RU2238554C1 - Method for determining total antioxidation activity of biologically active substances - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves preparing samples of reference substance and substance under analysis, electrochemically oxidizing them in amperometric cell, amplifying electric signals, recording them and calculating antioxidation activity with a proposed mathematical relation.

EFFECT: high accuracy and reproducibility in estimating total antioxidation activity.

4 cl, 1 dwg

Description

The invention relates to the field of medicine, pharmacology, pharmacy and can be used to evaluate the antioxidant activity of various drugs, food products and dietary supplements. The antioxidant activity (potential) of a drug is an indicator of the quality of the drug, characterizing its restoring properties, i.e. properties of reacting with reactive oxygen species (ROS), both in vivo and in vitro, which are formed in the body in the presence of oxygen, when it is incompletely reduced to form the following radicals:

- superoxide anion radical (O 1/2);

- hydroperoxide radical (CHOO ·)

- hydrogen peroxide (H ₂ O ₂ )

- hydroxyl radical (HO ·), etc.

In the case of hyperoxidation, ROS organisms can act as radicals attacking lipids in cell membranes, tissue proteins, enzymes, polysaccharides and DNA.

The aging process and serious diseases are associated with damage to the human body by active oxygen-containing radicals, especially with their excess or with a decrease in the activity of the endogenous antioxidant system of the human body.

To maintain the necessary concentration of free radicals in the body, drugs and biologically active substances are used, including in the form of food products or dietary supplements.

A known method for determining antioxidant activity, which measures the total chemiluminescent change after 3 seconds from the moment of adding sodium hypochloride to hydrogen peroxide in the presence of a biologically active substance in comparison with the control. The method allows to speed up the analysis and expand the range of the investigated substances (RU 2033609, 04/20/1995).

A known method for determining the antioxidant activity of a substance by spectrally evaluating their ability to suppress the appearance of OH radicals in the Fenton reaction, where the assessment is carried out by the intensity of the chemiluminescence of the Fenton formula A = (A0-A +) / A0, where A0 is the maximum amplitude of the chemiluminescence of the Fenton reaction at in the absence of the test substance, A + is the maximum amplitude of the glow of the reaction in the presence of the test substance and at values of A> 0.3, we conclude about the antioxidant properties of the test substance (RU 2163021, 02/10/2001).

A disadvantage of the known methods is the formation of luminescent substances also from individual compounds and the lack of selectivity for oxidizing substances in complex multi-component systems (complex drugs, extracts from medicinal plants, dietary supplements and food products).

A known method for determining the amount of antioxidant substances, such as phenolic compounds, using photometric titration according to Leventhal, based on oxidation in an acidic medium with potassium permanganate in the presence of an indigo carmine indicator, which is a regulator of the oxidation reaction. For the calculation, a coefficient of 6.792 mg of the amount of phenolic hop compounds and the corresponding 1 ml of 0.1 N potassium permanganate solution is used.

The disadvantage of this method is that phenolic compounds are not the only active ingredients of plants and phytopreparations based on them - infusions, decoctions, elixirs, tinctures that react with potassium permanganate. (Lyashenko N.I. et al. Methods for the quantitative determination of polyphenols in hops. Khmelovodstvo, v.2, Kiev, Urozhai, 1980, p. 57-64).

A known method for determining the antioxidant activity of individual substances, based on the electrochemical absorption of oxygen, for example, the activity of 1,5D-antifructose is determined in the emulsion of methyl ester of citric acid. An oxidative reaction in the system is initiated by the addition of methioglobin solution. Immediately after the initiation of the reaction, the sample is injected into a thermostatically controlled (25.0 ± 0.1 ° C) closed cell, while providing reliable protection against oxygen entering the system. Oxygen absorption is determined by the Clark electrode, which is connected to a computer-controlled database. The relative concentration (%) of oxygen is determined every 30 s (see description to RU 2140988, 11/10/1989). This method is not suitable for determining the total antioxidant activity of drugs. In general, methods that use spectroscopic detection at the final stage are, in most cases, used in combination with liquid chromatography methods and are used to quantify a specific substance with antioxidant properties. (Grosset C. Cantin P. Alarys. Quantitative determination in galenic forms of antioxidants by liquid chromatography. // Analusis, 1989, 17, No. 7, p. 409-412).

This method has the following disadvantages: the method of determining indirect, includes several stages, and, as a result, is quite long in time. Complexity of equipment requiring specialist service and an equipped installation site. A sufficiently high percentage of determination errors.

The closest in technical essence and the achieved result is a method that allows to determine the total antioxidant activity of biologically active substances, which consists in preparing a dose of the analyzed and standard substances, their oxidation and calculation of antioxidant activity according to the formula, in particular, that 0.05 H potassium permanganate solution in a 0.024 M sulfuric acid solution is titrated at room temperature with a solution of the analyzed sample until discoloration and the calculation of the concentration of biologically active substances is carried out according to the formula in terms of e by quercetin.

An indicator of relative AOA is the volume of the drug (object) in milliliters spent on titration of 1 ml of a 0.05 N potassium permanganate solution. The smaller the volume of the drug spent on titration, the higher the antioxidant activity of the drug. For a quantitative assessment of AOA of drugs (objects), an activity index is introduced - B. This value is the sum of a BAS of a restoring nature and is expressed by the number of milligrams of quercetin in 1 ml or 1 g of the drug (object). The higher the value of B, the higher the AOA has an object (RU 2170930, 07.20.2001). The disadvantage of the method chosen for the prototype is the titration of potassium permanganate in the preparation of substances that do not have a reducing potential, and therefore not involved in the inhibition of active radicals.

The objective of the present invention is to develop a method that allows with high accuracy and reproducibility to evaluate the total antioxidant activity of pharmaceuticals, medicinal plants, biologically active additives and food products (tea, wine, beer, juices). The method should be easy to use and not too expensive.

The problem is solved by the described method for determining the antioxidant activity of biologically active substances, which includes preparing doses of the analyzed and standard substances, their electrochemical oxidation by directly supplying the prepared doses to the thermostatically controlled electrochemical cell of the amperometric detector with receiving signals in the form of corresponding electric current pulses, signal amplification, registration in the form of output curves and calculation of the areas of the obtained peaks emogo (S) and standard (Sst) compounds, wherein the calculation parameter antioxidant activity (AOA) is carried out according to the following formula:

where S _a is the peak area of the analyzed drug; S _c is the peak area of the standard; m _with - a sample of the standard; V _c - standard solution volume, ml; P is the purity of the standard used,%; n - dilution of the analyzed drug.

Preferably, the preparation of the analyzed samples is carried out by mixing the appropriate substances with a solvent that does not have antioxidant ability. The method provides for the use of substances having high redox potential as standard substances. For example, substances selected from the range of quercetin, dihydroquercytin, and rutin are used as standard substances.

The proposed method is based on the property of antioxidants as active reducing agents having various "redox potential". For example: Redox potential of quercetin = 0.3 V. Redox potential of dihydroquercetin = 0.5 V. Redox potential of rutin = 0.6 V.

Using a standard substance, it is possible to accurately determine the redox potential of any multicomponent preparations that have an even greater difference in antioxidant activity.

The proposed method is applicable to the studied objects in a liquid state. Solids are converted to liquid forms before analysis.

To implement the method created by the installation, depicted schematically in the drawing.

Installation contains

1 - tank for solvent;

2 - pump;

3 - dispensing tap;

4 - amperometric detector with a thermostatically controlled electrochemical cell;

5 - electric signal amplifier;

6 - analog-to-digital Converter;

7 - recording device (computer with printer);

8 - input device of the analyzed and standard substances.

The installation works as follows: the pump (2) pumps the solvent from the tank (1) through the entire system. The test solution (8) is introduced into the metering valve using a standard 1 ml syringe. The solvent stream supplies a dose of the analyte to the detector cell (4). In the cell, electrochemical oxidation of the substance occurs on the surface of the working electrode. In this case, the current between the two electrodes increases. The emerging currents are small (10 ^-16 -10 ^-9 A), therefore they are amplified in the device (5), using an analog-to-digital converter (6), they are converted into a digital signal and recorded on the computer display (7).

The following are specific examples of the method using the above installation.

EXAMPLE 1

1 ml of the drug "Immunal" is introduced into a volumetric flask with a capacity of 100 ml, the volume of the flask is adjusted with water to the mark and mixed. 20 μl of the resulting solution manually or using an automatic dispenser is introduced into the dispensing tap of the installation, in which the sample is mixed with a passing solvent and the pump is fed directly to the thermostatically controlled electrochemical cell of the amperometric detector, with the receipt of the corresponding signal in the form of an electric current pulse, amplification of this pulse and its graphical registration with a differential curve (peak area S _n), both being prepared 100 ml of a standard solution vesche Twa antioxidant (rutin purity 98%) having a high redox reducing potential, which is also introduced into the faucet spout and the sample arrives at the same detector issuing pulse to a peak area (S _c), a quantitative evaluation of the antioxidant activity (AOA) the analyte is calculated by the following mathematical dependence:

where S _n is the peak area of the analyzed drug;

s _article - peak area of the routine standard;

n is the dilution of the analyzed drug;

m _c - sample standard rutin, mg;

V _c - the volume of the prepared solution of the standard antioxidant rutin, ml.

Found experimentally:

The peak area of the preparation "Immunal" S _n = 396,0

The peak area of the standard routine S _c = 165,0

EXAMPLE 2

GREEN TEA. 5 g (accurately weighed) of green tea is poured with 200 ml of distilled water heated to a boil, covered with a watch glass and infused for 5 minutes, then the resulting extract is filtered through a white ribbon filter, cooled and the volume is adjusted to 200 ml with water (solution for analysis). The solution through the dispenser is fed into the electrochemical cell of the detector. Obtaining the areas of the analyzed tea and the standard of rutin, as well as the quantitative calculation of the AOA value, are carried out, as in example No. 1, per 1 ml or 1 g of dry tea.

For the analyzed variety of green tea

AOA = 3.92 mg / ml or 157 mg / g dry green tea.

Similarly, studies have been conducted to evaluate the antioxidant activity of various substances. The results are presented in examples 3-5.

EXAMPLE 3

MEDICINAL PRODUCT ELIXIR "FITOIMMUNAL"

The determination of the peak areas of the drug and the standard of rutin, as well as the quantitative calculation of AOA, is carried out as in example 1.

For the analyzed sample of the drug "Fitoimmunal"

AOA = 7.42 mg / ml

EXAMPLE 4

Cognac "Dagestan"

The determination of the peak areas of the analyzed cognac and the rutin standard, as well as the quantitative calculation of AOA, are carried out as in example 1

For the analyzed sample cognac "Dagestan"

AOA - 1.18 mg / ml

EXAMPLE 5

BLACK TEA

Preparation of the extraction and determination of the studied extraction peaks and the standard of the routine, as well as the quantitative calculation of AOA, are carried out as in example 2.

For the analyzed variety of black tea.

AOA - 6.4 mg / l or 256 mg / g of black tea.

The measurements showed that the proposed method is simple to use and depending on the analyzed object has high reproducibility and accuracy, which exceeds the similar characteristics of the prototype method.

The results of the proposed method can be used in medical practice for the correct dosage of drugs and dietary supplements in the human body, as well as for reliable standardization of drugs, medicinal plants and dietary supplements. Since antioxidant activity is an integral indicator of quality, it would be advisable to introduce such an indicator in regulatory documents (GOST, TU, Pharmaceutical articles, etc.)

Claims (4)

1. The method of determining the antioxidant activity of biologically active substances, including the preparation of samples of the analyzed and standard substances, their oxidation and calculation of the indicator of antioxidant activity, characterized in that the oxidation of the analyzed and standard substances is carried out electrochemically by directly supplying prepared samples to a thermostatically controlled electrochemical cell of an amperometric detector with receiving signals in the form of corresponding pulses of electric current, signal amplification in, registered in the form of differential output curves and calculation of peak areas derived analyte (S) and standard (S _v) compounds, wherein the index calculation antioxidant activity (AOA) is carried out according to the following formula:

where S _a is the peak area of the analyzed drug; S _c is the peak area of the standard; m _with - a sample of the standard; V _c - standard solution volume, ml; P is the purity of the standard used,%; n - dilution of the analyzed drug. 2. The method according to claim 1, characterized in that the preparation of the analyzed samples is carried out by mixing the corresponding substances with a solvent that does not have antioxidant ability. 3. The method according to claim 1, characterized in that as the standard substances using substances with high redox potential. 4. The method according to claim 3, characterized in that as standard substances use substances selected from the series: rutin, quercetin, dihydroquercetin.