Exogenous biologically active substances contribuye

 

(57) Abstract:

The invention relates to medicine and applies polyuronic acids or their salts as a means of therapy for treatment of diseases associated with impaired metabolic processes, in particular conjugation reactions with glucuronic acid (coma renal, hepatic cell failure, hepatitis, jaundice, and others). Substitution treatment containing uronic acid, characterized in that as uronic acids contains polygalacturonase acid with a degree of esterification of 40.4% (sugar beet pectin) or a derivative of sodium salts polymannuronate and prigorodovoy acid (sodium alginate). The tool facilitates the efficient protection of patients with impaired metabolic processes. 5 table.

The invention relates to medicine and applies polyuronic acids or their salts as a means of therapy for treatment of diseases associated with impaired metabolic processes, in particular conjugation reactions with glucuronic acid. To such diseases include hemolytic disease, hepatic coma, hepatic cell failure, hepatitis, jaundice and other

Uridinediphosphate-a-D-glucose (UDPG)

Uridinediphosphate--D-glucoronosyl acid

Bilirubin glucuronide (R-configuration) (Vasilenko Y. K. Biological chemistry. - M.: high school, 1978. - S. 370-371; Balakhovskiy S. D., Balakhovskiy I. C. Methods of chemical analysis of blood. - M.: Medgiz, 1953. - S. 588).

Important physiological role of education glucuronidase (glycosides-D-glucuronic acid) is related to the fact that this reaction is one of the major conjugation reactions, leading to neutralization and excretion of drugs and toxic substances, and some metabolites. These include as many normal metabolites (bilirubin, steroids, thyroxine, and others) and foreign substances (products of metabolism of the intestinal microflora, medicines, chemical pollution). In the pathogenesis of geolit is koronida bilirubin, i.e. the form of bilirubin, which is excreted from the body; as a result of this enzymopathies bilirubin accumulates in the blood and tissues. The pathogenesis of the symptoms of liver cell failure and hepatic coma associated with the poisoning of the body with toxic substances as a result of violations of the mechanism of neutralization reactions in the liver, in particular the reactions of education glucuronidase (Great medical encyclopedia. - M.: Owls. Encyclopedia, 1976. - So 4. - Ed.3. - S. 325).

-D-Glucuronic acid is an endogenous substance, i.e. a substance produced in the human body, as a result of enzymatic oxidation of glucose.

Of pharmaceutical preparations containing glucuronic acid, known only one drug - honored (Chonsuridum) is a drug obtained from the box of cattle (Mashkovsky M. D. Drugs: In 2 so - So 2. - 10th ed., wiped. - M.: Medicine, 1986. - S. 156). Active substances honsurida is hondroitinsernaya acid, is built of molecules N-atsetilgalaktozamin-6-sulfate and glucuronic acid. This tool close to the claimed and selected for the prototype. The disadvantages of this drug are: 1) the impossibility of its application for conjugation reactions, sposoby acid linked to N-atsetilgalaktozamin-6-sulfate is very durable (14)-glycosidic bond, and to participate in the reaction conjugation of glucuronic acid must be connected in UFGC (complex with a high energy bond); 2) impossibility of the use of concorida inside, because he, along with hyaluronic acid is involved in the construction of the ground substance of connective tissue.

According to the chemical structure related glucuronic acid compounds are polyuronide: pectins, alginates. Pectins are biopolymers composed of residues of D-galacturonic acid, interconnected (14)-glycosidic bonds. Polygalacturonase acid partially etherification methanol, which helps dissolve the pectin in water (Komissarenko S. N., Spiridonov Century. N. Pectin - their properties and applications. The overview. / Raises. resources, 1998. - So 34. - Vol.1. - S. 111-119). Alginic acid are heteropolyacid, molecules are built from residues-D-mannurone and-L-guluronic acids linked in a linear chain 1-4-glycosidic bonds. Because of alginic acid is sparingly soluble in water, and their sodium salts are easily soluble in water, it is often used last (Chem. the encyclopedia. - M.: Science. publishing house of the “Great Russian encyclopedia”, 1998. - T. 1. - S. 192-193). Chemical formulas of monomers CLASS="ptx2">

As can be seen from the chemical formula hexuronate acid, glucuronic and galacturonase acids are epimerase on the hydroxyl group at C4; glucuronic and mannurone acid - epimer on the hydroxyl group at C2and guluronate and mannurone acid - epimere the hydroxyl at C5.

Similar structure mentioned above hexuronate acids suggests the possibility of replacing glucuronic acid conjugation reactions to other uronic acids or their salts of exogenous origin. As conjugating substances we have selected sugar beet pectin (JV) with a degree of esterification polygalacturonic acid 40,4% and sodium alginate (NA), isolated from kelp sugary.

The purpose of the invention is studying the possibility of using vegetable polyuronides as conjugating substances instead of glucuronic acid in violation of biosynthesis or metabolism in the body of the person.

There are various factors that hinder the progress of the conjugation reactions with glucuronic acid: a) blocking ions of heavy metals glucuronic acid, glucose-1-phosphate (educt for the synthesis of UDPG). With the nye and others) of biomolecules, forming a connection, deprived of many physico-chemical and biological properties, thereby disrupting the metabolism in General and glucuronid conjugation, in particular (Emergency treatment of acute poisoning. Handbook of toxicology. / Ed. by S. N. Golikova. - M.: Medicine, 1978. - S. 214-215); b) enzyme inhibition UDF-glucuronyltransferase and other enzymes and heavy metal ions, parakhlornitrobenzolom (Great medical encyclopedia), which inhibits the transfer of glucuronid part with UFGC on inactivating substance and causes poisoning of the body of toxic substances due to the accumulation in blood and tissues; C) genetic defects of enzymes (Biochemistry man. / Murray R., Granner D., Maes P., Rodwell C. - M.: Mir, 1993. - 2 so - So 1. - 382 S.).

This objective is achieved in that first creates a model of the disturbance of metabolic processes by daily oral administration white rats-males solution of acetate of lead (II) at a dose of 45 mg/kg in a week, and then daily orally for three months the animals injected solution SP or EN at a dose of 500 mg/kg In animals throughout the experiment examined the contents of the hexuronate acids and bilirubin (normal metal is bolism xenobiotics in particular aspirin, animals week after injection of lead acetate (II) and a monthly injection of polysaccharides daily for weeks after eating orally administered aspirin at a dose of 30 mg/kg as a suspension with water to a volume of 5 ml In comparison served as the group of intact animals.

The content of hexuronate acids in biological material determined after acid hydrolysis of sediment by reaction with carbazole in sulfuric acid medium by measurement of light absorption painted in pink-crimson color of the reaction product at a wavelength of 530 nm (research Methods in occupational disease (biochemical). A guide for physicians. / O. Arkhipova,, Sacca M. M., Semenova HP and others / edited by O. G. Arkhipova. - M.: Medicine, 1988. - 203 C.).

The detection of bilirubin in the urine held in the sample, rosin, based on the formation of bilirubin under the action of iodine biliverdin, colored green (Stroev, E. A., Makarova Century, Workshop on biological chemistry. - M.: high school, 1986. - S. 188).

The quantitative content of direct bilirubin in serum were determined by the method of cultivation, based on the interaction of vasoreactive with bilirubin in the diluted serum (Balakhovskiy S. D., Balahovskaia) in biological fluids has been determined using acid-base titration (the State Pharmacopoeia of the USSR. - M.: Medicine, 1968. - X ed. - Article.3).

Previously (before the creation of the model disturbances of metabolic processes) investigated the effects of SP and an on level hexuronate acids in various biological systems and organs of animals. The results are shown in table 1 (see end of description).

Table 1 data indicate that the oral administration to animals JV level and an hexuronate acids in all biosubstrate increases most quickly he rises in serum and soft tissue slowly in bone tissue. The increase in the content of hexuronate acids, especially in serum, liver suggests the possibility of participation of the input polyuronides in metabolic processes.

When determining the presence of bilirubin in the urine in the sample Rosina in each group of animals throughout the study period, the reaction for bilirubin was negative, indicating that the normal metabolic processes in the liver.

To create the model of the disturbance of metabolic processes in white rats male Wistar rats weighing 170-220 g were injected daily for 7 days oral solution of lead acetate (II) to within 12 weeks. Periodically in the blood and urine was determined by the content hexuronate acids (in terms of glucuronic acid and bilirubin. The results are shown in table 2.

The results in table 2 show that after the introduction of animals of a solution of acetate of lead (II) in blood and urine increases the content of bilirubin, the content of hexuronate acids in the blood drops, and in the urine - is growing. Apparently, this can be explained by the fact that under the influence of lead blocking transferring enzyme that catalyzes the transfer of glucuronid part with UFGC on bilirubin, i.e., the introduction of lead impairs the synthesis of glucuronide-conjugated, and therefore in the blood and in the urine content of unbound bilirubin increases. For the same reason, increases the content of glucuronic acid in the form of originaly coenzymes in the urine. The reduction in the content of glucuronic acid in the blood may occur due to the binding of ions of lead carboxyl groups of uronic acid or phosphate groups of the glucose-1-phosphate synthesis step UDFG.

On the background of the lead pathology subsequent long reception polyuronide drugs leads to the disappearance of urine bilirubin (after 8 weeks of supplementation polysaccharides) and reduced the slot in the serum and urine normalized after 4 weeks of taking polyuronides. Favorable action polyuronide drugs on the metabolism, particularly bilirubin, apparently, can be explained by the involvement of Monomeric fragments entered polyuronide drugs in the process of conjugation.

To establish a link between the content of hexuronate acids and direct bilirubin in serum, as well as to establish a relationship and quantify the reliability of the connection calculated the correlation coefficient (r) between the two above-mentioned features by well-known methods (Asatiani B. C. New methods of biochemical photometry. - M.: Nauka, 1965. - S. 507-510). Calculation of coefficient of correlation between the contents of hexuronate acids and direct bilirubin in serum after injection polyuronide drugs are shown in table 3.

The data of table 3 show that between content hexuronate acids after the introduction of polyuronides and direct bilirubin in serum there is a connection. The negative value of the correlation coefficient indicates a negative correlation between the considered traits. Values of coefficient very close to unity indicate a high probability of linkage between indicators in both cases, the applications of acids after giving polyuronide drugs and the content of direct bilirubin in the considered cases, there is a statistically significant inverse relationship. This confirms the assumption that the reduction of bilirubin concentration in the serum was due to the increase in the content of uronic acids by introducing polyuronide drugs.

Along with normal metabolites, which include bilirubin, attempt studying the relationship between the content of hexuronate acids after ingestion of polyuronides and content of any of xenobiotics that are metabolized in the body by glucuronid conjugation. As such xenobiotic was chosen aspirin. In the liver it forms a conjugate with glucuronic acid (Tyukavkina N. A., Baukov Y. I. Bioorganic chemistry. - M.: Medicine, 1985. - S. 376).

First, a model was created disturbances of metabolic processes by oral administration in one week to white rats males solution of lead acetate (II) (as the case above). Then on the background of the lead pathology one group of animals was administered orally in the course of a week day (after eating) was injected using a probe powder aspirin at the rate of 30 mg/kg of body weight as a suspension in 5 ml of water. Two other groups of animals after a week of the introduction of lead orally for 4 weeks daily is the group of animals). In the urine of animals was determined by the content oksibenzoynoy acid by the method of acid-base titration (the State Pharmacopoeia of the USSR. - M.: Medicine, 1968. - X ed. - Article.3). The results of quantitative determination of the content oksibenzoynoy acid in the urine of animals is presented in table 4.

Table 4 shows that as a result of the introduction of aspirin in the background lead pathology, when disturbed metabolic processes in the urine increases the content of free, unconjugated, oksibenzoynoy acid, because, apparently due to inhibition of lead ions enzyme transferase eronina component is not transferred with UFGC on inactivating substance (salicylates), which leads to increased content hexuronate acids in the urine. Presumably, glucuronic acid blocked ions of lead and may not participate in the conjugation of metabolites. The introduction of aspirin in the background lead pathology and a long reception of polyuronides indicates the normalization of metabolic processes: with reduced levels of salicylates is reduced and the content of hexuronate acids in the urine. To determine whether there is a relationship between two signs (the content of hexuronate acid and salicylates in the urine of the village is presented in table 5.

The data of table 5 show that between content hexuronate acids (after the introduction of polyuronides) and the content of salicylates in the urine there is a connection. A positive value of correlation coefficient indicates a direct correlation between indicators. In the case of the introduction of the Academy of Sciences of the correlation coefficient is almost 1 (0,992), indicating a high degree of probability of presence of dependencies between parameters. This confirms the assumption that the reduction in the content of oksibenzoynoy acid (salicylate) in urine was due to the reduction of the content of uronic acids by introducing polysaccharides.

Along with endogenous conjugating substance - glucuronic acid after administration of the polysaccharide drugs in biological organs and systems other uronic acids (galacturonic, mannurone, guluronate). We can assume that either these other uronic acid fulfill the same role conjugating substances, as glucuronic acid, or the conversion of exogenous uronic acids glucuronic, as it is observed in enzymatic transformations occurring in plants (Kretovich C. L. Biochemisty respective enzymes in the human body.

Along with described a pronounced biological effects of the use of SP and an harmless to humans, because both polysaccharides are components of food and biocompatible with the body.

Study of acute toxicity SP and EN carried out by the method of Cerberus (Sidorov, K. K. Methods for the determination of acute toxicity and hazards of chemicals (toxicomania). - M.: Medicine, 1970) 20 white rats-males weighing 180-220 g by oral administration of 5 ml solution SP or an dose of 3500 mg/kg body weight. After a single injection during the observation time (within 14 days) mortality was not observed, which allowed us to determine LD50when administered orally >3500 mg/kg (for both polysaccharides). According to the classification of toxic substances (Sidorov, K. K., 1970) both studied polysaccharide belong to the group of low-toxic substances.

The inventive method is illustrated by the following examples.

Example 1

The addition of lead acetate (II) mass 3,5313 g dissolved in 100 ml of water by stirring the flask with the solution on a magnetic stirrer, then the solution is quantitatively transferred into a volumetric flask with a capacity of 250 ml To suppress hydrolysis of ions of lead (II) and acetate ions to the resulting adjusted to the mark with water. 1 ml of the prepared solution of lead acetate (II) contains 9 mg of lead ions (II).

The experiments conducted on 60 adult white rats male Wistar rats weighing 180-220 g, divided into 6 groups of 10 animals each. The first group of animals - intact - served as a comparison. The rest of the groups (II-VI) animals daily for 7 days oral (with probe) is injected with a solution of lead acetate (II) in a volume of 1 ml at a dose of 45 mg/kg (in terms of lead). Group II animals served as a control. Then III-VI groups of animals daily oral injected with a solution of beet pectin (volume 5 ml) in a dose of 500 mg/kg for 2 weeks (group III), 4 weeks (group IV), 8 weeks (group V) and 12 weeks (group VI).

To prepare the solution of pectin was given a portion of the JV with a mass of 10 g, was transferred to a conical flask with a capacity of 1 l was added 500 ml of water, i.e., prepared 2% solution of the JV. For swelling and dissolution of pectin in water, the solution was left for a day at room temperature.

All animals during the experiment was kept on a standard diet. Animals in all groups studied the content of hexuronate acids (in terms of glucuronic acid) in serum and urine carbazole method (Methods and the Makarov Century, and others) and the content of direct bilirubin in serum (Balakhovskiy C. D. and others).

To obtain the urine of rats using probe was administered intragastrically in 5 ml of water, then animals were odarivali for a day in necesbary. Urine prior to analysis were filtered through a paper filter.

Blood collection animals were deceptional. The collected blood was defended in the refrigerator to separate the serum.

The results of biochemical analysis of biological fluids are shown in table 2. The probability of the differences in results compared to the control does not exceed 0.05, which is within the normal range and indicates the statistical validity of the data.

Example 2

Carried out analogously to example 1, but instead of a 2% solution of SP using 2% solution of sodium alginate (EN).

To prepare a 2% solution of an beat it EN mass 10 g is placed in a conical flask with a capacity of 1 l, add 500 ml of water and the solution is stirred until dissolution of the Academy of Sciences.

The results of biochemical analysis of blood serum and urine on the content of hexuronate acids and direct bilirubin are shown in table 2. The results obtained are statistically dostoverniye was carried out on 50 rats male Wistar rats, divided into 5 groups of 10 animals each. The first group of intact animals. The rest of the groups (II-V, in addition to (III) animals during the week daily oral injected with a solution of lead acetate (II) in a volume of 1 ml, at a dose of 45 mg/kg (in terms of lead). Then group IV (on the background of the lead pathology; control) and group III animals during the week daily using the probe after eating orally injected powder aspirin at the rate of 30 mg/kg of body weight as a suspension in 5 ml of water. Next group V animals (in the background lead pathology) daily oral introduced a 2% solution SP a volume of 5 ml at a dose of 500 mg/kg for 4 weeks and then a suspension of aspirin volume of 5 ml at a dose of 30 mg/kg for 1 week daily after a meal.

Animals in all groups at the end of the experience, and the V group daily for 4 days after administration of aspirin studied the content in the urine hexuronate acids (in terms of glucuronic acid) carbazole method (research Methods in occupational disease (biochemical)...) and oksibenzoynoy acid by the method of acid-base titration (the State Pharmacopoeia of the USSR...).

The results of biochemical studies statistically significant and are shown in table 4.

The use of the e described in example 2).

The results of biochemical studies are shown in table 4.

Thus, acidic polysaccharides, offered as conjugating substances, provide the following benefits:

1) when introduced into the body intact animals acidic polysaccharides (SP, EN) contents hexuronate acids in biological systems increases; in the soft tissues of the increase occurs earlier than in the bone tissue. The increase in the content of uronic acids in the serum, liver, urine suggests the participation of the input polyuronides together with glucuronic acid in the metabolism, in particular glucuronid conjugation.

2) In the weekly injection of lead acetate (II) violated the metabolic processes: the content of hexuronate acids in the serum is reduced by 1.7 times, and in the urine is increased in 3 times; the content of free bilirubin (normal to the body metabolite) in the serum is increased 2.9 times in urine also appears bilirubin. Perhaps this is due to the fact that ions of lead (II) block functional groups as glucose-1-phosphate, D.O.-glucuronic acid, and enzyme - transferase that catalyzes the transfer Wernigerode the form is in the urine and in the blood. In part, because of the blocking ions of lead (II), D.O.-glucuronic acid cannot konjugierte bilirubin. Subsequent long-term use SP and an normalizes metabolic processes: after 4 weeks of polysaccharides in the serum and urine of normal content hexuronate acids, which leads to normalization of bilirubin concentration in the blood, and after 8 weeks of supplementation polyuronide drugs bilirubin in the urine is not found. The beneficial effect of polyuronides on the metabolism of bilirubin is normally cleared from the body in the form of conjugates with glucuronic acid, apparently, can be explained by the formation of conjugates bilirubin with acidic polysaccharides, are introduced into the body with a lack of glucuronic acid.

3) have Established an inverse relationship between the content of hexuronate acids, administered at SP and EN, and bilirubin in the serum. Correlation coefficients respectively for JV and an amount -0,984 and -0,996, this indicates a high degree of probability of a connection between the content of hexuronate acids and bilirubin in the blood.

4) has a beneficial effect JV and an on metabolism not only normal metabolites, but also on the metabolism xenobiotic the holding in urine unconjugated oksibenzoynoy acid and glucuronic acid in comparison with the model without pathology, respectively, 1.4 and 3.7 times. If aspirin to enter after a week of taking the lead and 4 weeks of taking polysaccharide drugs, metabolic processes are normal. Therefore, the polysaccharide preparations are included instead of the glucuronic acid cycle conjugation with aspirin in urine is reduced and the content of hexuronate acids, and the content of aspirin.

5) Established a direct relationship between the content of hexuronate acids, administered at SP and EN, and content oksibenzoynoy acid (aspirin). Correlation coefficients respectively for SP and EN be +0,968 and +0,992, which proves that with high probability there is a direct correlation between these traits.

6) Acidic polysaccharides (SP, EN) is moderately soluble in water and therefore can be introduced into the body in the form of solutions, including injection. Solutions, having antimicrobial activity, stable (Pecto. Natural bactericidal enterosorbent. / Pogorelskiy L. C., tracina I. P., bonow S. C. guidelines. - M., 2000. - 16 S.).

7) As components of food (JV produced from pomace, sugar beet, an - of seaweed thalli) and being biocompatible with the human body, the SP and the Academy of Sciences of harachamim, containing uronic acid, characterized in that as uronic acids contains polygalacturonase acid with a degree of esterification of 40.4% (sugar beet pectin) or a derivative of sodium salts polymannuronate and prigorodovoy acid (sodium alginate).



 

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