The Immunomodulation Effect of Aronia Extract Lacks Association with Its Antioxidant Anthocyanins

Abstract

Polyphenols comprise a diverse group of molecules with antioxidative and anti-inflammatory activities. To compare the antioxidative and anti-inflammatory capacity of Aronia melanocarpa berries (chokeberries), recognized for their high content of anthocyanins, a noncytotoxic isolation method was developed to obtain high-purity anthocyanins in the extract. The antioxidative activity of the extract, the anthocyanin-rich fraction (AF) was determined by 1,1-diphenyl-2-picrylhydrazyl radical and ferric-reducing ability of plasma along with resveratrol as a reference. The immunomodulation properties were assessed in lipopolysaccharide (LPS)-stimulated human monocytes mono mac 6. The isolated AF, containing six different anthocyanins, exhibited a stronger antioxidative capacity compared to resveratrol. Resveratrol enhanced tumor necrosis factor-α and reduced interleukin-10 (IL-10) production by LPS, whereas AF only had a slight effect in reducing IL-10. These results demonstrated that there was no major relationship between the antioxidative effect and immunomodulation capacities of AF and resveratrol. The immunomodulatory activity of the extract is associated with bioactive compounds in Aronia other than its anthocyanins.

Introduction

B erries and red-colored fruits are important dietary sources of phenolic antioxidants that are known to have significant health benefits. Aronia melanocarpa (chokeberry) is widely used as a raw material in natural coloring for the food industry. Aronia berries have preventive potential toward various diseases, including uveitis,^1,2 gastric mucosal damage,³ and colon cancer.⁴ Extract of Aronia berries has been shown to inhibit platelet adhesion, reduce aggregation, and generation of O₂ ^−•, as well as reduce the production of inflammatory markers.^5,6

Anthocyanins represent one of the most potent groups of polyphenolic antioxidants⁷ and be an abundant compound in Aronia berries.⁸ It has been proposed that anthocyanins in the extracts of many berries and fruits are the components responsible for a large proportion of their anti-inflammatory activity.⁹ Studies on endothelial cell lines have shown that the anthocyanins cyanidin-3-O-β-glucoside and peonidin-3-O-β-glucoside inhibit CD40-induced activation of the cells,¹⁰ while tumor necrosis factor alpha (TNF-α)–induced endothelial dysfunctions were abrogated by cyanidin-3-O-β-glucoside.¹¹ The anti-inflammatory capacity of anthocyanins on oxLDL-induced endothelial injury were found to be significantly correlated with different substituents at C3′, C4′, and C3 positions.¹² Thus, a beneficial effect of anthocyanins on endothelial cells seems well established. In contrast, a single study found that cyanidin-3-O-β-glucoside upregulated TNF-α production in lipopolysaccharide (LPS)/interferon-γ-stimulated murine macrophage-like RAW 264.7 cells (RAW) macrophages.¹³ To our knowledge, this is the only study that has addressed the anti-inflammatory/immunomodulatory activity of the anthocyanin-rich fraction (AF) in immune cells that are often involved in proinflammatory processes, for example, monocytes or other phagocytosing cells.

Many other plant antioxidants have been shown to possess an anti-inflammatory activity in their pure form, for example, flavonoids and stilbenes¹⁴ and curcuminoids.¹⁵ One molecule, trans-3,5,4′-trihydroxystilbene, also known as resveratrol, has been shown to hold anti-inflammatory activities in many different cells, including monocytes, macrophages, and dendritic cells.^14,16

–19 The antioxidative activity of resveratrol is well known, although it is moderate compared to antioxidants in Aronia berries.²⁰ Resveratrol is abundant in the skin of grapes, in peanuts, and in many berry species, but it is not found in Aronia berries.²¹

For many years, it was believed that the antioxidative effect of plant polyphenols was also the cause of their anti-inflammatory activities. In recent years, polyphenols, however, were found to be involved in the proinflammatory signaling pathways (e.g., the Janus kinases 1 and 2),^22,23 resulted in inhibition of the activation of transcription factors such as nuclear factor kappa-B (NF-κB) and activator protein–1 (AP-1).²⁴ Accordingly, strong antioxidative activity may not necessarily imply strong anti-inflammatory activity of a polyphenolic compound, and vice versa.

With this in mind, the study aimed at characterizing the anthocyanin fraction of Aronia berries in regard to their antioxidant and anti-inflammatory capabilities, respectively, as compared to resveratrol, a well-described reference compound as a therapeutic agent for inflammatory diseases.^14,16

Materials and Methods

Resveratrol, piceid, chlorogenic acid, neochlorogenic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•), 2,4,6-tripyridil-s-triazine (TPTZ), and LPS from Escherichia coli (026:B6) were purchased from Sigma Aldrich (St. Louis, MO, USA). The organic Aronia crude extract was from Aronia melanocarpa (purchased from Berrifine ApS., Slagelse, Denmark). The extract was stored at −18°C. Anthocyanin standards were obtained from Polyphenols Laboratories A/S (Sandnes, Norway).

Characterization of anthocyanins

The purified anthocyanins and the anthocyanins present in Aronia crude extract were identified and quantified by high-performance liquid chromatography (HPLC; TSP Spectra System) with an UV 6000 LP diode array detector with a 5-cm flow cell with a mass spectrometry (MS) detector. Ten-microliter aliquots of Aronia crude extract were applied to a Phenomenex Luna C 18 column (Torrance, CA, USA; 150 mm×2.0 mm, 3 μm particle size) and eluted with a linear eluent gradient program of 5% formic acid in water (A) and methanol (B) with a flow rate 0.17 mL/min: 0–15 min, 8%–20% B; 15–35 min, 20%–27% B; 35–50 min, 27%–45% B; 50–60 min, decreasing from 45% to 8% B; 60–70 min, constant 8% B. The mass spectrometry was a LCQ-Deca ion-trap instrument from ThermoFinnigan (San Jose, CA, USA) equipped with an electrospray ionization interface run in a positive mode. A positive potential (+3 kV) was applied to the silica needle, discharge current 80 mA, capillary voltage 23 V, capillary temperature 225°C, tube lens offset −5 V, sheath gas (N₂) and auxiliary gas (N₂) 84 and 41 arbitrary units, respectively. The diode array detector (DAD) absorption spectra were recorded at wavelengths between 190 and 800 nm or more narrow intervals, with a fixed filter. The ion trap was run in the data-dependent tandem mass spectrometry (MS/MS) scanning mode and the first scan event was obtained in the m/z interval 250–1200. This was followed by a second scan event of the most intensive ion from the first scan in which a high-intensity MS² fragment of the MS/MS spectrum was obtained. Isolation m/z width=1, normalized collision energy 28, activation time 30 ms, minimum signal required 10⁵ counts.

Purification of anthocyanins

Aronia crude extract (1 mL) was diluted with an aqueous solution of 1% formic acid (60 mL). The solution was then eluted through a strong cation-exchange column (Varian MEGA-BE-SCX, 1 g, 6 mL; AA, Walnut Creek, CA, USA) binding the positively charged anthocyanins, while neutral molecules were removed by washing with water. The strong cation-exchange column based on –SO₃H groups had a capacity of 1–3 mg of anthocyanins (2–6 μmol/g) corresponding to 1%–3% of the available –SO₃H groups on the stationary phase may exchange their proton for a positive anthocyanin molecule. The anthocyanins were eluted from the column by concentrated hydrochloric acid/water/ethanol (8:12:80), diluted 10 times with water, and applied to a reverse-phase C18 sep-pack column (Varian MEGA-BE-C18, 1 g, 6 mL; Walnut Creek, CA, USA). The C18 column was carefully washed with water and the anthocyanins were eluted with 0.1 M formic acid in ethanol. Finally, the ethanol was removed by rotary evaporation and the product dried in a vacuum oven at 40°C.

Determination of total anthocyanin content

The total anthocyanin content of Aronia 25 crude extract was determined by the pH differential method²⁵ and HPLC (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/jmf). The total anthocyanin isolated from the extract was determined by dissolving 0.8 mg dry matter in 8 mL 0.1 M aqueous formic acid, which was then analyzed by the pH differential method and HPLC. Anthocyanins were quantified using the pelargonidin-3-O-glucoside calibration curve at 520 nm on HPLC. Commercially available pelargonidin-3-O-glucoside standard was dissolved in 0.1 M aqueous formic acid and thereafter diluted (0.04–0.6 mM) to make a standard curve (R ²≥0.99). The results were expressed as cyanidin-3-O-glucoside equivalents. The determination was performed in triplets.

Determination of antioxidative capacity using the DPPH^• assay

The DPPH^• method was performed according to the protocol described previously.²⁷ In brief, the antioxidant solution (100 μL) was transferred to a UV/Vis-cuvette containing 3 mL DPPH^• in methanol (1×10⁻⁴ mol/L), incubated for 30–300 min at room temperature, sealed in the dark, and monitored at 515 nm. The antiradical power (ARP=n _DPPH/n ₅₀) was obtained from a plot of % remaining DPPH^• (n _DPPH) versus n ₅₀, the amount of antioxidant necessary to remove 50% DPPH^• (Supplementary Fig. S2).²⁶

Determination of antioxidative capacity using the ferric-reducing ability of plasma assay

The ferric-reducing ability of plasma (FRAP) reagent was prepared by a 10:1:1 mixture of acetate buffer (300 mM, pH 3.6), 2,4,6-TPTZ (10 mM) dissolved in HCl (40 mM), and FeCl₃·6H₂O (20 mM). The measurements were performed according to Benzie and Strain.²⁷ The antioxidant solution (100 μL) was added to the FRAP reagent (3 mL) and the absorbance obtained after 4 min at 593 nm. FeSO₄·7H₂O solutions (5–20 mM) were used as standard antioxidant solutions (Supplementary Fig. S3).

Cell culture conditions

Mono Mac 6 cells (MM6) were obtained from German Collection of Microorganisms and Cell Cultures (DSMZ). The MM6 cells were cultured in the growth medium RPM1 1640 (Cambrex, East Rutherford, NJ, USA) with 10% fetal calf serum (BioWhittaker, Walkersville, MD, USA), 1% penicillin/streptomycin (Cambrex), 2 mM glutamine (Cambrex), 2 mM nonessential amino acids (Cambrex), 1 mM sodium pyruvate (Invitrogen^™; Gibco, Auckland, NZ), and 9 μg/mL bovine insulin (Sigma Aldrich, St. Louis, MO, USA). Twice weekly, the cells were harvested, counted, and diluted to 2×10⁵ cells/mL. The cells were incubated in a humidified atmosphere with 5% CO₂ at 37°C.

Stimulation of MM6 cells with plant extracts and resveratrol

Three-day old cultures of MM6 cells were harvested and cell density adjusted to 0.67×10⁶ cells/mL. Resveratrol dissolved in dimethyl sulfoxide (DMSO), Aronia crude extract, and the AF in 70% ethanol were diluted in Dulbecco's phosphate-buffered saline (DPBS; Biowhittaker), and added to MM6 cells for 30 min, and then LPS (final concentration 1 μg/mL) was added and the cells were incubated for 18 h in a humidified atmosphere with 5% CO₂ at 37°C. For the combinatory stimulations, Aronia crude extract or the AF diluted with resveratrol or DMSO/DPBS was added to the cells incubated under the conditions described above. The supernatants were harvested and the cytokine production (interleukin [IL]-6, IL-10, and TNF-α) was analyzed by enzyme-linked immunosorbent assay (DuoSet^® ELISA Development System, R&D Systems, Inc., Minneapolis, MN, USA; Supplementary Figs. S4–S6) according to the manufacturer's instructions.

Statistical analysis

Unless otherwise stated, all experiments were performed three times, and the results are given as mean values±standard deviation. No outliers were rejected from the data sets. One-way analysis of variance (ANOVA) followed with Tukey's multiple comparison test was performed to determine the different immunomodulation ability among single compounds or in combined. One-way ANOVA with Dunnett's test was used to determine whether the effect by single compounds is significantly different from the controls. For nonparametric analysis, ANOVA (Kruskal–Wallis test) was used, followed by Dunnett's multiple comparison test; analysis was performed using a significance level of P<.05. Graphpad Prism 5.0 for Windows (Graphpad Prism Software, San Diego, CA, USA) was used. Microsoft Excel 2010 was used for antioxidative capacity data statistics according to the protocol.²⁸ No outliers were removed from any data sets.

Results and Dissusion

Characterization and isolation of the anthocyanins from Aronia crude extract

To characterize the anthocyanin content in Aronia crude extract as well as in the AF isolated from Aronia crude extract, reversed-phase separation analysis was performed. Figure 1A and B show the HPLC-DAD chromatograms of aliquots from a single batch of Aronia crude extract obtained in the wavelength intervals 190–800 nm and in the maximum absorption mode 500–540 nm, respectively. In total, six anthocyanin derivatives (peaks 3–8, in Fig. 1) were present in Aronia crude extract, but comparison of the two chromatograms revealed two additional major nonanthocyanin components (peaks 1, 2, Fig. 1A). These two nonanthocyanin compounds were identified as neochlorogenic acid (peak 1) and chlorogenic acid (peak 2) based on retention time match with authentic reference samples. The six anthocyanin compounds were identified from their UV/Vis and electrospray molecular ion of [M⁺] and fragment ion of MS/MS spectra and comparisons with literature data²⁹ are summarized in Table 1 and their chemical structures in Figure 2. The four major anthocyanins (liquid chromatography peaks 3–6, Fig. 1B), corresponding to cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-xyloside, were all identified and extensively characterized in previous studies.³⁰ The remaining two species were identified as cyanidin pyruvates, commonly found in the skins of some deep frozen stored grapes and some red wines.³¹ Based on retention times and MS fragment patterns, the crude extract did not contain any resveratrol (fragment m/z 227), its glucoside, piceid (fragment m/z 389), or other resveratrol derivatives.³²

FIG. 1.

High-performance liquid chromatography–diode array detector (HPLC-DAD) chromatogram of Aronia extract and anthocyanin fraction purified from Aronia. (A) Aronia extract HPLC-DAD chromatogram obtained UV/Vis and mass spectrometry data 190–800 nm. (B) Aronia extract (λ _max mode 500–540 nm). (C) Cation-exchange purified anthocyanin-rich fraction (AF; λ _max mode 280–350 nm). (D) Like (C) with a wavelength interval 500–540 nm. Peaks 1–8 correspond to the compounds listed in Table 1.

FIG. 2.

Structures of compounds, which are part of this investigation and have been previously identified in Aronia. ²⁹ In this study, cyanidin glycosides, 5-carboxypyranocyanidin glycosides, neochlorogenic acid, and chlorogenic acid were identified in the extract used.

Table 1.

Retention Times, Ultraviolet/Visible and Mass Spectrometry Data of Compounds 1–8

	UV/Vis (nm)	MS¹ [M⁺] (m/z)	MS² [M⁺] (m/z)	Identification	Mole percent ^a
1	324	355	163	Neochlorogenic acid	22.0±1.0
2	326	355	163	Chlorogenic acid	18.0±1.0
3	514	449	288	Cyanidin-3-galactoside^b	39.0±3.0
4	515	449	287	Cyanidin-3-glucoside^b	2.33±0.19
5	515	419	287	Cyanidin-3-arabinoside^b	17.3±1.9
6	518	419	287	Cyanidin-3-xyloside^b	0.86±0.31
7	503	517	355	Cyanidin-3-O-hexo pyruvate	0.56±0.050
8	509	487	355	Cyanidin-3-O-pento-pyruvate	3.14±0.94

Total anthocyanin content in Aronia extract was 4.49±0.87 g/L determined by pH differential method and the high-performance liquid chromatography analysis. Mean values±SD, n=6.

The exact hexose/pentose structures were obtained from Wu et al. ²⁹

MS, mass spectrometry; SD, standard deviation.

Anthocyanins are positively charged at low pH (<2–3) and may therefore be conveniently isolated and separated from other neutral molecules in Aronia crude extract by means of a strong cation-exchange column based on –SO₃H groups on the stationary phase, which may exchange their proton for a positive anthocyanin molecule. To our knowledge, the same noncytotoxic separation method that is competent for the immunomodulation study has not been reported previously.

Figure 1C and D show the HPLC-DAD chromatograms of cation-exchange-purified Aronia AF obtained in the maximum absorption mode in the wavelength intervals 280–350 nm and 500–540 nm, respectively. Comparison of the chromatograms of AF with the anthocyanin chromatogram of Aronia crude extract (Fig. 1B) revealed almost identical profiles, indicating that our developed strong cation-exchange purification protocol for AF is capable of extracting the entire content of anthocyanins from Aronia crude extract. Furthermore, the analyzed cytokines TNF-α, IL-6, and IL-10 in the nonstimulated monocytes were not affected by the addition of AF (data not shown). This demonstrated the noncytotoxic quality of the purification methods and made it possible to evaluate immunomodulatory effects of various plant extracts with or without anthocyanins.

The efficiency of cation-exchange chromatography is shown to be comparable to the commonly used C18 solid-phase extraction. Compared to C18 extraction, strong cation-exchange chromatography is rapid, easy to modify, and low cost to obtain high-purity anthocyanins. Based on the color observed in the experiments, the chemical forms of anthocyanins are pH dependent and easily reduced at pH >6. To obtain high purity, the acidic solvent is favorable to keep anthocyanins in its flavylium cation form during the isolation process. The hydrophilic condition is more suitable for retaining anthocyanin species. However, strong cation-exchange chromatography does not eliminate the aqueous solvent, and rotary evaporation would need to be applied subsequently.

Aronia crude extract and AF were dehydrated and dry weights (DWs) were determined, three independent experiments were repeated. DW of Aronia crude extract was found to be 600±100 g/L.

The anthocyanin content was quantified by the pH differential method and HPLC analysis. The results obtained from these two methods were comparable. Using the pH differential method, the anthocyanin content in Aronia crude extract was found to be 4.0±1.0 g/L, whereas the HPLC analysis gave a concentration of 4.98±0.43 g/L. The two methods also showed that the 0.8±0.2 mg dehydrated isolation product contained 0.87±0.32 mg anthocyanins as measured by pH differential and 0.73±0.15 mg as measured by HPLC. Together, this showed that our purification protocol is capable of obtaining high-purity products. The total anthocyanins content in Aronia crude extract was determined to be 7.5 mg/g DW, which is consistent with previously reported numbers for extract from black chokeberry.² In other studies, the total content of anthocyanins from Aronia raw extract was reported to vary from 6.5 to 10.4 mg/g DW.^33,34 The difference may be explained by different plant origins.

Antioxidative capacity of AF and kinetic behavior

The antioxidative capacities of the extract, AF, and resveratrol were determined by DPPH^• and FRAP assay. The stock solutions used in cell experiments were measured and expressed as Trolox equivalent in μmol/mL for both DPPH^• and FRAP. The kinetic behavior was assessed according to the previously established protocol.^26,27

Table 2 shows that resveratrol had approximately half the molar antioxidative capacity of the vitamin E analog Trolox, while the AF exhibited 1.1–2.9 times the antioxidative activity as Trolox.^27,35,36 Table 3 shows the antioxidative capacities of the stock solutions used in cell experiments; Aronia crude extract, AF, and 44 μM (10 mg/L) resveratrol, expressed as Trolox equivalent (TE) in μmol/mL. Samples of 44 μM resveratrol exhibited an antioxidative capacity in the order of a 600× dilution of AF or to a 2000× dilution of Aronia crude extract as used in cell experiments.

Table 2.

Radical Scavenging Parameters of Resveratrol and Anthocyanin-Rich Fraction Determined by DPPH ^• and FRAP

	DPPH ^•	FRAP
Antioxidant	stoic. ratio ^a	relative to FeSO₄
Resveratrol	1.255±0.090 (1)^b	0.706±0.040
Trolox	2.10±0.10 (2.2)^b	1.53±0.16 (2.0)^c
Anthocyanin-rich fraction^d	6.085±0.035	1.72±0.16

Mean value±SD, n=3.

Number of DPPH^• radicals, which can be removed by one molecule of antioxidant. Stoichiometric ratio=antiradical power/2.³⁵

Value from Villano et al. ³⁶

Value from Benzie and Strain.²⁷

Anthocyanin-rich fraction isolated from Aronia.

DPPH^•, 1,1-diphenyl-2-picrylhydrazyl radical; FRAP, ferric-reducing ability of plasma; stoic., stoichiometric.

Table 3.

Antioxidative Capacity of Aronia Extract, Anthocyanin-Rich Fraction, and Resveratrol Stock Solutions Used in Cell Experiments

	DPPH^• (Trolox eq. μmol/g DW)	DPPH^• (Trolox eq. μmol/mL for the solutions used in cell experiments) ^a	FRAP (Trolox eq. μmol/g DW)	FRAP (Trolox eq. μmol/mL for the solutions used in cell experiments) ^a
Aronia extract	241±82.4	145^b±49.6	369±67.8	221±40.6^b
Anthocyanin-rich fraction	29,435±12,510	34^c±14.5	10,892±971.5	12^c±1.1
Resveratrol	3199±1223	0.6^d±0.23	2025±106.44	0.4^d±0.02

Mean values±SD, n=3.

Antioxidative capacity expressed in Trolox eq. μmol/mL=amount of test compound stocks in g/L×Trolox eq. μmol/g DW by DPPH^• or FRAP.

600±100 g/L Aronia extract stock used in cell experiments.

1.14±0.29 g/L anthocyanin-rich fraction stock used in cell experiments.

0.2 g/L resveratrol used before loading in cell wells.

DW, dry weight.

The DPPH^• analysis determined the antioxidative capacity of Aronia crude extract to be 241–369 μmol TE/g DW (Table 3). This result is consistent with the values recently reported^30,37 when normalized to the same DW (600 g/L). However, the antioxidative capacity of AF as measured by DPPH^• was 1.7-fold higher than the value measured by FRAP. Similar observations have been made in previous studies.³⁸ The DPPH^• method measures the capacity of antioxidants to transfer an H-atom to the DPPH^• radical, whereas the FRAP method expresses the reductive capacity of the antioxidant.^27,35 This may explain the discrepancies between values obtained in the two different assays. The percent contribution of the anthocyanin fraction to the total antioxidative capacity of Aronia crude extract, as determined by DPPH^• and FRAP were 81% and 33%, respectively. In contrast, Zheng and Wang found³⁰ 53.1% for the anthocyanin fraction in Aronia berries by the oxygen radical absorbance capacity assay. Thus, other antioxidative compounds, in addition to anthocyanins, are present in Aronia crude extract. The phenolic composition of the chokeberry analyzed by Zheng and Wang were similar to what we have observed.³⁰ He reported that two chlorogenic acids represented high proportions of antioxidative acitivities found in Aronia. ³⁰ The combination of anthocyanins and chlorogenic acids was hypothesized to contribute significantly to the total antioxidative capacity of Aronia crude extract. The presence of other compounds with antioxidant activities cannot be fully excluded based on the findings of this study or Zheng and Wang.³⁰

Characterization of the immunomodulatory effect of Aronia crude extract, anthocyanidin-rich fraction, and resveratrol in LPS-stimulated human monocytes

To test the immunomodulatory effect of Aronia crude extract, AF, and resveratrol, these samples were added to cultures of the human monocyte cell line, MM6, before stimulation with LPS. They were evaluated by measuring the production of proinflammatory cytokines TNF-α, IL-6, and the anti-inflammatory cytokine IL-10.

We first compared the immunomodulatory activity of resveratrol and Aronia crude extract added to cells in comparable antioxidant concentrations as determined in Figure 3 and Table 3. Resveratrol increased the LPS-induced TNF-α production and reduced the IL-10 production at the highest tested concentration (44 μM). The IL-6 production was not affected. Addition of Aronia crude extract in 600× and 2000× dilution led to a dose-dependent reduction in LPS-induced IL-10 and TNF-α production, and increase in IL-6. Thus, although the antioxidant concentration measured in the diluted extract and in the resveratrol sample were comparable, the modulatory effects on the LPS-stimulated monocytes were rather disparate: Aronia crude extract affected the production of each cytokine analyzed, led to an increase in the IL-6 production and, in contrast to resveratrol, decreased the TNF-α production.

FIG. 3.

The modulation of cytokine expression in Mono Mac 6 (MM6) cells by resveratrol. MM6 cells were exposed to increasing concentrations of pure compounds resveratrol (0.4–44 μM) (A), or Aronia extract (diluted in 2000×, 600× times) (B), 30 min before the exposure to 1 μg/mL lipopolysaccharide (LPS). Cells were incubated for additional 18 h and the culture supernatants were harvested, the levels of cytokines were determined by enzyme linked immunosorbent assay (ELISA). The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 are shown as means±standard deviation (SD) of three, four, or six experiments. Compared with LPS: **P<.01, ***P<.001.

The dose-dependent downregulation of the TNF-α and IL-10 production in the monocytes by Aronia crude extract is consistent with previous studies in RAW cells.¹ Similar results were also obtained from an extract of raspberries,³⁹ whereas increased TNF-α levels were observed when exposing RAW cells to the extracts of blue berries, black currants, blackberries, and Saskatoon berries in an experimental setup very similar to the present experiments.¹³

The results obtained from resveratrol are consistent with previous studies. Resveratrol has previously been shown to downregulate IL-10 and to upregulate TNF-α in macrophages and monocytes.^16,40 It should be noted, however, that resveratrol seems to affect cytokine production according to the cell type studied; In studies of, for example, dendritic cells, resveratrol led to an increase in LPS-induced TNF-α production.^39,41 Furthermore, resveratrol was reported to regulate transcription factors: For example, regulation of NF-κB in a cell-type-specific manner, discrepancy may be explained by that resveratrol regulates individual NF-κB subunits differently. For example, different roles of p50–p50 homodimers and p50–p65 heterodimers may consequently result in different cytokine production.⁴² Therefore, it will be interesting to study the effect of test groups on certain NF-κB subunits in future studies. The lack of immunomodulatory effects of anthocyanins corresponds well with earlier observations of Ohgami et al.,¹ while others found increased TNF-α levels when the RAW cells were exposed to increasing concentrations of pure anthocyanins such as cyanidin and cyanidin glycosides.¹³

As Aronia crude extracts may contain many other compounds with or without the antioxidative activity that might modulate the cytokine response, for example, chlorogenic acids or quercetin derivatives,¹³ it was important to assess the contribution of the AF in Aronia crude extract per se to the immunomodulation exerted by Aronia crude extract and furthermore, to assess the combined immunomodulatory effects of resveratrol and either Aronia crude extract or AF (Fig. 4). If the antioxidative activity in a sample relates directly to the immunomodulatory activity, the combined action of the two samples holding the antioxidative activity should enhance the immunomodulatory activity induced by the individual samples.⁴³ The cell experiment was repeated as indicated in figure captions. An average value of the cytokine expression was then calculated. In contrast to the addition of Aronia crude extract, addition of AF to the monocytes did not affect the LPS-induced TNF-α and IL-6 production, but it did reduce the IL-10 production dose dependently in basal cytokine measurements. Resveratrol or the AF alone did not induce cytokine production above background. The addition of Aronia crude extract alone led to the responses in TNF-α and IL-10 that did not exceed 200 pg/mL. Only Aronia crude extract induced a slight response in IL-6, which increased by fivefold when resveratrol was added to a 600× dilution of the extract (data not shown). Taken together, this demonstrated that AF only exhibits a weak reducing effect in IL-10 production in LPS-stimulated monocytes, and that Aronia crude extract contains bioactive compounds other than anthocyanidins. Furthermore, these other bioactive compounds affect the inflammatory response of the cells differently as compared to resveratrol.

FIG. 4.

The modulation of cytokine expression in MM6 cells by Aronia crude extract, anthocyanidin-rich fraction, resveratrol, and their combination. To investigate the contribution of the antioxidant effect of AF from Aronia extract in cytokine expression. MM6 cells were exposed to the Aronia crude extract (A, C, E) or anthocyanidin (B, D, F) at increasing concentrations with or without 44 μM resveratrol 30 min before the addition of 1 μg/mL LPS. After an additional 18 h of incubation, cytokine levels TNF-α (A, B), IL-6 (C, D), or IL-10 (E, F) in culture supernatants were determined by ELISA. The levels are means±SD of three end up to nine independent determinations. Compared with LPS: *P<.05, **P<.01, ***P<.001; Aronia extract/AF versus corresponding Aronia/AF+resveratrol: ^# P<.05, ^## P<.01, ^### P<.001; compared with resveratrol: ^R1 P<.05, ^R2 P<.01, ^R3 P<.001.

When resveratrol was added together with the extract, the enhanced TNF-α production caused by resveratrol was reduced dose dependently by the extract (Fig. 4A). Thus, as might be expected from the results obtained by the individual samples, the two samples conferred contrasting effects. The resveratrol-induced reduction of LPS-stimulated IL-10 production was further reduced dose dependently by Aronia crude extract (Fig. 4E). Interestingly, the IL-6 production, which was not affected by resveratrol and increased by Aronia crude extract, exhibited a dose-dependent decrease upon addition of both resveratrol and Aronia crude extract (Fig. 4C). In contrast, addition of AF, which only weakly decreased the IL-10 production, did not give rise to alterations in the TNF-α, IL-6, and IL-10 responses induced by resveratrol and LPS together (Fig. 4B, D, F).

The observed effect on the LPS-induced IL-6 by Aronia crude extract together with resveratrol (Fig. 4C) may be explained by the fact that the two compounds regulate LPS-induced IL-6 signaling through different modes of action. As this effect was not observed by combining resveratrol and AF, the role of the AF on the IL-6 production can be ruled out. TNF-α and IL-10 expression followed the same pattern as Aronia crude extract regardless of the presence of resveratrol (Fig. 4A, E), especially in the TNF-α responses, the reducing effect of Aronia crude extract became more pronounced when combined with resveratrol. This interestingly explains the French paradox,⁴⁴ as red wine is rich in both resveratrol and flavonoid. In contrast, the AF slightly increased the resveratrol-induced increase in TNF-α. No previous combinatory treatment by these two compounds has been studied; however, more studies would be necessary to further narrow the conclusions of the concentrations of Aronia crude extract to show whether the effect is dose dependent. Additional cytokine species as well as other models for the assessment of inflammation will be necessary for analysis to further confirm the results of this study.

Aronia berries are known to be a rich source of antioxidants, anthocyanins in particular;⁴⁵ it has been speculated that this property is the root cause behind many beneficial health effects reported for Aronia.^46,47 Recently, increasing evidence of polyphenols directly interfering with intracellular proinflammatory enzymes has been reported.^22,23 By using the noncytotoxic cation-exchange-purified method, it was possible to assess the immunomodulatory effects of adding resveratrol and AF or reservatrol and Aronia crude extract to stimulated monocytes. This study demonstrated no major relationship between the antioxidative activity and immunomodulatory activity. In addition, our results revealed that the combined immunomodulatory effects of distinct polyphenols cannot be predicted from the action of the individual compounds. These findings underlined the extra caution that should be given when interpreting the individual classes of polyphenols because the characterization of the major classes of polyphenols cannot be predicted based on testing complex extracts. Resveratrol and Aronia crude extract in combination, as contrasted to them as single compounds may to some extent have more health promotive effects in reducing the risk of diseases such as diabetes, cardiovascular, or inflammatory diseases⁵ or cancer.

Footnotes

Acknowledgments

The authors thank Dr. Hanne Frøkiær (BioCentrum, Technical University of Denmark, Kongens Lyngby, Denmark), Dr. Torben Lund (Department of Chemistry, Roskilde University), Dr. Jens E.T. Andersen (Department of Chemistry, Technical University of Denmark), and Dr. Bangjun Wang (Department of Biology, University of Fribourg, Fribourg, Switzerland) for providing us with critical comments in the process of manuscript writing; Dr. Ole Vang (Department of Science, Systems, and Models, Roskilde University) for providing us project coordination and testing materials; for excellent technical assistance, Ms. Louise Henningsen and Ms. Marianne K. Petersen (both from Technical University of Denmark) in the cellular experiments, and Mr. Jacob Krake (Roskilde University) for HPLC techniques; and Miss Katherine Chiang (Waterloo, ON, Canada) and Ms. Lisa Hughes (Roskilde, Denmark, and Greensboro, NC, USA) for helping with language corrections.

Author Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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The Immunomodulation Effect of Aronia Extract Lacks Association with Its Antioxidant Anthocyanins

Abstract

Introduction

Materials and Methods

Characterization of anthocyanins

Purification of anthocyanins

Determination of total anthocyanin content

Determination of antioxidative capacity using the DPPH• assay

Determination of antioxidative capacity using the ferric-reducing ability of plasma assay

Cell culture conditions

Stimulation of MM6 cells with plant extracts and resveratrol

Statistical analysis

Results and Dissusion

Characterization and isolation of the anthocyanins from Aronia crude extract

Antioxidative capacity of AF and kinetic behavior

Characterization of the immunomodulatory effect of Aronia crude extract, anthocyanidin-rich fraction, and resveratrol in LPS-stimulated human monocytes

Footnotes

Acknowledgments

Author Disclosure Statement

References

Supplementary Material

Determination of antioxidative capacity using the DPPH^• assay