The method of determining the regenerative capacity of fat-soluble antioxidants in vitro

 

(57) Abstract:

The invention relates to the food and pharmaceutical industries and can be used in assessing the quality of medicines and biologically active additives to food with antioxidant properties; selection of optimum process conditions in the allocation of natural fat-soluble antioxidants; in search of an effective system of antioxidant synergists. In the model system, consisting of an alcoholic solution of ferric chloride (III) and o-phenanthroline administered chloroform solutions of antioxidants, and subject to discoloration after 10 min of exposure determine the magnitude of the regenerative properties, spectrophotometric registration of the optical density of the resulting colored complex of Fe (II) with o-phenanthroline at a wavelength of 510 nm with subsequent calculation of the regenerative capacity of the mass concentration of the test substance according to the formula X=D/C, where C is the mass concentration of antioxidant, %; D is the optical density of the complex Fe (II) with o-phenantroline. Improved reproducibility and efficiency definitions. table 4.

The invention relates to the food and pharmaceutical industries and can be ¾ antioxidant properties. The method allows to use the result as an evaluation criterion in the selection of optimum process conditions in the allocation of natural fat-soluble antioxidants, and also to carry out rapid testing when searching for an effective system of antioxidant synergists.

Activation of free-radical oxidation processes on the background of the depletion of the antioxidant defense system (SAZ) of the body lies at the basis of many non-communicable diseases: atherosclerosis and its thrombectomies complications (myocardial infarction, stroke), diabetes, hypertension, cataracts, cancer, etc. It determines the necessity of application of antioxidants, including prophylactic, to maintain the functional activity of this system. Feature SAZ is the distribution of components between the hydrophilic and hydrophobic phases of the cell structures. When this key role in ensuring that the most important functions of the SAZ - protection of cell membranes - are fat-soluble antioxidants, penetrating into the hydrophobic zone of the phospholipid bilayer. This explains the appearance in the consumer market of a large number of products in the pharmaceutical and food purposes, C information about the degree of manifestation of antioxidant properties, even those substances traditionally regarded as antioxidants (-carotene, -tocopherol, and others), and the manifestation of synergism or antagonism in their joint application. Perhaps this inconsistency is not due to the real and the declared content of the active substance (concentration) in the tested drugs and biologically active food supplements and/or a status of active centers (pharmacophores) of these substances. In this regard, an effective and simple means of determining and controlling the activity of fat-soluble antioxidants.

We identified only source of information defining restorative properties (capacity) antioxidants (Dadali Y. C., Dadali C. A., Makarov Century, Quantum-chemical approach to the analysis of antioxidant activity of some natural antioxidants// Actual problems of creation of new medicinal preparations of natural origin: Materials of the IV international. Congress (Veliky Novgorod, 29 June - 1 July 2000) - SPb, 2000. - S. 135-143). The method provides for the assessment of antioxidant activity from the point of view of thermodynamic advantage of the process of electron transfer and recovery of molecules of substrate oxidation. For this purpose, conduct quantum is the situation on the basis of MO LCAO (method linear combinations of atomic orbitals) self-consistent field Hartree-Fock with all of the valence electrons and the neglect of the zero differential overlap.

Disadvantages: the method is unsuitable for practical use when defining restorative properties of complex compositions of natural fat-soluble antioxidants and use in evaluation of specific medicines and biologically active additives to food with antioxidant properties.

The task of the invention to provide an easily replicable and effective test to determine the restorative properties of fat-soluble antioxidants and containing preparations in the model system in vitro.

The technical result is the model definition of regenerative capacity, which as substrate contains iron ion (III). Simulated electron transport, a similar transfer in the antioxidant system of the body. Calculated value that characterizes the reduction activity of the mass concentration of the test substance and will allow for a preliminary evaluation of the quality of the medicinal product, the BAD: the quantitative content of the active substance, the degree of preservation of the active center (the degree of destruction, oxidation), responsible for the antioxidant and antihypoxant properties.

The definition of restorative approach, because it does not take into account: possible multiple interactions with the enzyme systems of the body and membrane structures and the stability of the formed products of the oxidation reaction. Reducing properties are determined only by the energy utility of electron transfer, and the degree of participation of the substance in the total antioxidant effect in the case of a combination with other antioxidants will be determined by the magnitude of the regenerative capacity and the effect of synergism or antagonism.

Technical novelty is that in the model system, consisting of an alcoholic solution of ferric chloride (III) and o-phenanthroline, administered chloroform solutions of antioxidants, and subject to discoloration after 10 min of exposure determine the magnitude of the regenerative properties spectrophotometric registration of the optical density of the resulting colored complex of Fe (II) with o-phenanthroline at a wavelength of 510 nm with subsequent calculation of the regenerative capacity of the mass concentration of the test substance according to the formula

X=D/C

where C is the mass concentration of antioxidant, %; D is the optical density of the complex Fe (II) with o-phenantroline.

Rating scale restoration with the antioxidants, regardless of the mechanism of action and the nature of the antioxidant, because the value of optical density in all cases is determined by the ion concentration of Fe (II) in solution and the formed complex of Fe (II) with o-phenantroline (red staining).

In turn, the optical density will depend on whether the test substance is capable of restoring Fe (III) Fe (II). The optical density of the solution of the complex Fe (II) with o-phenanthroline is directly dependent on the concentration of the latter, the calibration graph passes through the origin, thus, there is a subjection to the law of the Bouguer-Lambert-Bera.

The method is as follows.

To 1 ml of 0.5% alcohol solution of o-phenanthroline and 1 ml of 0.2% alcoholic solution of ferric chloride (III) was added 3 ml of a chloroform solution of the test substance. After exactly 10 min after the addition of a solution of the test substance measured optical density of a solution of red-orange color on the spectrophotometer SF-56 or equivalent) at a wavelength of 510 nm in a cell with a layer thickness of 10 mm as the reference solution using 1 ml of 0.5% alcohol solution of o-phenanthroline, 1 ml of 0.2% alcohol solution of chloride as the e

X=D/C

Determination was carried out repeatedly for different concentrations of antioxidants, then the expected average value of the regenerative capacity (HSR), which is characteristic of the regenerative properties of a particular fat-soluble antioxidant.

A comparative analysis of individual parameters (X) allows to evaluate the regenerative capacity and, accordingly, the degree of manifestation of the antioxidant properties in the range of the investigated substances. With the same purpose can be used, assuming 100% or 1.0, the regenerative capacity of ascorbyl palmitate, as the most pronounced antioxidant direct action, in addition to standardize the quality of fat-soluble antioxidants and products on their basis in conditional relative units.

The method is illustrated by the following examples.

Example 1. Analyzed the substance with the content of-carotene to 99.2%. Prepared chloroform solution with mass fraction-carotene- 0,001%, 0,00075%, 0,0005%, 0,00025%, 0,0001%. Next, 1 ml of 0.5% alcohol solution of o-phenanthroline and 1 ml of 0.2% alcoholic solution of ferric chloride (III) was added 3 ml of 0.001% chloroform solution-carotene. After exactly 10 min after the addition was measured od a layer thickness of 10 mm As the reference solution used 1 ml of 0.5% alcohol solution of o-phenanthroline, 1 ml of 0.2% alcoholic solution of ferric chloride (III) and 3 ml of chloroform. The magnitude of the regenerative capacity (X) in relative units is calculated by the formula

X=D/C

The determination was repeated for solutions with mass-carotene- 0,00075%, 0,0005%, 0,00025%, 0,0001%. Then the expected average reductive capacity (HSR), which is characteristic of the regenerative properties of a particular fat-soluble antioxidant. The determination results are presented in table 1.

The average recovery capacity (HSR) in relative units for a-carotene (99,2%) is

HSR=(1071+1073+1079+1069+1063)/5=1071.

Example 2. Analyzed the substance of content-tocopherol - 98,3%. Prepared chloroform solution with mass fraction-tocopherol- 0,01%, 0,0075%, 0,005%, 0,0025%, 0,001%. Next came analogously to example 1. The determination results are presented in table 2.

The average regenerative capacity in relative units for a-tocopherol is

HSR=327.

Example 3. Analyzed drug - retinol acetate 3,44% solution in oil. Prepared chloroformiate definitions are presented in table 3.

The average regenerative capacity in relative units for retinol acetate is

HSR=79.

Example 4. Analyzed the substance L-6-O ascorbyl palmitate containing ascorbyl palmitate - 98,0%. Prepared chloroform solution with a mass fraction of ascorbyl palmitate- 0,001%, 0,00075%, 0,0005%, 0,00025%, 0,0001%. Next came analogously to example 1. The determination results are presented in table 4.

The average regenerative capacity in relative units for ascorbyl palmitate is

HSR=2269.

Reduction activity, expressed in conventional “ascorbic” units for a-carotene on the in the data tables will be equal to 0.47(1071:22691,0), for a-tocopherol to 0.14, and retinol acetate - 0,035.

Finally, you can use the obtained parameters for the selection of the optimal combination of antioxidants, because the definition of the magnitude of the regenerative capacity of the composition and its comparison with the calculated theoretical (i.e., the sum of the scores of the individual components of the composition with regard to their concentration) will allow us to determine either the absence or the presence and magnitude of the effect of synergism or antagonism.

Aprilrain with their content in the analyzed sample, respectively 0,00005% and 0.0005% 0,603, while theoretical density will be:

DTeoret.=3270,00005+10710,0005=0,0164+0,536=0,552.

Since DTeoret.< Dfactit is possible to make a conclusion about the lack of effect of antagonism for the selected ratio and concentration of the analyzed antioxidants and a slight excess of the restorative properties of the composition above theoretically expected.

Technical result: a rapid method for the detection of antioxidant synergists, that allows you to combine them in one dosage form, BAA and to assess the quality of these drugs.

The method of determining the regenerative capacity of fat-soluble antioxidants in vitro, characterized in that in the model system, consisting of an alcoholic solution of ferric chloride (III) and o-phenanthroline, administered chloroform solutions of antioxidants, and, subject to discoloration, after 10 min of exposure determine the magnitude of the regenerative properties spectrophotometric registration of the optical density of the resulting colored complex of Fe (II) with o-phenanthroline at a wavelength of 510 nm with subsequent calculation of the regenerative capacity of the mass concentration of the test substance

 

Same patents:
The invention relates to medicine, in particular for dentistry
The invention relates to medicine, namely to diagnostic methods research
The invention relates to medicine, in particular to liquorrhea

The invention relates to medicine, in particular to rheumatology, Orthopaedics and traumatology

The invention relates to medicine, in particular to surgery

The invention relates to medicine (urology) and veterinary

The invention relates to the field of biological chemistry, in particular to Hematology, namely, laboratory methods of determining the rate of formation of products of lipid peroxidation in erythrocyte membranes of farm animals

The invention relates to medicine, in particular for laboratory diagnosis

The invention relates to diagnosis using the reagent for determining the concentration of the analyzed compounds in biological fluids containing hemoglobin, such as whole blood

The invention relates to diagnosis using reagent-based tetrazole for measuring the concentration of an analyte in containing hemoglobin biological fluid such as whole blood

The invention relates to the field of pharmaceutical and analytical chemistry and can be used for determination of papaverine, demerol and other alkaloids in medicinal forms

The invention relates to methods for the analytical determination of alkalis, alkaline salts and other substances having an alkaline reaction and reacts with acids, namely to a method of explosive used in the learning process and production

The invention relates to analytical chemistry, and in particular to compositions of water-sensitive pastes, and can be used to determine the boundary between the oil or oil and water reservoirs, tanks, tankers, settling treatment facilities of factories, where it is necessary to regularly monitor the level of oil products, oil and water

The invention relates to the field of determining the residual disinfectant in the water, in particular to aqueous solution of the azo dye for this purpose
The invention relates to the field of analytical chemistry of the elements, namely the methods of selection and determination of osmium, and can be used in the isolation and determination of osmium in objects of different material composition

The invention relates to analytical chemistry (test compounds), and can be used for the determination of Nickel (II) in aqueous solutions, in particular in waste water and industrial solutions

The invention relates to analytical chemistry (test compounds), and can be used for the determination of rhenium (VII) in aqueous solutions, in particular wastewater and industrial solutions

The invention relates to analytical chemistry (test compounds), and can be used for the determination of molybdenum (VI) in aqueous solutions, in particular in natural and waste waters

The invention relates to analytical chemistry (test compounds), and can be used for the determination of copper (II) in aqueous solutions, in particular in waste water and industrial solutions

The invention relates to analytical chemistry (test compounds), and can be used for the determination of palladium (II) in aqueous solutions, in particular in waste water and industrial solutions

FIELD: analytical methods.

SUBSTANCE: group of inventions is directed on detecting and quantitatively determining hydrogen radicals present in water or in aqueous solution and is characterized by adding sodium 3,5-dibromo-4-nitrosobenzenesulfonate to sample to detect hydrogen radicals from coloration appearing as a result of their absorption characteristics, and additionally characterized by blowing gaseous hydrogen through 1,1-diphenyl-2-picrylhydrazide solution absorbing near 517 nm and sodium 3,5-dibromo-4-nitrosobenzenesulfonate solution at constant velocity in presence of platinum black in order to quantitatively evaluate hydrogen radical concentration using calibration curve based on correlation between coefficient of absorption near 450 nm for azo compound of sodium 3,5-dibromo-4-nitrosobenzenesulfonate and concentration of hydrogen radicals appeared.

EFFECT: increased determination accuracy and enabled analysis of hydrogen radicals in various media.

6 cl, 15 dwg, 1 tbl, 5 ex

Up!