RU2677232C2 - Method for obtaining heterogeneous preparation of various dispersities based on bromelain and chitosan - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine and process for producing heterogeneous preparation of various dispersities based on bromelain and chitosan, including the immobilization of the enzyme preparation in a buffer solution, incubation and washing. Immobilization of bromelain is carried out on an acid-soluble chitosan matrix of medium molecular or high molecular weight; as a buffer solution for immobilization, a tris-glycine buffer with pH 8.5–9.0 for medium molecular weight and pH 8.5 for high molecular weight chitosan is used. Incubation is carried out at room temperature for 4 hours for medium molecular weight and 5 hours for high molecular weight chitosan; the precipitate formed is washed with tris-HCl buffer.EFFECT: invention provides reduction in consumption of the wound healing agent due to the high stability of the preparation at temperatures higher than physiological and prolonged action of the preparation.1 cl, 3 dwg, 2 ex

Description

The invention relates to biotechnology and can be used in the pharmaceutical industry. A method for the preparation of heterogeneous biocatalysts based on the bromeline cysteine protease is proposed. The enzyme preparation can be used in the field of medicine and veterinary medicine.

Proteolytic enzymes occupy a separate niche in the medical industry. Enzyme preparations are used as regenerative, wound healing, antiparasitic agents.

Bromelain (KF 3.4.22.32) - a proteolytic enzyme of plant origin derived from pineapple. In 1957, Heinik and Gortner reported that in all the plants of the Bromeliacea family they studied, active proteolytic enzymes were found not only in fruits, but also in other parts of plants. One common name has been proposed for all of these enzymes, bromelain. Bromelain and / or other cysteine proteases are part of oral care products that prevent plaque formation, destroy biological films, have anti-inflammatory effects, and slow down the development of oral diseases [Patent RU 2380084, IPC A61K 8/21, A61K 8 / 98, A61Q 11/00, publ. 01/27/2010]. Bromelain acts as one of the types of active compounds in the composition for the prevention and treatment of caries [Patent RU 2446208, IPC C12N 1/20, publ. 03/27/2012]. Cysteine proteases are part of the dental composition to reduce or remove surface deposits of staining substances from natural teeth and dentures, including contact areas of teeth and / or dentures [Patent RU 2139037, IPC A61K 7/20, publ. 10/10/1999]. Plant proteases are part of the identification strips for the rapid test for individual compatibility of the blood of the donor and recipient. It is intended to use these strips in all areas of medicine (surgery, traumatology, resuscitation, obstetrics, etc.) associated with transfusion of red blood cells and other blood components [Patent RU 2129281, IPC G01N 33/80, publ. 04/20/1999].

The use of soluble enzymes has a number of limitations: fast inactivation of enzymes under the influence of various environmental factors, the inability to separate the reaction product from the enzyme catalyst, narrow temperature limits and the acidity of the medium. These problems can be partially solved by enzymes immobilized on an insoluble matrix, for example, on chitosan.

Chitosans are copolymers of 2-amino-2-deoxy-β-D-glucosamine and 2-acetamido-2-deoxy-β-D-glucosamine. Chitosan molecules contain hydroxyl and amino groups; as a result, their matrix allows sorbing various agents within the network and on its surface. Chitosan derivatives are characterized by non-toxicity, biodegradability, biocompatibility and poor immunogenicity. A biologically active composition has been created for medical and cosmetic purposes, as well as for use as a food supplement containing chitosan gel or chitosan suspension with spherical nanogranules no more than 100 nm in size and noble metal ions in an amount of not more than 10 wt. % [Patent RU 2471477, IPC A61K 9/00, A61K 31/722, A61P 17/02, A61K 31/498, A61P 31/04, publ. 01/10/2013]. There is a composition of a complex of chitosan succinate and dioxidine with chlorhexidine, which has antibacterial and wound healing properties [Patent RU 2193895, IPC A61L 15/28, A61L 15/30, A61L 15/32, publ. 12/10/2002]. A monolayer film for covering wounds is proposed, consisting of chitosan, collagen, a plant-derived polysaccharide and rubber latex. Such a coating is proposed for the treatment of wounds, burns, trophic ulcers [RU 2476199, IPC A61K 6/00, A61K 31/14, A61K 33/38, A61K 31/722, A61P 31/04, publ. 02/27/2013]. Chitosan comes with a composition for disinfection and cleaning of removable laminar dentures. Improving the disinfection efficiency of partial and complete removable plate prostheses is achieved due to the combined effect of the introduced substances - chitosan succinate, silver ions and cetrimide. These substances provide a pronounced bactericidal effect against gram-positive, gram-negative microorganisms, fungicidal action against yeast-like fungi, including the genus Candida, antiviral effect (on enveloped and non-enveloped viruses), and are also effective against mycobacteria [Patent RU 2380084, IPC A61K 8 / 21, A61K 8/98, A61Q 11/00, publ. 01/27/2010].

A method is proposed that allows the production of suture surgical material with sufficient strength, good tying, biocompatibility and non-invasiveness. The above effects are achieved by the fact that the mylar is coated with a sheath of chitosan [Patent RU 2080126, IPC A61L 17/00, publ. 08/09/1994].

The prototype was a METHOD FOR PRODUCING A MEANS FOR TREATING PURULAR NECROTIC RAS (SU 1814764, IPC A61K 31/557, A61K 47/48, published March 20, 1995), including immobilization of the enzyme preparation, in order to increase the therapeutic effect of the drug, immobilization was carried out by treatment of a 2% chitosan gel with a 2% solution of glutaraldehyde in phosphate buffer at pH 7.5, taken in a 1: 1 ratio, keeping it at a temperature of 18-20 ° C for 30 minutes, after which the extract of hepatopancreas Kamchatka was passed through the gel enzyme complex crab: DNAase in the amount of 100-120 u / mg of protein, RNA-azu - 100-120 u / mg, alkaline phosphatase - 0.05-0.07 u / mg, type 1 phosphodiesterase - 0.05-0.06 u / mg and protease - 120 cd / mg, previously dissolved in phosphate buffer pH 7.5 with a ratio of chitosan: extract 1: 2.5-5 (by weight).

The technical result consists in the development of a method for producing a heterogeneous preparation of various dispersion based on bromelain and chitosan, which will reduce the consumption of wound healing agents due to the high stability of the drug at temperatures above the physiological and prolonged action of the drug.

The active ingredient of our product is bromelain, which has an anti-inflammatory effect, accelerates tissue repair as a result of depolymerization of intercellular structures and modification of vascular permeability. The anti-inflammatory and antiplatelet effects of the enzyme are due to its ability to alter the metabolism of arachidonic acid. The temperature optimum for the functioning of bromelain is about 60 ° C [H.J. Suh [et al.] Purification and characterization of bromelain isolated from pineapple // J. Han'guk Nonghwa Hakhoechi - 1992. - Vol. 35. - P. 300-307], immobilization of the enzyme on an insoluble polymer matrix gives the effect of additional drug stabilization [R. Mahmood [et al.] Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies // Biochemistry. - 2007. - Vol. 72. - P. 307-312]. In addition, as a carrier for the immobilization of bromelain (the base of the dressing), we selected chitosan of various molecular weights and degrees of dispersion, which itself has anti-inflammatory, antimicrobial, antifungal, and wound healing properties, without causing allergic reactions.

The technical result is achieved by the fact that in the method for producing a heterogeneous preparation of different dispersion based on bromelain and chitosan, including immobilizing an enzyme preparation in a buffer solution, incubating and washing according to the invention, immobilizing bromelain is carried out on an acid-soluble chitosan matrix of medium or high molecular weight enzyme in a ratio of 20 ml of solution at a concentration of 5 mg / ml per 1 g of carrier; as a buffer solution for immobilization using 50 mm Tris-glycine buffer with a pH of 9.0 for medium molecular weight and with a pH of 8.5 for high molecular weight chitosan; incubation is carried out for 4 hours for medium molecular weight and 5 hours for high molecular weight chitosan at room temperature; the precipitate formed is washed with 50 mM Tris-HCl buffer (pH 7.5) until there is no protein in the washings.

In FIG. Figure 1 shows a diagram of the protein content (mg / g of carrier) in bromelain preparations immobilized on an acid-soluble matrix of medium molecular weight (SM) and high molecular weight (BM) chitosans. In FIG. Figure 2 shows a diagram of the total catalytic activity (U / ml of a solution) in bromelain preparations immobilized on an acid-soluble matrix of medium molecular weight (SM) and high molecular weight (BM) chitosans. In FIG. Figure 3 shows a diagram of the specific catalytic activity (u / mg protein) in bromelain preparations immobilized on an acid-soluble matrix of medium molecular weight (SM) and high molecular weight (BM) chitosans.

The bromeline of the Sigma-Aldrich company was chosen as the object of the study. As an insoluble matrix for adsorption, we used acid-soluble medium molecular weight (Mr = 200 kDa, deacetylation degree - 82%) and high molecular weight (Mr = 350 kDa, SD = 94.85%) chitosans manufactured by Bioprogress CJSC. The following solutions were tested as an immobilization medium: 0.05 M glycine (pH 8.6-10.0), 0.05 M acetate (pH 4.0-5.0), 0.05 M borate buffer with the addition of KCl (pH 8.0-10.0) and 0.05 M Tris-glycine buffer (pH 8.5-9.0).

Bromelain was immobilized on a chitosan matrix by an adsorption method. To 1 g of chitosan was added 20 ml of a buffer solution of the enzyme at a concentration of 5 mg / ml (using various buffers in the pH range from 4.0 to 10.5), incubated for 4 hours with periodic stirring for medium molecular weight (SM) chitosan, and when using high molecular weight ( BM) chitosan was incubated for 5 hours. After the incubation time, the precipitate formed was washed with 50 mM Tris-HCl buffer (pH 7.5) until there was no protein in the washings (this fact was monitored on an SF-2000 spectrophotometer at λ = 280 nm).

The protein content in the immobilized preparations was determined by the Lowry method [Lowry O.N., Rosebrough N.J., Faar A.L., Randall R.J. Protein measurement with folin-phenol reagent // J. Biol. Chem. - 1951. - V. 193. - P. 265-275]. The proteolytic activity of immobilized bromelain was determined spectrophotometrically using azocasein (Fluka) as a substrate. 200 μl of Tris-HCl buffer, pH 7.5, 800 μl of azocasein (0.5% in 50 mM Tris-HCl buffer, pH 7.5) was added to 50 mg of the sample and incubated for 2 hours at 37 ° C. Next, 800 μl of TCA (5%) was poured, incubated for 10 minutes at -4 ° C, then centrifuged for 3 min at 13000 rpm to remove non-hydrolyzed azokasein. To 1200 μl of the supernatant, 240 μl of 3% NaOH was added to neutralize the acid, after which the optical density of the experimental sample was measured at 410 nm in a 1 cm cuvette. The control sample contained 800 μl of azocasein, 800 μl of trichloroacetic acid, 50 mg of sample and 200 μl of buffer (without preliminary incubation with the substrate). The amount of enzyme was taken as a unit of catalytic activity, which under experimental conditions hydrolyzes 1 μM of the substrate in 1 min. The specific proteolytic activity of bromelain was calculated by the formula:

PA = D * 1000/120/200 / C,

where PA is the proteolytic activity, μM / min per 1 mg of protein, D is the optical density at 410 nm, C is the protein concentration in the sample, mg / ml, measured by the Lowry method, 120 is the incubation time in minutes, 200 is the sample volume, 1000 - recalculation in microns.

Statistical processing of the results was carried out at a significance level of 5% using Student t-test.

Analysis of the protein content in heterogeneous preparations showed that the greatest amount of bromelain (in mg per g of carrier) is adsorbed on acid-soluble medium molecular chitosan using a borate buffer with addition of KCl with a pH of 8.0-10.0, acetate buffer with a pH of 4.5-5.8 and glycine buffer with a pH of 8.6 -10.5. When immobilized on high molecular weight chitosan, the greatest amount of bromelain is sorbed using a Tris-glycine buffer with a pH of 8.5-9.0, a glycine buffer with a pH of 8.6-10.5, a borate buffer with added KCl with a pH of 8.0-10.0, and an acetate buffer with a pH of 5.0-5.8 ( Fig. 1).

The total activity (in units per ml of solution) of bromelain immobilized on medium molecular chitosan was higher using the following buffers: acetate with pH 4.5, 5.8, borate with KCl in the pH range 8.0–10.0, and Tris-glycine with pH 9.0. When immobilized on high molecular weight chitosan, the total activity of bromelain was higher when using Tris-glycine buffer with a pH of 8.5 and acetate buffer in the pH range of 5.0-5.8 (Fig. 2).

The greatest specific activity was shown by bromelain preparations immobilized on a matrix of medium and high molecular weight chitosans when using Tris-glycine buffer with pH 8.5-9.0 and pH 8.5, respectively (Fig. 3).

The optimal ratio of protein content (mg per g of carrier), total activity (in units per ml of solution) and specific activity (in units per mg of protein) was revealed when bromelain was immobilized using Tris-glycine buffer on a medium molecular weight chitosan matrix with pH 8.5-9.0 and on a matrix of high molecular weight chitosan with a pH of 8.5.

From the above material it follows that the following two options are optimal for creating heterogeneous preparations based on bromelain:

Example 1. To 1 g of acid-soluble medium molecular weight chitosan was added 20 ml of a solution of bromelain in 50 mm Tris-glycine buffer with a pH value of 8.5-9.0, incubated for 4 hours. After incubation, the precipitate formed was washed with 50 mM Tris-HCl buffer (pH 7.5) until there was no protein in the washings (this fact was monitored on an SF-2000 spectrophotometer at λ = 280 nm).

Example 2. To 1 g of acid-soluble high molecular weight chitosan was added 20 ml of a solution of bromelain in 50 mm Tris-glycine buffer with a pH value of 8.5, incubated for 5 hours. After incubation, the precipitate formed was washed with 50 mM Tris-HCl buffer (pH 7.5) until there was no protein in the washings (this fact was monitored on an SF-2000 spectrophotometer at λ = 280 nm).

Thus, a methodology was developed for producing a heterogeneous biocatalyst of varying degrees of dispersion based on bromelain immobilized on an acid-soluble matrix of medium and high molecular weight chitosans for medical and veterinary use.

Claims (1)

A method of obtaining a heterogeneous preparation of varying dispersion based on bromelain and chitosan, including immobilizing an enzyme preparation in a buffer solution, incubating and washing, characterized in that the immobilization of bromelain is carried out on an acid-soluble chitosan matrix of medium or high molecular weight in a ratio of 20 ml of an enzyme solution in a concentration of 5 mg / ml per 1 g of carrier; as a buffer solution for immobilization using 50 mm Tris-glycine buffer with pH 8.5-9.0 for medium molecular weight and with pH 8.5 for high molecular weight chitosan; incubation is carried out at room temperature for 4 hours for medium molecular weight and 5 hours for high molecular weight chitosan; the precipitate formed is washed with 50 mM Tris-HCl buffer at pH 7.5 until there is no protein in the washings.

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