Determination of total antioxidant activity. Thesis: antioxidant properties of dihydroquercetin. The text of the scientific work on the topic "Chemiluminescent method for determining the total antioxidant capacity in medicinal plant materials"

19.09.2023

The invention relates to the food industry and can be used in determining 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) reacts with the same reagent, which is added in a ratio of 1:100. Next, incubate for at least 90 minutes and photometer at 510 ± 20 nm. After this, the dependence of the magnitude of the analytical signal on the amount of the substance is established and the value of the total AOA is calculated. The presented method makes it possible to less labor-intensively and more reliably determine the total antioxidant activity of plant materials and food products based on them. 2 salary f-ly, 1 ill., 5 tables.

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.

There is a 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. Budnikov. Coulometric assessment of the antioxidant capacity of tea extracts with electrically generated bromine // Journal of Analyst. Chemistry 2001. T.56. No. 6. P.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 at multiple bonds. This allows us to cover a wide range of biologically active tea compounds that have antioxidant properties. The disadvantages of this method are the possibility of a bromination reaction with substances that are not antioxidants, and the expression of the resulting value of total AOA in units of electricity (kC/100 g), which makes it difficult to evaluate the results obtained.

There is a known voltammetric method for determining the total antioxidant activity by the relative change in the current of electroreduction of oxygen in the potential range from 0.0 to -0.6 V (rel. sat. h.s.e.) on a mercury film electrode (Pat. 2224997, Russia, MPC 7 G 01 N 33/01. Voltammetric method for determining the total activity of antioxidants / Korotkova E.I., Karbainov Yu.A. - application 06.06.2004; The disadvantage of this method is that side electrochemical reactions occur, as a result of which the efficiency of determining antioxidants decreases, which leads to a decrease in the reliability of the results.

There is a known method for monitoring the total AOA of preventive and therapeutic antioxidant agents by lipid peroxidation to malonaldehyde with spectrophotometric or chemiluminescent detection (Pat. 2182706, Russia, IPC 7 G 01 N 33/15, 33/52. Method for monitoring the antioxidant activity of preventive and therapeutic antioxidants funds / Pavlyuchenko I.A., Fedosov S.R. - no. 2001101389/14; publ. Moreover, 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 only one group of antioxidants is determined - lipids.

There is a known method for determining the total AOA of a plant extract, which consists of incubating the extract with linetol and iron (II) sulfate, initiating an oxidation reaction by 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 general antioxidant activity / Rogozhin V.V. - Appl. 07/08/1997; When carrying out spectrophotometry, a mixture of ethanol and 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 pro-oxidant. The disadvantage of this method is the possibility of side reactions occurring during UV irradiation, which reduces the reliability of the obtained analysis results.

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 to and is not comparable with any generally accepted 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 during the reaction with luminol in the presence of the oxidizing agent hydrogen peroxide (M.H. Navas, A.M. Khiminets, A.G. Azuero Determination of the reducing ability of tinctures of Canary seed canary by chemiluminescence // Journal of analytical chemistry. 2004. T.59. P.84-86). To quantify the total AOA, the reducing ability of the extract of medicinal raw materials and the activity of a potent antioxidant - ascorbic acid in an amount of 25-110 μg were compared. In comparison with the listed methods, the prototype uses hydrogen peroxide as an oxidizing agent, which interacts 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 generally accepted antioxidant, which allows one 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 to develop a less labor-intensive 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 value on the amount of substance and calculating the value of the total AOA. In particular, the calculation can be carried out using 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 AC;

a", b" - coefficients in the regression equation for the dependence of the analytical signal on the amount of the object being studied;

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

The use of the proposed reagent under the specified conditions made it possible to expand the linear range and reduce the lower limit of the determined amounts of ascorbic acid. The proposed set of essential characteristics makes it possible to determine the total AOA of a wide range of plant materials and food products based on them.

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 test object under the condition of equal antioxidant activity.

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

Coefficients a and b are calculated using the formulas:

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

y AK = a x AK (mg) + b,

regression equation for the dependence of AC on the amount of the studied object (reducing agent):

y VOST =a" x VOST (mg)+b",

where at AK, at VOST is the optical density of the photometered solution;

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

then, equating the values of the functions, we obtain formula (I) for calculating the antioxidant activity of the studied object in units of quantity (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 using a KFK-2MP photoelectrocolorimeter.

It is known (F. Umland, A. Yasin, D. Tirik, G. Wünsch 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.

Optimization of the composition of the reagent and its quantity introduced into the reaction was carried out on the basis of the results of multifactorial experimental design using the “Latin square” method, which consisted in changing all the studied factors in each experiment, and each level of each factor occurs only once with different levels of other factors. This allows you to isolate and evaluate the effect caused by each factor under study separately.

The factors were: the amount 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 identify the following levels for each factor:

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

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

At the optimal concentration of the reagent, we studied the change in the dependence of AC 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 reducing agents studied, the dependence of AC on their content is linear in the range of 10-150 μg (see drawing) and the value of AC depends on the incubation time (Table 3).

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

Table 3 Change in AC of reducing agents over time
Test substance	m substance, mg/cm 3	Analytical signal
Test substance	m substance, mg/cm 3	Incubation time of the reaction mixture, min
30	60	90	120
Ascorbic acid	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 summative nature of the determined AOA value, the effect of the Fe (III) reagent - o-phenanthroline on model solutions, which included reducing agents: tannin, rutin, quercetin and ascorbic acid in various proportions, was studied. Table 4 presents the results of the analysis of model mixtures.

Table 4 Results of analysis of model mixtures (P=0.95; n=3)
Number of components in the mixture	Total AOA, calculated, µgAC	Total AOA, found, µgAC
introduced	Total AOA, calculated, µgAC	Total AOA, found, µgAC	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 using quantitative correspondence equations characterizing the antioxidant capacity of the studied reducing agent in relation 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 AC on the amount of ascorbic acid. From the results presented in Table 4, it is clear that the experimentally obtained AOA values are in satisfactory agreement with the theoretically calculated ones.

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

The proposed method was tested on real samples. To determine the total AOA of a real sample or its extract, calibration dependences of AC on the amount of analyte and ascorbic acid were obtained with 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 using known methods, assessing the content of ascorbic acid (GOST 24556-89 Processed products of fruits and vegetables. Methods for determining vitamin C) and the predominant reducing agents: tannin in tea (GOST 19885-74 Tea. Methods for determining content tannin and caffeine), in rose hips - the amount of organic acids (GOST 1994-93 Rose hips. Technical conditions) (Table 5).

graduate work

1.4 Antioxidant research methods

antioxidant activity are classified: according to methods of recording the manifested AOA (volumetric, photometric, chemiluminescent, fluorescent, electrochemical); by type of oxidation source; by type of compound being oxidized; by 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 equivalents 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 region of the spectrum (600 nm). A significant disadvantage of the method is the two-step reaction to produce the radical. This lengthens the analysis time and can increase the scatter of results, despite the fact that a standardized set of reagents is used for the analysis.

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

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

Iron reducing/antioxidant capacity (FRAP). The reaction used here is the reduction of Fe(III)-tripyridyltriazine to Fe(II)-tripyridyltriazine. However, this method cannot determine the determination of 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 reaction species generated in the reaction mixture. This method is simple, fast and low-cost to implement.

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 oxygen radical absorbance capacity (ORAC). In this method, the fluorescence of a substrate (phycoerythrin or fluorescein), which arises as a result of its interaction with ROS, is recorded. If the test sample contains antioxidants, then a decrease in fluorescence is observed compared to the control sample. This method was originally developed by Dr. Guohua Kao at the National Institute on Aging in 1992. In 1996, Dr. Kao teamed up with Dr. Ronald Pryer in a joint group at the USDA Aging Research Center, where the semi-automated method was created.

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

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

This method takes advantage of the ability of antioxidants to interact with the peroxyl radical 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH). TRAP modifications consist in methods for recording the analytical signal. Most often, at the final stage of the analysis, the peroxy radical AAPH 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 to evaluate antioxidant activity. These methods have high sensitivity and rapid analysis.

The assessment 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.

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. An electrochemical cell is a container containing a K-Na-phosphate buffer solution, a Fe(III)/Fe(II) mediator system and a complex electrode before measuring the redox potential. Antioxidant activity is assessed 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 the potential applied to it. The detection limit of the amperometric detector of polyphenols and flavonoids is at the nano-picogram level; at such low concentrations, there is a lower probability of mutual influence of different antioxidants when they are present together, in particular the manifestation of the phenomenon of synergy. 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 speed, prostate and sensitivity.

Galvanostatic coulometry method using electrically generated oxidants - the method is applicable for the analysis of fat-soluble antioxidants.

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

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

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

chemiluminescent determination of ascorbic acid can be carried out by the flow-injection method using 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 rapid and highly sensitive. Over the past few decades, a large number of varieties of methods for determining antioxidant activity using 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 tea varieties were compared
Pulido,Bravo,Saura-Calixto	Model solutions	t=30min. The influence of a non-aqueous solvent was revealed
	Plants
	Blood, tissue	PIA method. The influence of foreign substances has been tested.
Firuzi, Lacanna, Petrucci e.a.	Model solutions	The sensitivity of determination of different AOs was studied as a function of their structure and redox potential.
Katalinic, Milos,	Various wines
Temerdashev, Tsyupko and others.	Model mixtures
Loginova, Konovalova	Medicines Medicines	Test method
Temerdashev, Tsyupko and others.	Dry red wines	Correlation of AOA with other indicators of wine quality
Continuation of Table 1
	Model mixtures	The sensitivity of determining different AOs was studied
Vershinin, Vlasova, Tsyupko	Model mixtures	The signal was found to be non-additive when there was a lack of oxidizing agent
Anisimovich, Deineka and others.	Model solutions	Kinetic parameters for assessing AOA have been proposed.

Notes: Conventionally designated: FIA-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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], however, the definition of antioxidants as chemical compounds does not give a complete picture of the protective properties of the object being studied: they are determined not only by the amount of a particular antioxidant, but also by the activity of each of them. Antioxidant activity, or antioxidant activity, AOA, is the rate constant of the reaction of an antioxidant with a free radical (kInH). The chemiluminescence (CL) method allows you to determine the total amount of radicals that bind antioxidants in a 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, that is, AOA [, ,].

The most common modification of the chemiluminescence method for determining the total antioxidant capacity is based on the use of luminol as a chemiluminescence activator [, , ,]. A sample is placed in a chemiluminometer cuvette with the addition of luminol, hydrogen peroxide and a compound capable of forming 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 the peroxyl radical ROO: R + O 2 → ROO. Next, 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 intermediate substances (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 rate of photon production, and it, in turn, is proportional to the stationary concentration of LH in the system. By interacting with radicals, antioxidants interrupt the described chain of transformations and prevent the formation of a photon.

Compounds susceptible to thermolysis are not the only possible source of radicals when analyzing the antioxidant capacity of a sample using the chemiluminescent method. Alternatives are the systems horseradish peroxidase–hydrogen peroxide [, ], hemin–hydrogen peroxide, cytochrome With–cardiolipin–hydrogen peroxide, etc. The reaction scheme for the oxidation of luminol 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 increasing CL intensity and the stage of a plateau or gradual decline in luminescence, when the CL intensity is either constant or slowly decreases. The work describes two approaches to measuring total antioxidant capacity that take into account this feature of the curves. The TRAP (Total Reactive Antioxidant Potential) method is based on measuring 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 reaction rate constant with radicals and for this reason can be called strong antioxidants. During the latent period, their complete oxidation occurs. The TAR (Total Antioxidant Reactivity) method measures the degree of chemiluminescence quenching q at the plateau or 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 in comparison with the constant of luminol.

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

If the material under study, 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 v max). But plant materials contain a mixture of antioxidants of varying strengths. To solve this problem, some authors [ , , , ] used the change in the light sum of chemiluminescence over a certain time ∆S, calculated by the formula , where ∆ S 0 and ∆ S S- CL light sums for a given time t in the control and test samples, respectively. The time must 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 assumes that researchers must not only record the light sum of the glow, but also record the CL kinetics curve for a sufficiently long time, which is not always done.

Since all measured indicators depend on the device and measurement conditions, the antioxidant effect of the 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 the works [,] to estimate the amount of antioxidants in the samples, the latent period of CL (TRAP method) was used, and in the works [, ,] - the area under the CL development curve. However, the listed works do not provide a clear justification for the choice of one or another parameter for assessing OAU.

The purpose of the study was to determine how the ratio of different types of antioxidants affects TOA, and to modify the chemiluminescence method in such a way as to be able to more accurately determine TOA in plant materials. To do this, we set ourselves several tasks. First, 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 OAU of the studied objects. Secondly, calculate the OAE of the objects under study by measuring the decrease in the CL light sum under the influence of these objects in comparison with the effect of the antioxidant that provides the greatest contribution to the OAE.

MATERIALS AND METHODS

The objects of the study were industrial samples of hawthorn, rowan and rose hips produced by JSC Krasnogorskleksredstva (Russia), as well as raspberry fruits collected by the authors in the Moscow region under natural growth conditions and dried at a temperature of 60–80 ° C until they stopped releasing juice and deformation when pressed.

The reagents for analyzing the antioxidant capacity using the chemiluminescent method were: KH 2 PO 4, 20 mM buffer solution (pH 7.4); peroxidase from horseradish roots (activity 112 units/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 are manufactured by Sigma Aldrich (USA).

Decoctions of hawthorn, rowan and rosehip fruits and an infusion of raspberry fruits were prepared according to the methods of the State Pharmacopoeia of the USSR, set out in the general pharmacopoeial article “Infusions and decoctions”.

The determination of total antioxidant capacity was carried out by recording chemiluminescence on a Lum-100 chemiluminometer (DISoft, Russia) using PowerGraph 3.3 software. To determine OAE 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 decoction or infusion (depending on the concentration) and phosphate buffer in the amount required were placed in the cuvette of the device. 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 recording the background signal, 100 μl of H2O2 at a concentration of 1 mM was added to the cuvette and CL recording was continued for 10 min. Four samples were prepared with different concentrations of each plant object. CL was also recorded for solutions of ascorbic acid, quercetin and tocopherol at five different concentrations for each antioxidant. Subsequently, the OAU of the samples of decoctions and infusions was recalculated to 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 an acceptable 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, indicating the complete destruction of antioxidants in the system. Dilutions of the studied samples and concentrations of solutions of standard antioxidants (indicated in the legends to the figures) were selected in such a way that all CL kinetic curves were measured at the same sensitivity of the device.

Antioxidant capacity was calculated from the change in area (∆ S) under the kinetic curve of chemiluminescence (light sum) when adding a substance containing an antioxidant. For this purpose we calculated S 0 for a system without an antioxidant and subtracted the area from it 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 biological material under study in the cuvette, g/l, and V- cuvette volume, l) expresses the antioxidant capacity of 1 g of the material being studied, i.e. plant raw materials.

The antioxidant capacity ∆ was calculated in a similar way S A a solution of a standard antioxidant, for example 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 antioxidant.

For each of the standard antioxidants, the signal from solutions of several concentrations was recorded to ensure that the calculations were within 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 k A. According to the Fisher criterion, the values obtained for standard antioxidants k A 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 was obtained (∆ S = k·C), from which the stoichiometric coefficient was calculated k. With a probability of 0.975 (Fisher’s test), the k values obtained for plant samples are statistically significant. The total antioxidant capacity of plant material in terms of the mass of the standard antioxidant (mg%) was found using the formula.

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

RESEARCH RESULTS

Study of the kinetics of chemiluminescence in the presence of sodium ascorbate (Fig. 1. Effect of sodium ascorbate on the kinetics of chemiluminescence" 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 - 0.15 µM; 5 - 0.2 µM sodium ascorbate.">Fig. 1) showed that for this The 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 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.

Other researchers have also shown that the results of chemical analysis and the TAU value determined by the chemiluminescent method often do not coincide. 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 the object of study was another plant, they did not observe a correlation of OAE with the content of any group of substances, including flavonoids.

Such discrepancies are associated with at least three factors. Firstly, the activity of antioxidants is important, i.e. the rate of their interaction with radicals, which is different for different antioxidants included in the plant sample. According to Izmailov, the rate constants of the corresponding reactions for mexidol, tocopherol and quercetin correlate as 0.04: 2: 60. Secondly, each antioxidant molecule, entering 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 we, however, did not observe.

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

The stoichiometric coefficient of the antioxidant is even more important. Number of radicals n intercepted by it is equal to , where ρ is the 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 luminescence in the absence of an antioxidant and in its presence is proportional n. The total number of intercepted radicals is , where ρ i is the stoichiometric coefficient of a specific antioxidant, and m i- its concentration during measurement. The total number of intercepted radicals is obviously not equal to the total amount of antioxidants, since the coefficients ρ i not only are not equal to unity, but also differ significantly for different antioxidants.

Magnitude 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 discussed in the article allows us to determine the total antioxidant capacity, while chemical analysis allows us to determine the total content of antioxidants in the product. Therefore, the chemiluminescence method seems to be more informative than chemical analyses.

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 - 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 minutes. This measurement duration is convenient and ensures the required measurement quality.

Analysis of the kinetics of chemiluminescence showed that in the studied objects (decoctions of rowan fruits, rose hips, hawthorn and infusion of raspberry fruits) the main antioxidants are antioxidants of medium strength, including flavonoids, and weak strength (tocopherol, etc.). Based on the decrease in 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 that contain a mixture of various antioxidants. At the same time, it is characterized by simplicity and low cost of research. The combination of measuring chemiluminescence kinetics with mathematical modeling of reactions will make it possible not only to automate the process of determining TAU, but also to determine the contribution of individual groups of antioxidants to the indicator.

Antioxidants (AO)- substances that prevent oxidation. In a living organism, the leading factor of oxidation is the formation of free radicals, therefore the action of antioxidants in biological systems is considered primarily from the standpoint of preventing the oxidation of organic substances by free radicals.

Currently, there are a large number of different methods for determining antioxidants: photometric, chemical, electrochemical, etc. However, many of them have significant drawbacks that make it difficult to understand and further use the results obtained by these methods. The most common disadvantages include the following:

Artificial or uncharacteristic conditions for biological systems are used to measure the antioxidant effect. For example, instead of biological free radical reactions, purely chemical redox reactions are used, or the ability of a substance to give/accept electrons when exposed to electric current is measured. The measurement results obtained under such conditions do not allow us to say whether the substance under study will exhibit the same “antioxidant” effect in the body.
Determination of antioxidant effect is carried out by measuring the amount of accumulated oxidation products (oxidation markers). In this way, it is indeed possible to determine the amount of antioxidant in the test sample, but very important information about the activity of the antioxidant is missed. Ignoring the activity of an antioxidant, in turn, can lead to significant errors in determining its amount, for example, for “weak” antioxidants that act slowly but over a long period of time.

In general, in the field of antioxidant determination there is no standardization that allows the results obtained by different methods to be compared.

Chemiluminescent method is the most informative method for studying antioxidants and has a number of significant advantages:

Direct determination of antioxidant effect- the direct effect of antioxidants on free radicals is recorded. The chemiluminescent method uses a chemical system for generating free radicals, which gives a control chemiluminescent glow. An antioxidant is then added to such a system, which neutralizes free radicals, which leads to the suppression of control chemiluminescence.
A significant advantage of this approach is the possibility of using various chemical systems for the generation of free radicals, which makes it possible to further determine the specificity of antioxidants and the localization of their action.
Measuring quantitative and qualitative characteristics of antioxidants- the chemiluminescent method allows you to characterize any compound that has an antioxidant effect by two independent indicators:
- Antioxidant Capacity (AOE)- the total amount of free radicals that can neutralize a compound contained in a sample of a certain volume.
- Antioxidant Activity (AOA)- the rate of neutralization of free radicals, i.e. the number of radicals neutralized per unit time.

Chemiluminescent method gives an important understanding that the effect of antioxidants must be assessed by two indicators - quantitative (AOE) and qualitative (AOA).
The following figure demonstrates this situation:

Effect of different antioxidants on chemiluminescence
(the numbers next to the graphs indicate the antioxidant concentration):
on the left is a “strong” antioxidant, on the right is a “weak” antioxidant.

Antioxidants differ significantly in their activity. There are “strong” antioxidants, i.e. Highly active antioxidants that inhibit free radicals at a high rate and completely suppress chemiluminescence. Such antioxidants have a maximum effect even at low concentrations and are quickly consumed. On the other hand, there are “weak” antioxidants, i.e. Low potency antioxidants that inhibit free radicals at a low rate and only partially inhibit chemiluminescence. Such antioxidants have a significant effect only in high concentrations, but at the same time they are slowly consumed and act for a long time.

The chemiluminescent method can be used to determine antioxidant indicators:

biological fluids (plasma, saliva, urine);
pharmacological drugs and dietary supplements;
drinks and food additives;
cosmetics and care products;
and etc.

To implement the chemiluminescent method for determining antioxidants, it is recommended to use the following equipment:

Chemiluminometer Lum-100 - provides temperature control and registration of chemiluminescence of 1 sample.
Chemiluminometer Lum-1200 - provides temperature control and simultaneous registration of chemiluminescence for up to 12 samples.

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 the scientific work - 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 chemiluminescence analysis is widespread. In this work it was used to estimate total antioxidant capacity(OAE) decoctions of rowan, rosehip and hawthorn fruits and infusion of raspberry fruits. The kinetics were recorded in the experiment chemiluminescence in a system consisting of horseradish peroxidase, hydrogen peroxide and luminol. The concentrations and volume of system components in the sample were selected so that strong antioxidants (ascorbic acid) and moderate antioxidants (quercetin) were completely oxidized during the measurement time (10 min). A method for calculating OAE based on changes in light sum is proposed and justified chemiluminescence in the presence of plant samples. Kinetics analysis chemiluminescence showed that in the studied objects, medium-strength antioxidants predominate, including flavonoids, and weak antioxidants (tocopherol, etc.). A comparison of the calculated OAE 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 type may differ in their ability to protect the body from the harmful effects of free radicals. The described technique is promising for studying 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.

Text of 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

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

2 Department of Pharmacognosy, Faculty of Pharmacy,

First Moscow State Medical University named after I.M. Sechenov, 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 chemiluminescence analysis is widespread. In this work, it was used to assess the total antioxidant capacity (TAC) of decoctions of rowan, rosehip and hawthorn fruits and infusion of raspberry fruits. In the experiment, the kinetics of chemiluminescence was recorded in a system consisting of horseradish peroxidase, hydrogen peroxide and luminol. The concentrations and volume of system components in the sample were selected so that strong antioxidants (ascorbic acid) and moderate antioxidants (quercetin) were completely oxidized during the measurement time (10 min). A method for calculating OAE based on changes in the light sum of chemiluminescence in the presence of plant samples has been proposed and justified. Analysis of chemiluminescence kinetics showed that medium-strength antioxidants, including flavonoids, and weak antioxidants (tocopherol, etc.) predominate in the studied objects. A comparison of the calculated OAE 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 type may differ in their ability to protect the body from the harmful effects of free radicals. The described technique is promising for studying plant objects containing a mixture of antioxidants of various types.

Key words: free radical, antioxidant, antioxidant activity, total antioxidant capacity, chemiluminescence, luminol

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

Ex3 For correspondence: 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

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: Georgiy Vladimirov

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

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

The main methods for determining antioxidants are discussed 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 a particular antioxidant, but also by the activity of each of them. Antioxidant activity, or antioxidant activity, AOA, is the rate constant of 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 bind antioxidants in a sample (total antioxidant capacity, TCA), and when using the method of mathematical modeling of CL kinetics, also the rate of formation and reaction of radicals with antioxidants, that is, AOA.

The most common modification of the chemiluminescence method for determining the total antioxidant capacity is based on the use of luminol as a chemiluminescence activator. A sample is placed in a chemiluminometer cuvette with the addition of luminol, hydrogen peroxide and a compound capable of forming 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 the peroxyl radical ROO^:

ROO^ + LH2 ^ ROOH + LHv From LH, through the formation of intermediate substances (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 rate of photon production, and it, in turn, is proportional to the stationary concentration of LH in the system. By interacting with radicals, antioxidants interrupt the described chain of transformations and prevent the formation of a photon.

Compounds susceptible to thermolysis are not the only possible source of radicals when analyzing the antioxidant capacity of a sample using the chemiluminescent method. Alternatives are horseradish peroxidase-hydrogen peroxide, hemin-hydrogen peroxide, cytochrome c-cardiolipin-hydrogen peroxide, etc. The reaction scheme for the oxidation of luminol by peroxidases is discussed in the work of Cormier et al. .

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

CL intensity is either constant or slowly decreases. The work describes two approaches to measuring total antioxidant capacity that take into account this feature of the curves. The TRAP (Total Reactive Antioxidant Potential) method is based on measuring the latent period of CL t and can be used to determine antioxidants 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 (Total Antioxidant Reactivity) method measures the degree of chemiluminescence quenching q at the plateau or maximum of the chemiluminescent curve:

where I is the chemiluminescence intensity without an antioxidant, and 11 is the CL intensity 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 in comparison with the constant of luminol.

The effect of antioxidants is characterized not only by indicators t and c. As can be seen from the works, the effect of antioxidants such as uric acid in the hemin-H2O2-luminol system or tocopherol, rutin and quercetin in the cytochrome c-cardiolipin-H2O2-luminol system is characterized by a change in the maximum rate of increase in CL (utx). As the results of mathematical modeling of kinetics show, the values of the rate constants of interaction of these antioxidants with radicals are close to the value of the luminol constant, therefore such antioxidants can be called antioxidants of medium strength.

DE = DE0 - DE,

where DE0 and DE5 are the CL light sums for a given time? in the control and test samples, respectively. The time must 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 assumes that researchers must not only record the light sum of the glow, but also record the CL kinetics curve for a sufficiently long time, which is not always done.

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

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

the choice of one or another parameter for assessing OAU.

MATERIALS AND METHODS

The objects of the study were industrial samples of hawthorn, rowan and rose hips produced by JSC Krasnogorskleksredstva (Russia), as well as raspberry fruits collected by the authors in the Moscow region under natural growth conditions and dried at a temperature of 60-80 ° C until they stopped releasing juice and deformation when pressed.

The reagents for analyzing the antioxidant capacity using the chemiluminescent method were: KH2PO4, 20 mM buffer solution (pH 7.4); peroxidase from horseradish roots (activity 112 units/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 are manufactured by Sigma Aldrich (USA).

The determination of total antioxidant capacity was carried out by recording chemiluminescence on a Lum-100 chemiluminometer (DISoft, Russia) using PowerGraph 3.3 software. To determine OAE 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 decoction or infusion (depending on the concentration) and phosphate buffer in the amount required were placed in the cuvette of the device. 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 recording the background signal, 100 μl of H2O2 at a concentration of 1 mM was added to the cuvette and CL recording was continued for 10 min. Four samples were prepared with different concentrations of each plant object. CL was also recorded for solutions of ascorbic acid, quercetin, and tocopherol at five different concentrations for each of the antioxidants. Subsequently, the OAU of the samples of decoctions and infusions was recalculated to quercetin.

reached a plateau, indicating the complete destruction of antioxidants in the system. Dilutions of the studied samples and 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 sensitivity of the device.

The antioxidant capacity was calculated from the change in area (AS) under the kinetic curve of chemiluminescence (light sum) upon addition of a substance containing an antioxidant. To do this, we calculated S0 for a system without an antioxidant and subtracted from it the area SS, which characterizes the system to which the antioxidant was added. The value of AS 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 raw materials.

In a similar way, we calculated the antioxidant capacity ASa of a solution of a standard antioxidant, for example, quercetin, placed in the same volume of the reaction mixture. 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 antioxidant.

For each of the standard antioxidants, the signal from solutions of several concentrations was recorded to ensure that the calculations were within a linear relationship and the results obtained were reproducible. Indeed, a linear dependence (ASa = kA ■ CA) of the signal on the 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 the concentration was obtained (AS = k ■ C), from which the stoichiometric coefficient k was calculated. With a probability of 0.975 (Fisher’s test), the k values obtained for plant samples are statistically significant. The total antioxidant capacity of plant material in terms of the mass of the standard antioxidant (mg%) was found using the formula

OAE = k ■ 105. k

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

RESEARCH RESULTS

A study of the kinetics of chemiluminescence 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 effect of tocopherol (Fig. 2) was manifested by a decrease in the CL intensity on the 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, whereas the effect of tocopherol is usually studied in nonpolar media. In a study where the source of radicals was a complex of cytochrome c with cardiolipin and the reaction with luminol took place within this complex, tocopherol had the properties of a medium-strength antioxidant.

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 medium strength 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., when reaching

Time, min

Rice. 1. Effect of sodium ascorbate on chemiluminescence kinetics

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.

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 the infusion of raspberry fruits (Fig. 4, D), there is a noticeable decrease in chemiluminescence at the plateau level, which is typical for weak antioxidants, which in this case is tocopherol. In terms of quercetin and tocopherol, the raspberry infusion contains 4.7 ± 0.9 µmol/g quercetin and 11.9 ± 0.8 µmol/g 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 series: raspberry infusion (Fig. 4, D), rose hip decoction (Fig. 4, B), decoction of rowan fruits (Fig. 4, A), decoction of hawthorn fruits (Fig. 4, B). The AS values based on the concentration C of the studied substance in the cuvette and the values of the total antioxidant capacity in terms of quercetin are given in the table.

THE DISCUSSION OF THE RESULTS

The data obtained during the experiments and the OAE 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 the positive correlation between the total amount of antioxidants and TAU in different objects is undeniable, there are still 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 greater than the calculated TAU for all studied objects, except for the decoction of hawthorn fruits (table).

Other researchers have also shown that the results of chemical analysis and the TAU value determined by the chemiluminescent method often do not coincide. In operation, the total antioxidant capacity, determined

46 Time, min

I" "h chi----.

Rice. 2. Effect of tocopherol on chemiluminescence kinetics

Concentrations of 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. 3. Effect of quercetin on the kinetics of chemiluminescence Concentrations of 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. 4. Effect of decoctions of rowan fruits (A), hawthorn (B), rose hips (C) and infusion of raspberry fruits (D) on the kinetics of chemiluminescence. Concentrations of system components: luminol - 40 µM, horseradish peroxidase - 4 nM, hydrogen peroxide - 100 µM . (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. (B) Curves: 1 - control sample; 2 - 0.001 g/l; 3 - 0.0015 g/l; 4 - 0.002 g/l; 5 - 0.0025 g/l rosehip decoction. (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 raspberry infusion.

in the peroxidase-luminol-hydrogen peroxide system correlated with the content of triterpene compounds. However, in the work of the same authors, in which the object of study was another plant, they did not observe a correlation of OAE with the content of any group of substances, including flavonoids.

Such discrepancies are associated with at least three factors. Firstly, the activity of antioxidants is important, i.e. the rate of their interaction with radicals, which is different for different antioxidants included in the plant sample. According to Izmailov, the rate constants of the corresponding reactions for mexidol, tocopherol and quercetin correlate as 0.04: 2: 60. Secondly, each antioxidant molecule, entering 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-H2O2-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 leads to more

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

The stoichiometric coefficient of the antioxidant is even more important. The number of radicals n intercepted by it 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 the antioxidant and in its presence is proportional to n. The total number of intercepted radicals is equal to n = Y.p. m,

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

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

Rowan fruit decoction 8.87 ± 0.01 210.00 ± 10.00 0.67 ± 0.02 7.13 ± 0.96 56.53 ± 7.61

Rosehip decoction 4.66 ± 0.04 850.00 ± 20.00 3.70 ± 0.12 16.60 ± 3.40 131.63 ± 27.26

Hawthorn fruit decoction 3.01 ± 0.06 12.00 ± 3.00 0.23 ± 0.002 3.18 ± 0.29 25.20 ± 2.32

Infusion of dried raspberry fruits 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 light sum for the sample, rel. units, C - sample concentration in the cuvette, g/l. The calculated values are reliable at p<0,05. Число измерений для каждого образца - четыре.

Rhenia. The total number of intercepted radicals is obviously not equal to the total amount of antioxidants, since the pt coefficients 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.

We selected the conditions 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 - 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 minutes. This measurement duration is convenient and ensures the required measurement quality.

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Determination of total antioxidant activity. Thesis: antioxidant properties of dihydroquercetin. The text of the scientific work on the topic "Chemiluminescent method for determining the total antioxidant capacity in medicinal plant materials"

1.4 Antioxidant research methods

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

Related topics scientific works in chemical sciences, author of the scientific work - Georgy Konstantinovich Vladimirov, E. V. Sergunova, D. Yu. Izmailov, Yu. A. Vladimirov

Chemiluminescent determination of total antioxidant capacity in medicinal plant material

Text of scientific work on the topic “Chemiluminescent method for determining the total antioxidant capacity in medicinal plant materials”