RU2255758C2 - Biologically active carnosine-anserine-containing complex and method for its preparing - Google Patents

Abstract

FIELD: biotechnology, biochemistry.

SUBSTANCE: invention relates to producing the biologically active complex eliciting antioxidant and immunomodulating activity and used in medicine, cosmetics, veterinary science and food industry. The biologically active complex preparing by enzymatic hydrolysis of muscle tissue represents complex of biologically active compounds involving carnosine and anserine in the amount 85-97 wt.-% of the native content of these components in poultry muscle tissue, 1-7 weight parts of amino acids, 0.5-12 weight parts of oligopeptides of molecular mass 10 kDa, not above, and 0.1-15 weight parts of cyclic and polycyclic phenolic compounds as measured for 1 weight part of carnosine and anserine in the complex. This complex is prepared by enzymatic hydrolysis of milled and homogenized water muscle tissue in preferable dilution homogenate with water in the range 0.2-0.6 and with using proteolytic enzymes in the amount 2-5 wt.-% of the protein content and working at pH 4.5-8.5 and at enhanced temperature being preferably at 45-65°C. Product is isolated as extract or powder prepared in drying the extract. Invention provides enhancing effectiveness of the claimed complex.

EFFECT: improved method for preparing, valuable properties of complex.

7 cl, 6 tbl, 6 ex

Description

The invention relates to biotechnology and for the production of biologically active agents, including histidine-containing dipeptides with antioxidant, immunomodulating activity, obtained on the basis of natural raw materials - chicken muscle tissue, which can be used in the medical, food industries, cosmetics, veterinary medicine, animal husbandry.

It is known that both natural and synthetic products of various structures and compositions have antioxidant and immunomodulating activity. Such products include, for example, histidine-containing dipeptides, amino acids. In recent years, much attention has been paid to the study of histidine-containing dipeptides with antioxidant activity, namely carnosine having an Ala-His structure and its derivatives, such as anserine of the Ala-N-MeHis structure. Carnosine was first isolated and investigated already in 1900 (A.A. Boldyrev. Karnozin. Biological significance and possibilities of use in medicine. M., 1998). Currently, the natural dipeptide-carnosine contained in the skeletal muscles of humans and vertebrates has been characterized as an effective water-soluble antioxidant, immunomodulator and pH buffer, used as an antitumor, antihistamine, anti-stress, anti-ulcer, anti-cataract and anti-cancerous (RF) Patent No. 2084457, C 07 K 5/062, 1997). Natural carnosine was first isolated from an aqueous suspension of minced meat by planting with ethanol (Gulewitsh WS Amiradzibi S Uber das Carnosin eine neue organische Base des Fleischxtraktes Ber Deutsch Chem Ges 1900, 33, 1902-1903). In clinical practice, both natural and synthetic carnosine, as well as its homologues, in particular anserine, are currently used. Synthetic carnosine is currently produced by foreign companies, for example, such as SERUA, SIGMA (Italy). However, as is known from additional experimental studies, as well as from available information sources, synthetic histidine-containing dipeptides and their mixtures, due to the presence of products by-products from the synthesis, have sufficiently high toxicity, which limits the possibility of their use (RF Patent No. 2084457, С 07 K 5/062, 1997). In 1996, the industrial production of natural carnosine by extraction from beef meat was mastered in Russia. The product obtained by this method was entered into the Register of Medicinal Products of Russia No. 96/50/02. Natural carnosine is recognized as low toxic in comparison with its synthetic analogues. Its LD 50 was 21 g / kg body weight. However, carnosine itself in the absence of activating additives has a relatively low antioxidant activity, which limits its use as a therapeutic drug, and which led to the search for more active forms of this drug, for example, to the selection of mixtures containing carnosine. Thus, there is known a composition used as a pharmaceutical or dietary preparation containing carnosine and / or its derivatives, such as homocarnosine, anserine, opidine and / or their pharmacologically acceptable salts and / or their acyl derivatives in a mixture with branched chain amino acids selected from groups: leucine, isoleucine, valine, and also additionally containing salts of organic and inorganic acids, for example, carbonates, lactates, sulfates, gluconates of magnesium, zinc, iron, and other additives, such as vitamins, flavonoids (EP, Patent No. 0825871, A 61 K 38/05, 2001). In the known composition, carnosine and its homologs are 1-0.5 wt.%, Preferably 15-25 wt.% To the total weight of the whole mixture, and the mixture of three amino acids is 50-70 wt.%, And leucine is contained in the mixture at the level of 20 wt. % This composition is used as an oral preparation in the form of tablets, capsules, liquid solutions. The composition is obtained by mixing its constituent components, their heat treatment and subsequent isolation. As the starting dipeptides in this composition, synthetic products are used. The antioxidant effectiveness of this drug was tested in experimental animals on changes in lipoperoxides in blood plasma and liver, and it was found that carnosine in combination with branched amino acids reduces the content of peroxides by 35.5%. Dosages of this known drug depend on the nature and extent of the disease and are 0.1-500 mg / kg body weight. This composition is similar in composition and scope to the new tool and therefore is selected as its prototype. A disadvantage of the known composition of the prototype is its lack of effectiveness, which limits its scope.

In known publications there is no information regarding the preparation from natural raw materials of compositions containing both an amino acid complex and histidine-containing dipeptides. The use of natural protein raw materials is widely described for the preparation of biologically active amino acid complexes by enzymatic hydrolysis. As a source of raw materials for the production of known enzymatic protein hydrolysates, for example, mammalian animal skin splitters (RF Patent No. 2068879, С 12 N 1/00, 1992), internal organs of a bird subjected to hydrolysis under the action of endogenous enzymes of the intestine of a bird in an alkaline medium ( USSR, AS No. 878780, C 12 N 1/20, 1979), meat or meat and bone raw materials of slaughtered animals, crushed and subjected to enzymatic hydrolysis at pH 6.5-7.8 using protosubtilin G20X and proteolytic enzymes obtained from pancreas slaughter animals x (RF patent No. 2112397, A 23 J 1/10, 1998), muscle tissue of cattle or pigs (USSR, AS No. 1025722, C 12 N 1/00, 1983). In the last cited source, carefully chopped muscle tissue of cattle or pigs is subjected to enzymatic hydrolysis in the presence of proteolytic enzymes, such as the pancreas of cattle or pigs or pancreatin with an activity of 45 PIECES. Upon completion of the hydrolysis at a depth of 0.9-1.0, precipitation of high molecular weight compounds is carried out at the isoelectric point and subsequent filtration and drying of the preparation. The resulting known dry enzymatic hydrolyzate contains at least 18 amino acids, the total amount of which is 63-69%. However, this complex does not contain carnosine with anserine, which is the main goal of the new invention. An enzymatic dry hydrolyzate of muscle proteins in cattle or pigs has been used to create a culture medium for growing cell cultures and biological products. However, due to the fact that cattle is susceptible to a dangerous disease, the so-called mad cow disease, which can be transmitted to humans under certain conditions, the use of cattle and pig meat for the pharmaceutical and food industries has recently been limited.

As mentioned above, carnosine and its homologues are used as part of antioxidant compositions, in particular in cosmetic creams with a therapeutic effect. For example, it is known: a composition of carnosine with arnitine and other effective additives used in the treatment and prevention of photodermatosis (PCT патент 1175898, A 61 K 7/48, 2002), a cosmetic composition in the form of a cream that has anti-inflammatory effect and is used for the skin, prone to acne. The amount of carnosine in such a cream is 0.0009-0.0011 wt.% (RF patent No. 2045949, A 61 K 7/48, 1995).

To expand the range of biologically active preparations with increased efficiency and low toxicity, a new composition of a biologically active complex containing carnosine and anserine mixed with other biologically active substances in the following weight parts with respect to the content of carnosine with anserine was obtained from chicken muscle tissue by the enzymatic method: amino acids - 1-7 weight. parts, oligopeptides in an amount of 0.5-12 weight. parts, and cyclic and polycyclic phenolic compounds in an amount of 0.1-15 weight. parts. The biologically active complex is in the form of an extract or a solid powder.

This biologically active carnosine-anserine-containing complex is obtained by enzymatic hydrolysis of chicken muscle tissue, which, after grinding and homogenization in water, is treated with proteolytic enzymes at pH 4.5-5.5 or 8.0-9.0 at a temperature of 45-65 ° C using 2-5 wt.% the enzyme in relation to the protein content in the homogenate and subsequent cooling and filtration purification. In this case, the homogenized feedstock is diluted with water, preferably in a ratio range of 0.2-0.6. Before the stage of enzymatic hydrolysis, the homogenate diluted with water is preheated at 45-65 ° C. Upon receipt of the product in powder form, the isolated purified extract is further subjected to vacuum evaporation and / or spray or freeze drying.

The therapeutic and cosmetic composition containing the indicated biologically active complex and a cosmetically acceptable base contains the Bio Curator complex in an effective amount depending on the selected cosmetic form (cream, emulsion, lotion, etc. (and other factors), purpose, age customer, etc.)

The resulting biologically active complex contains a mixture of amino acids such as asparagine, tryptophan, serine, glutamine, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, hydroxylisine, lysine, histidine, arginine. The new tool, in contrast to the known prototype composition, always contains a mixture of two histidine-containing dipeptides (carnosine and anserine), as well as a wider spectrum of amino acids, which is most important, additionally contains oligopeptides, as well as cyclic and polycyclic phenolic compounds of indefinite structure, which provide significant synergistic effect, manifested in the increased effectiveness of the new complex, significantly exceeding the known drugs of a similar effect and composition. These properties of a new biologically active complex are created with a certain sequence of operations and observing certain modes in the process of its preparation. To study the antioxidant effect of the obtained complex, the method of chemiluminescence analysis of peroxidation of donor blood lipoproteins induced by iron was applied. The essence of the method is that when ferrous ions are added to the lipoprotein samples, the induction of reactive oxygen species occurs, causing a chain reaction of the formation of free radicals, accompanied by the ignition of chemiluminescence. The results of testing the antioxidant activity of a new biologically active complex depending on the concentration of biologically active substances in the sample are shown in Table 3. The final sample volume in the studies was 1.0 ml. Table 3 also shows the test results in which synthetic carnosine was used as a reference for comparison. The results indicate a high efficiency of the new complex. Already when a sample of the biologically active complex was added in an amount equal to 10 μl, a significant increase in the duration of the latent period and a decrease in the intensity of the main outbreak of chemiluminescence / N / were noted. For comparison, the effective concentrations for carnosine and anserine when used individually in this model are 500 mM and higher (A. Boldyrev - Carnosine and tissue protection against oxidative stress. Moscow, 1999). Thus, a preparation of the indicated composition obtained by a new method due to its effectiveness can be used in the pharmaceutical and cosmetic industries as a separate preparation or as an ingredient in compositions, for example, medical and cosmetic compositions with antioxidant, UV-reflecting activity.

The following are examples illustrating the new drug, and the method for its preparation and medical-cosmetic compositions based on it.

Example 1

The muscle tissue of chickens in the amount of 1 kg is ground in a meat grinder and homogenized. The resulting homogenate mixture was diluted with 3 liters of purified water with dilution in the range of 0.3 and aqueous extraction was carried out at 45 ° C. After that, the aqueous extract is separated from the precipitate by filtration, and the protein compounds in the resulting extract are subjected to enzymatic hydrolysis at pH 5.5 using the Flavourzime proteolytic enzyme, used in an amount of 4 wt.% And at a temperature of 50 ° C until the termination of the increase in amine nitrogen . After that, the extract is heated to inactivate the enzyme at 75 ° C for 30 minutes, then cooling and removal of the formed precipitate is carried out, and the packed liquid is filtered using depth filters. Get the product with the indicators shown in tables 1-3.

Example 2

Example 2 is carried out analogously to example 1 when using chicken breast fillet, while the water extraction process is carried out using 4 liters of water, and the enzymatic treatment is carried out at a temperature of 55 ° C and pH 8.5 using the same proteolytic enzyme in an amount of 5 wt. .% of the protein content in the extract. The resulting product is described in tables 1-3.

Example 3

The extraction and hydrolysis process is carried out analogously to example 1. At the end of the extraction, the final product is concentrated to a final concentration of at least 10 wt.% And dried by spray drying at 100 ° C.

Example 4

Muscle tissue in an amount of 1 kg is ground and homogenized, after which the resulting homogenate is mixed with purified water in an amount of 4 liters and placed in a reactor. The complex amyloproteolytic enzyme Protease “C” is used at pH 8.0 at a temperature of 50-55 ° C in an amount of 4 wt.% Of the protein content in the homogenate. Hydrolysis is carried out until the termination of the increase in the amount of amine nitrogen. The resulting product is characterized in tables 1-3.

Examples 5, 6. Therapeutic and cosmetic products based on the drug “Bio Curator” (above the biologically active complex considered).

An effective amount of an aqueous extract of the “Bio Curator” preparation is introduced into a cosmetically acceptable base containing the components listed below, the components are mixed until a homogeneous mass is obtained and packaged. The following are examples of formulations based on the drug “Bio Curator”. However, the above examples do not limit the possibility of creating other therapeutic and cosmetic products based on this drug.

Example 5. Sunscreen composition (wt.%):

Cetaret 25-2.0, Cetaret 6 - 2.0, cetearyl alcohol - 3.0, ethylhexylmethoxycinomat - 5.0, benzophenone 3-1.0, butylmethoxybenzoylmethane - 1.0, cetearylisonanoate - 6.0, cocoglycerides - 2.0, glycerol - 5.0, 0.3, triethanol tocopherol acetate - 0.5, glucan - 0.5, cyclomethicone - 1.5, dimethicone - 0.5, methyl paraben - 0.3, propyl paraben - 0.2, “BioCurator - 0.5 (in the form of a 7% aqueous solution), water - the rest is up to 100%.

Example 6. Anti-inflammatory balm composition, (wt.%):

Phytostearin - 1.0, lipid complex - 2.0, propyl paraben - 0.2. methyl paraben - 0.3, glycerol - 5.0, allantoin - 0.2, carbomer - 0.4, triethanolamine - 0.5, Bio Curator - 1.0 / in the form of a 5% aqueous solution, D-panthenol - 0.3, bisabolol - 0.3.

The tables below reflect the effectiveness of the biologically active complex.

Table 1.
The content of carnosine and anserine in the biologically active complex, depending on the method of its preparation. Name of indicator Examples 1-2 Example 4 Carnosine, mg / ml 1.5-4.5 1.6-6.5 Anserine, mg / ml 2.5-10 2.5-13.5

Table 2.
Physico-chemical characteristics of a biologically active complex, depending on the method of its preparation. Name of indicator Examples 1-2 Example 4 The solids content in the unpacked extract,% wt., Not less 4 7 Mass fraction of total nitrogen,% wt., Not less 2.6 4.7 Amine nitrogen, mg / ml, not less 1.6 3.5

Table 3.
A study of the antioxidant activity of a biologically active complex for various examples. Option Volume, μl / sample Anzerin, mcg / sample Carnosine, mcg / sample Hydroperoxides (h),% of control Latent period (τ),% of control Oxidation (H),% of control Example 1-2 10 3.2 2.5 60 125 96 Sample Composition: Carnosine: 2.45 mg / ml 25 8 6.25 54 175 85 Anserine: 3.19 mg / ml fifty 16 12.5 fifty 300 87 Solids: 45.8 mg / ml For analysis, the sample was diluted 10 times 100 32 25.0 33 325 65 Example 4 10 eleven 6.2 70 123 98 Sample Composition: Carnosine: 6.2 mg / ml 25 thirty 15.5 63.5 143 95 Anserine: 11 mg / ml fifty 55 33.75 fifty 154 91 Solids: 65.8 mg / ml For analysis, the sample was diluted 10 times 100 110 62.00 49 183 55 No extract The control 105 ± 12, mV 90 ± 8 sec 1524 ± 85, mV Carnosine 1 mM 226 40 160 90 0.5 mM 113 52 144 94 0.25 mM 56.5 57 125 95 0.1 mM 11.3 36 96 100

Table 4
Composition of preparations “BIOCURATOR” industrial designs Sample Dry in (%)
70 mg / ml Dipeptides (mg / ml) (according to HPLC) Amino acid balance (mg / ml) (according to data obtained on an amino acid analyzer) The effectiveness of hardware hydrolysis of the broth
(%) Anzerin Carnosine Amount Free I After complete hydrolysis II No. 1 7.2 1,452 0.807 2,259 10.29 36.80 28 Number 2 7.4 1,780 0.820 2.6 15.07 46.90 31 No. 3 7.2 1,845 0.735 2,58 28.57 47.04 44

According to the amino acid analysis, we can conclude that the process of hardware hydrolysis does not go through to the end: in the 1st and 2nd samples, the hydrolysis depth was 28 and 31%, respectively, in the 3rd - 44%. The yield of dipeptides in the 2nd and 3rd samples is almost the same.

Table 5
Antioxidant activity of the preparations “BIOCURATOR” Sample Dipeptides, concentration in samples (mM) Antioxidant activity The end of the test sample (mm) DPPH recovery (nmol / min) The end of the test sample (mm) Inhibition of MDA accumulation (%) Anzerin Carnosine Amount No. 1 6.02 3.57 9.59 0.19 1.76 0.96 16.4 ± 0.48 Number 2 7.38 3.63 11.01 0.22 1.71 1,1 26.8 ± 0.88 No. 3 7.65 3.25, 10.9 0.22 3.19 1.09 33.6 ± 1.16 Carnosine 20 mm 0.40 0.26 1,0 0% 0.8 0.39 2.0 16.5 ± 0.55

The antioxidant (AO) activity of the BIOCURATOR preparations was determined in two tests:

- By the rate of recovery of the stable radical - diphenylpicrylhydrazyl [Schlesier K., Harwat M., Bohm V, Bitsch R. Assessment of antioxidant activity by using different in vitro methods // Free Radic. Res., 36, pp. 177-187, (2002)]

- To suppress the accumulation of MDA in the reaction of iron-induced lipid protein peroxidation in the blood serum of donors [Ohkawa H., Ohishi N., Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.//Anal Biochem, 95 (10), 351-358 (1979)]

The obtained results indicate that, compared with carnosine, BIOCURATOR preparations provide greater AO activity than would be expected based on the total content of histidine-containing dipeptides (HSD) in them. When an aliquot of the BIOCURATOR preparations is introduced into the reaction system in the DPPH recovery test, they provide an effective concentration of GDS of 0.19-0.22 mM. AO activity is 20-40 times higher than that observed for carnosine. Considering the fact that the test used reveals the antioxidant activity of compounds with certain hydrophobic properties, it can be assumed that the AO activity of the BIOCURATOR preparation is largely dependent on the presence of other (not HSD) antioxidants in their composition. This is also indicated by the data obtained in the test to suppress the accumulation of MDA: in this test, the preparations of BIOCURATOR are 2-4 times superior in their AO activity to carnosine.

Claims (7)

1. Biologically active carnosine-anserine-containing complex, including amino acids, characterized in that it further contains oligopeptides and cyclic and polycyclic phenolic compounds and is a complex of biologically active substances of natural origin, obtained by enzymatic hydrolysis of chicken muscle tissue, characterized by the following weight ratio of mixture components, weight . hours: Carnosine and Anserine 1 Amino acids 1-7 Oligopeptides 0.5-12 Cyclic and polycyclic phenolic compounds 0.1-1 2. The biologically active complex according to claim 1, characterized in that it is made in the form of an aqueous extract or a solid powder product. 3. Biologically active complex according to claims 1 and 2, characterized in that it is an integral part of external cosmetic therapeutic agents. 4. A method of obtaining a biologically active carnosine-anserine-containing complex, characterized in that the raw material used is chopped homogenized muscle tissue of chicken, which is subjected to enzymatic hydrolysis, followed by cooling and filtration purification of the resulting final product, and after grinding and homogenization, the homogenate is diluted with water and subjected treatment with proteolytic enzymes working at a pH of 4.5-5.5 or 8.0-9.0 and a temperature of 45-65 ° C and used in an amount of -governing 2-5 wt.% of the protein content of the homogenate. 5. The method according to claim 4, characterized in that the crushed homogenized feedstock is diluted with water, preferably in a ratio range of 0.2-0.6. 6. The method according to claim 4, characterized in that before the stage of enzymatic hydrolysis, the homogenate diluted with water is heated at 45-65 ° C and subjected to filtration purification. 7. The method according to any one of claims 4 to 6, characterized in that upon receipt of the product in powder form, the isolated purified extract is further subjected to vacuum evaporation and / or spray or freeze drying.