CZ66492A3 - Preparation for inhibiting yeast candida albicans to mucosal epithelium - Google Patents

Abstract

A preparation for inhibiting Candida albicans yeast adherence to mucosal epithelium containing, as an active ingredient, a chitin-glucan complex, isolated from the cell wall of the mycelium Aspergillus niger. The preparation inhibits the virulence of yeast and can be used for treatment and prophylaxis of vaginal colpitis. The preparation according to the invention uses part of waste Aspergillus niger mycelia from production of citric acid and therefore contributes to the solution of ecological problems.

Description

The invention relates to a composition for inhibiting the adherence of yeast Candida albicans to mucosal epithelium. The composition comprises an active ingredient isolated from mycelium in the manufacture of citric acid.

BACKGROUND OF THE INVENTION

It is a known fact that Se out of vaginal colpitis is the most common fungal infection, most often leads to relapses and is the most common reason to visit a doctor. In addition, persistent relapses lead to somatopsychic and psychosomatic disorders in patients. Invasiveness of pathogenic yeasts is conditioned by their ability to adhere to the cell walls of the mucosal epithelium, which is realized by a chemical reaction between the cell wall components of yeast and epithelium. The formation of this bond appears to be a molecular biological signal for changing the yeast's growth pattern from ovoid to filamentous form, which easily penetrates into the deeper layers of the infected organ (Krogh, Holmstrup, Vedtofte and Pindborg; Special Symp, Yeasts Yeasts in study of Dormitory Smolenice iss:

Human Environment, Abstr. 62.). Yeast adherence to human epitheliums can be inhibited by, for example, amino sugars (Segal, Lehrer and Ofek).

Adherence o f C and n di d <

a 1bicans h u in a n v a g i n e a p helium 1 cellssInhibition by amino sugars. Exp. Cell Biol. 50, 13.

The essence of the invention

A novel means for inhibiting yeast adherence, e.g., Candida albicans to mucosal epithelium characterized by:

-i c i n i i ia 551 l c h i t i η ~ · g .1 li c a n o l o o * τι p 1 e χ mycsl i cell wall

Aspergillus niger. The composition utilizes the ability of chitin (N-acetyl-glucosamine) to inhibit the adherence of pathogenic yeasts to human mucosal epithelium, thereby reducing their virulence. It is obtained from a fungal cell wall that contains a large amount of chitin, a polymer of N-acetyl-D-glucosamine with β-1-4 bonds (Peberdy JFs Fungal cell walls. , PJ Kuhn, APJ Trinec, MS Yu.ng, MW Goosey, Eds.). It is produced as a waste product in the production of citric acid for food or pharmaceutical needs, when a large amount of mycelium producing mold Aspergillus niger, which is a difficult waste, is eliminated.

Chitin-glucan complex from the mycelium of the Asp strain. niger from citric acid production in the Lachema Kaznějov plant was obtained by alkaline hydrolysis of 1 kg wet mycelium in 11 N KOH at 100 ° C for 1 hour, thereby removing both cell cytoplasm and the cell wall protein component. The solid residue was washed until neutral and lyophilized. The substance gives a characteristic IR spectrum for glucan 920, 891 and 774 and for chitin 1618, 1379 and 781.

The product obtained was tested in an in vitro experiment for the ability to inhibit the adherence of a virulent C. albicans strain (isolated in the gynecological mucosa and ambulance) in an in vivo cell to experimental candidiasis in rabbits

DETAILED DESCRIPTION OF THE INVENTION

Example 1 human vaginal and buccal

Determination of the ability of the chitin-glucan complex to inhibit Candida adherence activity. albicans - cells of the vaginal and buccal mucosa in vitro.

The experiment was carried out in 4 variants:

var. 1: epithelium + c. Albicans + chitin-glucan (own experiment) var.2: epithelium + C. albicans + formalin (dead yeast) v a r. a 11t1 set u. m (yeast yeast) var.4: epithelium + C. albicans + buffer (live yeast)

Variants 2,3 and 4 were triple,

To perform the experiment:

suspension A: 25 mg chitin-g 1 ukan. / 1 ml phosph. buffer, pH = 7 suspension B; C, albicans, 24 h, grown on Sabouraud agar, suspended in phosph. buffer pH = 7, to density 5 according to Nc Farland (Tomšíkova A., Sandula J .: Immunology and pathogenicity. V: Yeast in research and practice. Academia 1786, D. Vraná, Eds., p. 265.) vaginal mucosal epithelium obtained by smear into fost. buffer pH = 7, washed 3 times with the same buffer and finally into 1 ml of the same buffer. resuspended.

1 ml of suspension A + 1 ml of suspension B are mixed and incubated for 24 hours at 4 ° C. Then 0.2 ml of the combined suspensions (A + B) are mixed with 0.2 ml of washed epitheliums (suspension C) and incubated with shaking for 90 min. Filtration through a Synpor 1 membrane filter followed by washing the sediment on a 100 ml phosphor filter. buffer pH = 7. The impression glass is prepared, after drying and flame fixation, stained by the Gram procedure and the number of G ⁺ yeasts touching or lying on irregularly shaped G-epitheliums is microscopically evaluated.

In the control variants, 0.9X formalin (variant 2) or a specific rabbit antiserum (variant 3) was used instead of phosphate buffer to prepare suspension B. 100 epitheliums were evaluated microscopically, see Table I.

Tab. AND

epi telie b C. albicans at 1 epithelium var. 1 var. 2 var. 3 v ar. 4 vaginal 1.18 2, 99 3.74 21.07 buccal 2.70 4.90 6.20 16.40

Evaluation ". 1. The different number of C. Albicans cells per epithelium in the control (variant 4) was 20 times the number of cells in the experiment (variant 1).

2. Virulent C. albicans cells adhere to the vaginal rather than buccal epithelium in the ball.

3. The chitin-glucan complex of Aspergillus niger mycelium inhibits C. albicans adherence to cells of the vaginal epithelium more than to cells of either epithelial cell.

Example 2

Determination of the ability of a chitin-glucan complex to inhibit the adherence activity of various Candida albicans strains isolated in a gynecological outpatient clinic in vitro.

The same experiment was performed with 4 C. albicans strains isolated from 4 patients. For these strains, the specific growth rate jj and the doubling time of the cell mass g were predetermined in Saudi ra, and the specific growth rate, j, and the doubling time of the cell mass g, in order to assess the relationship between growth rate and ability adherence, Growth 1 determined under 1 eoptical cleat 11 at a time interval of 0-24 hours. The value of jj was calculated according to the equation j.> - X. / t. 1 / X. The value of g was determined to be van and according to the formula g - 0.69 / jj (Aries et al.: Cell growth and multiplication. V: Yeast in research and practice. Ac and clem .ia 1986, Ei. , Ed., P. 169.) »The results are given in Tables II and III.

Tab. II

strain % increase in 24 hours i- ¹ g / hour 16 (v a g.) 4.76 0,002 345.0 17 (vag.) 1825,82 0, 120 cr -rer /, / · ._ » 18 / vag / 13 / ζΤζ, 33 0, 100 6.90 19 / vag / 1196,62 0, 106 6.50

The strains of C. albicans differ significantly from different donor plants in their growth characteristics. The test of adherence and inhibitory effect of chitin-glucan was arranged as in Tab. AND.

Tab. III

Strain number Bc albicans na 1 epithelium var. 1 var. 2 var. 3 var. 4 16 1.54 1.66 2,0 11.96 17 0.84 2.68 22, 56 13 1.44 3.96 1.9 20, So 19 Dec 1, 18 1.66 0, 86 4 cr no 1 J, 7 U

The cells of the slowly growing strain (strain 16) had a lower adherence ability to the cells of the rapidly growing strains. The inhibitory effect of chitin-glucan is less pronounced in slow-growing cells.

Example

Determination of the ability of the chitin-glucan complex to prevent experimental vaginal candidiasis in rabbit females.

The ability of chi tin-g bow. The ararium complex to prevent experimental candidiasis was tested in female rabbits injected with chitin-glucan into the vagina together with virulent C. albicans.

21 rabbits were used. A suspension was prepared from virulent C. albicans strain in distilled water according to McFarland scale # 10 and mixed with chitin-glucan at a ratio of 1 (chitin-glucan weighed 25 mg / ml saline). 7 rabbits were injected deep into the vagina once daily for 7 days (Group A). A further 7 rabbits were infected with C.albicans alone on the same schedule (group B) and finally the last 7 experimental animals were treated with chitin-glucan only (group C) in the same manner.

After 7 days all vaginal swabs were cultured on Sabouraud's medium for 7 days with glucose (control swab was performed in all females prior to the start of the experiment). At the same time, skin tests were performed on all animals with C. albicans mananprotein.

In the control group (Group B), the mycological finding was positive in all animals and the females suffered from discharge. In the experimental group in which the animals were co-administered with the infectious agent chitin-glucan (group A), even in the last group where the animals were treated only with chitin-glucan (group C), the mycological finding was negative in all females. Skin tests were negative in all 21 animals enrolled in the experiment.

Industrial applicability

The composition of the invention could provide a tool for the combined therapy and prophylaxis of vaginal mice.

If it is possible to detect part of the waste m e t i e

Π i r from citric acid production and thus contributes to solving eco problems. The introduction of the preparation into the medical practice would be a financial saving of the myocardia abroad.

o g i c k ý c h

Claims (1)

A composition for inhibiting yeast adherence, e.g., Candida albicans, to the mucosal epithelium, characterized in that it contains as an active ingredient a chitin-glucan complex from Aspergillus niger cell wall cells.