RU2426109C1 - Voltammetric method of determining activity of antioxidants - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method enables to measure activity of antioxidants with respect to OH radicals, which are most dangerous to the body. When realising the method during reduction of substance on an electrode in the solution of a background electrolyte, a model reaction is employed, which generates OH radicals. Cathode waves of reducing the substance are recorded in potential interval of -1.2 - -1.9 V relative a saturated silver chloride comparison electrode. A peak appears in the said interval, which is presumably of OH radicals and which falls in proportion to the amount of antioxidant added. Activity of antioxidants is determined from the relative drop of this peak.

EFFECT: invention increases sensitivity of the method of determining activity of antioxidants 277 times.

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Description

The invention relates to the field of determining the activity of antioxidants and can be used in the food, cosmetic, pharmaceutical industries, as well as in medicine and forensics.

A known method for determining the total antioxidant activity (AOA) of biologically active substances (RF patent No. 2238554, IPC ⁷ G01N 33/15, G01N 27/26, publ. 20.10.2004), including the preparation of samples of the analyzed and standard substances, their electrochemical oxidation in the cell amperometric detector, amplification of electrical signals, their registration and calculation of AOA according to the proposed mathematical dependence. In this method, the preparation of the analyzed samples is carried out by mixing the corresponding substances with a solvent that does not have antioxidant ability. In addition, substances with a high redox potential selected from the range of rutin, quercetin, dihydroquercetin are used as standard substances.

The main disadvantages of this method are "relatively low sensitivity, as well as the complexity of the hardware design, because the method requires a pump, a thermostatic electrochemical cell, a dispenser tap. In addition, in this method, the oxidation of antioxidants is carried out electrochemically on the surface of the working electrode, which occurs deep oxidation, not characteristic of processes occurring in the body.

Also known is the voltammetric method we selected for the prototype for determining the total activity of antioxidants (RF patent No. 2224997, IPC ⁷ G01N 33/00, publ. 02.27.2004), including the process of reducing a substance on an electrode in an electrolyte solution. In this method, the process of oxygen electroreduction is used as a model reaction, which is carried out on a mercury-film electrode against a background of 0.1 M Na ₂ SO ₄ in an aqueous medium, 0.1 M NaClO ₄ in an aprotic medium, or 0.9% NaCl in biological objects the cathode waves of one-electron oxygen reduction are recorded in differentiation mode at a potential sweep speed of 50-100 mV / s, the total antioxidant activity is determined by the relative change in the oxygen electroreduction current in the potential range from 0 to -0.6 V rel relatively saturated silver chloride reference electrode.

The main disadvantage of this method is the low sensitivity. In addition, the method does not objectively assess the activity of antioxidants in relation to processes occurring in the body, because using the model reaction indicated in the prototype, it is impossible to determine the activity of antioxidants in relation to ^. OH to the radicals. As shown in the work (Yu.A. Vladimir Vladimirov. Free radicals in biological systems // Soros Educational Journal. - 2000. - No. 12, t.6. - P.16), free harm is most harmful to health ^. OH radicals, because "In nucleic acids ^. It destroys carbohydrate bridges between nucleotides and, thus, breaks the DNA and RNA chains, as a result of which mutations and cell death occur. So the radical ^. HE is a radical destroyer, a radical killer. "

The main technical result of the proposed invention is to increase the sensitivity of the method for determining the activity of antioxidants by 277 times. In addition, the proposed method allows to measure the activity of antioxidants in relation to the most dangerous for the body ^. OH to the radicals.

The indicated technical result is achieved in that in the proposed voltammetric method for determining the activity of antioxidants, including the use of a model reaction in the process of reducing a substance on an electrode in a background electrolyte solution, registering cathodic waves of reduction of a substance in a given potential range relative to a saturated silver-silver reference electrode, adding an antioxidant and determination of antioxidant activity by relative change in current is electroreduced according to the proposed solution, a generating reaction is used as a model reaction ^. OH radicals, and cathode waves are recorded in the range of potentials -1.2 ÷ -1.9 V.

In the proposed method for determining the activity of antioxidants using the method of voltammetry. The measurement of the current-voltage dependences of the reduction of the substance is carried out using a voltammetric analyzer or polarograph of any type, which is connected to an electrochemical cell. The electrochemical cell consists of a container into which the background electrolyte solution is poured and two electrodes are lowered - a working one and a reference electrode. Well-conducting aqueous solutions of strong electrolytes are used as background solutions: KCl, Na ₂ SO ₄ , acetate or citrate buffers, as well as non-aqueous solutions of alcohols, in particular ethylene glycol with the addition of tetraalkyl ammonium bromide (TAAB) for electrical conductivity. As the electrodes, a silver-silver reference electrode is used, as a working electrode, an electrode inert in the working range of potentials of -1.2 ÷ -1.9 V, for example, mercury film, mercury spherical, film bismuth and film lead on graphite substrates. Typically, a mercury film electrode is used on a silver substrate. The prepared electrodes are connected to the analyzer, lowered into an electrochemical cell with 10-20 ml of a background solution, and a voltammogram of the background is recorded. Then conduct a model reaction generating ^. OH radicals using one of the model systems: Fe ²⁺ -EDTA, Cu ²⁺ -EDTA, Fe ²⁺ -HPO ₄ ^2- , Na ₂ SO ₃ -O ₂ and Fe ²⁺ -H ₂ O ₂ (Fenton reaction ) The experimental technique consists in measuring the voltammetric dependences of the cathodic current of electroreduction in the potential region of -1.2 ÷ -1.9 V. When carrying out a model generating reaction in the background electrolyte solution ^. OH radicals, a new cathode wave appears on voltammograms, presumably ^. OH radical. At the used sweep speeds, the cathode wave has a peak shape with a maximum of -1.4 ÷ -1.6 V, depending on the background. Then, a sample of soluble antioxidant or the measured volume of its solution of known concentration is added to the background electrolyte solution, and the voltammetric dependence is re-measured. In this case, the magnitude of the new cathode wave, presumably ^. OH radical decreases in proportion to the amount of antioxidant added.

Evaluation of antioxidant ability (K) is carried out by comparing the height of the cathode wave, presumably ^. OH radical, before (I ₀ , μA) and after (It, μA) the introduction of an antioxidant according to the formula:

K = X / C · V, g ^-1 , where X = (I ₀ -It) / I ₀ , C is the concentration of the applied antioxidant solution, g / ml, V is the volume of the applied antioxidant solution, ml

An example of a specific implementation 1.

Prepare the device - voltammetric analyzer type STA-1 (manufactured by ITM LLC, Tomsk), combined with a computer, to work. For this, the type of polarography is established: constant current, potential sweep speed of 50 mV / s, initial potential - 0 V, potential sweep end -1.9 V. Scan to the cathode region from 0 to -1.9 V. Mercury is used as a working electrode. -film, and silver chloride as a reference electrode.

When implementing the method, 10 ml of a background electrolyte solution, namely 0.1 M Na ₂ SO _{4 is} poured into a glass cup (electrochemical cell), the electrodes are lowered into it and connected to the device. After this, a voltammogram is recorded (Fig. 1) in the background solution in the indicated potential region, registering the cathode waves of oxygen and hydrogen peroxide (D. Skug, West D. Fundamentals of Analytical Chemistry / Transl. From English under the editorship of Yu.A. Zolotov. - M .: Mir, 1979.- P.69). Then conduct a model reaction generating ^. OH radicals using the model system Fe ²⁺ -EDTA. To do this, add 0.03 ml of a 0.01 M solution of ethylenediaminetetraacetic acid (EDTA) and 0.025 ml of a 0.01 M solution of Mohr's salt - FeSO ₄ · (NH ₄ ) 2SO ₄ · 6H ₂ O into the cell with a dispenser. Mix the solution by vibration of the electrodes in within 5 s. Moreover, according to (XFYang, XQGuo. Fe (II) -EDTA Chelate-Induced Aromatic Hydroxylation of Terephtalate as a New Method for the Evalution of Hydroxil Radical-Scavenging Ability // The Analist. - 2001. - # 126. - P. 926-932), generation ^. OH radicals flows from oxygen with the participation of iron (II) complexes with EDTA. After calming the solution for 5 s, a voltammogram is recorded and the disappearance of the cathode wave of H ₂ O _{2 is} recorded, and the appearance of a new cathode wave is possible ^. OH radical, in the range of potentials -1.3 ÷ -1.8 V, with a maximum at -1.52 V (figure 2). Then, 0.045 ml of a solution containing 45 μg of the antioxidant ascorbic acid is added to the electrochemical cell and mixed. Next, a voltammogram is recorded and a decrease in the cathode wave at -1.52 V and its shift by 20 mV to the positive region is recorded, i.e. at -1.50 V (figure 3). The height of the cathode waves is estimated at -1.52 ÷ -1.50 V before (Io, μA) and after (It, μA) application of the antioxidant and antioxidant activity is calculated by the formula:

K = X / C · V, g ^-1 ,

where X = (I ₀ -It) / I ₀ , C is the concentration of the introduced antioxidant solution, g / ml, V is the volume of the introduced antioxidant solution, ml

X = (4.41-2.46) / 4.4 = 0.44, C = 1 · 10 ^-3 g / ml, V = 0.045 ml,

K = 0.44 / 1 · 10 ^-3 · 0.045 = 9.8 · 10 ³ g ^-1 .

Using the Fe ²⁺ -EDTA model system, against the background of 0.1 M Na ₂ SO ₄ , the antioxidant activity of other antioxidants is also determined: sodium sulfite and glucose, and against the background of 0.1 M KCl, the activity of ascorbic acid, sodium sulfite, pyrogallol, pyrocatechol is determined and resorcinol (see table 1). The obtained AOA values for ascorbic acid, pyrogallol, pyrocatechol and resorcinol satisfactorily correlate with the prototype method.

Similar results were obtained at various concentrations of the background electrolyte solution of Na ₂ SO ₄ : 0.2 M, 0.5 M and 1 M.

An example of a specific implementation 2.

Prepare the device for operation as described in example 1.

10 ml of a background electrolyte solution, namely 0.1 M KCl, is poured into the electrochemical cell, the electrodes are lowered into it and connected to the device. After this, a voltammogram is recorded in the background solution, registering the cathode waves of oxygen and H ₂ O ₂ (Fig. 4, curve 1). Then conduct a model reaction generating ^. OH radicals using the model system Cu ²⁺ -EDTA. To do this, 0.03 ml of 0.01 M EDTA solution and 0.025 ml of 0.01 M copper salt solution - CuSO ₄ · 5H ₂ O are added to the cell with a dispenser. The solution is stirred for 5 s. With this generation ^. OH radicals flows from oxygen with the participation of copper complexes with EDTA (Christelle Hureau, Peter Faller. Aβ-mediated ROS production by Cu ions: Structural insights, mechanisms and relevance to Alzheimer's disease // Biochimie. - 2009. - V.91, Issue 10 . - P.1212-1217). After calming the solution for 5 s, a voltammogram is recorded and the disappearance of the cathode wave of H ₂ O _{2 is} recorded, and the appearance of a new cathode wave is possible ^. OH radical, in the range of potentials -1.2 ÷ -1.7 V with a maximum at -1.48 V (figure 4, curve 2). Then, 0.06 ml of a solution containing 60 μg of the antioxidant ascorbic acid is added to the electrochemical cell and mixed. The voltammogram is recorded and the decrease and shift of the cathode wave at -1.45 V are recorded (Fig. 4, curve 3). The height of the cathode waves is estimated at -1.48 ÷ -1.45 V before (I ₀ ) and after (It) application of the antioxidant, and antioxidant K activity is calculated by the formula given in Example 1:

K = X / C · V = (4.49-2.62) / 4.49 / 1 · 10 ^-3 · 0.06 = 6.9 · 10 ³ g ^-1 .

Using the model system Cu ^+2- EDTA, the activity of ascorbic acid is also determined against a different background - 0.1 M Na ₂ SO ₄ (see Table 1).

Table 1 Sample No. Background solution Model system Antioxidant Antioxidant. activity, K · 10 ³ , g ^-1 Interval of potentials of registration of cathode waves, V one 2 3 four 5 6 one Na ₂ SO ₄ Fe ²⁺ -EDTA Vitamin C 9.80 -1.3 ÷ -1.8 2 Na ₂ SO ₄ Fe ²⁺ -EDTA Glucose 0.51 -1.3 ÷ -1.8 3 Na ₂ SO ₄ Fe ²⁺ -EDTA Sodium sulfite 2.05 -1.3 ÷ -1.8 four Na ₂ SO ₄ Cu ²⁺ -EDTA Vitamin C 7.15 -1.3 ÷ -1.75 5 Kcl Cu ²⁺ -EDTA Vitamin C 6.90 -1.2 ÷ -1.7 6 Kcl Fe ²⁺ -EDTA Catechol 0.21 -1.2 ÷ -1.7 7 Kcl Fe ²⁺ -EDTA Vitamin C 8.20 -1.2 ÷ -1.7 8 Kcl Fe ²⁺ -EDTA Sodium sulfite 1.73 -1.2 ÷ -1.7 9 Kcl Fe ²⁺ -EDTA Resorcinol 0.13 -1.3 ÷ -1.7 10 Kcl Fe ²⁺ -HPO ₄ ^2- Vitamin C 4.09 -1.3 ÷ -1.65 eleven Kcl Fe ²⁺ -H ₂ O ₂ Vitamin C 5.48 -1.3 ÷ -1.7 12 Kcl Na ₂ SO ₃ -O ₂ Vitamin C 8.56 -1.2 ÷ -1.65 13 Acetate buffer Fe ²⁺ -EDTA Vitamin C 9.70 -1.4 ÷ -1.85 fourteen Ethylene Glycol with TAAB Fe ²⁺ -EDTA Vitamin C 4.30 -1.2 ÷ -1.9 fifteen Na ₂ SO ₄ Fe ²⁺ -EDTA Ethanol 0.007 -1.2 ÷ -1.7 16 Na ₂ SO ₄ Fe ²⁺ -EDTA Pyrogallol 1.13 -1.3 ÷ -1.7

Similar results for measuring the activity of ascorbic acid are obtained using other working electrodes: a mercury spherical electrode, a film of bismuth and film lead on graphite substrates, as well as in an aprotic medium using a non-aqueous electrolyte - ethylene glycol with the addition of 0.1 M tetraalkylammonium bromide (see table .one).

Table 1 also presents the results of determining the antioxidant activity of ascorbic acid against the background of KCl using model generating reactions ^. OH radicals using other model systems: Fe ²⁺ -H ₂ O ₂ , Fe ²⁺ -HPO ₄ ^2- and Na ₂ SO ₃ -O ₂ .

From table 1 it follows that the claimed range of potentials for recording cathode waves -1.2 ÷ -1.9 V.

Using the model system Fe ²⁺ -EDTA against a background of 0.1 M Na ₂ SO _{4, the} proposed method also determines the antioxidant activity of aqueous extracts of products of natural origin - garlic, mandarin and apple (see table 2).

table 2 Sample No. Background solution Model system Antioxidant Antioxidant activity, K, g ^-1 Interval of potentials of registration of cathode waves, V one 2 3 four 5 6 one Na ₂ SO ₄ Fe ²⁺ -EDTA Garlic 16.62 -1.3 ÷ -1.8 2 Na ₂ SO ₄ Fe ²⁺ -EDTA Mandarin 9.59 -1.3 ÷ -1.8 3 Na ₂ SO ₄ Fe ²⁺ -EDTA An Apple 6.20 -1.3 ÷ -1.8

An example of a specific implementation 3.

Determination of the sensitivity of the method.

The sensitivity of the proposed method for determining the activity of antioxidants is calculated using the Seidel criterion (A.N. Seidel. Fundamentals of spectral analysis. - M .: Nauka. - 1965). The analytical signal is the difference ΔI, μA between the peak current values ^. OH radical before (I ₀ ) the introduction of the antioxidant and after (It) the introduction of a certain concentration of C, mg / l antioxidant. First, the standard deviation σi is found when ΔI is determined in 3 parallel experiments (in 3 cells) with the same antioxidant additive. Then build a graph in the coordinates ΔI-C (Fig.5 and 6). The tangent of the slope of the straight line is determined and substituted into the formula: C _min = 2σi · C / ΔI _cp . Thus, the minimum antioxidant concentration (detection limit) is calculated at which a decrease in peak can be noticed.

We calculated the detection limit of the prototype method. For this, the ΔI value was measured - the decrease in the oxygen peak from ascorbic acid additives, a graph was plotted in the ΔI-C coordinates (Fig. 5), and the detection limit was calculated by the Seidel criterion. For the prototype method, it is equal to:

C _min = 2 · 0.078 · 400 / 0.623 = 100.2 (mg / l).

For the proposed method, the detection limit calculated using the graph (Fig.6) is equal to:

C _min = 2 · 0.069 · 3.15 / 1.194 = 0.36 (mg / l).

Thus, from figures 5 and 6, as well as from calculations using the example of ascorbic acid, it follows that the sensitivity of the proposed voltammetric method for determining the activity of an antioxidant is 277 times higher than the sensitivity of the prototype method. In addition, as can be seen from examples 1 and 2, the proposed method allows to determine the activity of antioxidants in relation to ^. OH to the radicals.

Claims (1)

A voltammetric method for determining the activity of antioxidants, including the use of a model reaction in the process of reducing a substance on an electrode in a background electrolyte solution, registering cathode waves of reducing a substance in a given range of potentials relative to a saturated silver-silver reference electrode, adding an antioxidant and determining antioxidant activity from a relative change in the electroreduction current, characterized in that as a model reaction using a reaction OH generating radicals, and cathode waves are recorded in the range of potentials -1.2 ÷ -1.9 V.

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