Antioxidant activities of Geranium sanguineum L. polyphenolic extract in chemiluminescent model systems

Inorganic Chemistry Communications 108 (2019) 107518

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Short communication

Antioxidant activities of Geranium sanguineum L. polyphenolic extract in chemiluminescent model systems

T

Elitsa Pavlovaa, , Lora Simeonovab, Julia Serkedjievab ⁎

a b

Biophysics & Medical Physics, Sofia University “St. Kliment Ohridski”, 5 James Boucher Blvd., 1164 Sofia, Bulgaria Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Georgi Bonchev Str., 1113 Sofia, Bulgaria

GRAPHICAL ABSTRACT

Table 1 Inhibitory constants for reactions with proven antioxidant / chelation effects: New data Comparative data (achieved earlier) [47]. hydroxyl radicals and hydroxide ions

type

H2O2 oxida!on

system Fenton`s Fenton`s Fenton`s Fenton`s Fenton`s Fenton`s Fenton`s Fenton`s H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS NAD.H-PhMS FeSO4 FeSO4 FeSO4 FeSO4 FeSO4 FeSO4

superoxide radicals

tested substance [10-4M] [1,29.10-3M] [2,1.10-4M] [1,75.10-5M] [10-4M] [1,29.10-3M] [2,1.10-4M] [1,75.10-5M] [10-4M] [1,29.10-3M] [2,1.10-4M] [1,75.10-5M] [0,1 mg/ml] [0,2 mg/ml] [1 mg/ml] [10-4M] [1,29.10-3M] [2,1.10-4M] [1,75.10-5M] [0,1 mg/ml] [0,2 mg/ml] [1 mg/ml] [10-4M] [1,29.10-3M] [2,1.10-4M] [1,75.10-5M] [0,1 mg/ml] [0,2 mg/ml] [1 mg/ml] [10-4M] [1,29.10-3M] [2,1.10-4M] [1,75.10-5M] [0,1 mg/ml] [0,2 mg/ml] [1 mg/ml] [0,1 mg/ml] [0,2 mg/ml] [1 mg/ml] [0,1 mg/ml] [0,2 mg/ml] [1 mg/ml]

Fe2+ ions

vit.C vit.C vit.E vit.E vit.C vit.C vit.E vit.E vit.C vit.C vit.E vit.E Polyphenols polyphenols polyphenols vit.C vit.C vit.E vit.E polyphenols polyphenols polyphenols vit.C vit.C vit.E vit.E polyphenols polyphenols polyphenols vit.C vit.C vit.E vit.E polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols polyphenols

pH 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 7.4 7.4 7.4

K7 [l / mol.s] 1,810 0,179 1,011 10,278 3,028 0,176 2,057 25,220 2,817 0,1809 0,658 2,2143

13,2 0,316 0,493 23,431

82,300 3,620 37,762 466,571

18,800 0,806 11,119 134,857

K7 [l / g.s] 1,027.10-2 0,102.10-2 0,190.10-2 1,936.10-2 1,719.10-2 0,099.10-2 0,387.10-2 4,751.10-2 1,599.10-2 0,103.10-2 0,124.10-2 0,417.10-2 0,055.10-3 -0,225.10-3 0,099.10-3 7,494.10-2 0,179.10-2 0,093.10-2 4,414.10-2 -5,85.10-3 -0,627.10-3 0,082.10-3 46,727.10-2 2,055.10-2 7,114.10-2 87,901.10-2 22,6.10-3 15,9.10-3 1,26.10-3 10,674.10-2 0,458.10-2 2,094.10-2 25,407.10-2 5,75.10-3 1,678.10-3 0,433.10-3 58,3.10-3 30,425.10-3 5,075.10-3 10,35.10-3 6,525.10-3 1,065.10-3

ARTICLE INFO

ABSTRACT

Keywords: Polyphenols Antioxidants Reactive oxygen species Superoxide dismutase Glutathione reductase Chemiluminescence

The aim of this study is to assess the antioxidant activities of Geranium sanguineum polyphenolic extract in chemiluminescent model systems by calculating the constants of inhibition towards different reactive oxygen species (ROS). The kinetics of lucigenin-enhanced chemiluminescence was examined in three chemical systems designed for generation of ROS (%OH, O2%−, H2O2) and in one system with FeSO4 added as a chelator. Antioxidant enzyme activities of superoxide dismutase and glutathione reductase were also evaluated. All the calculated constants of inhibition were compared to vitamin C and vitamin E values obtained earlier. Based on the results we can make the following major conclusions: 1) the polyphenol complex reacts against all studied ROS (H2O2, %OH, O2%−) with various efficacy. It exerts lower anti-oxidant effects when compared to standards such as vitamin C and vitamin E. 2) the polyphenol complex has pronounced chelation properties against Fe2+, which is of extreme biological importance.

Abbreviations: GR, glutathione reductase; LDL, low-density lipoprotein; NADPH, nicotinamide adenine dinucleotide phosphate oxidase; NADH, α-nicotinamide adenine dinucleotide; ROS, reactive oxygen species; RONS, reactive oxygen nitrogen species; SOD, superoxide dismutase ⁎ Corresponding author. E-mail address: [email protected] (E. Pavlova). https://doi.org/10.1016/j.inoche.2019.107518 Received 3 July 2019; Received in revised form 6 August 2019; Accepted 9 August 2019 Available online 15 August 2019 1387-7003/ © 2019 Elsevier B.V. All rights reserved.

Inorganic Chemistry Communications 108 (2019) 107518

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3) The reference concentration (0.1 mg/ml) is most appropriate for therapeutic purposes. All obtained data contribute to the elucidation on the therapeutic and prophylactic reactive mechanisms of polyphenols extracted from Geranium sanguineum L.

1. Introduction

these compounds to organic acids and sugars. The mechanisms by which these compounds act may vary depending on the concentration and types of compounds present in foods [14]. Some polyphenol hydroxyls are very reactive in neutralizing free radicals (–R%) by donating a hydrogen atom (-RH) or an electron (–R−), chelating metal ions in aqueous solutions and binding and precipitating of proteins, due to extensively coating of hydrophobic surfaces of peptides and then to cooperative bridge formation [15–18]. Polyphenols have powerful antioxidant activity in vitro by scavenging reactive oxygen, nitrogen, and chlorine species, such as superoxide anion, hydroxyl radical, peroxyl radicals, hypochlorous acid and peroxynitrous acid. They also chelate metal ions, thus decreasing their prooxidant activity. Since considerable evidence indicates that increased oxidative damage is associated with the development of most major age-related degenerative diseases, it has been speculated that polyphenols may have protective effects against such conditions. High polyphenol intake has allegedly been associated with decreased risks for cancers, cardiovascular diseases and neurodegenerative disorders. Such biological activity has been studied on purified enzymes, cultured cells, or isolated tissues using food aglycones or glycosides [19,20]. Because of their low redox potential, flavonoids thermodynamically reduce strong oxidizers, including peroxyl, hydroxyl, alkoxyl and superoxide free radicals [21]. Of all the reactive molecules, H2O2, O2%−, NO% and ONOO− are the most widely studied reactive oxygen species and play important roles in many complications [22]. Until these ROS are deactivated, uncontrolled free radical oxidation reactions may occur within milliseconds [23]. Reactive oxygen nitrogen species (RONS) also interact with lipids leading to lipid peroxidation in biological membranes by attacking the double bonds of polyunsaturated fatty acids [24]. Lipid peroxides may interact with biological targets also inducing cytotoxicity [25]. Furthermore, the formation of lipid peroxides may lead to oxidation of low-density lipoprotein (LDL). The sensitivity of LDL to oxidative damage depends on an appropriate balance between the amount of polyunsaturated fatty acids and antioxidant concentrations [26]. The LDL particle itself contains various antioxidants (e.g. tocopherols, b-carotene, ubiquinol 10, cryptoxanthin) that protect it from non-enzymatic oxidation [27]. ROS producers such as macrophages and neutrophils, which contain the membrane bound nicotinamide adenine dinucleotide phosphate oxidase (NADPH) complex, are capable of generating substantial amounts of superoxide anion and also play important roles in the destruction of invading pathogens [28]. RONS can participate in many potentially beneficial events in vivo, including energy production, pathogen destruction and intercellular signaling regulation. However, when the balance of RONS production exceeds antioxidant defenses, reactive species predominate and attack biological macromolecules, namely lipids, proteins and DNA, inducing oxidation and causing membrane damage, enzyme inactivation and DNA damage [29]. Under these conditions, an external source of antioxidants may be required to combat oxidative stress-related damage. Antioxidants play an important role in terminating radical chain reactions by donating electrons to free radicals and their intermediates. Simple antioxidants (vitamin A, C, E, bilirubin, taurine, caffeine, uric acid) and enzymatic antioxidants (superoxide dismutase, glutathione peroxidase, catalase) work together to neutralize oxidants and RONS in biological systems [31–34]. In addition, chelating agents that bind metal ions are also classified as antioxidants as a result of their ability to inhibit reduction–oxidation reactions [35]. Polyphenol bioavailability is influenced by the phenolic structure, food processing and matrix, host and other factors and together with the antioxidant activity are assessed using the total antioxidant capacity

Polyphenols are the most numerous and abundant secondary plant bioactive molecules grouped in five general classes – flavonoids, phenolic acids, such as hydrobenzoic acids and hydroxycinnamic acids, stilbenes and lignans. Among these, flavonoids are subdivided into flavones, flavanones, flavonols, flavanols, isoflavones and anthocyanidins. All the compounds display a great variety of structures, ranging from simple substances containing a single aromatic ring, to highly complex polymers such as tannins and lignin [1–3]. Geranium sanguineum L. (Geraniaceae) is a medicinal plant which has been used in ethnopharmacotherapy in Bulgaria for ages and its root extracts are still being administered to treat various infectious diseases and inflammatory conditions. The individual constituents have been identified and analyzed by HPLC. The variety of biologically active compounds, as well as the possible synergistic interactions between them seem to be decisive for the overall effects. The total soluble phenolic constituents of the extract, measured by Folin-Ciocalteu reagent comprised 34.60% (w/w) and were fractioned as 16.15% tannins, 0.126% flavonoids and 2.12 mg/kg catechins and proanthocyanidines, 12% free sugar moieties and a small amount of aminoacids. No proteins and saponins have been detected [4]. Another study on Geranium sanguineum extract determined the following constituents: caftaric acid (1.30 mg/g), caffeic acid (2.41 mg/g), hyperoside (1.64 mg/g), isoquercitrin (2.58 mg/g), rutin (1.71 mg/g), quercitrin (0.42 mg/g), quercetin (0.82 mg/g), kaempferol (0.19 mg/g). This confirmed the presence of other polyphenols such as caftaric acid and quercitrin, which hadn't been identified before [3]. Phenols are not found in the same chemical form in food products and biological fluids, as they are first converted to a great extent into glucuronides, sulfates or methylated forms in the organism [5,6]. The bioavailability of phenolic compounds differs largely, which suggests that the most abundant polyphenols are not necessarily those leading to the highest levels of active metabolites in target tissues [7]. Absorbed flavonoids are bound to serum albumin and are transported to the liver via the portal vein, where they are subjected to extensive Phase II metabolism before entering the systemic circulation [8]. The most effective enzymes to metabolize phenols are found in the small intestine, liver and kidneys [5]. The polyphenols that are mostly absorbed are isoflavones and gallic acid, followed by catechins, flavanones, and quercetin glucosides, having different kinetics. The leastabsorbed polyphenols are the proanthocyanidins, the galloylated tea catechins, and the anthocyanins. Data on the absorption of other polyphenols are still limited [9]. Phenolic compounds have been reported to have multiple biological effects, including antioxidant activity, as they can scavenge free hydroxyl and peroxyl radicals [10–12]. Occurrence, position, structure and total number of sugar moieties in flavonoids (flavonoids glycosides) play an important role in their antioxidant activity, as aglycones being more potent antioxidants than their corresponding glycosides [13]. The main factor on antioxidant capacities of phenolic compounds is the number and position of hydroxyl groups. Flavonoids possess more hydroxyl groups and higher antioxidant activity. Moreover, the solubility and stearic effects of each molecule may be affected by the structure of the molecule; for example, the presence of glycosylated derivatives of other adducts, can increase or decrease the antioxidant activity of phenolic compounds. Flavonoid compounds are commonly present in plants as glycosides, but can be released by the action of enzymes to its corresponding aglycone. The antioxidant activity of phenolic acids is also based on the binding of 2

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E. Pavlova, et al.

in the plasma. However, this method is not free from flaws since other micronutrients, such as vitamins E and C exert greater antioxidant activity and the scavenging capacity does not directly correlate to in vivo mechanisms of defense, which are mostly enzymatic [36–38]. The mechanism of action of polyphenols in vivo and in vitro might differ. In fact, their classical antioxidant activity is unlikely to be the principal explanation for cellular effects in humans, where non-specific protein/enzyme modulating mechanisms are primarily involved. This hypothesis is based on two lines of reasoning. First, polyphenol metabolism significantly alters their redox potentials. Polyphenol conjugates and metabolites show weaker antioxidant capacity than their parental aglycones. Second, despite that the concentration of polyphenols in plasma and organs is lower than that of proved antioxidant micronutrients, such as ascorbic acid and a-tocopherol, their efficacy is higher against oxidative stress at tissue level. This hypothesis suggests that the polyphenol activity against several types of cancer, proliferative diseases, inflammation, neurodegeneration and other diseases is mainly exerted through the inhibition and modulation of a wide range of receptors, enzymes and transcription molecules. Interestingly, some oxidized polyphenol metabolites act as pro-oxidants, but induce apoptosis by production of toxic ROS against cancer cell mitochondria. On the other hand, such pro-oxidant activity suggests that high dietary polyphenol intake could be potentially more of an oxidative risk than a benefit - a concern corroborated by one epidemiological study reporting a direct association between flavonoid intake and colon cancer. This suggests that dietary supplementation with large amounts of single polyphenols may actually be deleterious to human health [39]. The aim of this study is to assess the antioxidant activities of Geranium sanguineum polyphenolic extract in chemiluminescent model systems by calculating the constants of inhibition towards different reactive oxygen species.

2.3. Statistics All results are received as triple reproducible measurements, p ≤ 0.07, statistically processed by Origin 8.5 and Microsoft Office Excel 2010. 2.4. Chemiluminescent method and calculations The kinetics of lucigenin-enhanced chemiluminescence was examined for three chemical systems designed for the generation of ROS and one system with applied FeSO4. All four systems were tested at 25 °C/pH 7.4 – physiological, and 25 °C/pH 8.5 – for better differentiation of the chemiluminescent response. The total volume of each sample in the measuring cuvette was 2 ml, and the kinetics was registered for 3 min using chemiluminometer LKB 1251 (Sweden). The first seconds of fast flash luminescence are most informative for the inhibition antioxidant or prooxidant effects. Each sample was prepared as a mixture, containing the buffer medium, chemiluminescent probe lucigenin, the chemical system for ROS-generation or FeSO4 and the tested polyphenol complex. The utilized in vitro buffer solutions were prepared from Na2HPO4 [0.05 M] and C8H8O7·H2O [0.1 M]. The control systems contained all the presented reagents in the absence of the expected inhibitor ([InH]0 = 0) – the tested polyphenol complex: 1) Fenton's reagent for the generation of %OH and −OH species (FeSO4 [5 · 10−4 M]-H2O2 [2,5 · 10−3 M]) It is well known that the interaction between Fe2+-ions and Н2О2 results in highly reactive, short-living %ОН-radicals:

Fe2 + + H2 O2

Fe3 + + OH +

Fe3 + + H2 O2

Fe2 + + OOH + H+

(1)

OH

(2)

2) H2O2 oxidation (H2O2 [2,5 · 10−3 M]) 3) FeSO4 reduction (FeSO4 [5 · 10−4 M]) 4) reduced α-nicotinamide adenine dinucleotide (NADH) [10−4 M]phenazine methosulfate [10−6 M] - for the generation of superoxide radicals (O2%−) [40,41]:

2. Materials and methods 2.1. Reagents 2.1.1. Luminescent analysis Iron sulfate FeSO4 (Merck, Germany), phenazine methosulfate C13H11N2·CH3SO4 (PMS) (N-methyldibenzopyrazine methyl sulfate salt) (Merck, Germany), hydrogen peroxide H2O2 (Boron, Bulgaria), disodium hydrogen phosphate Na2HPO4 (Boron, Bulgaria), citric acid C8H8O7·H2O (Boron, Bulgaria), lucigenin C28H22N4O6 (bis-N-methylacridinium nitrate) (Aldrich, USA), α-nicotinamide adenine dinucleotide, reduced form C21H27N7O14P2Na2 (NАD·Н) (Boehringer, Germany), dimethyl sulfoxide C2H6OS (DMSO) (Aldrich, USA).

+

NAD·H + PMS +

PMS·

2

PMS·

+ PMS +

2

PMS·

2

(3)

+ NAD+

(4)

2PMS· PMS +

2

+

(5)

+ −4

The chemiluminescent probe lucigenin [10 M] is dissolved in DMSO. The tested substance was added in the following final concentrations, as water solutions: 1×, 2× and 10× according to the prescribed physiologically active concentration equal to 10 mg/kg for in vivo application.

2.1.2. Enzyme analysis Superoxide Dismutase Assay Kit, Glutathione Reductase Assay Kit (Cayman Chemical Company, USA).

2.4.1. Calculations The integral evaluation of the content of free radicals and ROS as well as the registration of the kinetic parameters of the oxidation reaction could be easily done by the lucigenin-enhanced chemiluminescent method. The constants of inhibition k7 can be calculated according to the kinetics of chemiluminescence, in correlation to the concentration of ROS [42–44]:

2.2. Polyphenolic extract from Geranium sanguineum, L. Was prepared from ground, air-dried air rhizomes collected in the area of Iskar Dam, Bulgaria in blooming time, which were degreased with petroleum ether and treated with methanol to fully recover the polyphenol components. The extract is then lyophilized (yield 16%); the resulting substance is a dark red, odorless, water-soluble powder. The phytochemical analysis shows that it contains tannins (34%), flavonoids (0.17%), catechins and proanthocyanidins (2 mg/kg). It contains flavonoids - aglycans and glycosides (quercetin, quercetin 3-Ogalactoside, morin, myriacetin, campherol, rhamazin, retusin, apigenin), polyphenolic acids (caffeine, chlorogenic, elagone, quinine), galotanins, catechins and maltol.

(I / I0 ) =

[ROS ] [ROS ]0

2

(6)

where I is the measured light intensity [mV], [ROS]0 is the initial concentration of the reactive oxygen species in the absence of inhibitors (t = 0). The constant of inhibition k7 [l/(mol ∙ s)] can be calculated from

the slope of the linear part

{ } (t ) during the first period of fast cheI I0

miluminescent emission, as follows: 3

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d (I / I0 ) = dt

2k 7 [InH]0

3.1. Chemiluminescent response to ROS after the application of the polyphenol complex isolated from Geranium sanguineum L.; calculations of the inhibition constants

(7)

where I0 is the registered light intensity [mV] at t = 0, and [InH]0 is the initial concentration of the inhibitor. The accuracy of k7 was determined mainly by the accuracy of the light intensity measurements and less than [InH]0. For convenience, k7 can be expressed in [l/(g ∙ s)], when taking into account the molecular mass of the inhibitor Table 1.

• System 1 generated highly reactive, very short-living %ОН radicals.

2.5. Enzyme analysis and calculations

• The activity of superoxide dismutase (SOD) was measured according

to the dismutation of superoxide radicals, generated by the reaction between xanthine and hypoxanthine (Fig. 1). The activity was estimated with a variation coefficient about the analyzed samples which was lower than 4%. The calculations were done according to the following formula:

SOD (U/ml) = {[(sample LR

y intercept)/slope] × 0.23/0.01} × sample dilution

• Glutathione reductase (GR) activity was measured according to the

oxidation of NADPH to NADP+ which was accompanied with decrease of the absorption at 340 nm (Fig. 2). The rate of the decrease of the absorption at 340 nm was proportional to the activity of glutathione reductase in the sample. The activity was assessed with a variation coefficient about the analyzed samples which was lower than 4%. The calculations were performed according to the following formula:

GR Activity

nmol min

ml

=

A 0.19 sample dilution 0.00373 0.02

3. Results and discussion

•

The application of flavonoids and their derivatives can stabilize and stimulate the body's antioxidant protection system. Various species have been studied from the Geraniaceae family. Positive results are found for Geranium macrorrhizum and Geranium herba. That plant is one of the most popular medical plants applied and described for a millennium. There are data by other authors on the antiviral, antibacterial and antimycotic activity of complexes isolated from the Geranium genus. However, there is no evidence on the antioxidant activity of Geranium sanguineum, L., with regards to the controversial free-radical oxidation reactions that would explain some of its therapeutic effects [2,4–7,10,11]. Hydrogen peroxide, superoxide anion radicals, singlet oxygen and other ROS are discussed as agents attacking polyunsaturated fatty acids in cell membranes, resulting in lipid peroxidation [29, 30, 45]. Many authors suggest that lipid peroxidation can lead to destabilization and degradation of the cell membranes, resulting in cellular damage and development of so-called “free-radical” diseases, and last but not least, aging and susceptibility to cancer. However, as a rule, cell membranes are protected from lipid peroxidation by the extremely effective antioxidant mechanisms in the body. These mechanisms include enzymatic inactivation by superoxide dismutase, glutathione peroxidase and catalase, as well as non-enzymatic protection of polyunsaturated fatty acids ensured by physiological and biological antioxidants like vitamin E, vitamin C, beta-carotene, uric acid, etc. [29, 30, 46]. It is important antioxidant effects to be evaluated by their strength and duration over time with measurement of the intensity and kinetics of model free-radical reactions and further calculation of the inhibition constants (antioxidant activity).

The chemiluminescent emission from this reaction was higher than other reactive mixtures. In the control sample, at pH 8.5, the measured chemiluminescence was stable and very slow, attenuating over time, with values above 1200 mV. The addition of the polyphenol complex at a concentration for in vivo application (1×) as well as 2 times higher showed an inhibitory effect on the generation of hydroxyl radicals between 5% and 7%; and when applied as 10-times higher concentration, the inhibitory effect reached 62%. It should be noticed that this effect is quickly exhausted over time. At physiological pH level (7.4), the luminescence in the control samples was significantly lower (about 26–27 mV), which was expected and was due to the higher acidity of the medium. The signal was stable and very slow over time, with similar kinetics to the reaction at pH 8.5. The addition of the polyphenol complex at concentration 2 and 10 times higher than the reference for application in vivo also showed a reproducible inhibitory effect on the generation of hydroxyl radicals - about 9% and 33%, respectively. However, the application of a reference dose (1×) resulted in an increase of the chemiluminescent response over time and revealed prooxidant activity of the complex of about 20%. There is evidence in the literature on pronounced prooxidant activity of the complex when administered to a healthy organism but on a prominent antioxidant and therapeutic effect when administered to patients with a pathological condition. The investigation on specific free radicalgenerating reactions and ROS enables to elucidate those contradictory effects. Our results, with respect to the Fenton's reaction, showed and confirmed that the application of low dosages of the polyphenol complex for prophylaxis is not a reasonable approach. Similar results for this concentration are also obtained in some of the other systems studied below. System 2 In this system hydrogen peroxide played a double role of an oxidant as well as a reactive oxygen species. At pH 8.5, in the control sample, the measured chemiluminescence was stable and very slow attenuating over time, with values above 1300 mV. The addition of the polyphenol complex at concentrations 2 times and 10 times higher than the reference for application in practice (1×), demonstrated a potent inhibitory effect towards hydrogen peroxide (a ROS), respectively about 10% and 35%; the effects are analogous to those recorded in System 1 under the same reaction conditions (Fig. 3A, B). It is important to notice, however, that the inhibitory effect at the highest administered dose of the complex did not only deplete over time but was significantly

Fig. 1. Generation of superoxide radicals and the effect of superoxide dismutase (SOD). 4

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Fig. 2. Glutathione reductase reaction.

•

increased. However, the application of a reference dose (1×) results in a slight increase in the luminescent signal and prooxidant activity of the complex about 8% (Fig. 3A, B), indicating that this concentration of the complex is not suitable for prophylaxis. Hydrogen peroxide is also a standard player in the inflammation cascade of the oxidative stress. At physiological pH values (7.4), luminescent control levels were significantly lower (about 31–33 mV), which resulted from the higher acidity of the medium. The chemiluminescence was unstable, with fluctuations, but with gradual attenuation over time, after the registration of the so-called fast flash about the 20-th second after the start of the reaction. Analogous rapid flashes were also recorded for the reactions including the polyphenols, but their kinetics were characterized by great instability and wide fluctuations. The addition of the polyphenol complex at concentrations 2× and 10× higher than the reference (1×) produced an initial inhibitory effect towards hydrogen peroxide (as a ROS) reaching 10%, which declined very quickly and transformed into a strong prooxidant effect about the 2× concentration. When applying a reference dose (1×) the luminescence response and oxidation were also enhanced (Fig. 3A, B). It can be concluded that the polyphenol complex has no antioxidant properties at physiological pH and against H2O2. Considering that in inflammation, significant concentrations of hydrogen peroxide are easily accumulated in the organism as a signaling molecule, special attention should be paid to the application of the polyphenol complex for prophylactic and therapeutic purposes. System 3 The iron sulfate in that chemiluminescent system is a reducing agent and a source of iron ions that could be chelated. Large amounts of iron circulate in the blood stream, transported and deposited by different proteins. Free Fe2+ ions of the so-called “active iron” are released only in pathological processes and are very dangerous as initiators and participants in the Fenton's response - the initiation of free-radical oxidation and accumulation of ROS in the bloodstream. At pH 8.5, in the control sample, the reaction started with a fast flash till 20-th second and a subsequent gradual attenuation of the signal. The registered chemiluminescence was lower in comparison to System 1 and System 2, with control peaks about 3.4 mV. For all tested concentrations, the reactions followed the control kinetic

•

curve. The polyphenol complex demonstrated a strong inhibitory effect towards Fe2+ ions in that system. Obviously, the complex acts as a chelator and inhibits the oxidation respectively 38%, 31% and 55% for the reference 1×, 2× and 10× concentrations (Fig. 4A, B). Interestingly, the suppression observed was more pronounced at the reference than at the double dosage at pH 7.4. At physiological pH, the registered chelation effect was 9.7%, 7.2% and 11%, respectively, from the lowest to the highest dose. The curves of kinetics at lower acidity of the medium followed an analogous course of those at pH 8.5 but with constant fluctuations (Fig. 4A, B). Probably, the inhibitory effects and antioxidant activity recorded in System 3 were due to chelation effects of the complex towards Fe2+ ions. System 4 In the control sample, at pH 8.5, the measured chemiluminescent signal was stable, starting with a fast flash for 20 s and values of 8 mV, decreasing gradually over time. The addition of 1× polyphenols, 2 times and 10 times higher, showed a strong inhibitory effect towards the generation of superoxide (O2%−) radicals, respectively 23%, 56% and 87% (Fig. 5A, B). At physiological pH, the antioxidant effects of 2× and 10× concentrations were also strong 20% and 34%, respectively. However, the application of the polyphenol complex as 2 times higher concentration than the reference, did not result in a stable suppressing effect, and over time the oxidation was elevated. In general, the kinetic curves at pH 7.4 were similar to those at pH 8.5, but the reactions showed constant fluctuations, with overall level of chemiluminescence about 8 times lower than at higher acidity (Fig. 5A, B).

3.2. Inhibition constants and comparison to standard antioxidants Vitamins C and E are standard antioxidants and their inhibition constants against specific oxygen species we have calculated before [47]. Ascorbic acid is a natural organic compound with antioxidant properties. The antioxidant effects of vitamin C are only a small part of its inherent activity. The ascorbate ion is the predominant species for typical biological pH values. Vitamin C is a weak reducing agent. It is oxidized with the loss of an electron and forms a radical anion, then with a loss of a second electron forms dehydroascorbic acid [48,49]. Frequently, free radicals initiate a chain reaction. The ascorbate can stop these radical chain reactions by an electron transfer. Ascorbic acid is special because it can transfer a single electron due to the resonancestabilized nature of its own radical ionic form, which is called semidehydroascorbate. The oxidized forms of ascorbate are relatively inert

Fig. 3. Inhibitory effect towards hydrogen peroxide (H2О2) at рН 8.5 and рН 7.4 and the application of polyphenols from Geranium sanguineum L., at reference (1×), 2 times higher (2×) and 10 times higher (10×) concentration, evaluated by the chemiluminescent method. 5

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Fig. 4. Inhibitory effect towards active iron (Fe2+) at рН 8.5 and рН 7.4 and the application of polyphenols from Geranium sanguineum L., at reference (1×), 2 times higher (2×) and 10 times higher (10×) concentration, evaluated by the chemiluminescent method.

and do not cause cell damage but as a good electron donor its excess in the presence of free metal ions can not only enhance but also initiate free radical generation reactions, thus making vitamin C a potentially hazardous compound in some metabolic conditions. Studies of the dosedependent effects of vitamin C in healthy people show sigmoidal ratios between the oral dose and the plasma and tissue concentrations of vitamin C. Consequently, optimal dosing is crucial for the observed effects of vitamin C [50]. Vitamin C is administered as a 10−4 M dose reference with proven antioxidant activity or 1.29.10−3 M, which is the mean physiological concentration. Vitamin E refers to a group of eight fat soluble compounds that include tocopherols and tocotrienols. Alpha-tocopherol, the most active form of vitamin E, is the second most commonly present form. It can be found abundantly in wheat germ oil, sunflower oil, saffron and other oils. As a fat-soluble antioxidant, it suppresses the production of ROS in the oxidation of fat and fights mainly against lipid-oxide radicals. It has been used as a commercially available antioxidant for ultra-high molecular weight polyethylene, used in hip and knee joint replacements, making it resistant to oxidation. Vitamin E is well known for its protection of the lipids and polyunsaturated fatty acids from oxidation [51,52].

Vitamin E is administered as a water-soluble form of α-tocopherol succinate (2.1.10−4 M physiological concentration or 1.75.10−5 M, representing 1/6 of the mean physiological vitamin C concentration). We can make the following conclusions about the effects of Geranium sanguineum L. extract in the free-radical oxidation model systems from the calculated constants of inhibition (Table 1): - In the Fenton's system (against %OH radicals), the polyphenol complex has a strong antioxidant effect when administered at 10 times higher than the reference dose, which however is rapidly depleted over time. - In all tested model systems, the polyphenol complex has anti-oxidant effects that are lower when compared to standard antioxidants such as vitamin C and vitamin E. - The polyphenol complex presents most effective and stable activity over time when it is administered at 0.1 mg/ml against superoxide radicals O2%− (System 4) and as a chelating agent against Fe2+ (System 3).

Fig. 5. Inhibitory effect towards superoxide radicals (О2%−) at рН 8.5 and рН 7.4 and the application of polyphenols from Geranium sanguineum L., at reference (1×), 2 times higher (2×) and 10 times higher (10×) concentration, evaluated by the chemiluminescent method.

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Table 1 Inhibitory constants for reactions with proven antioxidant/chelation effects. New data. Comparative data (achieved earlier) [47]. Tested substance −4

Vit. C [10 M] Vit. C [1,29 · 10−3 M] Vit. E [2,1 · 10−4 M] Vit. E [1,75 · 10−5 M] Vit. C [10−4 M] Vit. C [1,29 · 10−3 M] Vit. E [2,1 · 10−4 M] Vit. E [1,75 · 10−5 M] Vit. C [10−4 M] Vit. C [1,29 · 10−3 M] Vit. E [2,1 · 10−4 M] Vit. E [1,75 · 10−5 M] Polyphenols [0,1 mg/ml] Polyphenols [0,2 mg/ml] Polyphenols [1 mg/ml] Vit. C [10−4 M] Vit. C [1,29 · 10−3 M] Vit. E [2,1 · 10−4 M] Vit. E [1,75 · 10−5 M] Polyphenols [0,1 mg/ml] Polyphenols [0,2 mg/ml] Polyphenols [1 mg/ml] Vit. C [10−4 M] Vit. C [1,29 · 10−3 M] Vit. E [2,1 · 10−4 M] Vit. E [1,75 · 10−5 M] Polyphenols [0,1 mg/ml] Polyphenols [0,2 mg/ml] Polyphenols [1 mg/ml] Vit. C [10−4 M] Vit. C [1,29 · 10−3 M] Vit. E [2,1 · 10−4 M] Vit. E [1,75 · 10−5 M] Polyphenols [0,1 mg/ml] Polyphenols [0,2 mg/ml] Polyphenols [1 mg/ml] Polyphenols [0,1 mg/ml] Polyphenols [0,2 mg/ml] Polyphenols [1 mg/ml] Polyphenols [0,1 mg/ml] Polyphenols [0,2 mg/ml] Polyphenols [1 mg/ml]

System

Type

pH

K7 [l/mol·s]

Fenton's Fenton's Fenton's Fenton's Fenton's Fenton's Fenton's Fenton's H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 H2O2 NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS NAD·H-PhMS FeSO4 FeSO4 FeSO4 FeSO4 FeSO4 FeSO4

Hydroxyl radicals and hydroxide ions

8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 8.5 8.5 8.5 8.5 7.4 7.4 7.4 7.4 7.4 7.4 7.4 8.5 8.5 8.5 7.4 7.4 7.4

1810 0,179 1011 10,278 3028 0,176 2057 25,220 2817 0,1809 0,658 22,143

H2O2 oxidation

Superoxide radicals

Fe2+ ions

3.3. Ex vivo evaluation of the superoxide dismutase activity after the application of low, medium and high concentrations of polyphenols from Geranium sanguineum L.

1.0 mg/ml 0.2 mg/ml 0.1 mg/ml

SOD activity [U/ml] 3.358 4.439 5.343

82,300 3620 37,762 466,571

18,800 0,806 11,119 134,857

1,027 · 10−2 0,102 · 10−2 0,190 · 10−2 1,936 · 10−2 1,719 · 10−2 0,099 · 10−2 0,387 · 10−2 4,751 · 10−2 1,599 · 10−2 0,103 · 10−2 0,124 · 10−2 0,417 · 10−2 0,055 · 10−3 −0,225 · 10−3 0,099 · 10−3 7,494 · 10−2 0,179 · 10−2 0,093 · 10−2 4,414 · 10−2 -5,85 · 10−3 −0,627 · 10−3 0,082 · 10−3 46,727 · 10−2 2,055 · 10−2 7,114 · 10−2 87,901 · 10−2 22,6 · 10−3 15,9 · 10−3 1,26 · 10−3 10,674 · 10−2 0,458 · 10−2 2,094 · 10−2 25,407 · 10−2 5,75 · 10−3 1,678 · 10−3 0,433 · 10−3 58,3 · 10−3 30,425 · 10−3 5,075 · 10−3 10,35 · 10−3 6,525 · 10−3 1,065 · 10−3

- The polyphenols from Geranium sanguineum L. exhibit glutathionereductase activity that is inversely proportional to the administered concentration. The polyphenol complex limits the rate of that reaction, the rate of absorption reduction and is directly proportional to the activity of glutathione reductase (polyphenols complex) in the sample, i.e. a lower reaction rate is indicative of stronger antioxidant properties.

3.3.1. Ex vivo evaluation of the glutathione reductase activity after the application of low, medium and high concentrations of polyphenols from Geranium sanguineum L. All measurements of the enzymatic activity are performed in triplicate; the obtained values are averaged and used for calculation: Polyphenols from Geranium sanguineum L. concentration

13,2 0,316 0,493 23,431

K7 [l/g·s]

From the results obtained, the reference concentration (0.1 mg/ml) is the most appropriate dose for therapeutic purposes.

GR activity [nmol/ml/min]

4. Conclusions

6.365 5.092 1.273

Abundant evidence shows that plant polyphenols exhibit strong antioxidant and radical scavenging properties. It is shown in other studies that the extract significantly restored and stimulated the antioxidant activities. The treatment by the polyphenol complex reduced to normal levels the elevated SOD and catalase activities as well [53]. It has been reported that plant polyphenols are naturally occurring antioxidants but they also exhibit prooxidant properties under certain conditions. Antioxidant properties of polyphenols arise from their high reactivity as hydrogen or electron donors and from the ability of the polyphenol derived radical to stabilize and delocalize the unpaired

We can withdraw that: - The polyphenols from Geranium sanguineum L. exhibit superoxide dismutase activity inversely proportional to the applied concentration, i.e. the use of a reference concentration (0.1 mg/ml) for therapeutic purposes is the most appropriate dose. 7

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electron (chain-breaking function), and from their ability to chelate transition metal ions [54]. Using various methods to assess the antioxidant properties of a polyphenol complex extracted from Geranium sanguineum L., we can make the following major conclusions:

[19] C. Manach, A. Scalbert, C. Morand, C. Remesy, L. Jimenez, Polyphenols: food sources and bioavailability, Am. J. Clin. Nutr. 79 (2004) 727–747. [20] B. Halliwell, J. Rafter, A. Jenner, Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am. J. Clin. Nutr. 81 (2005) 268S–276S. [21] P.G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 63 (2000) 1035–1042. [22] S.V. Lakshmi, G. Padmaja, P. Kuppusamy, V.K. Kutala, Oxidative stress in cardiovascular disease, Indian J. Biochem. Biophys. 46 (2009) 421–440. [23] A. Kaliora, G. Dedoussis, H. Schmidt, Dietary antioxidants in preventing atherogenesis, Atherosclerosis 187 (2006) 1–17. [24] B. Halliwell, M.A. Murcia, S. Chirico, O.I. Auroma, Free radicals and antioxidants in food and in vivo: what they do and how they work? Crit. Rev. Food Sci. Nutr. 35 (1995) 7–20. [25] W. Bors, W. Hellers, C. Michel, M. Saran, Flavonoids as antioxidants: determination of radical-scavenging efficiencies, in: L.P.A.N. Glazer (Ed.), Methods in Enzymology, Academic Press, San Diego, CA, 1990, pp. 343–355. [26] A.B. Santhakumar, A.C. Bulmer, I. Singh, A review of the mechanisms and effectiveness of dietary polyphenols in reducing oxidative stress and thrombotic risk, J. Hum. Nutr. Diet. 27 (1) (2014) 1–21. [27] H. Esterbauer, J. Gebicki, H. Puhl, G. Jurgens, The role of lipid peroxidation and antioxidants in oxidative modification of LDL, Free Radic. Biol. Med. 13 (1992) 341–390. [28] M.T. Quinn, M.C. Ammons, F.R. Deleo, The expanding role of NADPH oxidases in health and disease: no longer just agents of death and destruction, Clin. Sci. (Lond.) 111 (2006) 1–20. [29] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd edn, Clarendon Press, Oxford, 1999. [30] M. Valko, M. Izakovic, M. Mazur, C.J. Rhodes, J. Telser, Role of oxygen radicals in DNA damage and cancer incidence, Mol. Cell. Biochem. 266 (2004) 37–56. [31] F. Gultekin, N. Delibas, S. Yasar, I. Kilinc, In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats, Arch. Toxicol. 75 (2001) 88–96. [32] N. Abdilla, M.C. Tormo, M.J. Fabia, F.J. Chaves, G. Saez, J. Redon, Impact of the components of metabolic syndrome on oxidative stress and enzymatic antioxidant activity in essential hypertension, J. Hum. Hypertens. 21 (2007) 68–75. [33] A.C. Bulmer, J.T. Blanchfield, I. Toth, R.G. Fassett, J.S. Coombes, Improved resistance to serum oxidation in Gilbert's syndrome: a mechanism for cardiovascular protection, Atherosclerosis 199 (2008) 390–396. [34] A. B. Santhakumar, M. D. Linden, I. Singh, Taurine in lower concentration attenuates platelet activity, Food Pub. Health 2 (2012) 58–64. [35] M. Percival, Antioxidants, Clin. Nutr. Insights 31 (1998) 1–4. [36] H. Sies, Total antioxidant capacity: appraisal of a concept, J. Nutr. 137 (2007) 1493–1495. [37] C.A. Rice-Evans, N.J. Miller, G. Paganga, Structure-antioxidant activity relationships of flavonoids and phenolic acids, Free Radic. Biol. Med 20 (1996) 933–956. [38] E.P. GutiErrez-Grijalva, D.L. Ambriz-Pere, N. Leyva-Lopez, R.I. Castillo-Lopez, J.B. Heiedia, Review: dietary phenolic compounds, health benefits and bioaccessibility, Arch. Latinoam. Nutr. 66 (2) (2016) 87–100. [39] S. Petti, C. Scully, Polyphenols, oral health and disease: a review, J. Dent. 37 (2009) 413–423. [40] J.T. Hancock, R. Desikan, S.J. Neill, Role of reactive oxygen species in cell signalling pathways, Biochem. Soc. Trans. 29 (2) (2001) 345–350. [41] D. Armstrong, R. Browne, The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress as applied to the clinical chemistry laboratory, Adv. Exp. Med. Biol. 366 (1994) 43–58. [42] I. Rusina, On the absolute values of constants of speed of interoperability of the inhibitors with peroxide radicals, Izv. Akad. Nauk. SSSR 224 (3) (1975) 646–649 (in Russian). [43] Е. Pavlova, М. Lilova, V. Savov, Study of free radical peroxidation processes in urine by enhanced chemiluminescence, Annuaire de L'universite de Sofia “St.Kliment Ohridski”, Faculte de Physique 95 (2002) 57–69. [44] E. Pavlova, V. Savov, Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton's reaction in urine, Biochem. Mosc. 71 (8) (2006) 861–863. [45] J.W. Moffet, R.G. Zika, The reaction kinetics of hydrogen peroxide with copper and iron in seawater, Environ. Sci. Technol. 21 (1987) 804–810. [46] T. Akaike, M. Suga, H. Maeda, Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO, Proc. Soc. Exp. Biol. Med. 217 (1998) 64–73. [47] E. Pavlova, N. Zografov, L. Simeonova, Comparative study on the antioxidant capacities of synthetic influenza inhibitors and ellagic acid in model systems, Biomed. Pharmacother. 83 (2016) 755–762. [48] http://en.wikipedia.org/wiki/Ascorbic_acid. [49] Y. Tsujimoto, H. Hashizume, M. Yamazaki, Superoxide radical scavenging activity of phenolic compounds, Int. J. BioChemiPhysics 25 (1993) 491–494. [50] S.J. Padayatty, A. Katz, Y. Wang, P. Eck, O. Kwon, J.H. Lee, S. Chen, C. Corpe, A. Dutta, S.K. Dutta, M. Levine, Vitamin C as an antioxidant: evaluation of its role in disease prevention, J. Am. Coll. Nutr. 22 (1) (2003) 18–35. [51] N. Hide, H. Toyoda, Antioxidant therapy as a potential approach to severe influenza-associated complications, review, Molecules 16 (2011) 2032–2052, https:// doi.org/10.3390/molecules16032032, ISSN 1420-3049. [52] http://en.wikipedia.org/wiki/Vitamin_E. [53] J. Serkedjieva, Antioxidant effects of plant polyphenols; a case study of a polyphenol-rich extract from Geranium sanguineum L. in: S.D. Gupta (Ed.), Reactive Oxygen Species and Antioxidants in Higher Plants, Science Pub, USA, 2009, pp. 275–294. [54] E. Haslam, Natural polyphenols (vegetable tannins) as drugs: possible modes of action, J. Nat. Prod. 59 (1996) 205–215.

1) the polyphenol complex reacts against all studied ROS (H2O2, %OH, O2%−) with various efficacy. It exerts lower anti-oxidant effects when compared to standards such as vitamin C and vitamin E. 2) the polyphenol complex has pronounced chelation properties against Fe2+, which is of extreme biological importance. 3) The reference concentration (0.1 mg/ml) is most appropriate for therapeutic purposes. All data obtained contribute to the elucidation of the therapeutic and prophylactic reactive mechanisms of polyphenols extracted from Geranium sanguineum L. The extract can be used as an accessible source of natural antioxidants with consequent health benefits. Acknowledgments We acknowledge Prof. Atanas Pavlov and his team from the Department of Applied Microbiology, The Stephan Angeloff Institute of Microbiology, BAS for the preparation of the root extract from the Geranium sanguineum L. We greatly acknowledge University of Sofia “St Kliment Ohridski” for the financial support of project under Contract Number: 80-1072/ 19.04.2018. Antioxidant properties of polyphenol complex from Geranium sanguineum. References [1] W.E. Hardman, Diet components can suppress inflammation and reduce cancer risk, Nutr. Res. Pract. 8 (3) (2014) 233–240. [2] M. Abbas, F. Saeed, F.M. Anjum, M. Afzaal, T. Tufail, M.S. Bashir, A. Ishtiaq, S. Hussain, H.A.R. Suleria, Natural polyphenols: an overview, Int. J. Food Prop. 20 (8) (2017) 1689–1699. [3] S. Leucuta, L. Vlase, S. Gocan, L. Radu, C. Fodorea, Determination of phenolic compounds from Geranium sanguineum by HPLC, J. Liq. Chromatogr. Relat. Technol. 28 (2005) 3109–3117. [4] A. Pantev, S. Ivancheva, L. Staneva, J. Serkedjieva, Biologically active constituents of a polyphenol extract from Geranium sanguineum L. with anti-influenza activity, Z. Naturforsch. 61c (2006) 508D516. [5] R.F. Guerrero, M.C. García-Parrilla, B. Puertas, E. Cantos-Villar, Wine, resveratrol and health: a review, Nat. Prod. Commun. 4 (5) (2009) 635–658. [6] S. Scholz, G. Williamson, Interactions affecting the bioavailability of dietary polyphenols in vivo, Int. J. Vitam. Nutr. Res. 77 (3) (2007) 224–235. [7] R. Banc, C. Socaciu, D. Miere, L. Filip, A. Cozma, O. Stanciu, F. Loghin, Benefits of wine polyphenols on human health: a review, Bull. UASVM Food Sci. Technol. 71 (2) (2014). [8] C. Manach, F. Regerat, O. Texier, G. Agullo, C. Demigne, C. Rémésy, Bioavailability, metabolism and physiological impact of 4-oxo-flavonoids, Nutr. Res. 16 (1996) 517–544. [9] C. Manach, G. Williamson, C. Morand, A. Scalbert, C. Rémésy, Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies, Am. J. Clin. Nutr. 81 (2005) 230S–242S. [10] B. Yang, A. Kotani, K. Arai, F. Kusu, Estimation of the antioxidant activities of flavonoids from their oxidation potentials, Anal. Sci. 17 (2001) 599–604. [11] D.E. Pratt, Phenolic Compounds in Food and Their Effects on Health II, Amer. Chem. Soc., Washington, DC, 1992, pp. 54–72. [12] S.R. Husain, J. Cillard, P. Cillard, Hydroxyl radical scavenging activity of flavonoids, Phytochemistry 26 (9) (1987) 2489–2491. [13] S. Kumar, A.K. Pandey, Chemistry and biological activities of flavonoids: an overview, Sci. World J. (2013) 162750. [14] L. Bravo, Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance, Nutr. Rev. 56 (11) (1998) 317–333. [15] M. Leopoldini, N. Russo, M. Toscano, Gas and liquid phase acidity of natural antioxidants, J. Agric. Food Chem. 54 (2006) 3078–3085. [16] N. Lavid, A. Schwartz, O. Yarden, E. Tel-Or, The involvement of polyphenols and peroxidise activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae), Planta 212 (2001) 323–331. [17] M. Zhu, J.D. Phillipson, P.M. Greengrass, N.E. Bowery, Y. Cai, Plant polyphenols: biologically active compounds or nonselective binders to protein? Phytochemistry 44 (1997) 441–447. [18] A.J. Charlton, N.J. Baxter, M.L. Khan, A.J. Moir, E. Haslam, A.P. Davies, et al., Polyphenol/peptide binding and precipitation, J. Agric. Food Chem. 50 (2002) 1593–1601.

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