbodiimide. After swelling of chitosan in a solution of acetonitrile during the day, the reaction mixture was heated with periodic stirring at a temperature of 40 ° C to complete the reaction. The modified chitosan was separated by filtration, then it was washed with 50 ml of distilled water on a filter and dried in air.

The resulting guanidine chitosan modifier is a light yellow bulk powder, hardly soluble in water and slightly better in a 2% solution of acetic acid.

Example 2

A portion of 25 g of commercial chitosan succinate (n = 150) was purified by reprecipitation. To this end, it was dissolved in 1.5 l of a 2% aqueous solution of acetic acid. Complete dissolution of a portion of chitosan requires ~ 48 hours with periodic stirring. The resulting homogeneous solution of chitosan acetate was decanted from a small precipitate, which may include unhydrolyzed chitin, as well as protein impurities. A clear solution was added with vigorous stirring to 2 L of a 15% aqueous ammonia solution. The reaction mixture was left for a day and after that the upper clear solution was carefully decanted. 1.5 l of distilled water was added to the precipitate, which was in the form of white cotton wool, mixed thoroughly and left again for a day. The washing and decanting operation was repeated three times until the smell of ammonia completely disappeared. After this, the precipitate of the chitosan base was dehydrated in a centrifuge and neutralized by dropwise adding 5 g of glacial acetic acid, thoroughly mixing in a mortar and drying in air to constant weight. Yield 20 g.

The obtained chitosan acetate was used in the reaction with carbodiimide according to the procedure of Example 1. The degree of guanidation of purified chitosan is higher (75%) than the non-precipitated (60%), according to colorimetric determination with eosin.

Samples of 0.5 g of modified and initial chitosan were dissolved in 100 ml of a 2% aqueous solution of acetic acid and the reduced specific viscosity of the obtained solutions was measured. Close values of the obtained viscosities η _ref = 0.4 and η _mode = 0.3 indicate the absence of destruction of the chitosan chain during guanidation.

Example 3

A portion of 5 g of commercial chitosan succinate (n = 1400) was modified by treating 1 g of carbodiimide in 20 ml of acetonitrile according to the procedure of Example 1. The modified chitosan was washed on a filter and dried to constant weight.

Samples of 0.1 g of the modified and initial chitosan were dissolved in 100 ml of a 2% aqueous solution of acetic acid and the reduced specific viscosity of the obtained solutions was measured. Close values of the obtained viscosities η _ref = 1.0 and η _mod = 0.9 indicate the absence of destruction of the chitosan chain during guanidation.

The anti-tuberculosis activity of guanidized chitosan A drug Dose% Time of action 30 minutes 1 hour 24 hours Guanidized Chitosan Succinate 0.5 + - - 0.25 + + - Guanidized Chitosan Acetate 0.5 - - - 0.25 + - - Streptomycin 1,0 + - - 0.5 + + - Unmodified chitosan 2.0 + + - (succinate) 1,0 + + +

Claims (1)

Modified chitosan of the following structure:

where n = 150-1400.