Antioxidant capacity and cadmium accumulation in parsley seedlings exposed to cadmium stress

Antioxidant Capacity and Cadmium Accumulation in Parsley

Seedlings Exposed to Cadmium Stress

Y. Ulusu

, L. Öztürk

*, and M. Elmastaş

a_{Department of Bioengineering, Faculty of Engineering, Karamanoğlu Mehmetbey University, Karaman, 70100 Turkey} b_{Department of Biology, Faculty of Science and Arts, Gaziosmanpaşa University, Tokat, 60150 Turkey} c_{Department of Chemistry, Faculty of Science and Arts, Gaziosmanpaşa University, Tokat, 60150 Turkey}

*e-mail: lokman.ozturk@gop.edu.tr

Received April 19, 2016

Abstract⎯Parsley (Petroselinum hortense L.) plants cultivated under controlled conditions were exposed to different doses of cadmium to investigate the antioxidant capacity and cadmium accumulation in the sam-ples. Two-months-old parsley seedlings were treated with four different concentrations of CdCl2 (0, 75, 150, and 300 μM) for fifteen days. Cadmium level in leaves increased significantly by increasing the Cd contami-nation in the soil. Total chlorophyll and carotenoid content declined considerably with Cd concentration. Cd treatment caused a significant increase lipid peroxidation in tissue of leaf. Superoxide dismutase activity (SOD, EC 1.15.1.1) increased partially at 75 and 150 μM CdCl2 concentrations whereas the activity decreased at 300 μM CdCl2. Catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were reduced by Cd application. Total phenolic compound amount increased significantly with increasing Cd concentration, as ferric reduction power, superoxide anion radical, and DPPH˙ free radical scavenging activ-ities elevated slightly by the concentration. These results suggest that antioxidant enzymes activactiv-ities were repressed depending on accumulation of cadmium in leaves of parsley while the non-enzymatic antioxidant activities slightly increased.

Keywords: Petroselinum hortense, ascorbate peroxidase, antioxidant activity, cadmium, catalase,

photosyn-thetic pigment, polyphenol contents, superoxide dismutase

DOI: 10.1134/S1021443717060139

INTRODUCTION

Parsley is widely used in Middle Eastern, Euro-pean, and American cooking and often used as a gar-nish. In the central and eastern Europe and in western Asia, many traditional dishes are served with fresh green chopped parsley, a source of phytochemicals known to exert a variety of biological effects.

Heavy metal pollution as a result of agricultural and industrial activities, one of the major problems of human being recently, is increasing and contaminat-ing to soil and water [1]. Cadmium is widely dispersed to the environment because of its usage in many areas such as mining, smelting, electroplating, and produc-tion of paints and pigments [2].

Cadmium is a mobile element and has a high solu-bility in water; therefore it can be readily taken up by plant root systems and eventually suppresses plant growth [3, 4]. Cadmium is fairly toxic to plants since it affects many physiological and biochemical

parame-ters such as growth, photosynthesis, nutrition, and water status [1, 4]. Cadmium has high affinity to thiol groups, and so directly affects the sulf hydryl groups in proteins which leads to inhibition of activity or disrup-tion of structure [5]. Although Cd does not participate directly in cellular redox reactions, displacement of an essential element of protein leads indirectly formation of free radical and reactive oxygen species (ROS) and causes oxidative damage such as lipid peroxidation [5]. Plants have evolved enzymatic and non-enzymatic antioxidant mechanism to protect their cellular and sub cellular component from the effects of the reactive oxygen species. ROS scavenging system comprises enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase as well non-enzy-matic compound: ascorbate, carotenoids α -tocopher-ols and glutathione. Antioxidant enzymes and com-pounds are modulated by heavy metal-induced oxida-tive stresses and reduce stress damage [3].

Plants induce the synthesis of phenolic compounds when exposed to heavy metals. Cadmium accumulates in plant tissues and also increases the amount of phe-nolic compounds due to its concentration in the soil [3, 6, 7]. Phenolic compounds have various functions 1_{The article is published in the original.}

Abbreviations: APX—ascorbate peroxidase; CAT—catalase; DPPH˙—2,2-diphenyl-1-picryl-hydrazil radical; FRAP—Fe3+ reducing antioxidant power; SOD—superoxide dismutase.

including antioxidant, antimicrobial and antimuta-genic activity. The antioxidant properties of phenolic compounds are due to their high metal chelating potential and ability to capture lipid alkoxyl radicals that initiate lipid peroxidation. Phenolic compounds act as a barrier to entry of heavy metals into the cell from the cell surface and also play an important role in Cd capture [1, 6, 8]. Because of its long biological life-time and retention in soil, Cd easily accumulates in plant originated foods which eventually affects human health by means of nutrition [2, 5]. Parsley can easily be grown in areas open to heavy metal pollution such as small gardens in residential areas, road sides, and industrial districts which is commonly consumed throughout the year in Turkey. The aim of this study was to investigate Cd accumulation and its effects on enzymatic and non-enzymatic antioxidant capacity of parsley leaves.

MATERIALS AND METHODS

Plant growth conditions and cadmium treatments. Parsley (Petroselinum hortense L.) seeds were surface ster-ilized with 5% hypochlorite (v/v). The seeds were sown into 2 kg capacity pots containing 40% turf, 40% soil and 20% sand. Seedlings were grown at 13.14 J m–2 _s–1 _for three months in green house. CdCl2 (0, 75, 150 and 300 μM) were applied to three-months-old seedlings for 15 days, and then leaves of seedlings were harvested.

Determination of cadmium content. Dried leaf tis-sue (1 g) was homogenized with 6 mL of a 65% nitric acid and 2 mL of a 30% hydrogen peroxide. Samples were boiled for 1–2 h until completely digested. The pellet was dissolved with 1 mL of nitric acid and added 4 mL of deionized water [9]. Cd concentrations in the tissue extract were determined by atomic absorption spectrophotometry (Perkin Elmer Analyst 700, United States).

Chlorophyll and carotenoids determination. Total chlorophyll and carotenoids were determined by the methods of Arnon [10] and Jaspar [11]. Leaf tissues (0.2 g) were homogenized with 80% (v/v) acetone and centrifuged at 3000 g for 5 min. The absorbency of supernatant was measured by spectrophotometer at 450, 645 and 663 nm wavelengths.

Determination of lipid peroxidation. The concentra-tion of lipid peroxidaconcentra-tion products was determined in leaves in terms of thiobarbituric acid according to the method of Velikova et al [12]. Fresh leaf (0.5 g) was homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA), and then centrifuged at 10000 g for 20 min. 0.5 mL of the supernatant was added to 1 mL 0.5% (v/v) thiobarbituric acid (TBA) in 20% TCA and the mixture was incubated in boiling water for 30 min, and the reaction stopped by immersion of reaction tubes into an ice bath. Then the samples were centri-fuged at 10000 g for 5 min, and the absorbance of supernatant was read at 532 nm and corrected for

non-specific turbidity by subtracting the absorbance value at 600 nm wavelength. An extinction coefficient of 1.55 × 105 _M–1_cm–1 _{was used to quantify lipid} perox-ides and it was expressed as μmol malondialdehyde (MDA)/g fresh weight.

Enzyme extractions and assays. Leaf tissues (0.5 g) from control and treated plants were grounded with liquid nitrogen and homogenized in 3 mL of buffer containing 50 mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, and 1% polyvinylpolypyrrolidone (PVPP) (w/v). The homogenates were centrifuged at 15 000 g for 15 min at 4°C and resulting supernatants were freshly used for the determinations of SOD, CAT, and APX activities. Enzyme activity was expressed as enzyme unit (U) for mg protein, per g of fresh leaf.

Superoxide dismutase activity. SOD activity was measured by the modified method of Beyer and Fridovich [13]. 3 mL of the reaction mixture con-tained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 60 μM nitroblue tetrazolium (NBT), 0.1 mM EDTA, and 0.1 mL enzyme extract. Reaction was started by adding 60 μM ribof lavine and placing the tubes under two cool white f luorescent lamps (60 W) for 30 min. Reaction was stopped by switching off the light and putting the tubes into dark. Control reaction mixture was prepared to include the same content except enzymes. A non-irradiated reaction mixture served as a blank. The absorbance for all sam-ples was measured at 560 nm. SOD activity unit was defined as the amount of enzyme inhibiting NBT reduction by 50%.

Catalase activity. To determine the CAT activity, the reaction mixtures were used containing 50 mM KH2PO4 (pH 7), 13 mM H2O2, and 30 μL enzyme extract. The decrease in the absorbance of H2O2 was recorded at 240 nm for 3 min by using spectrophotom-eter (Schimadzu UV/VIS-1208, Japan). One unit of activity was defined as the amount of enzyme catalyz-ing the decomposition of 1 μmol H₂O₂ per min, calcu-lated from the extinction coefficient (0.036 cm2 _μmol–1₎ for H2O2 at 240 nm [14].

Ascorbate peroxidase activity. The activity of APX was measured according to the method of Wang et al. [15]. The reaction mixture was composed of 50 mM phosphate buffer (pH 6), 1.47 mM H2O2, 0.5 mM ascorbic acid, and 50 μL enzyme extract. The reaction was started by the addition of H2O2, and the oxidation of ascorbate was measured for 3 min at 290 nm. The enzyme activity was calculated using the extinction coefficient of ascorbate (2.8 mM–1 _cm–1 _{at 290 nm).}

Determination of total phenolic content. Total phe-nolic content was determined using Folin-Ciocalteu’s reagent. Fresh leaf tissue (1.5 g) was dried at 50°C in incubator for two days and extracted with 95% etha-nol. Stock solution was prepared as 2 mg dry matter per mL ethanol. 100 μL of the solution were diluted with 9 mL water and 1 mL of Folin-Ciocalteu’s reagent

and shaken vigorously. Then 3 mL of 2% sodium car-bonate solution was added and the volume was made up to 14 mL with distilled water. The contents were mixed thoroughly and the absorbance was recorded at 760 nm using a spectrophotometer. Total phenolics were calculated from the gallic acid standard curve and expressed as mg/g fresh weight [16].

Ferric ions (Fe3+_{) reducing antioxidant power}

(FRAP). The reducing power of samples was deter-mined by the method of Oyaizu [17] with slight mod-ification. Different concentrations of the samples (10–30 μg/mL) in 1 mL of distilled water were mixed with 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide (K3Fe(CN)6) (1%) and then the mixture was incu-bated at 50°C for 20 min. Aliquots (2.5 mL) of trichlo-roacetic acid (10%) were added to these mixtures. The 2.5 mL of this solution was mixed with distilled water (2.5 mL) and FeCl3 solution in water (0.5 mL, 0.1%), and the absorbance was measured at 700 nm in a spec-trophotometer.

Superoxide anion radical scavenging activity. Super-oxide radicals were generated according to Beau-champ and Fridovich [18] with slight modification. Reagents and samples were prepared in phosphate buffer (0.05 M, pH 7.8). Reactants were ribof lavin, methionine and NBT at concentrations of 1.33 × 10–5_{, 4.46 × 10}–5 _{and 8.15 × 10}–8 _M, respec-tively which constitutes a total of 3 mL volume. The photochemical reaction for 35 minutes at room tem-perature caused reduction of ribof lavin and generation of . The absorbance of formazan was measured at 560 nm and the inhibition percentage of superoxide anion radical generation determined according to the following formula:

scavenging activity (%) = (1– As/Ac) × 100, where Ac – absorbance of the control, As – absor-bance of samples.

DPPH free radical scavenging activity. The free radical scavenging activity of the samples were mea-sured by 2,2-diphenyl-1-picryl-hydrazil (DPPH˙) using the method of Blois [19]. 0.1 mM solution of

2 Oi−

2 Oi−

DPPH˙ in ethanol was prepared and 1 mL of this solu-tion was added to 3 mL of samples at different concen-trations. The mixture was shaken vigorously and allowed to stand at room temperature for 30 minutes. Then the absorbance of mixture was measured at 517 nm in a spectrophotometer. The DPPH˙ radical scavenge capacity was calculated using the following equation:

where Ac – absorbance of the control, As – absor-bance of samples.

Experimental design and statistical analysis. The experiment was set up as completely randomized design with four replications per treatment. Differ-ences between the mean values were analysed by one-way ANOVA, taking P < 0.05 as significance level, according to Duncan’s multiple range tests.

RESULTS AND DISCUSSION

Cd accumulations in leaves have increased 5.2, 7.4 and 25.7 folds in 75, 150 and 300 μM CdCl₂ concen-trations, respectively (Table 1). Cadmium content increased considerably in Cd stressed seedlings as compared with control seedlings (P < 0.05). Although the levels of metal accumulation in leaves increased remarkably with the increase of cadmium concentra-tion, no visual stress symptoms were observed on the parsley plants for the concentrations of CdCl₂ used during our experimental process. The increase of cad-mium concentration in the soil leads parsley plant to accumulate more Cd. These results are in agreement with the results of previous reports [9, 20].

Cd applications caused a reduction between 35.8– 47.5% in total chlorophyll quantity in the leaves. How-ever, carotenoid content decreased only at 150 and 300 μM cadmium concentrations and content declined between 18.1 and 30.9%, respectively (Table 1). Chlo-rophyll and carotenoids have a central role for the photosynthesis of every green plant. Therefore, any significant alteration in photosynthetic pigments is likely to cause a marked effect on the entire metabo-lism of the plants. Besides carotenoids are antioxidant

(

)

DPPH scavenging effect (%)= 1−A A ×100,

Table 1. Effect of cadmium treatment on contents of cadmium, chlorophyll, carotenoid and malondialdehyde in leaves of

parsley

Data are mean ±SE, n = 4, Different letters in the same column indicate significant differences, 5% level, Duncan’s multiple range test.

CdCl2 concentration, μM Cd content in leaves, μg/g fr wt Total chlorophyll content, mg/g fr wt Carotenoid content, mg/g fr wt Malondialdehyde content, μmol/g fr wt 0 (Control) 0.10 ± 0.03d 12 ± 0.1a 2.1 ± 0.04a 22.6 ± 0.25c

75 0.52 ± 0.19c 7.7 ± 0.08b 2.2 ± 0.04a 35.9 ± 0.4a

150 0.74 ± 0.2b 7.6 ± 0.1c 1.6 ± 0.03b 29.6 ± 1.8b

substances and they protect chlorophyll from oxida-tive damage. Cd applications to parsley plants caused decomposition of total chlorophylls more than carot-enoids in leaves which is consistent with the report about reduction in chlorophyll contents in the sun-f lower and spinach by excess Cd [20, 21]. High Cd concentration may inhibit the formation of chloro-phyll by interfering with activation of the enzymes responsible for chlorophyll biosynthesis [21].

Cd treatment enhanced lipid peroxidation signifi-cantly as compared to the control seedlings (P < 0.05). The relative increases of lipid peroxidation increase were 58.8, 30.9 and 23.4% respectively (Table 1). Increased production of malondialdehyde by Cd exposure had been observed in some plants such as pea [3], tomato [22] and wheat seedlings [7]. Lipid perox-idation increase in this research was probably due to the harmful effect of ROS derivatives in the cellular component. Excessive levels of ROS may have resulted in peroxidation of membrane lipids, ulti-mately leading to severe cellular damage [7].

Cd treatment has affected antioxidant enzyme activities differently. Antioxidant enzyme activities were generally decreased by Cd treatment in parsley seedlings except SOD activity at 75 and 150 μM CdCl2 (Table 2). However, Cd stress has further reduced CAT activity in parsley leaves. It was reported that cadmium toxicity decreased CAT activity in wheat [7] and pine roots [23]. The reduction in CAT activity may be due to the fact that Cd causes substitution of Fe in active site of the catalase and inadequate iron uptake from soil. Cadmium may also lead to enzyme inhibition by interacting with thiol groups in their structure. A

decline in the uptake of elements like Fe, Mn, and Zn due to increase of Cd concentration in soil may cause activity loss in antioxidant enzymes, since they are found on active sites of enzymes [22]. The involve-ment of antioxidative enzymes for protecting plants from heavy metal stress conditions has been docu-mented through various studies [20, 22]. In these reports, antioxidant enzyme activities were varied depending on the type of metal, concentration, plant variety and plant tissue type.

Cd application caused an increase in the phenolic content of parsley leaves (Table 3) which were 7.1, 39.2 and 32.1% at 75, 150 and 300 μM cadmium concen-trations, respectively. Foregoing results agree with the phenolic constitution of cadmium treated Erica

ande-valensis [24]. Phenolic compounds have various

bio-logical activities such as playing an important role in adsorbing and neutralizing free radicals, quenching singlet oxygen, or decomposing peroxides by contrib-uting to antioxidant activity [1, 25].

FRAP, superoxide anion radical and DPPH˙ free radical scavenging activity were used to determine the non-enzymatic antioxidant capacity of parsley. The reduction of ferric ion (Fe3+_{) to ferrous ion (Fe}2+_{) in} the presence of the leaf extracts was used for determi-nation of FRAP in which the reduction power was increased to 15 and 6.19% at 150 and 300 μM CdCl2, respectively (Table 3). The scavenging activity of superoxide anion radical was determined by the con-sumption of superoxide anion produced from reaction mixture. Superoxide anion scavenging activity of leaves in ethanol extracts increased inappreciably at all

Table 2. Effect of cadmium treatment on antioxidant enzymes activities in leaves of parsley

Data are mean ± SE, n = 4, Different letters in the same column indicate significant differences, 5% level, Duncan’s multiple range test.

CdCl2 concentration, μM Superoxide dismutase, U/g fr wt Catalase, U/g fr wt Ascorbate peroxidase, U/g fr wt

0 (Control) 109.6 ± 3.2a 85.5 ± 4a 1.29 ± 0.04a

75 112.8 ± 2.7a 71 ± 3.3b 1.26 ± 0.03a

150 113 ± 2.3a 80 ± 1.6ab 1.17 ± 0.02a

300 107.2 ± 2.1a 78.5 ± 3.2ab 1.17 ± 0.04a

Table 3. Effect of cadmium treatment on non-enzymatic antioxidant activities in leaves of parsley

Data are mean ±SE, n = 4. Different letters in the same column indicate significant differences, 5% level, Duncan’s multiple range test.

CdCl₂ concentration, μM Total phenolic compounds, mg/g fr wt Reduction power (FRAP) A₇₀₀ Superoxide scavenging activity, % DPPH˙ scavenging activity (IC₅₀), μg/mL 0 (Control) 0.28 ± 0.02b 0.11 ± 0.01a 98.96 ± 0.13a 4.52 ± 0.05a

75 0.30 ± 0.01b 0.10 ± 0.03a 99.27 ± 0.08a 4.32 ± 0.04a

150 0.39 ± 0.02a 0.13 ± 0.01a 99.25 ± 0.26a 4.25 ± 0.04a

Cd concentrations (Table 3). Radical scavenging activity has vital importance due to the deleterious role on free radicals formed in foods and in biological sys-tems. The stable DPPH˙ radical is widely used to eval-uate the free radical scavenging activity of antioxidants in several plant extracts. The reduction of IC50 value depending on the increase in cadmium concentration in our study is an indication of free radical scavenging activity (Table 3).

Cd treatment increased slightly the power of reduc-tion, superoxide anion and free radical scavenging activities in leaves of parsley. The results are consistent with previous report [26].

Plants synthesize a large number of phenolic com-pounds with different antioxidant properties. Cad-mium decreased the amount of caffeic and ferulic acids and partially increases the content of rutin and chlorogenic acid in maize [8]. It was reported that Cd application reduced phenolic compounds such as gal-lic, protocatechuic and vanillic acids amount but sig-nificantly increased the contents of ferulic and chloro-genic acids in Matricaria chamomilla [6]. The antioxi-dant activity of phenolic compounds varies depending on the number and position of hydroxyl groups in the molecules, and structure of phenolic molecules [1]. In this study, although the amount of total phenolic com-pounds increased significantly in high cadmium con-centration, the partial increase in non-antioxidant activity may result from the composition of the pheno-lic compounds.

Superoxide anions are precursors for active free radicals reacting to biological macromolecules and thereby inducing tissue damage. They play an import-ant role in the formation of other reactive oxygen spe-cies (ROS) such as hydrogen peroxide, hydroxyl radi-cal, and singlet oxygen, which induce oxidative dam-age in lipids, proteins and DNA. Free radical scavengers and antioxidants are useful compounds for protection against Cd toxicity [27].

Although a partial decrease observed in antioxidant enzyme activities due to the Cd accumulation in leaves non-enzymatic antioxidant activity increased slightly. However, the increase in the non-enzymatic activity is not enough to prevent oxidative damage. Oxidative damage increased in parsley leaves as a result of imbal-ance between the production and consumption of reactive oxygen species. In the future studies, it would be beneficial to analyse individual phenolic com-pounds in understanding the effects of environmental stress on their antioxidant capacities.

ACKNOWLEDGMENTS

This work was supported by the Gaziosmanpasa University Unit for Scientific Research Projects, proj-ect no. 2005/25.

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