RU2750377C1 - Method for producing immobilized enzyme preparation based on bromelain, hyaluronic acid and polysaccharides modified with vinyl monomers - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry; medicine.

SUBSTANCE: present invention relates to the field of chemical and pharmaceutical industry and medicine, namely, to a method for producing an immobilized enzyme preparation based on bromelain, polysaccharides modified with vinyl monomers, and hyaluronic acid. The method includes the dissolution of bromelain in an aqueous solution of low-molecular-weight hyaluronic acid 300 kDa or medium-molecular-weight hyaluronic acid 500 kDa or high-molecular-weight hyaluronic acid 800 kDa in a ratio of 10 mg of bromelain per 2 ml of an aqueous solution of low-molecular-weight hyaluronic acid 300 kDa or medium-molecular-weight hyaluronic acid 500 kDa or high-molecular-weight hyaluronic acid 800 kDa at a concentration of 1.5%. Mixing is carried out until complete dissolving at room temperature. Bromelain is then immobilized by adding to the resulting mixture of a graft copolymer of carboxymethylcellulose 50-100 kDa (CMC) or chitosan 50-100 kDa (CHTS) with N-vinylimidazole (VI) or N,N-dimethylaminoethylmethacrylate (DMAEMA) in an amount of 100 to 290 mg to obtain a liquid preparation or 300 to 500 mg to obtain a gel. Due to the invention, bromelain solutions can be stored at temperatures from 4 to 25ºC for 21 days, and the enzyme becomes more stable in comparison with the native one and it is able to penetrate into the deep layers of the skin. The introduction of modified polysaccharides into the composition of the preparation allows not only to vary the viscosity and consistency of the samples (from the liquid state to the gel with different fluidity), but also promotes the formation of conjugates with an enzyme with a large number of bonds and interactions between the polysaccharide and the enzyme, compared with unmodified carboxymethylcellulose and chitosan. This further increases the stability of the preparation and the concentration of the active substance in the affected area of the skin, and provides a controlled (portion) release of bromelain, maintaining its necessary concentration for a long time.

EFFECT: increased stability of the preparation.

1 cl, 3 dwg, 1 ex

Description

The invention relates to biotechnology, namely to a method for stabilizing bromelain with solutions of hyaluronic acid and graft copolymers with a polysaccharide backbone of carboxymethylcellulose (CMC) or chitosan (CTS) with side chains from homopolymers of N-vinylimidazole (VI) or Ν,-dimethyllate DMAEMA). The invention can be used in the chemical-pharmaceutical industry, medical practice, cosmetology.

Bromelain (EC 3.4.22.4) is a proteolytic enzyme found in various plants of the bromeliad family (Bromeliaceae). The enzyme isolated from Ananas comosus is the most studied. The bromelain molecule from the stem of this plant contains 285 amino acid residues and is a polypeptide chain linked by five disulfide bonds and bearing one sulfhydryl group. The optimum catalytic activity of the enzyme lies at pH 7-10, depending on the substrate, the isoelectric point is 9.55. The Michaelis constant for bromelain from different sources ranges from 0.007 to 6.6 mM. The optimum temperature is 62 ° C.

Bromelain is widespread in medical practice, cosmetology, in addition, this enzyme is used in the food industry in the tenderization (softening) of meat products, to improve its quality and properties. Bromelain is used in cosmetic treatment to destroy pigmentation and scar tissue [Patent RU 2429028 C2, IPC A61M 35/00, A61K 31/60, A61P 17/02, A61P 17/12, publ. 20.09.2011, Bul. No. 26], is used for the manufacture of drugs for the treatment and / or prevention of eye diseases, which provides a decrease in angiogenesis [Patent RU 2472523 C2, IPC A61K 38/48, A61P 27/02, publ. 01/20/2013, Bul. No. 2], is part of the means for the prevention of oral diseases [Patent RU 2293551 C1, A61K 8/19, A61K 8/34, A61K 8/36, A61K 8/37, A61K 8/40, A61K 8/55, A61K 8/99, A61Q 11/00, publ. 02/20/2007, Bul. No. 5], prevention of the spread of wrinkles, expression lines, hyperpigmentation, lethargy and dryness of the skin [Patent RU 2558848 C1, IPC A61K 8/98, A61Q 19/08, publ. 08/10/2015, Bul. No. 22]. Bromelain is used in a composition comprising hydrolyzed yeast proteins to ensure cosmetic activity and stability over time [Patent RU 2491910 C2, IPC A61K 8/02, A61K 8/99, A61Q 5/12, A61Q 7/00, A61K 36/064, А61Р 17/06, А61Р 17/08, А61Р 17/10, publ. 10.09. 2013, Bul. No. 25].

Hyaluronic acid is a natural linear unsulfated glycosaminoglycan, which is a large polysaccharide molecule consisting of D-glucuronic acid and D-N-acetylglucosamine residues, connected alternately by β-1,4- and β-1,3-glycosidic bonds.

There are many drugs in medical practice, cosmetology, and the pharmaceutical industry that include hyaluronic acid. Described is a composition based on hyaluronic acid, which belongs to soft tissue fillers, for example, dermal and subcutaneous fillers [Patent RU 2496473 C2, A61K 8/73, A61L 27/20, A61K 47/36, publ. 10/27/13, Bul. No. 30]. Hyaluronic acid is used to treat eye diseases, is part of an implant intended for the treatment of glaucoma [Patent RU 2532333 C2, A61K 31/5575, A61P 27/06, publ. 10.11.2014, Bul. No. 31]. In ophthalmology, high molecular weight hyaluronic acid is part of an ointment for treating damage to the anterior segment of the eyeball, which enhances the wound healing effect [Patent RU 2173136 C1, A61K 9/06, 31/728, A61P 27/00, publ. 09/10/2001]. Described is a method for producing a sterile viscoelastic biopolymer based on high molecular weight hyaluronic acid for use in medicine [Patent RU 2501811 C2, C08B 37/00, publ. 12/20/2013, Bul. No. 35]. Hyaluronic acid is used as an anabolic agent and is used in pathological conditions associated with muscular dystrophy [Patent RU 2378007 C1, A61K 38/51, A61P 21/06, publ. 10.01.2010, Bul. No. 1]. In cosmetology, a composition with a rejuvenating effect and a lifting effect based on hyaluronic acid is known, which allows achieving a pronounced aesthetic result due to the physiological mechanism of stimulating the production of its own hyaluronic acid in the skin [Patent RU 2543651 C1, A61K 8/44, A61K 8/64, A61Q 19/00, publ. 03/10/2015, Bul. No. 7].

Graft copolymers of carboxymethylcellulose or chitosan with side chains of N-vinylimidazole or Ν, Ν-dimethylaminoethylmethacrylate are water-soluble in a wide range of compositions, in addition, aqueous solutions of copolymers containing Ν, Ν-dimethylaminoethyl- and pH-sensitive characterized by the presence of a lower critical dissolution temperature (LCST) [VA Kuznetsov, Α.V. Sorokin, Μ.S. Lavlinskaya. Synthesis of graft copolymers of carboxymethyl cellulose and Ν, Ν-dimethylaminoethyl methacrylate and their study as Paclitaxel carriers. 2020. Polymer Bulletin. DOI: 10.1007 / s00289-020-03250-z]. When the content of Ν, Ν-dimethylaminoethyl methacrylate units is from 45% higher and close to neutral pH, the LCST is in the physiological temperature range, which makes it possible to use such graft copolymers as carriers for targeted drug delivery [V.A. Kuznetsov, A.V. Sorokin, Μ.S. Lavlinskaya et al. Graft copolymers of carboxymethyl cellulose with N-vinylimidazole: synthesis and application for drug delivery. 2019. Polymer Bulletin. Vol.76. P. 4929-4949; V.A. Kuznetsov, Α.V. Sorokin, M.S. Lavlinskaya. Synthesis of graft copolymers of carboxymethyl cellulose and N, N-dimethylaminoethyl methacrylate and their study as Paclitaxel carriers. 2020. Polymer Bulletin. DOI: 10.1007 / s00289-020-03250-z].

At the moment, there is no information on complexes of the plant enzyme bromelain with hyaluronic acid and polysaccharides modified with vinyl monomers, such as graft copolymers of carboxymethyl cellulose (CMC) or chitosan (ChTZ) with N-vinyl imidazole (VI) or Ν, Ν-dimethylaminoethylMA) ...

A known method of obtaining a heterogeneous drug based on bromelain, which has wound healing properties [Patent RU 2677343 C2, IPC A61L 15/38, A61K 38/56, C12N 11/08, A61P 17/02, publ. 16.01.2019, Bul. No. 3], which includes the immobilization of bromelain in a buffer solution on a matrix of ion-exchange fibers VION KN-1 or VION AN-1 in a ratio of 20 ml of an enzyme solution at a concentration of 2 mg / ml per 1 g of fiber; 0.05 Μ glycine buffer with pH 9.0-10.5 or 0.05 Μ borate buffer with pH 8.0 without addition of KCl is used as a buffer solution for immobilization on VION KN-1, and 0.2 Μ acetate buffer with pH is used for immobilization on VION AN-1 5.0; incubation is carried out for 24 hours at room temperature, the formed precipitate is washed with the buffer used during immobilization until there is no protein in the washings.

The disadvantage of this method is the production of an enzyme preparation in the solid phase, which does not allow the penetration of bromelain molecules into the deep layers of the skin.

A patented method for stabilizing enzymes - pepsin, chymotrypsin, trypsin, pancreatin, papain and other proteases, by adding polysaccharides to their solutions - guar gum, xanthan gum, locust bean gum, starch, dextran, pullulan, alginic acid, hyaluronan pectin, chitosan and other polysaccharides, as well as cellulose derivatives: carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose [JPH034791A].

A known method of obtaining a preparation of bromelain in a gel based on food-grade chitosan and chitosan succinate, which includes immobilization of bromelain in a buffer solution on a chitosan matrix in a ratio of 20 ml of an enzyme solution at a concentration of 5 mg / ml per 1 g of carrier; incubation at room temperature with occasional stirring; washing the formed precipitate with 50 mM Tris-HCl buffer (pH 7.5) until there is no protein in the washings, characterized in that the immobilization is carried out on a matrix of food-grade chitosan with a molecular weight of less than 100 kDa or chitosan succinate; as a buffer solution for immobilization, either 0.05 Μ glycine buffer with a pH of 8.6 for edible chitosan or with a pH of 9.0-9.5 for chitosan succinate is used; either 0.05 Μ acetate buffer with pH 5.5 for edible chitosan or 5.0 for chitosan succinate; incubation is carried out for 2 hours. The invention makes it possible to increase the speed of the enzymatic reaction and to increase the efficiency of using the obtained preparation, including when carrying out the reaction on hard surfaces [Patent RU 2691611, IPC C12N 11/04, C12N 11/10, publ. 06/14/2019 Bul. No. 17].

In contrast to these two methods, the proposed use of polysaccharides (carboxymethylcellulose and chitosan) modified with vinyl monomers (N-vinylimidazole or Ν, Ν-dimethylaminoethyl methacrylate) provides a greater number of bonds between the polysaccharide and the enzyme during immobilization, which increases the strength of the complex formed and prolongs release of the enzyme to the area of damaged tissues. In addition, the addition of hyaluronic acid to the preparation, which has the property of deep penetration into various layers of the skin, will contribute to better cleansing of the surface layers of the skin and mucous membranes from protein contaminants with bromelain.

It is known that polymers based on poly-β-glycosides (cellulose and its derivatives, chitosan, hyaluronic acid, etc.) tend to form films that can protect the skin from drying out or provide additional protection on the wound surface. However, the ability to form conjugates in such polymers is somewhat limited by a number of factors: surface charge, type of functional group, etc. Therefore, to impart a number of new properties, it is advisable to carry out chemical modification of natural polysaccharides. For example, the introduction of azole substituents increases the complexing ability of polysaccharides and reduces the surface negative charge of carboxymethylcellulose [V.A. Kuznetsov, Α.V. Sorokin, Μ.S. Lavlinskaya et al. Graft copolymers of carboxymethyl cellulose with N-vinylimidazole: synthesis and application for drug delivery. 2019. Polymer Bulletin. Vol. 76. P. 4929-4949], and the introduction of units of Ν, Ν-dimethyaminoethyl methacrylate not only reduces the surface charge, but gives an aqueous solution of the modified polysaccharide stimulus-sensitive properties [V.A. Kuznetsov, Α.V. Sorokin, Μ.S. Lavlinskaya. Synthesis of graft copolymers of carboxymethyl cellulose and N, N-dimethylaminoethyl methacrylate and their study as Paclitaxel carriers. 2020. Polymer Bulletin. DOI: 10.1007 / s00289-020-03250-z].

A topical antimicrobial dermatological composition [Patent RU 2668827 C2, IPC A61K 38/08, A61K 31/728, A61P 31/00, A61P 17/00, A61K 31/722, publ. 02.10.2018, Bul. No. 28], proposed as a drug in medicine and veterinary medicine, comprising a combination of at least one positively charged antimicrobial peptide combined with a lipid and hyaluronic acid with an average molecular weight of 100 kDa to 800 kDa or one of its salts, and the antimicrobial the peptide is a palmitic acid hexapeptide containing disulfide bridges.

Unlike the prototype, we use not an antimicrobial peptide as an active substance, but the enzyme bromelain, which, in addition to antimicrobial properties, has an immunomodulatory effect, accelerates tissue repair processes as a result of depolymerization of intercellular structures and modification of vascular permeability, is used to alleviate inflammatory processes in trauma, relieve edema soft tissues, as well as to accelerate their recovery from damage, has anti-cancer properties and the ability to prevent the formation of blood clots.

In addition, the addition of carboxymethylcellulose (CMC) or chitosan (ChTS) with N-vinylimidazole (VI) or да, Ν-dimethylaminoethyl methacrylate (DMAEMA) to the solution of graft copolymers of carboxymethylcellulose (CMC) or chitosan (ChTS) allows stabilize bromelain and provide it with prolonged action.

The technical result of the claimed invention consists in the development of a method for obtaining an immobilized liquid or gel-like enzyme preparation based on bromelain, polysaccharides modified with vinyl monomers, and hyaluronic acid of low molecular weight (300 kDa), medium molecular weight (500 kDa) or high molecular weight (800 kDa solution), due to which can be stored at temperatures from 4 to 25 ° C for 21 days, while the enzyme becomes more stable in comparison with the native and is able to penetrate into the deep layers of the skin, while the introduction of modified polysaccharides into the preparation allows not only varying the viscosity and consistency of the samples (from a liquid state to a gel with different fluidity), but also promotes the formation of conjugates with an enzyme with a large number of bonds and interactions between the polysaccharide and the enzyme, compared to unmodified carboxymethylcellulose and chitosan, which further increases the stability of the preparation rat and the concentration of the active substance in the affected area of the skin, and also provides a controlled (portioned) release of bromelain, maintaining its required concentration for a long time. In addition, the ability of modified polysaccharides to form stable films protects the enzyme and the treated area of the skin from drying out, makes it easy to remove the drug along with pus and exudate.

The technical result is achieved by the fact that in the method of obtaining an immobilized enzyme preparation based on bromelain, polysaccharides modified with vinyl monomers, and hyaluronic acid, including dissolving bromelain in an aqueous solution of low molecular weight hyaluronic acid 300 kDa or medium molecular weight hyaluronic acid 500 kDa the ratio of 10 mg bromelain per 2 ml of an aqueous solution of low molecular weight hyaluronic acid 300 kDa or medium molecular weight hyaluronic acid 500 kDa or high molecular weight hyaluronic acid 800 kDa at a concentration of 1.5%, stirring until completely dissolved at room temperature; then immobilization of bromelain is carried out by adding to the resulting mixture of graft copolymer carboxymethylcellulose 50-100 kDa (CMC) or chitosan 50-100 kDa (ChTZ) with N-vinylimidazole (VI) or Ν, Ν-dimethylaminoethyl methacrylate (DMAEMA) in an amount from 100 to 290 mg for a liquid preparation or 300 to 500 mg for a gel.

FIG. 1. Diagram of the values of the catalytic activity (A) of bromelain in the presence of hyaluronic acid of various molecular weights after storage at 4 ° C.

FIG. 2. Diagram of the values of the catalytic activity (A) of bromelain in the presence of hyaluronic acid of various molecular weights after storage at 25 ° C.

FIG. 3. Table 1. Viscosity of preparations containing bromelain, hyaluronic acid and polysaccharides modified with vinyl monomers.

An example of the implementation of the method.

Bromelain from Sigma-Aldrich was chosen as the object of the study; azocasein from Sigma-Aldrich was used as a substrate for hydrolysis. As stabilizing agents, we used three types of hyaluronic acid (Laboratory Gialika LLC) with molecular weights of 300 (NMHC), 500 (SMHC), and 800 (HMGC) kDa. Graft copolymers of carboxymethylcellulose (CMC) or chitosan (ChTS) with N-vinylimidazole (VI) or Ν, Ν-dimethylaminoethyl methacrylate (DMAEMA) with a molecular weight of 50-100 kDa were used as a matrix for immobilizing bromelain.

Bromelain was stabilized by dissolving its 10 mg weighed portion in 2 ml of an aqueous solution of hyaluronic acid with a molecular weight of 300 (LMHC), 500 (SMHC), or 800 (HMHC) kDa at a concentration of 1.5%, followed by stirring at room temperature until complete dissolution. Then to the resulting mixture was added to immobilize bromelain graft copolymer of carboxymethyl cellulose (CMC) or chitosan (ChTZ) (with a molecular weight of the polysaccharide of 50-100 kDa) with N-vinylimidazole (VI) or Ν, Ν-dimethylaminoethyl methacrylate in the amount of DMA up to 290 mg for a liquid preparation or from 300 to 500 mg for a gel (Table 1).

F. L. The digestive proteases of langostilla (Pleuroncodes planipes, Decapoda): their partial characterization and the effect of feed on their composition // Comparative Biochemistry and Physiology Part B: Comparative Biochemistry - 1992. - V. 103. - P. 575-578]. К 200 мкл образца добавляли 200 мкл трис-HCl буфера (рН 7.5), 800 мкл азоказеина (0.5% в 50 мМ трис-HCl буфере, рН 7.5) и инкубировали 2 часа при 37°С. Далее добавляли 800 мкл трихлоруксусной кислоты (ТХУ) (5%), инкубировали 10 минут при минус 4°С, затем центрифугировали в течение 3 мин при 13000 об/мин для удаления негидролизованного азоказеина. К 1200 мкл супернатанта добавляли 240 мкл 3% NaOH для нейтрализации кислоты, после чего измеряли оптическую плотность опытной пробы при 410 нм в 1 см кювете. Контрольная проба содержала 800 мкл азоказеина, 800 мкл ТХУ, 200 мкл трис-HCl буфера и 200 мкл образца, который вносили после инкубации смеси в течение 2 часов при 37°С. За единицу каталитической активности бромелайна принимали количество фермента, которое в условиях эксперимента гидролизует 1 мкМ субстрата за 1 мин. Удельную протеолитическую активность бромелайна рассчитывали по формуле:The protein content in immobilized bromelain preparations was determined by the Lowry method [Lowry ON, Rosebrough NJ, Faar AL, Randall RJ Protein measurement with folin-phenol reagent // J. Biol. Chem. - 1951. - V. 193. - P. 265-275]. Measurement of the level of proteolytic activity of the enzyme was carried out on the substrate azocasein [

FL The digestive proteases of langostilla (Pleuroncodes planipes, Decapoda): their partial characterization and the effect of feed on their composition // Comparative Biochemistry and Physiology Part B: Comparative Biochemistry - 1992. - V. 103. - P. 575-578] ... To 200 μL of the sample, 200 μL of Tris-HCl buffer (pH 7.5), 800 μL of azocasein (0.5% in 50 mM Tris-HCl buffer, pH 7.5) were added and incubated for 2 hours at 37 ° C. Then, 800 μl of trichloroacetic acid (TCA) (5%) was added, incubated for 10 minutes at minus 4 ° C, then centrifuged for 3 min at 13000 rpm to remove unhydrolyzed azocasein. 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 TCA, 200 μl of Tris-HCl buffer and 200 μl of the sample, which was added after incubation of the mixture for 2 hours at 37 ° C. The unit of catalytic activity of bromelain was defined as the amount of enzyme that, under the experimental conditions, hydrolyzes 1 μM of the substrate in 1 min. The specific proteolytic activity of bromelain was calculated using the formula:

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

where PA is proteolytic activity, μM / min per 1 mg of protein,

D is the optical density of the sample at 410 nm,

С - protein concentration in the sample, mg / ml, measured by the Lowry method,

120 - incubation time in minutes,

200 - sample volume, in μl,

1000 - recalculation in μM.

The results were statistically processed at a 5% significance level using Student's t-test.

Studies have been conducted on the stability of bromelain in hyaluronic acid solutions (300, 500 and 800 kDa) in comparison with the native enzyme. The preparations were incubated at 4 and 25 ° C for 0, 1, 2, 3, 7, 14, and 21 days with further measurement of proteolytic activity. The results are shown in FIG. 12.

In the course of our experiments, it was revealed that hyaluronic acid does not inhibit bromelain. FIG. 1 shows a diagram of the dependence of the catalytic activity of the enzyme on the time of its storage at 4 ° C. It was found that the proteolytic activity of native bromelain decreases from day 1 to day 7 of incubation from 80% to 35%. After 21 days at 4 ° C, the enzyme retains 32% of its activity. Bromelain in a solution of low molecular weight hyaluronic acid after incubation at 4 ° C on days 1, 2 and 3 shows 95, 87, 65% of the initial proteolytic activity. The enzyme, which is in a solution of low molecular weight hyaluronic acid at 4 ° C, has an activity of 37% on day 21. It was found that bromelain in a solution of medium molecular weight hyaluronic acid after 21 days retains 59% of its activity at 4 ° C. The decrease in catalytic capacity in this case occurs gradually without sharp jumps. The activity of bromelain in a solution of high molecular weight hyaluronic acid after incubation in a refrigerator for 21 days is 76%.

FIG. 2 shows a diagram of the change in the catalytic activity of bromelain after storage at 25 ° C. At 25 ° C, the native enzyme reduces the activity from 77% to 37% from 1 to 7 days of incubation; after 21 days, the activity of bromelain is 34%. The enzyme in a solution of low molecular weight hyaluronic acid after incubation at 25 ° C for 1, 2 and 3 days retains its catalytic ability at 88, 77, 57% of the initial proteolytic activity, on day 21 the bromelain preparation has 36% of the enzymatic ability. Bromelain in a solution of medium molecular weight hyaluronic acid after 21 days retains 57% of its activity at 25 ° C. It was shown that the activity of bromelain in a solution of high molecular weight hyaluronic acid after storage at 25 ° C on the 21st day of incubation decreases to 44%.

Thus, a method was developed for preparing an immobilized enzyme preparation based on bromelain, low molecular weight (300 kDa), medium molecular weight (500 kDa), and high molecular weight (800 kDa) hyaluronic acid, followed by the addition of carboxymethyl cellulose graft copolymer (CMC) or chitosan -vinylimidazole (VI) or Ν, Ν-dimethylaminoethyl methacrylate (DMAEMA) for the immobilization of bromelain.

In this case, the drug obtained by the proposed method can be stored at temperatures from 4 to 25 ° C for 21 days, while bromelain becomes more stable in comparison with native and is able to penetrate into the deep layers of the skin. The introduction of modified polysaccharides into the preparation allows not only to vary the viscosity and consistency of samples (from a liquid state to a gel with different fluidity), but also promotes the formation of conjugates with an enzyme with a large number of bonds and interactions between the polysaccharide and the enzyme, in comparison with unmodified carboxymethylcellulose and chitosan , which further increases the stability of the drug and the concentration of the active substance in the affected area of the skin, and also provides a controlled (portioned) release of bromelain, maintaining its required concentration for a long time. In addition, the ability of modified polysaccharides to form stable films protects the enzyme and the treated area of the skin from drying out, makes it easy to remove the drug along with pus and exudate.

Claims (1)

A method of obtaining an immobilized enzyme preparation based on bromelain, polysaccharides modified with vinyl monomers, and hyaluronic acid, including dissolving bromelain in an aqueous solution of low molecular weight hyaluronic acid 300 kDa, or a medium molecular weight ratio of hyaluronic acid 500 kDa, or high molecular weight hyaluronic acid in 10 mg kDa hyaluronic acid 2 ml of an aqueous solution of low molecular weight hyaluronic acid 300 kDa, or medium molecular weight hyaluronic acid 500 kDa, or high molecular weight hyaluronic acid 800 kDa at a concentration of 1.5%, while stirring until completely dissolved at room temperature; then immobilization of bromelain is carried out by adding to the resulting mixture of graft copolymer carboxymethylcellulose 50-100 kDa (CMC) or chitosan 50-100 kDa (ChTZ) with N-vinylimidazole (VI) or Ν, Ν-dimethylaminoethyl methacrylate (DMAEMA) in an amount from 100 to 290 mg for a liquid preparation or 300 to 500 mg for a gel.

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