Oxidative stress due to overproduction of reactive oxygen species (ROS) is often associated with the development of chronic and degenerative oncological, cardiological, and autoimmune diseases [1,2,3,4,5,6]. The advisability of using synthetic antioxidants in therapeutic practice is currently under discussion because of the toxicity of several compounds of this class [7,8,9]. Medicinal plants may be the most environmentally benign source of antioxidants [10]. Herbal antioxidants were proposed to be capable of acting directly (in vivo) on the body and indirectly by inducing mechanisms related to antioxidant protection [11]. Thus, the total antioxidant capacity (TAC) is a suitable indicator for determining the additive antioxidant properties of plants [12].

Currently known methods give variable results for the determined TAC [11,12,13,14,15,16,17,18,19,20,21,22]. Therefore, the TAC must be determined by more than one method to obtain the true value [20].

The aim of the present study was to develop a photochemical method for determining the total content of water-soluble antioxidants based on titration of the reductants with a solution of photogenerated iodine (I2) produced via irradiation of potassium iodide (KI) in the presence of a sensitizer (sodium eosinate) at pH 5.6. The photochemical titration was accompanied by a decrease of the current in the amperometric cell circuit and its stabilization indicating that the reaction was finished because the titrant content was monitored by voltammetry. Further irradiation of the solution and measurement of the generation time required to destroy the titrant enabled quantitative determination of the antioxidants.

Experimental Part

Samples of plant raw material (PRM) collected in middle Volga republics were used in the work. Samples were collected starting mainly from the fringe of their habitat. Raw material was collected and average specimens were selected according to GPM.1.5.0001.15 [23] and GPM.1.1.005.15 [24], respectively. Plants were collected 500 m from highways to avoid anthropogenic contamination. A study of the morphological signatures of the collected samples did not reveal any structural changes. Commercial pharmaceutical herbal substance (OAO Krasnogorskleksredstva) was used as a standard.

Hydrochloric acid (HCl, ρ = 1.19 g/mL, chemically pure), KI (analytically pure), ascorbic acid (AA, analytically pure), sodium eosinate (analytically pure), and oxalic acid (analytically pure) were used in the work. The acidity required for photogeneration of the titrant was maintained by adding acetate buffer solution (pH 5.6) to the absorber system. Water-soluble antioxidants were titrated on an apparatus that was previously described [25].

Quantitative determination of water-soluble antioxidants. The aqueous extract (infusion, decoction) obtained by the recommended method (GPM.1.4.1.0018.15 [26]) from PRM (3.00 g) was filtered, transferred quantitatively to a 100-mL volumetric flask discarding the first 5 mL of filtrate, and adjusted with dialyzed H2O to the mark (solution A).

Polyphenolic compounds (PC) were determined by treating solution A (20 mL) with HCl (2 mL, ρ = 1.19 g/mL) and EtOH (20 mL, 96%) and purging with air (atmospheric oxygen). The resulting precipitate of lipophilic substances was filtered off. The EtOH was distilled off. The resulting solution was transferred quantitatively to a 25-mL volumetric flask and adjusted to the mark with dialyzed H2O [27].

Ascorbic acid (AA) was determined by grinding a weighed portion of PRM with oxalic acid (0.5 g) in a mortar, transferring quantitatively to a perforated infusion cup, and adding H2O (30 mL). The aqueous infusion obtained according to GPM.1.4.1.0018.15 [26] was transferred quantitatively to a 100-mL volumetric flask and adjusted to the mark with dialyzed H2O (solution C).

Solutions of KI (40 mL, 0.5 M), sodium eosinate (10 mL, 10%), and acetate buffer (20 mL, pH 5.6) were placed beforehand into the cell for photochemical titration. The resulting mixture was purged with air and irradiated with light for 1 – 2 min to generate the titrant at a rate of 3.2·10–5 mmol/min until its content was 3.28·10–5 mmol (threshold value). Photogeneration of the titrant in the cell was monitored by voltammetry. Test solution (A, B, C; 0.5 – 5.0 mL) was added to the cell after the threshold value was reached, noting the change of galvanometer reading. When the reaction was finished, the current in the amperometric circuit became constant. Then, the auxiliary solution was again purged with air for 1 – 2 min and irradiated with light. The time required to destroy the titrant in the cell was measured. The solution in the cell was again irradiated with light, generating titrant to its threshold value, to perform subsequent determinations. A single absorber solution allowed 10 – 20 determinations to be made.

The TAC was calculated. Water-soluble antioxidants were determined using the formula (recalculated as AA and pyrogallol):

$$ X=\frac{sf\cdot \Delta \Delta \overline{I}\cdot M\cdot {V}_{\mathrm{f}}}{m\cdot \left(1-W\right)\cdot {V}_{\mathrm{a}}}\cdot \left(\frac{V_{\mathrm{c}1}}{V_{\mathrm{a}1}}\right)\mathrm{mg}/\mathrm{kg} $$

(from the current change in the amperometric cell circuit);

$$ X=\frac{sf\cdot \Delta \overline{\uptau}\cdot M\cdot {V}_{\mathrm{f}}}{m\cdot \left(1-W\right)\cdot {V}_{\mathrm{a}}}\cdot \left(\frac{V_{\mathrm{f}1}}{V_{\mathrm{a}1}}\right)\mathrm{mg}/\mathrm{kg} $$

(from the generation time), where sf is the apparatus scale factor calculated from the generation time (5.33 × 10–7 mmol/s) and current change (6.67 × 10–7 mmol/μA), respectively; \( \Delta \Delta \overline{I}/\Delta \overline{\uptau} \), change of current/generation time of titrant considering an apparatus blank test (μA/s); M, molecular mass of AA or pyrogallol (176.12 and 126.11 g/mol, respectively); Vf, volumetric flask volume (mL); Va, aliquot volume (mL); Vf1/Va1, dilution for calculating the PP content in the aqueous infusion; W, mass fraction of moisture (fraction) (GPM.1.5.3.0007.15 [28]); and m, PRM mass (g).

The difference between the results for photochemical titration of solutions C and B (if the PP content in solution B was estimated by recalculating for AA) was used to calculate the AA content in the aqueous infusions.

The accuracy of the results was monitored using methods recommended in the literature [29] and the SP RF [30, 31].

Results and Discussion

TAC. Currently, natural antioxidants from medicinal plants are interesting as potential therapeutic agents that prevent consequences from ROS [32, 33].

They possess broad spectra of biological activity including anti-inflammatory, antiatherosclerosis, and antitumor activity [34]. Water-soluble antioxidants, namely AA, N-acetylcysteine [35, 36], glutathione [37], and PP [12, 38, 39] are especially interesting because they are responsible for the TAC of aqueous infusions.

Table 1 presents results for the photochemical titration of PRM water-soluble antioxidants. The accuracy of the results was monitored by voltammetry [29]. According to the results, the highest content of water-soluble antioxidants was observed in the sample of Gemmae Betulae (birch buds) collected in Mari El Republic. Furthermore, the contents of water- soluble antioxidants in infusions and decoctions prepared from PRM collected in Mari El Republic showed a slight tendency to increase, most probably because of a slight effect from anthropogenic factors on their growth and development.

Table 1 Determination of Water-soluble Antioxidants in PRM (Data from 2018 – 2019, n = 7, p = 0.98)
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AA (vitamin C) is involved in several physiological processes including immune stimulation; synthesis of collagen, hormones, and neurotransmitters; and absorption of iron [40]. The main intake sources of vitamin C are plants and their products because humans are one of few mammalian species that cannot synthesize it [41].

Table 2 presents results for photochemical titration of AA in the aqueous infusions. The accuracy of the results was monitored by an arbitrary method [30]. In this instance, samples of PRM with high TAC values were investigated. PRM samples collected in Mari El Republic had the highest amounts of AA. This could also be related to a low-level effect of anthropogenic factors on the plant growth and development (Table 2). Furthermore, AA was found to contribute most (46 – 82%) to the PRM TAC value.

Table 2 Determination of Water-soluble Antioxidants in PRM (Data from 2018 – 2019)
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PP, including tanning agents, turned out to be a very important group of biologically active ingredients for the PRM TAC [42]. They could slow the progression of several types of cancer, neurodegenerative diseases, and diabetes and could also reduce the risk of cardiovascular diseases [43].

Table 2 presents results for photochemical titration of water-soluble PP in the aqueous infusions. The accuracy of the results was monitored by the method recommended in the SP RF [31]. A small amount of PP was detected in a sample (birch buds) collected in Mari El Republic and made up 29.8% of the TAC. It could be proposed that the PP content was so high because of anthropogenic contamination. Furthermore, the PP content in aqueous infusions prepared from the buds was slightly less than in those from leaves (Table 2).

The method of additions was used to prove that the proposed methods lacked systematic error. AA and pyrogallol were added to a decoction immediately before photochemical titration (Table 3). According to the results, the determination error in tests with the additives did not exceed the relative error of a single determination and deviated to both positive and negative values. This indicated that systematic error was absent.

Table 3. Determination of AA and PP in Nettle Leaves Collected in Mari El Republic by Method of Additions (p = 0.98)
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Detection limits (DL) and limits of quantitation (LOQ) of the water-soluble antioxidants from PRM were calculated based on equations of calibration curves. Table 4 lists the main analytical characteristics of the photochemical method.

Table 4 Analytical Characteristics of Photochemical Determination Method
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Thus, the developed method for determining water-soluble antioxidants was simple to use, did not require expensive equipment, and; therefore, could be recommended for determining them in any analytical monitoring laboratory.