Analyses of Metals Impact on Humulus lupulus Strobili Antioxidant Capacity

This paper presents results of analyses of metal ions effects on hop strobili antioxidant characteristics. Determination of total phenols (TP), total flavonoids (TF) and fifteen phenolic compounds, as well as the antioxidant activity (DPPH, ABTS and FRAP) of hop extracts from eight samples was conducted using UV/Vis spectrophotometry and HPLC method.


1.Introduction
Hop (Humulus lupulus L) is a plant widely known for its usage in brewing industry, precisely in beer production. All parts of this plant are a source of polyphenolic compounds such as flavonoids, catechins, flavanones, phenolic acids, etc. Phenolic compounds or polyphenols are a wide group of plant metabolites and represent nonessential dietary components. Antioxidant action of plant extract is usually related to the presence of these compounds [1]. Large number of studies shows that among all phenolic compounds, flavonoids are of the greatest significance for antioxidant activity of plant. Hydroxyl groups, especially their position, play an important role for antioxidant activity of flavonoids, since they are responsible for donating hydrogen atom and thus scavenge free radicals [2,3]. According to biological importance (antioxidant activity above all), phenolic compounds with two or three vicinal hydroxyl groups at C-atoms of benzene rings especially protrude as stated by Heim et al. [3] and Rice-Evans et al. [4,5]. One of the most important properties of these compounds is that they get easily oxidized by molecular oxygen under physiological conditions. In these autooxidation reactions, semiquinones, quinones and reactive oxygen species (ROS) are produced.
Metals are omnipresent in various biological systems. The uptake of metals, both by roots and leaves of plants, increases with metal concentration in external medium. Shah et al. reported that Cd, Cr, Pb, As, Sb and Hg are toxic for plants [6]. They can inhibit electron transport, reduction of CO2 fixation and affect plant growth through generation of free radicals and reactive oxygen species. However, plants use complex processes which activate the cells to adopt their metabolism to metal stress. Some heavy metals are essential for biological and physiological functions of plants, however, these metals become toxic depending on the nature and species of metal and plants. As stated by Lechno et al., Hernandez and Almansa, sodium ions can also be toxic for some plants [7,8].

2.1.Plant material
Strobili of eight hop samples were purchased at local markets. Each paper-aluminium bag contained 500 g of hop. Five bags of each sample were bought and mixed. In order to prepare samples for analysis, 2 grams from each mixed sample were taken in triplicate for each preparation method and further analyzed. Samples were stored in polyethylene bottles upon preparation.
Polyethylene bottles had been previously cleaned using 20% nitric acid and washed with ultra-pure water (0.05 μS/cm specific conductivity) in order to avoid contamination. All the glassware used was cleaned in the ethanol KOH solution, aqueous solution of HCl (1:1) and then thoroughly rinsed with tap, distilled and finally with ultra-pure water.

2.3.Instruments
The recording of spectra and absorbance readings were done using Agilent 8453 UV/Vis spectrophotometer, with 1 cm optical path quartz cuvettes. The pH measurements were carried out using Hanna Instruments pH-meter with glass electrode. The model 1200 Agilent Technologies, with analytical column C18 (Zorbax Eclipse XDB-C18, 5 μm, 4.6 × 150 mm), was used for performing HPLC analysis. Muffle furnace VIMS Serbia equipped with microprocessor temperature program IVIGOS3123 (±1 ºC) was used for dry mineralization. Inductively coupled plasma optical emission spectrometer iCAP 6000 (Thermo Scientific Inc., Cambridge, UK) was applied for multielement https://doi.org/10.37358/RC. 20.7.8241 analysis. This instrument uses glass concentric nebulizer, an Echelle optical design (52.91 grooves/ mm, 63.5° blaze angle) and a charge injection device solid-state detector (RACID86). High precision variable volume micropipettes (Lab Mate + ) were used for pipetting the solutions. Ultra-pure water was obtained using MicroMed high purity water system, TKA Wasseraufbereitungssystem GmbH. Thermo-stating of refrigerated solutions and extracts was done in Julabo MP 5A Open Bath Circulations.

2.4.Sample preparation
Two methods were applied for the sample's preparation: extraction-maceration (for UV/vis and HPLC methods) and dry mineralization (for ICP-OES method).
Extraction of sample (2 g) was performed with 60 mL of solvent (ethanol/water, 30/70, v/v %) during 2 h at t = 25ºC. Suspension was filtered through Büchner funnel and Whatman No. 1 filter paper. Extracts were stored in polyethylene bottles and refrigerated till they were analyzed. The procedure was based upon methods reported by Iacopini et al. [25], Borowska et al. [26] and Katalinić et al. [27].
Dry mineralization of sample (2 g) was done in porcelain crucible in the electrical furnace at the increasing temperature interval from 50 to 450ºC during the first 8 h, after it was held constant for the next 12 h. Cold ash-sample was dissolved in 2.5 mL HNO3 (1:1. v/v), filtered (filter paper Whatman, 6 µm pore size) and filled with deionized water to 50 mL. The procedure was done according to the method by Radojević and Bashkin [28].

2.5.Spectrophotometric analysis 2.5.1.TP content determination
Sample extract volume of 0.1 mL was transferred into the 10 mL volumetric flask; after that it was added 2.5 mL of deionized (DI) water, 0.5 mL of Folin-Ciocalteu reagent solution, 2 mL of 20% Na2CO3 solution and filled with water to the mark. TP content was determined by measuring absorbance at λ=760 nm after 2 h against water as a blank [29,30]. The measurements were compared to the calibration line obtained using gallic acid (GA) standard solution and the results of TP content were expressed as milligrams of gallic acid equivalents (GAE) per grams of hop (mg GAE/g ± SD). The stated procedure was repeated in triplicates for each sample.

2.5.2.TF content determination
Reaction solution was prepared by mixing 0.5 mL of hop extract, 3 mL of DI water and 0.3 mL of 5% NaNO2 according to the method by Zhishen et al. [31]. After incubation at room temperature for 5 min, 3 mL of 1% AlCl3 was added. The mixture was left at room temperature for next 5 min, then 2 mL NaOH (1 mol/L) and water to the 10 mL were added. Absorbance was measured at λ=510 nm against water as a blank. According to the measured absorbance, the content of TF was determined using catechin calibration line and displayed as milligrams of catechin equivalent per g of hop (mg CE/g ± SD). The stated procedure was repeated in triplicates for each sample.

2.6.Antioxidant activity determination
Antioxidant activity of hop extracts was evaluated applying three independent in vitro assays: DPPH, ABTS and FRAP.
The method by Brand-Williams et al., conducted with slight modifications, was used for DPPH assay [32]. The solution of DPPH radical (1×10 -4 mol/L) was prepared in methanol. Initially, 5.0 mL of this solution and 0.1 mL of hop extract were mixed in 10 mL volumetric flask. Methanol was added to the mark. The discoloration of this radical was recorded by absorbance measurement at 520 nm, 30 min after the beginning of the reaction against methanol as a blank. Total antioxidant activity (μmol/L) was determined using Trolox calibration curve. It was plotted as a function of the absorbance decrease (∆A=Ablank -Asample) of DPPH radical scavenging activity and the results were expressed as https://doi.org/10.37358/RC.20.7.8241 micromoles of Trolox equivalent (TE) per g of hop (μmol TE/g ± SD). The stated procedure was repeated in triplicates for each sample.
The assay of ABTS radical cation scavenging activity was performed according to the method described by Re et al. [33] and Arts et al. [34] with slight modifications [35]. ABTS was dissolved in methanol to the concentration of 7×10 -3 mol/L. ABTS radical cation was produced by reacting ABTS stock solution with 2.4×10 -3 mol/L K2S2O8 and allowing the mixture to stand in the dark at room temperature 12-16 h prior use. ABTS radical cation solution was diluted with methanol to obtain the absorbance of 0.7±0.02 at 734 nm. An aliquot of each hop extract (0.1 mL) was mixed with 3.9 mL of diluted ABTS radical cation solution. The reduction in absorbance at 734 nm was measured after the reaction time of 6 min at room temperature. The Trolox calibration curve was plotted as a function of the decrease in absorbance (∆A=Ablank -Asample) of ABTS radical cation scavenging activity. The results were expressed as micromoles of Trolox equivalents (TE) per gram of hop (μmol TE/g ± SD). The stated procedure was repeated in triplicates for each sample.
FRAP working reagent was prepared daily by mixing following constituents: 25 mL of acetic buffer (0.3 mol/L, pH=3.6), 2.5 mL of TPTZ (10 mmol/L) in 40 mmol/L HCl and 2.5 mL of FeCl3 (20 mmol/L). The reaction mixture was prepared in a 10 mL volumetric flask with 1 mL of hop extract, 2.1 mL of FRAP reagent and water to the mark. After the incubation time of 5 min at 37 ºC, the absorbance at 595 nm was measured. The concentration (μmol/L) of Fe 2+ equivalents was determined directly from the calibration curve. The final FRAP results were presented as micromoles of Fe 2+ equivalents per g of hop (μmol Fe/g ± SD). This assay was performed according to the method given by Benzie and Strain [35]. The stated procedure was repeated in triplicates for each sample.

2.7.HPLC analysis
Separation and identification of individual phenolic components are of importance in order to give information about real differences in phenolic profiles of studied hop samples. Chromatographic analyses of hop extracts was carried out using the gradient procedure with the mobile phase containing solvent A (5% formic acid in water) and solvent B (80% acetonitrile + 5% formic acid in water) as follows: 0-28 min, 0% B; 28-35 min, 25% B; 35-40 min, 50% B; 40-45 min, 80% B and for the last 10 min, 0% B. The temperature of column was set at 30 ºC. The solvent flow rate was 0.8 mL/min and the injection volume was 5 μL. HPLC chromatograms were recorded at 360 and 320 nm, as well as using fluorescence detector (λex/λem = 275/322 nm). The identification of individual phenolic components was performed by comparing their retention times and spectral data with those of standards. Quantification of each phenolic compound was done using external standard method. Results were expressed as mg per g of hop.

2.8.ICP-OES analysis
The operating conditions for conducting ICP-OES analysis were: power of RF generator-1150 W, pump speed -50 rpm, flush pump speed -100 rpm, cooling gas flow -12 L/min, nebulizer gas flow -0.7 L/min, auxiliary gas flow -0.5 L/min. The direction of plasma observation was dual.

2.9.Calibration parameters
The calibration line was plotted for each analyzed element. Linearity was considered satisfactory if the correlation coefficient (r) exceeded 0.9990, except for Al, Ca, Fe, K and Mg (r < 0.9990).
Selection of emission line in both axial and radial plasma viewing modes of each element was done based on the absence of spectral interferences and adequate sensitivity for determining low and high elements concentrations. Detection (LOD) and quantification (LOQ) limits were calculated using the following equations (1 and 2): were σ is residual standard deviation of the regression line and s is a slope of calibration curve [36,37]. Both limits were expressed in µg/g. The selected wavelengths, correlation coefficients (r), detection limits (LOD) and limits of quantification (LOQ) were listed in Table 1.

2.10.Statistical analysis
The aim of statistical analyses was to find correlations between the content of TP, TF, antioxidant capacity and mineral content in hop strobili samples. Principal component analysis was used to evaluate datasets and to establish the relationship among variables. Cluster analysis was performed in order to group samples based on their proximity. Analyses were performed using a statistical package Statistica 8.0. StatSoft, Inc., Tulsa, OK, USA. A probability of p<0.05 was considered statistically significant [36].

3.1.TP, TF content and antioxidant activity of hop extracts
The spectrophotometric results of TP, TF content and antioxidant activity of hop were presented in Table 2. The content of TP in hop samples was in the range 9.54 mg GAE/g-13.33 mg GAE/g. The data obtained in this study are comparable to the results reported by Kirca and Arslan [24] and by Lermusieau et al. [38] while the results published by Proestos et al. [39], Mudura et al. [22] and Wojdylo et al. [19] were significantly lower. Higher TP content (23.1 mg/g and 151.42 mg/g) in hop extract was determined by Kahkonen et al. [20] and by Arsene et al. [18]. The content of TF in hop samples was in the range 3.57 mg CE/g-5.54 mg CE/g, which is 36%-44% from TP content.
Ethanol hop extracts showed relatively good radical-scavenging properties toward stable DPPH radical. The obtained results of DPPH radical scavenging activity ranged from 32.25 μmol TE/g to 37.5 μmol TE/g, for ABTS from 41.67 μmol TE/g to 46.8 μmol TE/g and for FRAP assay from 30 μmol Fe/g to 75 μmol Fe/g. The highest values of TP, TF, DPPH and ABTS were obtained for sample no. 2, while the highest values for FRAP were measured for sample 8. The lowest values of TP, DPPH, ABTS were determined for sample 1, while sample 5 has the lowest values for TF and FRAP.
Total phenolic acids content was in the interval 0.286 mg/g-0.752 mg/g. Chlorogenic acid with concentration interval 0.152 mg/g-0.349 mg/g is the major phenolic acid in all hop extracts. The phenolic acid found in the lowest concentration is 4-hydroxybenzoic acid (0.018 mg/g-0.124 mg/g). pcoumaric acid (0.019 mg/g-0.203 mg/g) and ferulic acid (0.039 mg/g-0.098 mg/g) are detected as well. Proestos et al. [39] published very similar results for 4-hydroxybenzoic acid (0.015 mg/g) and pcoumaric acid (0.038 mg/g), but ferulic acid was not detected. The higher contents of p-coumaric acid (0.228 mg/g) and ferulic acid (0.143 mg/g) in hop were reported by Wojdylo et al. [19].
Total quantity of flavan-3-ols was determined in the range 0.429 mg/g-0.996 mg/g. In all hop extracts, procyanidin B2 (0.132 mg/g-0.416 mg/g) was the major, while epicatechin (nd-0.159 mg/g) was the minor phenolic component. The similar content of catechin (0.238 mg/g), but significantly higher quantity of epicatechin (1.438 mg/g) was reported by Callemien et al. [42]. Magalhaes et al. confirmed presence of catechin, epicatechin and procyanidin in hop extracts [43]. Proestos et al. reported that catechin is the most common flavan-3-ol in plant materials [39].

3.3.ICP-OES analyses of hop strobili mineral content
Experimental values of hop mineral content were expressed as mean value (μg/g) ± SD for three replicate measurements and three groups of elements were established for obtained data (Tables 4, 5 and 6).  Potassium concentration ranged from 9688 μg/g to 11007 μg/g, while sodium was the metal present in the lowest concentrations (78.7 μg/g-89.67 μg/g). Considering this, Na/K coefficients values were significantly low for all eight samples due to the high K content in all samples. The evaluated Ca content was in the interval of 1097 μg/g-1255.4 μg/g, while Mg concentrations were in the range of 1874.6 μg/g-2176.4 μg/g. Ghiselli et al. published elements concentrations in hop that are mostly in accordance with results of presented research, except for Ca that are 10 times higher (9380 mg/kg-15515 mg/kg) [44]. Liu et al. quantified approximately the same content of Ca and Na as it was determined in this study, but two times higher concentration of Mg and 5 times smaller concentration of K [16]. Our research revealed the major elements content in hop samples decreased as following: K>Mg>Ca>Na.
The results of minor elements content in hop extracts are presented in Table 5. The minor elements content in hop samples decreased as following: Fe>Zn>Mn>Cu>Si>V>Mo> Ni≈Cr≈Sn>Se>Co. The highest content among these elements in all samples was determined for Fe (65.9 μg/g-85.1 μg/g). Zinc was measured in concentration range 31.7 μg/g-42.74 μg/g, while manganese content in studied samples was about 30 μg/g. Copper concentration range was 7.47 μg/g-9.776 μg/g, while cobalt was quantified in the interval of 0.035 μg/g-0.091 μg/g. The intervals for other elements are: 8.98 μg/g-12.67 μg/g (Si), 7.12 μg/g-9.02 μg/g (V), 1.125 μg/g-2.056 μg/g (Mo), 0.564 μg/g-0.792 μg/g (Ni), 0.456 μg/g-0.871 μg/g (Cr), 0.46 μg/g-0.792 μg/g (Sn) and 0.17 μg/g-0.667 μg/g (Se). Ghiselli et al. presented partly similar or, for some of these elements, higher results (Si, V, Mo and Se were not examined in hop samples), while Cr and Ni content was higher, four times and ten times, respectively [44]. Liu  had up to 4 times higher content of Ni, but about 7 times lower content of V compared to the results reported in this study [16]. Melandrino et al. analyzed hop for the presence of Se and Sn but did not quantified these elements in samples they used [45]. A thorough search of the relevant literature yielded published articles that analyzed and quantified Sn and Se in hops.
The results of nonessential elements determination are shown in Table 6. The mean concentrations of third group elements were increasing in the following order: Cd<As<Pb<Sb<Ba<Li<Sr<Al. Among all nonessential elements, the highest contents of Al (13.88 μg/g-16.66 μg/g) and Sr (6.98 μg/g-9.234 μg/g) were determined. The metals such as Cd (0.055 μg/g-0.098 μg/g), As (0.11 μg/g-0.38 μg/g), Pb (0.157 μg/g-0.50 μg/g), Sb (0.378 μg/g-0.565 μg/g), Ba (2.871 μg/g-4.319 μg/g) and Li (5.876 μg/g-7.83 μg/g) were also quantified. Two times higher concentrations for Cd, twenty times for Ba and about five times for Sr, were reported by Ghiselli et al. [44]. Seven times higher content of Al, up to two times higher content of Cd, 35 times higher amount of Sr and approximately the same amount of As and Pb were presented by Li et al. [16]. A thorough search of the relevant literature yielded published articles that analyzed and quantified Sb in hops.

3.4.Statistical analyses of quantities of total phenols, total flavonoids, antioxidant capacity and mineral content of hop strobili 3.4.1.Principal component analysis (PCA)
PCA was applied to the entire data set and six principal components with eigenvalues higher than 1 were obtained, explaining 97.264% of variability. The first component explained 36.881%, the second 21.863%, the third 16.186%, the fourth 9.525%, the fifth 7.431% and the six component 5.378%. The PCA results are displayed in Table 7 and Figure 2 Table 7 and Figure 2 it can be observed that there is a strong positive relationship between DPPH, FRAP, ABTS, TP and TF. Group of elements (Pb, Sb, Co, Na and Cr) with highly positive factors is strongly negatively correlated with polyphenols and antioxidant tests.

3.4.2.Cluster analyses
Upon standardization of variables, Ward's method with squared Euclidian distance was used as a measure of proximity between the samples. Obtained dendrogram (Figure 3) presents clustering of hop strobili samples. Horizontal cluster analyses (HCA) showed that all the samples could be grouped into two main clusters. Cluster I was consisted of samples 7, 8, 6 and 2, while cluster II was formed by samples 4, 3, 5 and 1. The samples of cluster I are characterized by lower content of Pb, Sb, Co, Na and Cr and higher content of TP and TF and higher antioxidant capacity. The samples of cluster II are characterized by higher content of these metals and lower content of TP and TF and lower antioxidant capacity. Further analyses showed better similarity of samples by grouping them into the subclusters (S1 and S5 are similar to each other, and the nearest samples to them are S3 and S4, which are also similar to each other). The samples S8 and S6 are close to each other, while the sample S7 does have some similarities to either S8 or S6 (or both), but also with S2.

4.Conclusions
The results of mineral content effect on TP and TF content, as well as on antioxidant activities of hop strobili are presented in this paper. The content of TP was determined in interval 9.54-13.33 mg GAE/g. TF were quantified in the range 3.57-5.54 mg CE/g. Hop extracts showed ability of neutralization DPPH (32.25-37.52 µmol TE/g) and ABTS (41.67-46.85 µmol TE/g) radicals, as well as high potential to reduce Fe 3+ measured by FRAP assay (30.50-75.48 µmol Fe/g). Results obtained for these antioxidant assays have following trend FRAP>ABTS>DPPH. Hop extracts that do not follow this trend are sample 1 (ABTS>FRAP>DPPH) and sample 5 (ABTS>DPPH>FRAP). These two samples have the lowest content of TP and TF and the lowest antioxidant capacity, but have the highest total content of Pb, Sb, Co, Na and Cr. The best results considering TP, TF, DPPH and ABTS values were obtained for sample 2 due to the lowest total content of Pb, Sb, Co, Na and Cr. This study also included identifications and quantification of following phenolic compounds: seven flavonols (quercetin-3-galactoside, quercetin-3-rutinoside, quercetine-3-glucoside, quercetine-3-glycoside, kaempferol-3-rutinoside, kaempferol-3-glucoside, kaempferol-3-glycoside), four phenolic acids (4hydroxybenzoic acid, chlorogenic acid, p-coumaric acid and ferulic acid) and four flavan-3-ols (procyanidin B1, catechine, procyanidin B2 and epicatechine). The content of these compounds was 0.290-0.965 mg/g for total quercetin-glycosides, 0.048-0.546 mg/g for total kaempferol glycosides, 0.285-0.752 mg/g for total phenolic acids and 0.429-0.996 mg/g for total flavan-3-ols.
Furthermore, this is the first time the mineral content (24 elements) effect on TP and TF quantities and antioxidant capacity of hop strobili was analyzed. Statistical results showed strong positive https://doi.org/10.37358/RC.20.7.8241 relationship between DPPH, FRAP, ABTS, TP and TF. Group of elements (Pb, Sb, Co, Na and Cr) with highly positive factors is strongly negatively correlated with polyphenols and antioxidant tests (i.e. higher concentrations of polyphenolic compounds and higher values for antioxidant capacity were associated with lower concentrations of these, for hop, potentially toxic metals).