SK65893A3 - Sodium salt carboxymethyl-beta-(1-3)-d-glucanchitosamine and method of its preparation and using - Google Patents

Abstract

The solution relates to the sodium salt of carboxymethyl-P- (1-3) -D-glucan-chitosan, which is to be prepared, ie mycelium Aspergillus niger is subjected to alkaline hydroxide hydrolysis metal at 80 to 100 ° C for 0.5 to 2 h and the polysaccharide obtained is activated e.g. concentrated alkali metal hydroxide at temperature 40 to 70 ° C for 1 to 3 h or by ultrasonication at aqueous polysaccharide suspension. Activated polysaccharide % is resuspended in 10 to 25 wt. hydroxide solution alkali metal, sodium chloroacetate is added in an amount of corresponding to 1 to 2 times the amount of activated polysaccharide and the mixture is left for 2-4 h at room temperature temperature. The mixture is then neutralized with inorganic acid, the low molecular weight fraction is removed e.g. dialysis and the sodium salt of carboxymethyl-P- (1- [3] -D-glucan chitosan) is obtained it is dried e.g. lyophilization. The compound is used as a means of inhibiting pathogenic adherence microorganisms to mucosal epithelial cells.

Description

It is a well-known fact that fungal diseases are increasing all over the world. This is related to immune (anti-immune) and endocrine disorders after cancer chemotherapy, long-term administration of broad-spectrum antibiotics and radiotherapy.

The invasiveness of pathogenic yeasts is determined by their ability to adhere to the mucosal epithelium. On the surface of pathogenic candies there is a protein that specifically binds to some carbohydrates on the epithelial cell surface, such as fucose, N-acetyl-D-glucosamine [Cutler, J.E .: Ann. Rev.

Microbiol. 45, 187 (1991)]. Segal et al. [Exp. Cell Biol.

50, 13, 1982)] found that yeast adherence to human epithelium can be inhibited by amino sugars. The binding site of the protein in question is saturated and the pathogenic microbe loses its ability to attach to the surface of the epithelial cell.

Aminosugars are found predominantly in yeast and fungi in the form of chitin, which is a polymer of β-1,4-Ν-acetylglucosamine [Gow, N.A., Gooday, G.W .: Protoplasma 115, (1983)]. Chitin, in the ovoid form of yeast, is found in small amounts and only in whispers that remain on the cell wall after cell division. Mycelial forms of yeast cells and fungi have a higher chitin content, which is distributed throughout the cell wall. In these microorganisms, chitin is bound by a solid covalent bond to the glucans. The G-glucanine complex has great stability to various chemical agents, particularly alkali [Mol, P.C., Wessels, J.G.H .: FMS Microbiol. Letters 41, 95 (1907)].

Yeast and fungal β-1,3-D-glucans appear to be non-specific immunostimulators. Increases host resistance to bacterial, viral, fungal and para-responsive diseases by macrophage activation

Williams, D.L.

immune cells (D and Luzo, N.R.:

the host system, Immunophat. 8, 387

Res. 44, 54 (1988)] separately (1985): Glucan et al. : Int. J.

fungi in the fibrillar in fungi reaches X. Native fibrillar for application purposes in a mold. Glucanite has been shown to be the preferred carrier carriers of Kery, in Biochem. 22, 1203 (1990)]. β-Glucans or glucan-chitin occur in yeast, mold and mold. The fibril content of the cell wall 30-35 X, while in yeast only 5-6 glucan-chitin is insoluble in water, medicine is required its soluble derived from fungi is rigid in nature and resists conventional carboxmethylation procedures used in the literature, sodium chloroacetate to glucan or chitin [Whistler, RL: Methods in Carbohydr. Chem. 3, 322 (1963); Muzzarelli, R.A .: Carbohydr. Polvmers 8,1 (1988);

Wirth, S. J., Wolf, G. A., J. Microbiol. Meth. 12, 197 (1990)].

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a novel carboxymethyl-β- (1-> 3) -D-glucan-chitosan sodium salt which is prepared according to the present invention by subjecting the mycelium of Aspergillus niger to hydrolysis with an alkali metal hydroxide at 80 to 100 ° C for 0.5 to 2 h. The polysaccharide obtained is activated e.g. by treatment with concentrated alkali metal hydroxide at 40 to 70 ° C for 1 to 3 hours or by sonication on an aqueous suspension of the polysaccharide. The activated polysaccharide is resuspended in 10-25X (w / w) alkali metal hydroxide solution, sodium chloroacetate is added in an amount corresponding to 1-2 times the amount of activated polysaccharide, and the mixture is left at room temperature for 2-4 h. It is then neutralized with an inorganic acid, and the mixture is discarded. The salt of the salt of the salt 1 - β - (1 - 3) -1) -glucan is dried, e.g. 1 yo f i i z i c.

From an economic point of view, it is advantageous to use the waste mycelium Aspergillus niger, which remains after the production of citric acid, to produce the compound of the invention.

The compound of the invention may be advantageously used as a pathogenic agent Because sodium viscosity, inhibiting the adherence of microorganisms to mucosal epithelial cells, the carboxymethyl glucan-chitosan salt has a high potential for use in cosmetics as a retention agent in the food industry.

as a thickener, in the pharmaceutical industry as a drug carrier, and the like. The product is non-toxic. It breaks down glucose and glucosamine, substances that are part of the metabolic processes of living organisms.

The sodium carboxymethyl glucan-chitosan of the invention is well soluble in water and dilute acids. The ratio of glucose to glucosamine after thermal hydrolysis is 9: 1 to 8: 2. The substance shows characteristic IR spectrum of the glucan 920, 891 and 774, the chitin in 1618, 1379 and 781. The molecular weight is between 6 and 8.1O ⁵ Da, the intrinsic viscosity% = 1.2 to 2.0.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of the glucan-chitin complex kg wet mycelium A. niger from citric acid waste is digested with 114% (w / w) potassium hydroxide at boiling. The residue is washed with water until neutral, centrifuged, the sediment is dried and ground to a fine powder.

Activation of the glucan-chitin complex g of the polysaccharide is suspended in 125 ml of 60% w / w sodium hydroxide and stirred at 60 ° C for 3 hours. The mixture obtained is centrifuged.

Carboxmetal

The sediment is resuspended in 125 ml of 20% (w / w) sodium hydroxide, gradually adding 15 g with stirring.

- 4 sodium chloroacetate and the mixture is stirred at room temperature for 3 h. The mixture is then made up to 500 ml with water, neutralized with concentrated (37%) hydrochloric acid and centrifuged. The supernatant containing the water-soluble carboxymethyl derivative of glucan-chitosan is dialyzed and lyophilized.

The resulting product - carboxymethylated glucan-chitosan is in the form of sodium salt, is well soluble in water, yield is degree of substitution 0.45, carbohydrate composition

5.1 g, total hydrolysis viscosity% = 1.7.

% glucose, 9.5% glucosamine. internal

Example

The procedure is as in Example 1, except that the activation of the glucan-chitin complex is performed with 1 sonic.

g of the glucan-chitin complex powder is suspended in 125 ml of 20% w / w sodium hydroxide and the suspension is treated for 20 minutes with a UGA 20452 ultrasonic apparatus at a frequency of 20 W, 150 W power. Then 15 g of sodium chloroacetate is added. as in Example 1.

The yield is 1.8 g carboxymethy 1 g 1 sample. tozanu.

Example 3

Inhibition of adherence of pathogenic Candida albicans cells to epithelial cells using polysaccharides

Prepare 1 ml of a suspension of virulent C. albicans strain in phosphate buffer pH 7 to a density of 5 according to Mc Farland and mix with 1 ml of test substance (25 mg / ml). The mixture is incubated at 5 ° C with gentle shaking and 24 h. Next, a suspension of buccal cells is prepared in phosphate buffer (ph = 7) and washed three times with buffer.

0.2 ml of Ca 1 bicans cells after incubation with the polysaccharide were mixed. For a further 0.2 ml of the epithelial suspension and at 37 ^° C, 90 inin were incubated. Filtering is followed by a Synpor 1 membrane filter and the sediment on the filter is washed with 100 ml of phosphate buffer. A glass slide preparation is made and after drying and flame fixation it is stained by the Gram procedure.

The number of G ⁺ yeasts lying or touching the G 'epithelial cells of irregular shape is evaluated microscopically.

The results of the inhibition achieved by the control are shown in Table 1. In each experiment, the sodium carboxymethyl adherence inhibitory effect, even at lower concentrations. The mixture of polysaccharides after carboxymethylation contains, in addition to a soluble derivative and an insoluble residue, a partially soluble gelling agent.

tested polysaccharides and.

rated 100 epithelia. Soluble glucan-chitosan (Na-KM-G-Ch) has

Table 1

preparation horses. Test substance [mg / m1] C. albicans cell number / 1 ml epit. check 1 a 0 10.18 On-KM-G-Ch 6 2.63 On-KM-G-Ch 12 2.60 On-KM-G-Ch 25 2.50 mixture insol. a rozp. G-Ch 25 3.93 Insoluble. G-Ch 25 7.44 glucan Saccharo- myces cerevisiae 25 9.58

G-Ch = glucan-chitozan

Claims (3)

1. Sodium salt of 1-β- (1-> 3) -D-glucan-chitane carboxymethyl. Process for the preparation of sodium carboxymethyl-β- (1-3) -D-glucanchitosan according to claim 1, characterized in that the myergy of Aspergillus niger is subjected to hydrolysis by alkali metal hydroxide at a temperature of 80 to 100 ° C for 0.5 to 2 hours, the polysaccharide obtained is activated e.g. by treatment with concentrated alkali metal hydroxide at 40 to 70 ° C for 1 to 3 hours or by sonication on an aqueous polysaccharide suspension, the activated polysaccharide is resuspended in a 10-25% (w / w) alkali metal hydroxide solution, sodium hypochlorite is added in an amount corresponding to 1 to 2 times the amount of activated polysaccharide and the mixture is left for 2 to 4 hours at room temperature, then neutralized with inorganic acid, the low fluid fraction is removed by dialysis and the sodium salt of 1-β- (1-> 3) -D- The glucan-chi tozane is dried. The compound of claim 1 for use as a composition for inhibiting the adherence of microorganisms to mucosal epithelial cells.