1. Introduction
Antioxidants that compensate for deleterious effects of free radicals on cells and their relations to certain diseases continue to stimulate the research of the antioxidant and antiradical properties of components contained in various natural products and dietary supplements. Among the most important antioxidants and free radicals scavengers are polyphenols, such as phenolic acids and flavonoids.
For the in vitro evaluation of antioxidant activity, a large number of biochemical assays have been developed. Most of them are based on the scavenging of artificial reactive oxygen or nitrogen species. The most frequently used are 2,2′-Azino-
bis(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS), Oxygen Radical Absorption Capacity (ORAC) [
1]. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is a colorimetric, rapid, and sensitive reaction based on the neutralization of a nitrogen radical. 2,2′-Azino-
bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS) assay is based on the spectrophotometric measurement of specific cation radical neutralization, and in ORAC assay, the oxygen radical absorption capacity is measured kinetically with fluorimetric detection.
However, many of these methods do not exhibit a good correlation with the ability of the compounds to inhibit oxidative deterioration in vivo [
2]. This is mainly associated with the fact that the biological manifestation of antioxidant activity depends not only on the chemical reactivity of the antioxidant, but also on its pharmacokinetics. The important factors are bioavailability/bioaccessibility, target location in the organism, related environment, and interaction with other components present. To compensate (at least partially) for these aspects, utilizing cellular assays is usually the preferred option. Nevertheless, in general, due to the complexity of mixtures of antioxidants occurring in complex foods, and different reaction targets, there is no single method capable of accurately describing antioxidant activities, and individual models should be developed for particular food matrices.
In our paper, we assessed the antioxidant properties of silymarin, a complex of bioactive flavonolignans and their flavonoid precursor taxifolin, which are a significant part of the milk thistle (
Silybum marianum (L.)) plant. The major components of the silymarin complex are silybin A and B, isosilybin A and B, silydianin, silychristin, and isosilychristin, accounting, together with taxifolin, for approximately 70% of the silymarin complex (the remaining part is an undefined yet potentially bioactive polyphenolic fraction) [
3,
4]. Besides its significant antioxidative potential, the popularity of silymarin is steadily increasing due to its putative chemopreventive and hepatoptotective effects [
5,
6,
7]. However, it should be noted that although most experimental reports and some clinical data suggest that it does play a beneficial role [
8,
9,
10], the clinical importance of silymarin is negligible [
5,
7,
11,
12]. Moreover, the results of clinical studies conducted to date are often rather controversial and non-reproducible [
7,
11,
13]. This is probably related to the ambiguously defined chemical composition of the silymarin complex [
14] and possible contaminants [
15].
The aim of this study was to characterize chemical composition and antioxidation activity of 26 commercial milk thistle-based dietary supplements by a panel of antioxidant activity assays.
2. Materials and Methods
2.1. Analytical Standards and Chemicals
2,2′-Azo-bis-(2-methylpropionamidine) dihydrochloride (AAPH, Sigma-Aldrich, USA); 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Sigma-Aldrich, USA); 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, USA); 2′,7′-dichlorfluorescein diacetate (DCFH-DA, Sigma-Aldrich, USA); Antibiotic Antimycotic Solution (Sigma-Aldrich, USA); Fetal Bovine Serum (FBS, Sigma-Aldrich, USA); Fluorescein (Sigma-Aldrich, USA); Minimum Essential Medium Eagle (EMEM, Sigma-Aldrich, USA); Potassium persulfate (K2S2O8, Sigma-Aldrich, USA); Quercetin (Sigma-Aldrich, USA); Trolox (Sigma-Aldrich, USA).
Silymarin (defined as’ flavonolignan mixture extracted from the seeds of Silybum marianum’, product number S0292, Lot BCBM3466V, declared content of silybin A/B 42.6%, Sigma-Aldrich, USA; further referred to as silymarin SA); Silibinin (mixture of silybin A and B diastereoisomers, product number S0417, Lot BCBP6193V, declared purity 99.1%, Sigma-Aldrich, USA; further referred to as silibinin SA).
The analytical standards of silybin A; silybin B; isosilybin A; isosilybin B; 2,3-dehydrosilybin; silychristin; silydianin; and taxifolin isolated from commercially available silymarin (purchased from Liaoning Senrong Pharmaceutical, Panjin, People’s Republic of China, batch no. 120501) according to a published method (29) were provided by the Laboratory of Biotransformation (Institute of Microbiology of the CAS, Prague, Czech Republic).
The internal reference sample of dried milk thistle extract, containing 139 ± 17 mg/g of silybin A, 179 ± 23 mg/g of silybin B, 38 ± 5.2 mg/g of isosilybin A, 8.8 ± 0.9 mg/g of isosilybin B, 2.5 ± 0.3 mg/g of 2,3-dehydrosilybin, 180 ± 31 mg/g of silychristin, 72 ± 8.3 mg/g of silydianin, and 13.5 ± 6.2 mg/g of taxifolin, was available from our previous study [
15]; the reference values were calculated as mean values from repeated analyses (
n = 40) by the methods described below, obtained over a long period of time.
2.2. Samples and Standards Preparation
The milk thistle-based dietary supplement samples investigated in the antioxidation tests were purchased on the Czech and US market between 2016 and 2017, and their characterization as provided by manufacturers, is summarized in
Table 1. For each of the supplements, the internal content of twenty capsules was weighed separately and then mixed together to obtain a homogenized representative sample.
For the quantitative analysis of silymarin flavonoid/flavonolignans, the method previously described in Fenclova et al. [
15] was followed. Briefly, 1 g of representative sample of the dietary supplement was weighed into a 50 mL PTFE centrifuge tube and 3-times repeatedly extracted by shaking with 15 mL of ethanol, to assure the 100% recovery. The extracts were collected and pooled into a volumetric flask and made up to 50 mL with ethanol. Prior to the analysis, the final extract was diluted 10-, 100-, 1000- and 10,000-fold with ethanol. The analytical standards of silybin A; silybin B; isosilybin A; isosilybin B; 2,3-dehydrosilybin; silychristin; silydianin; and taxifolin were dissolved in ethanol, mixed together and further diluted with ethanol to obtain a set of calibration standards at concentrations of 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, and 2500 ng/mL.
For the purpose of the targeted screening of antioxidant compounds, 0.5 g of the representative sample of the dietary supplement was weighed into 15 mL PTFE centrifuge tube and extracted by shaking with 5 mL of methanol. The silymarin SA was also extracted by the same procedure.
For the purpose of the antioxidant activity determinations, 30 mg of the representative sample of the dietary supplement was weighed into a 50 mL PTFE centrifuge tube and extracted by shaking with 30 mL of methanol. The silymarin SA was dissolved in methanol and further diluted with methanol to obtain a 1 mg/mL solution. To reflect the composition of this solution, the model mixture of analytical standards of silybin A; silybin B; isosilybin A; isosilybin B; 2,3-dehydrosilybin; silychristin; silydianin; and taxifolin in methanol was also prepared. The representation of individual flavonoid / flavonolignans in the silymarin complex determined by the ultra-high performance liquid chromatography high-resolution tandem mass spectrometry (U-HPLC-HRMS/MS), thus in the model mixture, is depicted in
Table 2. The silibinin SA purchased from Sigma-Aldrich was dissolved in methanol and further diluted to obtain a set of calibration standards at concentration levels of 10, 25, 50, 100, 250, 500, 750, 1000, 1200, 1750, and 2500 mg/L.
2.3. Quantitative Analysis of Silymarin Flavonoid/Flavonolignans by U-HPLC-HRMS/MS
The quantitative analysis of silymarin flavonolignans and flavonoid taxifolin by ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (U-HPLC-HRMS/MS) in dietary supplements and the silymarin SA was performed according to [
15].
Dionex UltiMate 3000 ultra-high performance liquid chromatograph (Thermo Scientific, Sunnyvale, CA, USA) with a reversed phase Accucore; aQ analytical column (150 mm × 2.1 mm; i.d. 2.6 µm; Thermo Scientific, San Jose, CA, USA) and gradient elution in water—methanol—ammonium formate / formic acid system, and a Q-ExactiveTM high resolution tandem mass spectrometer (Thermo Scientific, Bremen, Germany) were used. The following exact masses were considered for detection: m/z 303.0510 ([M-H]−ions of taxifolin), 479.0984 ([M-H]-ions of 2,3-dehydrosilybin) and 481.1140 ([M-H]−ions of the other isomeric flavonolignans). The limits of quantification of silymarin components were estimated as the lowest concentration levels of the calibration batch providing long-term stable signals, and were 0.75, 0.75, 0.5, 0.5, 0.25, 2.5, 1.25, and 1.25 μg/g for silybin A, silybin B, isosilybin A, isosilybin B, 2,3-dehydrosilybin, silychristin, silydianin, and taxifolin, respectively. The reproducibility of the method, expressed as a relative standard deviation (RSD), was assessed by repeated analyses (n = 7) of an internal reference sample of milk thistle-based dietary supplement, and was 2.7%, 2.9%, 3.6%, 4.2%, 5.4%, 3.1%, 2.8%, and 3.2% for silybin A; silybin B; isosilybin A; isosilybin B; 2,3-dehydrosilybin; silychristin; silydianin; and taxifolin, respectively.
2.4. Antioxidant Activity Determinations
The antioxidant activity of milk thistle-based dietary supplements was studied using several different assays.
2.4.1. ABTS Radical Scavenging Assay
The fresh ABTS
+ radicals solution was prepared according to [
16]. Milk thistle-based dietary supplement extracts were binary diluted in order to determine their individual concentration of a supplement that gives half-maximal response (EC
50). The tested concentration range was from 0.26 to 66.66 mg/L. The quenching of the ABTS
+ radicals by the extracts was monitored spectrophotometrically (734 nm), based on the changes in the absorption spectrum of the ABTS radical using the SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices, San Jose, CA, USA). The percentage of inhibition was calculated according to the formula: 100 × (NC absorbance − sample absorbance)/(NC absorbance). The experiment was performed in 3 repetitions.
2.4.2. Oxygen Radical Absorption Capacity (ORAC)
For each experiment, fluorescien freshly diluted with PBS was prepared according to [
17]. The binary dilution of the milk thistle-based dietary supplement extracts was done to provide the final tested concentration range of 0.78–200 mg/L. The ability of extracts to quench AAPH radicals was monitored by measuring the fluorescence (excitation/emission 485/535 nm), recording for 2 h at 5 min intervals using the SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices, San Jose, CA, USA). The kinetic parameters were calculated as usual. The relative activity was evaluated as a percentage according to the formula: 100 × (slope of sample fluorescence − average slope of PC)/(average slope of NC − average slope of PC). The experiment was performed in 3 repetitions.
2.4.3. DPPH Radical Scavenging Assay
The DPPH solution was freshly prepared two hours before each measurement according to [
18]. The samples were binary diluted in order to determine EC
50 values. The tested concentration range was from 0.65 to 166.66 mg/L. The quenching of the DPPH-H radicals was recorded using the SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices, USA) as an absorbance difference (at 517 nm). The percentage of inhibition was calculated according to the formula: 100 × (NC absorbance − sample absorbance)/(NC absorbance). The reaction mixture where the sample was replaced with equal amount of solvent (methanol) served as a negative control. The experiment was performed in three independent repetitions.
2.4.4. Cellular Antioxidant Activity (CAA) Assay
The CAA assay was slightly modified according to [
19]. Human hepatoblastoma HepG2 cells (ATCC, HB-8065) were seeded at a density of 5 × 10
5/mL in EMEM medium supplemented with 10% FBS and Antibiotic Antimycotic Solution. After 24 h, the cells were washed with PBS and DCFH-DA (0.0125 mg/mL in medium without FBS) was added. The sample extracts were added to the final concentrations of 0.1–25 mg/L. After 1 h of co-incubation, the medium was replaced with AAPH (44 µM in PBS) and fluorescence (ex./em. 485/540 nm) was immediately recorded at 5min intervals for 1 h using the SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices, USA). The experiment was done in four replicates. The evaluation procedure and controls were the same as in the ORAC assay.
The highest testing concentration of the dietary supplement extracts, 25 mg/L, was chosen intentionally to reflect the real daily dose of overall silymarin when following the producers’ consumption recommendations (i.e., approximately 500 mg, [
15]). Taking into account the average volume of human blood of 4 L, the approximately 10% bioavailability of silymarin in the organism [
20], and approximately 50% of silymarin in the capsules, we calculated the approximate concentration of 25 mg per liter of a biological fluid.
2.5. Targeted Screening of Antioxidants by U-HPLC-HRMS/MS
The targeted screening of non-silymarin antioxidants reported in the literature for all of the plants occurring in the dietary supplements (
Table S1) was performed by the U-HPLC-HRMS/MS method using the 1290 Infinity LC system (Agilent Technologies, Sant Clara, CA, USA) coupled with Agilent 6560 QTOF mass spectrometer (Agilent Technologies, USA). The reversed phase Acquity UPLC BEH C18 analytical column (100 mm × 2,1 mm; i.d. 1,7 μm; Waters, USA) was used for the gradient elution, where the mobile phases consisted of H
2O: MeOH (95:5, v/v) (A) and
i-PrOH:MeOH: H
2O (65:30:5, v/v/v) (B), both containing 5 mM ammonium formate and 0.1% formic acid. The gradient was linear from 100% of A (initial; kept for 1 min) to 100% of B within 14 min, which was held for 5 min and followed by column equilibration for 2 min under the initial conditions. The mobile phase flow rate was 0.35 mL/min and the injection volume 1 µL. The mass spectrometer was operated in Auto MS/MS mode with the following parameters: electrospray ionization in positive and negative mode (ESI
+ and ESI
−; separate injections), drying gas (N
2) temperature 280 °C and flow rate 12 L/min, nebulizer 35 psig, sheath gas (N
2) temperature 350 °C and flow rate 12 L/min, capillary voltage 3 500 V, nozzle voltage 400 V. The following parameters were used in Auto MS/MS mode: mass range 100–1100
m/z (MS) and 50–1100
m/z (MS/MS), acquisition rate 3 spectra/s (MS) and 12 spectra/s (MS/MS), collision energy 20 V. The antioxidants were tentatively identified based on exact masses of the particular ions, their isotopic patterns, and the compliance of MS/MS spectra. For some of the compounds, more chromatographic peaks fulfilling the HRMS identity criterions were identified, referring probably to structural isomers of analysed bioactive compounds. The details about the peaks identity, mainly retention times and degrees of certainty of compounds identification, are presented in
Table S2. For purposes of correlations of occurrence of these compounds with antioxidant activities (as described in details in the
Section 2.6), the peak areas of all isomers were summed up together.
2.6. Statistical Analysis and Correlation
The EC50 values were obtained using the GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). Both standard errors of the mean (SEM) and standard deviations (SDs) were calculated as usual. Data were analysed by one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test (p > 0.05). All computations were done using the statistical software STATISTICA 10. The correlation coefficients were calculated using the automatic function “CORREL” in Microsoft® Office Excel (i.e., “matrix I” and “matrix II” explained below were correlated against each other). The following variables were used as matrix I: (i) concentrations of particular flavonoid/flavonolignans and overall silymarin, (ii) the peak areas of non-silymarin Silybum marianum antioxidants identified by targeted U-HPLC-HRMS/MS screening, namely phenolics, flavones, flavone glycosides, isoflavonoids, flavonolignans, and their sums, and (iii) the peak areas of other non-Silybum marianum antioxidants identified by targeted U-HPLC-HRMS/MS screening, namely phenolics, coumarins, lignans, lignan glycosides, flavones, isoflavonoids, saponines, terpenes, and their sums. The analytical standards of non-silymarin components were not available, so we correlated the sum of areas of the peaks belonging to the respective chemical class. The results of the antioxidant capacity of 26 dietary supplements obtained from all assays investigated (i.e., (i) ABTS assay, (ii) ORAC assay, (iii) DPPH assay, (iv) CAA assay) were utilized as matrix II. The significance of the correlation coefficient was evaluated using a comparison of coefficients and the critical values (α = 0.05), which were determined using the degrees of freedom (df = n − 2).
4. Discussion
Dietary supplements based on the milk thistle (
Silybum marianum) are among the most common preparations used by the EU and US adult population [
22]; in fact, those are among the six best-selling herbal-based products in the US [
23], but unfortunately with an insufficient level of composition control. The increasing popularity of silymarin for the treatment of liver and chemoprevention has generated scientific interest in this topic [
6,
7,
8], and it can be said that despite the suggested beneficial role of silymarin [
7,
8,
9], its clinical importance has still not been clearly proven [
2,
8,
10,
11]. The main limitations of the clinical studies conducted so far seem to be the lack of properly controlled clinical trials, especially in terms of the vaguely defined chemical composition of the therapeutic agents, silymarin preparations [
14,
24].
In our study, where 26 milk thistle-based dietary supplements were investigated, a significant variability in the content of total silymarin was observed, as well as in the composition of the silymarin complex (which is somewhat in line with the results of only two previous studies on this topic [
25,
26]). Our results showed relatively significant differences in the ratios of the most abundant flavonolignans silybin B, silybin A, and silychristin. Moreover, the content of 2,3-dehydrosilybin and silydianin, a minor flavonolignan possessing potent biological activities [
4,
27,
28,
29], differed approximately six- and 60-fold, respectively, across the positive samples (
Table 3). It is also important to note that the correspondence of the determined concentrations of silymarin with the producers’ declarations on the packaging is very low, as evidenced in our recent study on an identical set of samples [
15].
Because the antioxidant capacity of silymarin preparations, as determined by different analytical methods, reflect not only the chemical composition, but also the mechanisms of antiradical reactions, we compared the four most frequently used antioxidant activity assays (ABTS, ORAC, DPPH, and CAA), and evaluated the relationships between their results and the composition of complex samples. Silymarin has been previously tested for its radical scavenging ability in several separate studies [
11,
19,
20]. Even the chemical assays for antioxidant evaluation can be considered simple, rapid, sensitive, and reproducible [
19,
21], the cellular assays are more relevant considering such parameters as the bioavailability and bioaccessibility of the tested compounds [
18]. The best correlations of antioxidant capacities with concentrations of silymarin flavonoid/flavonolignans was demonstrated for the sum of silymarin components, followed by silychristin as one of the strongest antioxidants of the silymarin complex [
16]. The total concentrations of all flavonoid/flavonolignans present in the supplements plotted against the relative radical scavenging activity gave the significant correlation with ORAC (R
2 = 0.65) and ABTS (R
2 = 0.52) followed by non-significant correlation with CAA (R
2 = 0.10) and DPPH (R
2 = 0.13). The inappropriateness of DPPH for some antioxidants was previously published [
30]. Many samples that have the ability to reduce radicals in chemical assays failed to do so in cellular assays [
25], which corresponds to our results, where 38% of the tested supplements failed to scavenge 50% of cellular radicals up to a concentration of 25 mg/L (
Table 4). The highest tested concentration in CAA (25 mg/L) corresponds to a molar concentration of 26 µM of silymarin. In fact, such a large concentration is not expected in plasma, where the usual concentration is mainly in the nanomolar range and only in rare cases reaches the micromolar level [
16]. Taking into account the silymarin bioavailability of 1% (as reported for silybin A/B and rats [
16]), and the recommended daily dose of capsules, only two of the tested supplements (17 and 20) can lead to such concentrations in cells that can scavenge 50% of oxygen radicals inside.
The antioxidant activity of the commercially available silymarin SA extract determined by all of the used assays was significantly higher than the activity determined in the flavonolignans mixture mimicking silymarin SA, pointing to the presence of other non-silymarin antioxidants. A significant number of compounds with described biological activities (not only antioxidative) was also determined in the investigated commercially based dietary supplements (
Supplementary Tables S3 and S4). The best correlation, especially in the cellular assay, was achieved for the sum of total phenolics (simple phenolics and flavonoids) occurring in
Silybum marianum. These flavonoids have been reported many times in the literature for their antioxidant properties, which have been summarized e.g., in comprehensive reviews [
31,
32,
33] or in publications focused on individual compounds, e.g., quercetin [
34], rutin [
35], luteolin [
36], apigenin [
37], and genistein [
38]. In contrast to silymarin, these flavonoids are more bioavailable, i.e., rapidly absorbed from the small intestine and found in plasma [
39]. Although the silymarin complex is beneficial for its antioxidant capacity, the effect of other antioxidants originating from the
Silybum marianum should not be omitted when assessing the results of in vivo tests and / or clinical studies. To verify the presented results, a detailed in vivo evaluation of the samples must be perforSmed.