Trap method for determining antioxidant activity. Method for determining total antioxidant activity

The invention relates to the food industry and can be used to determine the total antioxidant activity. The method is carried out as follows: the analyte interacts with the reagent 0.006 M Fe(III) - 0.01 M o-phenanthroline. Ascorbic acid (AA) interacts with the same reagent, which is added in a ratio of 1:100. Then incubated for at least 90 minutes and photometered at 510±20 nm. After that, the dependence of the value of the analytical signal on the amount of the substance is established and the value of the total AOA is calculated. The presented method allows less time-consuming and more reliable determination of the total antioxidant activity of plant materials and food products based on it. 2 w.p. f-ly, 1 ill., 5 tab.

The invention relates to analytical chemistry and can be used in determining the total antioxidant activity (AOA) of plant materials and food products based on it.

Known coulometric method for determining the total AOA of tea, based on the interaction of aqueous extracts of the product with electrically generated bromine compounds (I.F. Abdulin, E.N. Turova, G.K. Chemistry, 2001, vol. 56, no. 6, pp. 627-629). The choice of electrogenerated bromine compounds as a titrant is due to their ability to enter into various reactions: radical, redox, electrophilic substitution and addition by multiple bonds. This makes it possible to cover a wide range of biologically active tea compounds with antioxidant properties. The disadvantages of the method are the possibility of the bromination reaction with substances that are not antioxidants, and the expression of the resulting value of the total AOA in units of the amount of electricity (kC/100 g), which makes it difficult to evaluate the results.

A known voltammetric method for determining the total antioxidant activity by the relative change in the current of oxygen electroreduction in the potential range from 0.0 to -0.6 V (rel. sat. c.s.e.) on a mercury-film electrode (Pat. IPC 7 G 01 N 33/01 Voltammetric method for determining the total activity of antioxidants / E. I. Korotkova, Yu. The disadvantage of this method is the occurrence of side electrochemical reactions, which reduces the efficiency of the determination of antioxidants, which leads to a decrease in the reliability of the results.

A known method for controlling the total AOA of prophylactic and therapeutic antioxidant agents for lipid peroxidation to malonic aldehyde with spectrophotometric or chemiluminescent detection (Pat. 2182706, Russia, IPC 7 G 01 N 33/15, 33/52. funds / Pavlyuchenko I.I., Basov A.A., Fedosov S.R. - No. 2001101389/14; application 15.01.2001; publ. 20.05.2002). At the same time, antioxidant activity is inversely proportional to the level of lipid peroxidation products. The disadvantage of this method can be considered a limited range of analyzed objects, since under these conditions, antioxidants of only one group, lipids, are determined.

A known method for determining the total AOA of a plant extract, which consists in incubating the extract with linetol and iron (II) sulfate, initiating the oxidation reaction with UV irradiation and subsequent interaction with thiobarbituric acid in the presence of triton X-100 (Application 97111917/13, Russia, IPC 6 G 01 N 33/00 Method for determining the total antioxidant activity / Rogozhin VV - Appl. 08.07.1997; publ. 10.06.1999). When carrying out spectrophotometry, a mixture of ethanol with chloroform in a ratio of 7:3 is used. The AOA value of a biological material is determined by the ratio of the accumulation of the reaction product - malondialdehyde in a sample containing an extract to a sample with a prooxidant. The disadvantage of this method lies in the possibility of side reactions during UV irradiation, which reduces the reliability of the results of the analysis.

The listed methods for determining the total AOA have a number of disadvantages: high labor intensity, low reliability, the measured value of the total AOA is not related and is not comparable with any conventional substance.

The closest analogue to the claimed invention is a method for determining the total AOA of medicinal plants by measuring the chemiluminescence that occurs when reacting with luminol in the presence of an oxidizing agent hydrogen peroxide (M.Kh. canary grass by chemiluminescence // Journal of Analytical Chemistry, 2004, V.59, No. 1, P.84-86). For a quantitative assessment of the total AOA, the reducing ability of the extract of medicinal raw materials and the activity of a potent antioxidant - ascorbic acid in the amount of 25-110 μg were compared. Compared with the above methods, in the prototype, hydrogen peroxide is used as an oxidizing agent, interacting with a wide range of antioxidants, and the measured value of the total AOA of the object is determined and expressed relative to ascorbic acid, which is a common antioxidant, which makes it possible to obtain reliable results while maintaining other disadvantages. The disadvantages also include the complexity of the equipment used in the method.

The technical objective of the claimed invention is the development of a less time-consuming and reliable method for determining the total antioxidant activity of plant materials and food products based on it.

To solve the technical problem, it is proposed to interact the analyte with the reagent 0.006 M Fe (III) - 0.01 M o-phenanthroline, and ascorbic acid (AA) with the same reagent, which is added in a ratio of 1:100, incubated for at least 90 minutes, photometered at 510±20 nm, followed by establishing the dependence of the analytical signal on the amount of substance and calculating the total AOA. In particular, the calculation can be carried out according to formula (I), derived from the equation of quantitative correspondence between the object under study and ascorbic acid:

where a, b are the coefficients in the regression equation for the dependence of the analytical signal on the amount of AA;

a", c" - coefficients in the regression equation for the dependence of the analytical signal on the amount of the object under study;

x sun. - mass of the studied reducing agent (sample), mg.

The use of the proposed reagent under these conditions allowed us to expand the linear range and reduce the lower limit of the determined amounts of ascorbic acid. The proposed set of essential features allows you to determine the total AOA of a wide range of plant materials and food products based on it.

Quantitative correspondence equations connect the dependence of the analytical signal on the amount of ascorbic acid and the dependence of the analytical signal on the amount of the object under study, provided that the antioxidant activity is equal.

After processing the results of photometric measurements of the magnitude of the analytical signal by the least squares method (K. Derffel Statistics in analytical chemistry. - M .: "Mir", 1994. S. 164-169; A.K. Charykov Mathematical processing of the results of chemical analysis - L .: Chemistry, 1984. S.137-144) these dependencies were described by a linear regression function: y=ax+b, where a is the regression coefficient, b is a free member. The coefficient a in the regression equation is equal to the tangent of the slope of the straight line to the x-axis; coefficient b - distance along the y-axis from the origin (0,0) to the first point (x 1 , y 1).

The coefficients a and b are calculated by the formulas:

The regression equation for the dependence of AS on the amount of ascorbic acid at a given time has the form:

y AK \u003d a x AK (mg) + b,

regression equation for the dependence of AS on the amount of the object under study (reducing agent):

y VOST \u003d a "x VOST (mg) + b",

where for AK, for VOST is the optical density of the photometric solution;

x AK (mg), x VOST (mg) - concentration of ascorbic acid (reducing agent) in solution;

then, by equating the values of the functions, we obtain formula (I) for calculating the antioxidant activity of the object under study in units of the amount (mg) of ascorbic acid.

The drawing shows the dependence of the analytical signal on the amount of reducing agent.

The optical density of the analyzed solutions was measured on a KFK-2MP photoelectric colorimeter.

It is known (F. Umland, A. Yasin, D. Tirik, G. Vunsch Complex compounds in analytical chemistry - M.: Mir, 1975. - 531 p.) that o-phenanthroline forms a water-soluble chelate with iron (II) red-orange color, which is characterized by an absorption maximum at λ=512 nm. Therefore, in the proposed method, photometry is carried out at λ=510±20 nm.

The optimization of the composition of the reagent and its amount introduced into the reaction was carried out on the basis of the results of multifactorial planning of the experiment using the Latin Square method, which consisted in changing all the studied factors in each experiment, and each level of each factor only once meets different levels of other factors. This allows you to identify and evaluate the effect caused by each factor under study separately.

The following factors were used: the amounts of Fe(III), o-phenanthroline, and the volume of the reagent introduced into the reaction. The combination of factors should provide a wide range of linearity of the analytical signal (AS) with sufficient sensitivity, on the one hand, and stability of the reagent over time, on the other. This made it possible to single out the following levels for each factor:

the amount of Fe(III): 0.003 M (A 1); 0.006 M (A 2); 0.009 M (A 3);

amount of o-phenanthroline: 0.01 M (B 1); 0.02 M (B 2); 0.03 M (B 3);

reagent volume: 0.5 ml (C 1); 1.0 ml (C 2); 2.0 ml (C 3) (Table 1).

To select the optimal combination of factor levels, calibration dependences of AS on the amount of ascorbic acid were obtained in the range from 10 to 150 μg (which is necessary to confirm the linearity of the function), the regression equation of the obtained dependence was calculated, and then the value of AS at a given amount (120 μg) of ascorbic acid. Thus, for each composition of the reagent (factors A, B), the volume (factor C) was selected, at which the AC value is maximum. This made it possible to reduce the number of considered combinations to nine (Table 2).

Comparing the total AS for each level, the amounts with the maximum value were identified: ΣA 2 (0.991); ΣB 1 (1.066); ΣC 2 (1.361). This made it possible to conclude that the reagent composition is optimal: 0.006 M Fe (III) - 0.01 M o-phenanthroline with its volume introduced into the reaction, 1.0 ml per 100 ml of solution.

At the optimal concentration of the reagent, we studied the change in the dependence of AS on the concentration of ascorbic acid and some reducing agents common in natural objects (tannin, rutin, quercetin) at different incubation times of the reaction mixture (30, 60, 90, 120 min). It was found that for all the studied reducing agents, the dependence of AS on their content is linear in the range of 10-150 μg (see drawing) and the AS value depends on the incubation time (table 3).

It can be seen from the drawing that the change in AC under the action of rutin is insignificant, tannin approaches, and quercetin exceeds the same dependence for ascorbic acid. When considering the change in AC from the time of incubation for all the studied reducing agents (table 3), it was found that the stabilization of the analytical signal over time is observed from 90 minutes.

Table 3 Change in AS of reducing agents over time
Test substance	m substances, mg / cm 3	Analytical signal
Test substance	m substances, mg / cm 3	Time of incubation of the reaction mixture, min
30	60	90	120
Vitamin C	10	0,038	0,042	0,044	0,044
100	0,340	0,352	0,360	0,363
Tannin	10	0,029	0,037	0,042	0,043
100	0,280	0,295	0,303	0,308
Rutin	10	0,013	0,016	0,019	0,019
100	0,150	0,166	0,172	0,175
Quercetin	10	0,031	0,044	0,051	0,053
100	0,420	0,431	0,438	0,442

To prove the summing nature of the determined AOA value, the effect of the reagent Fe (III) - o-phenanthroline on model solutions, which included reducing agents: tannin, rutin, quercetin, and ascorbic acid in various ratios, was studied. Table 4 presents the results of the analysis of model mixtures.

Table 4 Results of the analysis of model mixtures (P=0.95; n=3)
The number of components in the mixture	Total AOA, calculated, mcgAA	Total AOA, found, mcgAA
introduced	Total AOA, calculated, mcgAA	Total AOA, found, mcgAA	in terms of AK
AK	Tannin	Rutin	Quercetin	AK	Tannin	Rutin	Quercetin
-	20	20	20	-	16,77	9,56	32,73	59,06	57,08
-	10	10	10	-	8,35	4,77	16,41	29,53	26,95
-	50	10	10	-	42,02	4,77	16,41	63,20	55,04
-	10	50	10	-	8,35	23,93	16,41	48,69	50,06
-	10	10	50	-	8,35	4,77	81,70	94,82	91,61
-	30	10	10	-	25,19	4,77	16,41	46,37	39,24
-	10	30	30	-	8,35	14,35	49,06	71,76	73,47
20	20	20	20	20	16,77	9,56	32,73	79,06	96,29
50	10	10	10	50	8,35	4,77	16,41	87,95	93,07
10	50	10	10	10	42,02	4,77	16,41	73,20	78,15
10	10	50	10	10	8,35	23,93	16,41	58,69	78,74
10	10	10	50	10	8,35	4,77	81,70	104,82	121,45
30	30	10	10	30	25,19	4,77	16,41	76,37	84,59
10	10	30	30	10	8,35	14,35	49,06	81,76	103,31

The calculation of the theoretical value of the total AOA was carried out according to the equations of quantitative correspondence characterizing the antioxidant capacity of the studied reducing agent with respect to ascorbic acid, under conditions of equal antioxidant activity: .

The value of the experimental (found) AOA was calculated using the averaged regression equation for the dependence of AS on the amount of ascorbic acid. From the results presented in Table 4, it can be seen that the experimentally obtained AOA values agree satisfactorily with the theoretically calculated ones.

Thus, the determined value of AOA is a total indicator, and the determination of its value using the equations of quantitative correspondence is correct.

The proposed method has been tested on real samples. To determine the total AOA of a real sample or its extract, calibration dependences of AS on the amount of analyte and ascorbic acid were obtained at an incubation time of the reaction mixture of at least 90 minutes. The calculation of the total AOA was carried out according to formula (I) and expressed in mg of ascorbic acid per gram of the test object (mgAA/g).

To confirm the correctness of the proposed method, these samples were tested according to known methods, evaluating the content of ascorbic acid (GOST 24556-89 Processed products of fruits and vegetables. Methods for determining vitamin C) and the prevailing reducing agents: in tea - tannin (GOST 19885-74 Tea. Methods for determining the content tannin and caffeine), in rosehips - the amount of organic acids (GOST 1994-93 Rosehips. Specifications) (table 5).

], however, the definition of antioxidants as chemical compounds does not give a complete picture of the protective properties of the object under study: they are determined not only by the amount of one or another antioxidant, but also by the activity of each of them. Antioxidant activity, or antioxidant activity, AOA, is the rate constant for the reaction of an antioxidant with a free radical (kInH). The chemiluminescence (CL) method makes it possible to determine the total amount of radicals that antioxidants bind in the sample (total antioxidant capacity, TAU), and when using the method of mathematical modeling of CL kinetics, also the rate of formation and reaction of radicals with antioxidants, i.e., AOA [ , , ].

The most common modification of the chemiluminescent method for determining the total antioxidant capacity is based on the use of luminol as a chemiluminescence activator [ , , , ]. A sample is placed in the cuvette of the chemiluminometer with the addition of luminol, hydrogen peroxide and a compound capable of generating radicals as a result of spontaneous decomposition (thermolysis), for example, 2,2'-azobis-(2-amidinopropane) dihydrochloride (ABAP): ABAP → 2R. In the presence of molecular oxygen, the alkyl radical R forms a peroxyl radical ROO : R + O 2 → ROO . Further, the peroxyl radical oxidizes the chemiluminescent probe luminol (LH 2), and the luminol radical (LH ) is formed: ROO + LH 2 → ROOH + LH . From LH, through the formation of intermediates (luminol hydroperoxide and luminol endoperoxide), a molecule of the final product of luminol oxidation, aminophthalic acid, is formed in an electronically excited state, which emits a photon, and as a result, chemiluminescence is observed. The CL intensity is proportional to the photon production rate, which, in turn, is proportional to the stationary LH concentration in the system. Interacting with radicals, antioxidants interrupt the described chain of transformations and prevent the formation of a photon.

Compounds subject to thermolysis are not the only possible source of radicals in the analysis of the antioxidant capacity of a sample by the chemiluminescent method. Alternatives are systems horseradish peroxidase–hydrogen peroxide [ , ], hemin–hydrogen peroxide, cytochrome with–cardiolipin–hydrogen peroxide, etc. The scheme of reactions of luminol oxidation by peroxidases is considered in the work of Cormier et al. .

The CL kinetic curves for these systems reflect two stages of the reaction: the stage of an increase in the CL intensity and the stage of a plateau or a gradual decrease in luminescence, when the CL intensity is either constant or slowly decreases. The paper describes two approaches to measuring the total antioxidant capacity that take into account this feature of the curves. The TRAP (Total Reactive Antioxidant Potential) method is based on the measurement of the latent period of CL τ and can be used to determine antioxidants such as trolox or ascorbic acid: they are characterized by a high value of the reaction rate constant with radicals and for this reason can be called strong antioxidants. During the latent period, their complete oxidation occurs. The TAR method (Total Antioxidant Reactivity) measures the degree of quenching of chemiluminescence q at the plateau or at the maximum of the chemiluminescent curve: formula , where I is the intensity of chemiluminescence without an antioxidant, and I 1 is the intensity of CL in the presence of an antioxidant. This method is used if the system contains predominantly weak antioxidants with low rate constants of interaction with radicals - much lower compared to the luminol constant.

The action of antioxidants is characterized not only by indicators τ and q. As can be seen from [ , ], the effect of such antioxidants as uric acid in the hemin–H2O2–luminol or tocopherol system, rutin, and quercetin in the cytochrome with–cardiolipin–H 2 O 2 –luminol, characterized by a change in the maximum rate of CL rise ( vmax). As the results of mathematical modeling of kinetics show, the values of the rate constants of the interaction of these antioxidants with radicals are close to the value of the luminol constant, therefore, such antioxidants can be called medium-strength antioxidants.

If the studied material, in particular plant raw materials, contained only one type of antioxidants, then their content could be characterized by one of the three indicators listed above ( τ , q or vmax). But plant raw materials contain a mixture of antioxidants of different strengths. To solve this problem, some authors [ , , , ] used the change in the chemiluminescence light sum over a certain time ∆S, calculated by the formula , where ∆ S0 and ∆ S S- CL light sums for a given time t in the control and test samples, respectively. The time should be sufficient for the oxidation of all antioxidants in the system, that is, for the CL curve of the test sample to reach the level of the CL curve of the control sample. The latter suggests that researchers should not only record the light sum of the luminescence, but also record the CL kinetics curve for a sufficiently long time, which is far from always done.

Since all measured indicators depend on the device and measurement conditions, the antioxidant effect of a substance in the system under study is usually compared with the effect of an antioxidant taken as a standard, for example, Trolox [ , ].

The horseradish peroxidase–hydrogen peroxide system has been used to analyze the total antioxidant capacity of plant materials by many authors. In works [ , ] the latent period of CL (TRAP method) was used to estimate the amount of antioxidants in samples, and in works [ , , ] the area under the CL development curve was used. However, the listed works do not provide a clear rationale for choosing one or another parameter for estimating TAU.

The aim of the study was to determine how the ratio of antioxidants of various types affects the TAU, and to modify the chemiluminescence method in such a way as to be able to more accurately determine the TAU in plant materials. To do this, we have set ourselves several tasks. First, to compare the CL kinetics of the studied objects with the kinetics of standard antioxidants of three types (strong, medium, and weak) in order to understand which type of antioxidants make the main contribution to the TAE of the studied objects. Secondly, to calculate the TAU of the studied objects by measuring the decrease in the CL light sum under the action of these objects in comparison with the action of the antioxidant, which provides the greatest contribution to the TAU.

MATERIALS AND METHODS

The objects of the study were industrial samples of fruits of hawthorn, mountain ash and wild rose produced by Krasnogorskleksredstva JSC (Russia), as well as raspberry fruits collected by the authors in the Moscow region under conditions of natural growth and dried at a temperature of 60–80 ° C until they stop extracting juice and pressure deformations.

The reagents for the analysis of antioxidant capacity by the chemiluminescent method were: KH 2 PO 4 , 20 mM buffer solution (pH 7.4); peroxidase from horseradish roots (activity 112 U/mg, M = 44 173.9), 1 mM aqueous solution; luminol (5-amino-1,2,3,4-tetrahydro-1,4-phthalazinedione, 3-aminophthalic acid hydrazide, M=177.11), 1 mM aqueous solution; hydrogen peroxide (H 2 O 2 , M = 34.01), 1 mM aqueous solution; solutions of antioxidants (ascorbic acid, quercetin, tocopherol). All reagents were manufactured by Sigma Aldrich (USA).

Decoctions of hawthorn, mountain ash and wild rose fruits and an infusion of raspberry fruits were prepared according to the methodology of the State Pharmacopoeia of the USSR, set out in the general pharmacopoeial article "Infusions and decoctions".

The total antioxidant capacity was determined by registering chemiluminescence on a Lum-100 chemiluminometer (DISoft, Russia) using PowerGraph 3.3 software. To determine the TAU in plant materials, 40 µl of luminol at a concentration of 1 mM, 40 µl of horseradish peroxidase at a concentration of 0.1 µM, from 10 to 50 µl of a decoction or infusion (depending on the concentration) and phosphate buffer in the amount required to bring the total sample volume to 1 ml. The cuvette was installed in the device and CL was recorded, observing the background signal. After 48 s of registration of the background signal, 100 µl of H2O2 at a concentration of 1 mM was added to the cuvette, and CL registration was continued for 10 min. Four samples were prepared with different concentrations of each of the plant objects. CL was also recorded for solutions of ascorbic acid, quercetin, and tocopherol at five different concentrations for each of the antioxidants. Subsequently, the TAU of the samples of decoctions and infusions was recalculated for quercetin.

The concentrations of luminol, horseradish peroxidase, and hydrogen peroxide were selected so as to determine the antioxidant capacity of aqueous extracts from medicinal plant materials in a reasonable time (no more than 10 min). During this time, the chemiluminescence curves for the antioxidants ascorbate and the flavonoid quercetin (the main antioxidants of plant materials) reached a plateau, which indicated the complete destruction of antioxidants in the system. The dilutions of the studied samples and the concentrations of solutions of standard antioxidants (indicated in the captions to the figures) were selected so that all CL kinetic curves were measured at the same instrument sensitivity.

The antioxidant capacity was calculated from the area change (∆ S) under the kinetic curve of chemiluminescence (light sum) with the addition of a substance containing an antioxidant. For this, we counted S0 for the system without antioxidant and subtracted from it the area S S characterizing the system to which the antioxidant was added. ∆ value S depends on the sensitivity of the chemiluminometer and measurement conditions. Ratio ∆ S/C V(where C- concentration of the studied biological material in the cuvette, g/l, and V- cuvette volume, l) expresses the antioxidant capacity of 1 g of the studied material, i.e., plant materials.

The antioxidant capacity ∆ S A a solution of a standard antioxidant, such as quercetin, placed in the same volume of the reaction mixture. Ratio ∆ S A /C A V(where C A- weight concentration of the antioxidant in the cuvette, g/l) expresses the antioxidant capacity of 1 g of the antioxidant.

For each of the standard antioxidants, the signal from solutions of several concentrations was recorded to make sure that the calculations were carried out within the limits of a linear relationship, and the results obtained were reproducible. Indeed, a linear dependence was obtained (∆ S A = k A C A) signal from the concentration from which the stoichiometric coefficient was calculated kA. According to the Fisher criterion, the values obtained for standard antioxidants kA statistically significant with a probability of 0.975. Next, the signal from four concentrations was recorded for each of the four plant samples, and for all samples a linear dependence of the signal on concentration (∆ S = k C), which was used to calculate the stoichiometric coefficient k. With a probability of 0.975 (Fischer's test), the k values obtained for plant samples are statistically significant. The total antioxidant capacity of the plant material in terms of the weight of the standard antioxidant (mg%) was found using the formula .

Values were presented as arithmetic mean ± standard deviation (M ± δ) at p

RESULTS OF THE STUDY

Study of chemiluminescence kinetics in the presence of sodium ascorbate (Fig. 1. Effect of sodium ascorbate on chemiluminescence kinetics" data-note="Concentrations of system components: luminol - 40 µM, horseradish peroxidase - 4 nM, hydrogen peroxide - 100 µM. Curves: 1 - control sample; 2 - 0.05 µM; 3 - 0.10 µM; 4 - 0.15 µM; 5 - 0.2 µM; 6 - 0.25 µM sodium ascorbate. antioxidant is characterized by a latent period when CL is almost completely suppressed. Its duration is proportional to the amount of antioxidant in the system. At the same time, neither the slope of the CL curves nor the intensity of CL on the plateau changes. This is due to the fact that ascorbic acid is a strong antioxidant that intercepts all radicals formed in the system, including luminol radicals, and CL does not develop until all ascorbate is oxidized.

Other researchers have also shown that the results of chemical analysis and the TAU value determined by the chemiluminescent method often do not match. In the work, the total antioxidant capacity determined in the peroxidase–luminol–hydrogen peroxide system correlated with the content of triterpene compounds. However, in the work of the same authors, in which another plant was the object of study, no correlation was observed between TAU and the content of any group of substances, including flavonoids.

These discrepancies are related to at least three factors. First, the activity of antioxidants is important, i.e., the rate of their interaction with radicals, which is different for different antioxidants that make up the plant sample. According to Izmailov, the rate constants of the corresponding reactions for mexidol, tocopherol and quercetin are related as 0.04: 2: 60. Secondly, each antioxidant molecule, entering into a chemical reaction, can intercept a different number of radicals. According to the work , quercetin, uric and ascorbic acids intercepted 3.6 ± 0.1, 1.4 ± 0.1 and 0.5 ± 0.2 radicals per reacted antioxidant molecule, respectively (the hemin–H 2 O 2 system was used – luminol). Thirdly, the results of the study could be influenced by the presence of peroxidase activity in the plant samples themselves, as in the work, as well as the presence of calcium in the samples, which, as shown in the work, is capable of increasing the activity of horseradish peroxidase under certain conditions. This usually causes a higher CL intensity on the plateau than on the control curves, which, however, we did not observe.

The first factor sharply limits the use of such a parameter as a change in the light sum, since the time of chemiluminescence measurement should be longer than the time of consumption of all antioxidants in the test sample. The approach of this moment can be judged only by measuring the chemiluminescence kinetics. In addition, the contribution of weak antioxidants to OAE is sharply underestimated, since the time of their complete oxidation is many times longer than the acceptable measurement time (10–20 min).

Of even greater importance is the stoichiometric coefficient of the antioxidant. Number of radicals n, intercepted by it, is equal to , where ρ - stoichiometric coefficient, and ∆ m- change in the concentration of the antioxidant during the measurement, in our case - the initial concentration of the test substance in the test sample.

The difference in the light sum of the glow in the absence of an antioxidant and in its presence is proportional to n. The total number of intercepted radicals is , where ρ i is the stoichiometric coefficient of a particular antioxidant, and m i- its concentration during the measurement. The total number of intercepted radicals is obviously not equal to the total amount of antioxidants, since the coefficients ρ i are not only not equal to unity, but also differ significantly for different antioxidants.

Value n is proportional to the difference in light sums measured over a certain time between a sample containing an antioxidant and a control sample containing no antioxidants: S = k n, where k- coefficient constant under the same measurement conditions.

The method considered in the article allows determining the total antioxidant capacity, while chemical analysis allows determining the total content of antioxidants in the product. Therefore, the chemiluminescence method seems to be more informative than chemical analyses.

The conditions we have selected for assessing the total antioxidant capacity of plant raw materials by recording the kinetics of chemiluminescence in a system consisting of horseradish peroxidase, hydrogen peroxide and luminol (component concentrations are 4 nM, 100 μM and 40 μM, respectively; 20 mM phosphate buffer, pH 7.4), provided oxidation of strong antioxidants (ascorbic acid) and moderate antioxidants (quercetin) in 10 min. This duration of measurement is convenient and ensures the required quality of measurements.

An analysis of the chemiluminescence kinetics showed that in the studied objects (decoctions of rowan, wild rose, hawthorn fruits and raspberry fruit infusion), the main antioxidants are medium-strength antioxidants, including flavonoids, and weak-strength antioxidants (tocopherol, etc.). Based on the decrease in the chemiluminescence light sum, the total antioxidant capacity for the studied objects was calculated. Comparison of the obtained TAU values with the results of chemical analysis showed that products containing the same amount of antioxidants with different ratios may differ in their ability to effectively protect the body from the harmful effects of free radicals. The described technique is promising for studying plant objects containing a mixture of various antioxidants. At the same time, it is characterized by simplicity and low cost of research. Combining the measurement of chemiluminescence kinetics with mathematical modeling of reactions will not only automate the process of determining TAU, but also determine the contribution of individual groups of antioxidants to the index.

graduate work

1.4 Research methods for antioxidants

antioxidant activity are classified: according to the methods of registration of the manifested AOA (volumetric, photometric, chemiluminescent, fluorescent, electrochemical); by type of oxidation source; by type of oxidized compound; according to the method of measuring the oxidized compound.

However, the most well-known methods for determining antioxidant activity are:

1 TEAC (trolox equivalent antioxidant capacity): the method is based on the following reaction:

Metmyoglobin + H 2 O 2 > Ferrylglobin + ABTS > ABTS * + AO.

The Trolox Equivalence Method (TEAC) is based on the ability of antioxidants to reduce 2,2-azinobis radical cations (ABTS) and thereby inhibit absorption in the long wavelength part of the spectrum (600 nm). A significant disadvantage of the method is the two-stage reaction of obtaining a radical. This lengthens the time of analysis and may increase the scatter of results, despite the fact that a standardized set of reagents is used for analysis.

2 FRAP (ferric reducing antioxidant power): the method is based on the following reaction:

Fe(III)-Tripyridyltriazine+AO>Fe(II)-Tripyridyltriazine.

Iron reducing/antioxidant capacity (FRAP). Here, the reduction reaction of Fe(III)-tripyridyltriazine to Fe(II)-tripyridyltriazine is used. However, this method cannot determine some antioxidants, such as glutathione. This method allows the direct determination of low molecular weight antioxidants. At low pH, the reduction of the Fe(III) tripyridyltriazine complex to the Fe(II) complex is accompanied by the appearance of an intense blue color. The measurements are based on the ability of antioxidants to suppress the oxidative effect of the reaction particles generated in the reaction mixture. This method is simple, fast and low cost in execution.

3 ORAC (oxygen radical absorbance capacity): the method is based on the following reaction:

Fe (II) + H 2 O 2 > Fe (III) + OH * + AO> OH * + Luminol.

Determination of the ability to absorb oxygen radicals (ORAC). In this method, the fluorescence of the substrate (phycoerythrin or fluorescein) is recorded, which occurs as a result of its interaction with ROS. If there are antioxidants in the test sample, then a decrease in fluorescence is observed compared to the control sample. This method was originally developed by Dr. Guohua Cao at the National Institute of Aging in 1992. In 1996, Dr. Cao joined with Dr. Ronald Pryer in a joint group at the USDA Research Center for Aging, where a semi-automated method was developed.

4 TRAP (total radical trapping antioxidant parameter): the method is based on the following reaction:

AAPH+AO>AAPH* + PL (PE).

This method uses the ability of antioxidants to interact with the peroxyl radical 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH). TRAP modifications consist in methods for registering an analytical signal. Most often, at the final stage of the analysis, the AAPH peroxy radical interacts with a luminescent (luminol), fluorescent (dichlorofluorescein diacetate, DCFH-DA), or other optically active substrate.

The water-soluble vitamin E derivative Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxy acid) is used as a standard for the TEAC, ORAC and TRAP methods.

Recently, interest in the use of electrochemical methods has increased for the evaluation of antioxidant activity. These methods are highly sensitive and fast analysis.

The evaluation of the antioxidant activity of some food products is carried out by the potentiometry method, based on the use of the property of antioxidant substances to participate in redox reactions due to enol (-OH) and sulfhydryl (-SH) groups.

The determination of the antioxidant properties of solutions is based on the chemical interaction of antioxidants with the mediator system, which leads to a change in its redox potential. The electrochemical cell is a container containing a K-Na-phosphate buffer solution, a mediator system Fe(III)/Fe(II) and a complex electrode before measuring the redox potential. Antioxidant activity is estimated in g-eq/L.

The amperometric method for determining antioxidant activity is based on measuring the electric current that occurs during the oxidation of the test substance on the surface of the working electrode, which is under a certain potential. The sensitivity of the amperometric method is determined both by the nature of the working electrode and by the potential applied to it. The detection limit of the amperometric detector of polyphenols, flavonoids at the level of nano-picograms, at such low concentrations, there is a lower probability of mutual influence of different antioxidants in their joint presence, in particular, the manifestation of the phenomenon of synergism. The disadvantages of the method include its specificity: under these conditions, antioxidants that themselves are oxidized or reduced in the region of oxygen electroreduction potentials cannot be analyzed. The advantages of the method include its rapidity, prostate and sensitivity.

Galvanostatic coulometry method using electrogenerated oxidants - the method is applicable to the analysis of fat-soluble antioxidants.

Various methods have been developed for the determination of ascorbic acid:

an amperometric method using an aluminum electrode modified with a film of nickel(II) hexacyanoferrate by a simple solution immersion method;

a method for solid-phase spectrophotometric and visual test determination of ascorbic acid using silicic acid xerogel modified with Wawel's reagent and copper (II) as an indicator powder;

chemiluminescent determination of ascorbic acid can be carried out by the flow-injection method according to the chemiluminescent reaction of rhodamine B with cerium (IV) in a sulfuric acid medium.

determination of ascorbic acid in the range of 10 -8 -10 -3 g/cm 3 by anodic voltammetry in aqueous and aqueous-organic media.

The most common is the FRAP method, as it is express, highly sensitive. Over the past few decades, a large number of varieties of methods for determining antioxidant activity by the FRAP method have been developed (table 1).

Table 1 Development of the FRAP method and its application to determine the antioxidant activity of various objects

	Objects of analysis	Notes
	blood plasma	t=4min. The reaction stoichiometry and additivity were studied.
	Tea, wine	Determination of AOA due to polyphenols
		AOA values of different types of tea are compared
Pulido, Bravo, Saura-Calixto	Model solutions	t=30min. The influence of non-aqueous solvent was revealed
	Plants
	blood, tissue	PIA method. The influence of foreign substances was checked.
Firuzi, Lacanna, Petrucci e.a.	Model solutions	The sensitivity of determination of different AOs as a function of their structure and redox potential was studied.
Katalinic, Milos,	Various wines
Temerdashev, Tsyupko and others.	Model mixtures
Loginova, Konovalova	Medicines. Preparations	test method
Temerdashev, Tsyupko and others.	Red dry wines	Correlation of AOA with other indicators of wine quality
Table 1 continued
	Model mixtures	The sensitivity of the determination of different AO
Vershinin, Vlasova, Tsyupko	Model mixtures	The non-additivity of the signal with a lack of an oxidizing agent was revealed
Anisimovich, Deineka and others.	Model solutions	Kinetic parameters for AOA estimation are proposed.

Notes: conventionally labeled: PIA-flow-injection analysis, TPTZ-tripyridyltriazine, DIP-2,2, -dipyridyl, PHEN-o-phenanthroline, DPA-pyridinedicarboxylic acid, FZ-ferrozine, AA-ascorbic acid, CT-catechol, t - exposure time, min.

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Keywords

free radical/antioxidant/ antioxidant activity / total antioxidant capacity / chemiluminescence/ luminol / free radical / antioxidant / antioxidant activity / total antioxidant capacity / chemiluminescence / luminol

annotation scientific article on chemical sciences, author of scientific article - Georgy Konstantinovich Vladimirov, E. V. Sergunova, D. Yu. Izmailov, Yu. A. Vladimirov

Medicinal plant materials are one of the sources of antioxidants for the human body. Among the methods for determining the content of antioxidants in plant objects, the method of chemiluminescent analysis is widespread. In the present work, it was used to estimate total antioxidant capacity(OAU) decoctions of rowan, wild rose and hawthorn fruits and infusion of raspberry fruits. Kinetics were recorded in the experiment chemiluminescence in a system consisting of horseradish peroxidase, hydrogen peroxide and luminol. The concentrations and volumes of the system components in the sample were chosen so that strong antioxidants (ascorbic acid) and moderately strong antioxidants (quercetin) were completely oxidized during the measurement time (10 min). A method for calculating the TAU based on a change in the light sum is proposed and justified. chemiluminescence in the presence of plant samples. Kinetic analysis chemiluminescence showed that in the studied objects, antioxidants of medium strength, including flavonoids, and weak antioxidants (tocopherol, etc.) predominate. Comparison of the calculated TAU values for the studied objects and the data of their chemical analysis showed that products containing the same amount of antioxidants with different ratios by types may differ in their ability to protect the body from the harmful effects of free radicals. The described technique is promising for the study of plant objects containing a mixture of antioxidants of various types.

Chemiluminescent determination of total antioxidant capacity in medicinal plant material

Medicinal plant material is one of the sources of antioxidants for the human body. Chemiluminescence analysis is one of the common methods of determining the content of antioxidants in plant materials. In our work, chemiluminescence analysis was used to determine the total antioxidant capacity (TAC) of fruit decoctions of mountain-ash, rose and hawthorn, as well as raspberry fruit infusion. Experiments established the kinetics of the chemiluminescence of a system consisting of horseradish peroxidase, hydrogen peroxide and luminol . Concentrations and volumes of components of the system were chosen such that strong antioxidants (ascorbic acid) and antioxidants of average force (quercetin) were completely oxidized during measurement (10 minutes). A method for TAC calculation based on changes in chemiluminescence light sum in the presence of plant samples was proposed and substantiated. Analysis of chemiluminescence kinetics showed that antioxidants of average force dominate in the objects studied, including flavonoids and weak antioxidants (tocopherol and others). Comparison of the calculated TAC values for the objects under study and their chemical analysis data showed that products containing the same amount of antioxidants with different ratios of antioxidants by types might vary in their ability to protect the body against the harmful effects of free radicals. The technique described is a promising one for the study of plant objects containing a mixture of different types of antioxidants.

The text of the scientific work on the topic "Chemiluminescent method for determining the total antioxidant capacity in medicinal plant materials"

chemiluminescent method for determining the total antioxidant capacity in medicinal plant materials

G. K. Vladimirov1^, E. V. Sergunova2, D. Yu. Izmailov1, Yu. A. Vladimirov1

1 Department of Medical Biophysics, Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow

2 Department of Pharmacognosy, Faculty of Pharmacy,

I. M. Sechenov First Moscow State Medical University, Moscow

Medicinal plant materials are one of the sources of antioxidants for the human body. Among the methods for determining the content of antioxidants in plant objects, the method of chemiluminescent analysis is widespread. In the present work, it was used to assess the total antioxidant capacity (TOA) of decoctions of rowan, wild rose, and hawthorn fruits and raspberry fruit infusion. In the experiment, the kinetics of chemiluminescence was recorded in a system consisting of horseradish peroxidase, hydrogen peroxide, and luminol. The concentrations and volumes of the system components in the sample were chosen so that strong antioxidants (ascorbic acid) and moderately strong antioxidants (quercetin) were completely oxidized during the measurement time (10 min). A method for calculating the RAE based on the change in the chemiluminescence light sum in the presence of plant samples is proposed and justified. An analysis of the chemiluminescence kinetics showed that moderately strong antioxidants, including flavonoids, and weak antioxidants (tocopherol, etc.) predominate in the studied objects. Comparison of the calculated TAU values for the studied objects and the data of their chemical analysis showed that products containing the same amount of antioxidants with different ratios by types may differ in their ability to protect the body from the harmful effects of free radicals. The described technique is promising for the study of plant objects containing a mixture of antioxidants of various types.

Keywords: free radical, antioxidant, antioxidant activity, total antioxidant capacity, chemiluminescence, luminol

Funding: This work was supported by the Russian Science Foundation, grant no. 14-15-00375.

Ex3 Correspondence should be addressed: Georgy Konstantinovich Vladimirov

119192, Moscow, Lomonosovsky pr-t, 31, building 5; [email protected]

Article received: 03/10/2016 Article accepted for publication: 03/18/2016

chemiluminescent determination of total antioxidant capacity in medicinal plant material

1 Department of Medical Biophysics, Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow, Russia

2 Department of Pharmacognosy, Faculty of Pharmacy,

The First Sechenov Moscow State Medical University, Moscow, Russia

Medicinal plant material is one of the sources of antioxidants for the human body. Chemiluminescence analysis is one of the common methods of determining the content of antioxidants in plant materials. In our work, chemiluminescence analysis was used to determine the total antioxidant capacity (TAC) of fruit decoctions of mountain-ash, rose and hawthorn, as well as raspberry fruit infusion. Experiments established the kinetics of the chemiluminescence of a system consisting of horseradish peroxidase, hydrogen peroxide and luminol. Concentrations and volumes of components of the system were chosen such that strong antioxidants (ascorbic acid) and antioxidants of average force (quercetin) were completely oxidized during measurement (10 minutes). A method for TAC calculation based on changes in chemiluminescence light sum in the presence of plant samples was proposed and substantiated. Analysis of chemiluminescence kinetics showed that antioxidants of average force dominate in the objects studied, including flavonoids and weak antioxidants (tocopherol and others). Comparison of the calculated TAC values for the objects under study and their chemical analysis data showed that products containing the same amount of antioxidants with different ratios of antioxidants by types might vary in their ability to protect the body against the harmful effects of free radicals. The technique described is a promising one for the study of plant objects containing a mixture of different types of antioxidants.

Keywords: free radical, antioxidant, antioxidant activity, total antioxidant capacity, chemiluminescence, luminol

Funding: this work was supported by the Russian Science Foundation, grant no. 14-15-00375.

Acknowledgments: authors thank Andrey Alekseev from Lomonosov Moscow State University for his assistance in conducting the experiment. Correspondence should be addressed: George Vladimirov

Lomonosovskiy prospekt, d. 31, k. 5, Moscow, Russia, 119192; [email protected] Received: 03/10/2016 Accepted: 03/18/2016

Free radicals generated in the body disrupt the structure of cell membranes, which, in turn, leads to the development of various pathological conditions. The destructive oxidative effect of radicals is prevented by the body's antioxidant defense system, in which low molecular weight compounds - radical scavengers (traps) play an important role. One of the sources of antioxidants is medicinal plant raw materials, as well as preparations based on it, the study of the antioxidant potential of which helps to increase their preventive and therapeutic effect.

The main methods for determining antioxidants are considered in the works, however, the definition of antioxidants as chemical compounds does not give a complete picture of the protective properties of the object under study: they are determined not only by the amount of one or another antioxidant, but also by the activity of each of them. Antioxidant activity, or antioxidant activity, AOA, is the rate constant for the reaction of an antioxidant with a free radical (kInH). The chemiluminescence (CL) method makes it possible to determine the total amount of radicals that antioxidants bind in the sample (total antioxidant capacity, TAU), and when using the method of mathematical modeling of CL kinetics, also the rate of formation and reaction of radicals with antioxidants, i.e. AOA.

The most common modification of the chemiluminescent method for determining the total antioxidant capacity is based on the use of luminol as a chemiluminescence activator. A sample is placed in the cuvette of the chemiluminometer with the addition of luminol, hydrogen peroxide, and a compound capable of generating radicals as a result of spontaneous decomposition (thermolysis), for example, 2,2"-azobis-(2-amidinopropane) dihydrochloride (ABAP):

In the presence of molecular oxygen, the alkyl radical R^ forms a peroxyl radical ROO^:

ROO^ + LH2 ^ ROOH + LHv From LH, through the formation of intermediate substances (luminol hydroperoxide and luminol endoperoxide), a molecule of the end product of luminol oxidation, aminophthalic acid, is formed in an electronically excited state, which emits a photon, and as a result, chemiluminescence is observed . The CL intensity is proportional to the photon production rate, which, in turn, is proportional to the stationary LH concentration in the system. Interacting with radicals, antioxidants interrupt the described chain of transformations and prevent the formation of a photon.

Compounds subject to thermolysis are not the only possible source of radicals in the analysis of the antioxidant capacity of a sample by the chemiluminescent method. Alternatives are horseradish peroxidase-hydrogen peroxide, hemin-hydrogen peroxide, cytochrome c-cardiolipin-hydrogen peroxide, etc. The scheme of reactions of luminol oxidation by peroxidases is considered in the work of Cormier et al. .

The CL kinetic curves for these systems reflect two stages of the reaction: the stage of an increase in the CL intensity and the stage of a plateau or a gradual decrease in luminescence, when

CL intensity is either constant or slowly decreasing. The paper describes two approaches to measuring the total antioxidant capacity that take into account this feature of the curves. The TRAP (Total Reactive Antioxidant Potential) method is based on measuring the CL latency t and can be used to determine anti-oxidants such as trolox or ascorbic acid: they are characterized by a high reaction rate constant with radicals and for this reason can be called strong antioxidants. . During the latent period, their complete oxidation occurs. The TAR method (Total Antioxidant Reactivity) measures the degree of quenching of chemiluminescence q at the plateau or at the maximum of the chemiluminescent curve:

where I is the intensity of chemiluminescence without an antioxidant, and 11 is the intensity of CL in the presence of an antioxidant. This method is used if the system contains predominantly weak antioxidants with low rate constants of interaction with radicals - much lower compared to the luminol constant.

The action of antioxidants is characterized not only by indicators of t and c. As can be seen from the works, the action of antioxidants such as uric acid in the hemin-H202-luminol system or tocopherol, rutin and quercetin in the cytochrome c-cardiolipin-H202-luminol system is characterized by a change in the maximum rate of CL rise (utx). As the results of mathematical modeling of kinetics show, the values of the rate constants of the interaction of these antioxidants with radicals are close to the value of the luminol constant, therefore, such antioxidants can be called medium-strength antioxidants.

DE = DE0 - DE,

where DE0 and DE5 are CL light sums for a given time? in the control and test samples, respectively. The time should be sufficient for the oxidation of all antioxidants in the system, that is, for the CL curve of the test sample to reach the level of the CL curve of the control sample. The latter suggests that researchers should not only record the light sum of the luminescence, but also record the CL kinetics curve for a sufficiently long time, which is far from always done.

Since all measured indicators depend on the instrument and measurement conditions, the antioxidant effect of a substance in the system under study is usually compared with the effect of an antioxidant taken as a standard, such as Trolox.

The system horseradish peroxidase-hydrogen peroxide was used to analyze the total antioxidant capacity of plant materials by many authors. In the works, the amount of antioxidants in the samples was estimated using the CL latent period (TRAP method), and in the works, the area under the CL development curve was used. However, these works do not give a clear justification

the choice of one or another parameter for estimating the OAU.

The aim of the study was to determine how the ratio of antioxidants of various types affects the TAU, and to modify the chemiluminescence method in such a way as to be able to more accurately determine the TAU in plant materials. To do this, we have set ourselves several tasks. First, to compare the CL kinetics of the studied objects with the kinetics of standard antioxidants of three types (strong, medium, and weak) in order to understand which type of antioxidants make the main contribution to the TAE of the studied objects. Secondly, to calculate the TAE of the studied objects by measuring the decrease in the CL light sum under the action of these objects in comparison with the action of the antioxidant, which provides the greatest contribution to the TAE.

MATERIALS AND METHODS

The objects of the study were industrial samples of hawthorn, mountain ash and wild rose fruits produced by Krasnogorskleksredstva JSC (Russia), as well as raspberry fruits collected by the authors in the Moscow region under natural growing conditions and dried at a temperature of 60-80 ° C until they stop isolating juice and pressure deformations.

The reagents for the analysis of antioxidant capacity by the chemiluminescent method were: KH2PO4, 20 mM buffer solution (pH 7.4); peroxidase from horseradish roots (activity 112 U/mg, M = 44 173.9), 1 mM aqueous solution; luminol (5-amino-1,2,3,4-tetrahydro-1,4-phthalazinedione, 3-aminophthalic acid hydrazide, M=177.11), 1 mM aqueous solution; hydrogen peroxide (H2O2, M = 34.01), 1 mM aqueous solution; solutions of antioxidants (ascorbic acid, quercetin, tocopherol). All reagents were manufactured by Sigma Aldrich (USA).

The total antioxidant capacity was determined by recording chemiluminescence on a Lum-100 chemi-luminometer (DISoft, Russia) using PowerGraph 3.3 software. To determine the TAU in plant materials, 40 µl of luminol at a concentration of 1 mM, 40 µl of horseradish peroxidase at a concentration of 0.1 µM, from 10 to 50 µl of a decoction or infusion (depending on the concentration) and phosphate buffer in the amount required to bring the total sample volume to 1 ml. The cuvette was installed in the device and CL was recorded, observing the background signal. After 48 s of registration of the background signal, 100 μl of H2O2 at a concentration of 1 mM was added to the cuvette, and CL registration was continued for 10 min. Four samples were prepared with different concentrations of each of the plant objects. CL was also recorded for solutions of ascorbic acid, quercetin, and tocopherol in five different concentrations for each of the antioxidants. Subsequently, the TAU of the samples of decoctions and infusions was recalculated for quercetin.

reached a plateau, which indicated the complete destruction of antioxidants in the system. The dilutions of the studied samples and the concentrations of solutions of standard antioxidants (indicated in the captions to the figures) were selected in such a way that all CL kinetic curves were measured at the same instrument sensitivity.

The antioxidant capacity was calculated from the change in the area (AS) under the chemiluminescence kinetic curve (light sum) upon addition of a substance containing an antioxidant. To do this, we calculated S0 for the system without an antioxidant and subtracted from it the area SS, which characterizes the system to which the antioxidant was added. The AS value depends on the sensitivity of the chemiluminometer and measurement conditions. The ratio AS/C ■ V (where C is the concentration of the studied biological material in the cuvette, g/l, and V is the volume of the cuvette, l) expresses the antioxidant capacity of 1 g of the studied material, i.e., plant material.

The antioxidant capacity ASa of a solution of a standard antioxidant, for example, quercetin, placed in the same volume of the reaction mixture was calculated in a similar way. The ratio AS/CÄ ■ V (where CA is the weight concentration of the antioxidant in the cuvette, g/l) expresses the antioxidant capacity of 1 g of the antioxidant.

For each of the standard antioxidants, the signal from solutions of several concentrations was recorded to make sure that the calculations were carried out within the limits of a linear relationship, and the results obtained were reproducible. Indeed, a linear dependence (ASa = kA ■ CA) of the signal on concentration was obtained, from which the stoichiometric coefficient kA was calculated. According to the Fisher criterion, the kA values obtained for standard antioxidants are statistically significant with a probability of 0.975. Next, the signal from four concentrations was recorded for each of the four plant samples, and for all samples a linear dependence of the signal on concentration (AS = k ■ C) was obtained, from which the stoichiometric coefficient k was calculated. With a probability of 0.975 (Fischer's test), the k values obtained for plant samples are statistically significant. The total antioxidant capacity of the plant material in terms of the weight of the standard antioxidant (mg%) was found by the formula

OAU = k ■ 105. k

Values were presented as arithmetic mean ± standard deviation (M ± 5) at p<0,05.

RESULTS OF THE STUDY

The study of chemiluminescence kinetics in the presence of sodium ascorbate (Fig. 1) showed that this antioxidant is characterized by a latent period, when CL is almost completely suppressed. Its duration is proportional to the amount of antioxidant in the system. In this case, neither the slope of the CL curves nor the CL intensity on the plateau changes. This is explained by the fact that ascorbic acid is a strong antioxidant that intercepts all radicals formed in the system, including luminol radicals, and CL does not develop until all ascorbate is oxidized.

The action of tocopherol (Fig. 2) was manifested by a decrease in the intensity of CL on a plateau, which is typical for weak antioxidants, although tocopherol is considered one of the most

powerful antioxidants. Perhaps this discrepancy is due to the fact that in our experiment, free radicals were in an aqueous solution, while the effect of tocopherol is usually studied in nonpolar media. In the work , where the complex of cytochrome c with cardiolipin served as a source of radicals and the reaction with luminol proceeded within this complex, tocopherol had the properties of an antioxidant of medium strength.

Having studied the effect of various concentrations of quercetin on our system (Fig. 3) and comparing the kinetic curves for it and sodium ascorbate and tocopherol, it can be noted that the main effect of quercetin is manifested in a change in the slope of the curves, i.e., the rate of development of CL, which is typical for moderate antioxidants.

The CL curves for all the studied decoctions (Fig. 4) resemble the curves for quercetin with a slight decrease in the CL intensity at the end, i.e., upon reaching

Time, min

Rice. 1. Effect of sodium ascorbate on chemiluminescence kinetics

The concentrations of the system components: luminol - 40 μM, horseradish peroxidase - 4 nM, hydrogen peroxide - 100 μM. Curves: 1 - control sample; 2 - 0.05 μM; 3 - 0.10 μM; 4 - 0.15 μM; 5 - 0.2 μM; 6 - 0.25 μM sodium ascorbate.

plateau. As shown in the work , this behavior is typical for antioxidants of medium strength, which in our case include polyphenols - flavonoids and tannins. For an infusion of raspberry fruits (Fig. 4, D), a decrease in chemiluminescence at the plateau level is noticeable, which is typical for weak antioxidants, which in this case is tocopherol. In terms of quercetin and tocopherol, raspberry fruit infusion contains 4.7 ± 0.9 µmol/g of quercetin and 11.9 ± 0.8 µmol/g of tocopherol.

When comparing the chemiluminescence curves obtained for different concentrations of the four studied aqueous extracts from plant materials, it was shown that the contribution of medium and weak antioxidants to the total antioxidant capacity of the samples decreased in the following order: raspberry fruit infusion (Fig. 4, D), rosehip fruit decoction (Fig. 4, C), a decoction of rowan fruits (Fig. 4, A), a decoction of hawthorn fruits (Fig. 4, B). The AS values in terms of the concentration C of the studied substance in the cuvette and the values of the total antioxidant capacity in terms of quercetin are shown in the table.

THE DISCUSSION OF THE RESULTS

The data obtained during the experiments and the TAU values of the studied objects calculated on their basis were compared with the content of the main antioxidants in them, determined using chemical methods of analysis. Despite the fact that a positive correlation between the total amount of antioxidants and TAU in different objects is undeniable, there are noticeable differences between these indicators. For example, if we take the sum of the content of flavonoids, tannins and ascorbic acid, then it turns out to be more than the calculated TAU for all the studied objects, except for the decoction of hawthorn fruits (table).

46 Time, min

I" "h chi----.

Rice. 2. Effect of tocopherol on chemiluminescence kinetics

The concentrations of the system components: luminol - 40 μM, horseradish peroxidase - 4 nM, hydrogen peroxide - 100 μM. Curves: 1 - control sample; 2 - 0.01 μM; 3 - 0.025 μM; 4 - 0.06 μM; 5 - 0.1 μM; 6 - 0.2 μM tocopherol.

46 Time, min

Rice. Fig. 3. Effect of quercetin on chemiluminescence kinetics Concentrations of the system components: luminol - 40 μM, horseradish peroxidase - 4 nM, hydrogen peroxide - 100 μM. Curves: 1 - control sample; 2 - 0.02 μM; 3 - 0.03 μM; 4 - 0.04 μM; 5 - 0.05 μM; 6 - 0.06 μM quercetin.

Time, min

46 Time, min

120 I 100 80 \ 60 40 20

46 Time, min

Rice. Fig. 4. Influence of decoctions of rowan fruits (A), hawthorn (B), wild rose (C) and raspberry fruit infusion (D) on the chemiluminescence kinetics . (A) Curves: 1 - control sample; 2 - 0.002 g/l; 3 - 0.004 g/l; 4 - 0.006 g/l; 5 - 0.008 g/l decoction of rowan fruits. (B) Curves: 1 - control sample; 2 - 0.005 g/l; 3 - 0.0075 g/l; 4 - 0.01 g/l; 5 - 0.0125 g/l decoction of hawthorn fruits. (C) Curves: 1 - control sample; 2 - 0.001 g/l; 3 - 0.0015 g/l; 4 - 0.002 g/l; 5 - 0.0025 g/l decoction of rose hips. (D) Curves: 1 - control sample; 2 - 0.001 g/l; 3 - 0.003 g/l; 4 - 0.004 g/l; 5 - 0.005 g/l infusion of raspberries.

in the system peroxidase-luminol-hydrogen peroxide correlated with the content of triterpene compounds. However, in the work of the same authors, in which another plant was the object of study, no correlation was observed between TAU and the content of any group of substances, including flavonoids.

These discrepancies are related to at least three factors. First, the activity of antioxidants is important, i.e., the rate of their interaction with radicals, which is different for different antioxidants that make up the plant sample. According to Izmailov, the rate constants of the corresponding reactions for mexidol, tocopherol and quercetin are related as 0.04: 2: 60. Secondly, each antioxidant molecule, entering into a chemical reaction, can intercept a different number of radicals. According to the work, quercetin, uric and ascorbic acids intercepted 3.6 ± 0.1, 1.4 ± 0.1 and 0.5 ± 0.2 radicals per reacted antioxidant molecule, respectively (gemin-H202-luminol system was used) . Thirdly, the results of the study could be influenced by the presence of peroxidase activity in the plant samples themselves, as in the work, as well as the presence of calcium in the samples, which, as shown in the work, is capable of increasing the activity of horseradish peroxidase under certain conditions. This usually results in more

higher CL intensity on the plateau than on the control curves, which, however, we did not observe.

Of even greater importance is the stoichiometric coefficient of the antioxidant. The number of radicals n intercepted by them is equal to

where p is the stoichiometric coefficient, and Am is the change in the concentration of the antioxidant during the measurement time, in our case, the initial concentration of the test substance in the test sample.

The difference in the light sum of the luminescence in the absence of an antioxidant and in its presence is proportional to n. The total number of intercepted radicals is n = Y.p. m,

where is the stoichiometric coefficient of a particular antioxidant, and m is its concentration during the change

Object of study Flavonoids, mg%* Tannins, mg%* Ascorbic acid, mg%* AS/C ■ 10-8, arb. units OAU, mg% quercetin

Decoction of rowan fruits 8.87 ± 0.01 210.00 ± 10.00 0.67 ± 0.02 7.13 ± 0.96 56.53 ± 7.61

Decoction of rose hips 4.66 ± 0.04 850.00 ± 20.00 3.70 ± 0.12 16.60 ± 3.40 131.63 ± 27.26

Decoction of hawthorn fruits 3.01 ± 0.06 12.00 ± 3.00 0.23 ± 0.002 3.18 ± 0.29 25.20 ± 2.32

Infusion of dried raspberries 90.00 ± 4.00 40.00 ± 20.00 3.91 ± 0.08 6.65 ± 1.21 52.69 ± 9.56

Note: * - literature data, . AS - change in the light sum for the sample, rel. units, C - concentration of the sample in the cuvette, g/l. The calculated values are reliable at p<0,05. Число измерений для каждого образца - четыре.

rhenium. The total number of intercepted radicals is obviously not equal to the total amount of antioxidants, since the coefficients pt are not only not equal to unity, but also differ significantly for different antioxidants.

The value of n is proportional to the difference in light sums measured over a certain time between a sample containing an antioxidant and a control sample containing no antioxidants:

where k is a coefficient that is constant under the same measurement conditions.

The conditions we selected for assessing the total antioxidant capacity of plant raw materials by recording the kinetics of chemiluminescence in a system consisting of horseradish peroxidase, hydrogen peroxide, and luminol (component concentrations are 4 nM, 100 μM, and 40 μM, respectively; 20 mM phosphate buffer, pH 7.4 ),

ensured the oxidation of strong antioxidants (ascorbic acid) and medium-strength antioxidants (quercetin) in 10 min. This duration of measurement is convenient and ensures the required quality of measurements.

Literature

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11. Izmailov DYu, Demin EM, Vladimirov YuA. Opredelenie aktivnosti antioksidantov metodom izmereniya kinetiki khemilyuminestsen-tsii. Fotobiologiya i fotomeditsina. 2011; 7(2):70-6. Russian.

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14. Lissi EA, Escobar J, Pascual C, Del Castillo MD, Schmitt TH, Di Mascio P. Visible chemiluminescence associated with the reaction between methemoglobin or oxyhemoglobin with hydrogen peroxide. Photochem Photobiol. Nov 1994; 60(5):405-11.

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of the luminescent peroxidation of luminol by stopped flow techniques. J Biol Chem. 1968 Sep 25; 243(18): 4706-14.

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Lekarstvennoe rastitel "noe syr" e", Moscow: Medltsina, 1987, p. 147-8. Russian.

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26. Sergunova EV, Sorokina AA, Avrach AS. Izuchenie plodov boyaryshnika razlichnykh sposobov konservatsii i vodnykh izvlechenii. Farmatsia. 2010; (5): 16-8. Russian.

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28. Avrach AS, Samylina IA, Sergunova EV. Izuchenie biologicheski aktivnykh veshchestv plodov boyaryshnika - syr "ya dlya prigotovleniya nastoek gomeopaticheskikh matrichnykh. Proceedings of the 14th Moscow International Homeopathic Conference "Razvitie gomeopaticheskogo metoda v sovremennoi meditsine"; 2014 Jan 24-25; Moscow. M.; 7. Russian.

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30. Aleksandrova EYu, Orlova MA, Neiman PL. Izuchenie peroksidaznoi aktivnosti v ekstraktakh iz kornevishcha i kornei khrena i ee stabil "nosti k razlichnym vozdeistviyam. Moscow University Chemistry Bulletin. 2006; 47 (5): 350-2. Russian.

1 Milentiev V.N. 2Sannikov D.P. 3Kazmin V.M. 2

1 Oryol State Institute of Economics and Trade

2 Federal State Budget Institution "Center for Chemicalization and Agricultural Radiology "Orlovsky"

3 Federal State Budgetary Educational Institution of Higher Professional Education "State University - Educational, Scientific and Industrial Complex"

The possibility of using chemiluminescence to assess the antioxidant activity of food substances was studied. The proposed method is based on the chemiluminescence of luminol in an alkaline medium, the intensity of which depends on the amount of peroxides in the chemiluminescent sample. Chemiluminescence was recorded using a developed setup containing a dosing pump, a light-tight chamber, a glass vacuum photomultiplier tube, and a computer system. To enhance chemiluminescence, a solution of potassium ferricyanide was added to luminol. Changes in the intensity of chemiluminescence were recorded at the moment of introduction of the analyzed sample into the luminol solution. Dandelion extract obtained by dry low-temperature distillation was used as the analyzed sample. It contains phenolic compounds known for their high antioxidant activity. It has been established that the chemiluminescence method can be used to determine the antioxidant properties of various food compounds.

chemiluminescence

antioxidant activity

peroxides

nutrients

1. Vasiliev R.F. Chemical glow // Chemistry and chemists, 21.01.10. – URL: http://chemistry-chemists.com. (date of access: 22.08.13).

2. Vladimirov Yu.A. Free radicals and antioxidants // Vestn. RAMN. - 1998. - No. 7. - P. 43-51.

3. Kondrashova E.A. Chemiluminescence as the most sensitive method of enzyme immunoassay and its application. Clinical laboratory diagnostics. - 1999. - No. 9. - P. 32.

4. Lyubimov, G.Yu. Chemiluminescent analysis // Immunology. - 1991. - No. 1. - P. 40–49.

5. Mayansky A.N., Nevmyatullin A.L., Chebotar I.V. Reactive chemiluminescence in the phagocytosis system // Microbiology. - 1987. - No. 1. - S. 109–115.

6. Sherstnev M.P. Calcium-dependent and calcium-independent pathways of cell chemiluminescence generation. Chemiluminescence Issues. - 1991. - No. 2. - S. 1–4.

Today, chemiluminescence is a large area of science located at the interface between chemistry, physics and biology. With chemiluminescence, there is a direct conversion of chemical energy into the energy of electromagnetic oscillations, i.e. into the world. Using chemiluminescence, one can learn about how the reaction proceeds, what is its mechanism, which is necessary for the efficient and rational conduct of technological processes. If the technological process of obtaining any chemical product is accompanied by chemiluminescence, then its intensity can serve as a measure of the rate of the process: the faster the reaction, the brighter the glow. During the chemiluminescence reaction, energy-rich products are obtained, which then give off energy by emitting light, i.e., chemical energy is converted into electromagnetic radiation energy.

The aim of the study was to explore the possibility of using chemiluminescence to assess the antioxidant activity of food substances.

Research results and discussion

The problem of assessing the antioxidant activity of food substances is very relevant. The use of the term "antioxidant activity" in order to show the usefulness of a particular product is often done without any chemical and biochemical argument. As a rule, the antioxidant activity of any substance means the effectiveness of reducing the peroxide value. The very concept of peroxide value also does not fully reveal its chemical essence, since it does not fully correspond to the kinetics and thermodynamics of the stages of metabolism of a particular food product. In addition, this value is used to characterize lipids in the form of fats. However, the processes of oxidation and the formation of peroxides in the body occur not only with the use of fats, but also with other products. In other words, the content of peroxide in a particular product can be said to be “weighed” on a kind of balance, where the “reference weight” is a unit of concentration in an acidic environment of an iodide ion oxidized by peroxides, as a result of which molecular iodine is formed:

I- - e → I; (one)

I + I → I20. (2)

When molecular iodine is titrated with a solution containing sodium thiosulfate, its concentration is established and, consequently, the amount of oxidizing agents of iodide ions is determined, i.e. peroxide compounds, which is actually called peroxide number. Determining the peroxide value using this kind of "weighing" is based on the reaction shown in fig. one.

Rice. 1. Determination of peroxide value using sodium thiosulfate

Thus, the concentration of peroxides is determined from the equation

С(I2) = ϒ(C[-O-O-]), (3)

where ϒ is the correlation coefficient between the concentration of molecular iodine and the concentration of peroxides.

The proposed method for determining peroxides in products is based on the chemiluminescence of luminol (C[lm]) in an alkaline medium, the intensity (Ichl) of which depends on the concentration of peroxides (C[-O-O-]), in a chemiluminescent sample:

IHL. = Ϧchl ω, (4)

where Ϧchl is the quantum yield of chemiluminescence; ω - reaction rate involving peroxides:

khlC[-O-O-] C[lm] = ω, (5)

where kchl is the reaction rate constant or at:

C[lm] kchl Ϧchl = K, (6)

IХЛ = K C[-O-O-]. (7).

The amount of peroxides (-O-O-) is determined by the light sum (S):

The value of S depends on the degree of completeness of peroxide consumption in the chemiluminescent reaction.

To determine the constant K, a calibration curve is constructed for the dependence of the light sum S on the peroxide concentration, which is determined by titration:

S = f(C[-O-O-]). (nine)

Hydrogen peroxide H2O2 is used as peroxides.

Then the data obtained from equation (3) and (9) are compared. Based on the comparison of ϒ and K, a conclusion is made about the coordination of the reaction mechanisms underlying the determination of peroxides by these methods. It was found that in this range of peroxide concentrations ϒ and K indeed agree with each other and therefore they can be used to determine the peroxide value .

Chemiluminescence was observed in an alkaline medium containing luminol (5-amino-1,2,3,4-tetrahydro-1,4-phthalazinedione, 3-aminophthalic hydrazide, H2L). It was recorded using a chemiluminescent setup, including a glass vacuum photomultiplier. The photomultiplier is powered by a high-voltage rectifier (7) coupled to a block (9) that amplifies the photomultiplier signal, which is recorded on the computer monitor display (5).

Rice. 2. Registration of chemiluminescence of the analyzed product: 1 - dosing pump; 2 - lightproof chamber; 3 - mirror; 4 - cuvette; 5 - computer system; 6 - photomultiplier; 7 - high voltage rectifier; 8 - a device that allows you to determine the spectral region of chemiluminescent radiation; 9 - block amplifying the photomultiplier signal

A dosing pump (1) is required to introduce the analyzed sample into a cuvette (4) containing a chemiluminescent solution of luminol. This dispenser acts as a stirrer for the injected sample with a chemiluminescent solution. To enhance the reaction rate and intensity of chemiluminescence, a solution of potassium ferricyanide was added to luminol. Mixing is carried out by air bubbles obtained by pumping air through the solution liquid with a pump. The mirror (3) located in the opaque chamber (2) serves for better light collection of the chemiluminescent radiation incident on the photocathode of the photomultiplier (6) mounted in the opaque chamber. The dispenser allows you to enter the desired components of the liquid into the cuvette without opening the light-tight chamber (2) during the experiments. In this case, these liquids enter the cuvette (4) through glass or plastic tubes. The computer system allows you to register the dependence of the luminescence intensity I on time t, that is, the chemiluminescence kinetics:

The computer system reflects the rise and fall constants in the function I = f(t), which are conjugated with the rate constants of the reactions that cause chemiluminescence, that is, with their kinetics. A device (8) is included in the chemiluminescent chamber, which makes it possible to determine the spectral region of chemiluminescent radiation, that is, the dependence:

I = f1(λ). (eleven)

This block is a cassette in the form of a disk, in which boundary filters are mounted. The change of light filters is carried out by turning the disc cassette about the horizontal axis connecting the centers of the plane of the light filters and the plane of the photocathode of the photomultiplier.

The measurement process is carried out as follows:

1. The response of the photomultiplier to changes in its supply voltage and to changes in the intensity of the reference light source that falls on its cathode is set.

2. The cuvette is filled with a solution of luminol in an alkaline medium.

3. The dispenser is filled with the analyzed sample.

4. The dependence of the intensity of chemiluminescence on time t is recorded. Chemiluminescence is monitored until the time t1, at which the change in I1 from time t is minimal: I1 = f1(t).

5. A portion of the analyzed solution is fed using a dispenser.

6. Chemiluminescence of the analyzed sample is observed, the kinetics of which is I = f(t).

On fig. Figure 3 shows a graph of the dependence of functions (I1 = f1(t)), conjugated with a graph (I = f(t)), after the introduction of the analyzed solution.

As can be seen from fig. 3, the intensity of luminol chemiluminescence changes: a sharp rise is followed by a sharp decrease in luminescence after the addition of the analyzed sample.

Since the enhancement of chemiluminescence during the oxidation of luminol is associated with the formation of peroxides, the decrease in the intensity of chemiluminescence after the introduction of the analyzed sample indicates a decrease in their number. Therefore, we can speak about the presence of antioxidant activity in the compounds that make up the analyzed sample.

It should be noted that the dandelion extract obtained by dry low-temperature distillation, which contains phenolic compounds known for their high antioxidant activity, was used as the analyzed sample.

Rice. Fig. 3. Dependence graph of functions (I1 = f1(t)), conjugated with the graph (I = f(t)), after the introduction of the analyzed solution

In addition, during the experiment it was found that using chemiluminescence it is possible to determine the amount of peroxides in superdiluted systems, which is important for assessing the onset of oxidation of products, for example, during their storage.

Thus, the conducted studies have shown that the method for determining peroxides in products, based on the chemiluminescence of luminol in an alkaline medium, makes it possible to evaluate the antioxidant activity of food substances and can be used to establish the antioxidant properties of various food compounds.

Reviewers:

Litvinova E.V., Doctor of Technical Sciences, Professor of the Department of Technology, Organization and Food Hygiene, OrelGIET, Orel;

Kovaleva O.A., Doctor of Biological Sciences, Director of INITs, FSBEI HPE "Oryol State Agrarian University", Orel.

The work was received by the editors on November 08, 2013.

Bibliographic link

Panichkin A.V., Bolshakova L.S., Milentiev V.N., Sannikov D.P., Kazmin V.M. USE OF CHEMILUMINESCENCE FOR EVALUATION OF ANTIOXIDANT PROPERTIES OF NUTRIENTS // Fundamental Research. - 2013. - No. 10-11. – S. 2436-2439;
URL: http://fundamental-research.ru/ru/article/view?id=32810 (date of access: 12/17/2019). We bring to your attention the journals published by the publishing house "Academy of Natural History"

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