(red clover), which belongs to the bean family Fabaceae (or Leguminosae), is a medicinal plant that improves various health conditions such as asthma, whooping cough, cancer, and gout [1
]. Phytoestrogenic isoflavones of red clover such as daidzein, genistein, biochain A, and formononetin improved menopausal symptoms and vaginal cytology on menopausal women [2
]. Although red clover is a rich source of isoflavones, it is expected to have anthocyanins due to its color. However, anthocyanins of red clover are not identified. Anthocyanins are the plants’ pigments ranging from orange and red to purple and blue, which is classified as a subgroup of flavonoids with various health benefits such as antioxidants and anti-inflammation [6
]. Inflammation is a response against infection, illness, and injury by producing cytokines such as tumor necrosis factor (TNF)α, interleukin (IL)1β, and IL6, and further by generating reactive oxygen species (ROS) [9
]. Moreover, inflammation and oxidative stress are positively correlated with the development of chronic diseases such as obesity, diabetes, and cardiovascular disease, which attracts scientists to find new sources that have anti-inflammatory and antioxidant effects. Of natural sources, anthocyanins have been suggested as the potential candidate to ameliorate health issues related to inflammation and oxidative stress [10
]. Nuclear factor kappa B (NF-κB) is a major regulator for anti-inflammatory and antioxidant effects of anthocyanins [11
]. Malvidin-3-glucoside, a major anthocyanin of blueberries and grapes, suppressed TNFα- and IL4-stimulated inflammatory markers in human umbilical vein endothelial cells and peripheral blood mononuclear cells by inhibiting nuclear translocation of p65 of NF-κB [12
]. Cyanidin-3-glucoside reduced production of nitric oxide (NO), prostaglandin E2, and IL8, as well as the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)2 without IkB-α degradation and NF-κB activation in cytokine-stimulated human intestinal HT-29 cells [14
]. Cyanidin-3-O-sophoroside and cyanidin-3-O-sambubioside from black peanut ameliorated UV-irradiated oxidative injury through the action of the nuclear factor erythroid 2-related factor 2 (NRF2) by interaction with the NF-κB signaling pathway in human keratinocyte cells and mice skin [15
Red clover showed anti-inflammatory effect [16
]. Although its isoflavones seem to be responsible for this effect [16
], anthocyanins of red clover may also support this effect. However, the effects of the anthocyanins of red clover on inflammation are not explored yet. Thus, the purpose of this study was to examine anti-inflammatory and antioxidant effects of red clover’s anthocyanins and further identify and quantify anthocyanins and other dietary compounds of red clover.
2. Materials and Methods
2.1. Preparation of Anthocyanin Fractions from Red Clover
Red clover (Trifolium pratense
) flowers were purchased from Starwest Botanicals Inc (Sacramento, CA, USA). Petals of the red clover were cleaned and dried at room temperature. Dried petals (50 g) were extracted with 1 L of 80% aqueous methanol (v/v) by homogenization and sonication [18
]. Red clover extract (RC) yield was 13.2%. To isolate red clover anthocyanins fraction (RCA), RC was loaded into a C-18 SPE cartridge (Waters, Milford, MA, USA) and 10 mL of 0.01 N HCl was added to remove sugar, acids, and water-soluble compounds. The cartridge was dried with N2
gas for 10 min and then washed with 40 mL of ethyl acetate to remove non-anthocyanin flavonoids. Anthocyanins were eluted with 6 mL of acidic methanol. Solvents of RCA were evaporated using a rotary evaporator (Buchi RE120 Rotovapor, Flawil, Switzerland). RCA yield was 0.36%. RC and RCA were stored at −20 °C until use.
2.2. Cell Culture
Mouse monocyte RAW 264.7 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) in a humidified culture incubator containing 5% CO2
at 37 °C. Cells were seeded into 12-well plates (Corning Inc., Corning, NY, USA) at a density of 0.5 × 106
and incubated for 24 h. Media was then replaced to serum-free media and cells were treated with RC or RCA. After incubation for 12 h, cells were stimulated with 1 or 0.5 μg/mL lipopolysaccharide (LPS) (Thermo Fisher Scientific, Waltham, MA, USA) for different hours depending on experiments in the presence and absence of RC or RCA. For the gene expression, cells were treated with 1 μg/mL LPS for 3 h. On the completion of the incubation, total RNA and protein were extracted. Cytotoxicity of RC, RCA, and LPS was determined as previously described [18
2.3. Measurement of Intracellular ROS
To determine the cellular antioxidant effects of RC and RCA, cellular ROS levels were measured using a 2’-7’-dichlorofluorescein diacetate (DCFDA), a fluorogenic dye. Cells were seeded in a black-24-well plate (Corning Inc) at a density of 2.5 × 105 cells per well and incubated with serum-free culture media containing RC or RCA for 12 h. The cells were then stimulated with LPS for 1 h in the presence and absence of RC or RCA. After cells were washed with 1M HEPES buffer (Thermo Fisher Scientific), they were incubated with phenol red- and serum-free culture media containing 10 μM DCFDA for 1 h at 37 °C in the dark. DCF fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a Biotek Synergy H1 microplate reader (Biotek, Winooski, VT, USA). After the fluorescence was measured, total protein was extracted, and protein concentrations were used to normalize fluorescent intensity. ROS levels were expressed as an arbitrary unit of fluorescence intensity/μg total cell protein.
2.4. Quantitative Polymerase Chain Reaction (qPCR) Analysis
Total RNA was extracted using a Trizol and then cDNA was synthesized using XLAScript cDNA MasterMix (Exella GmbH, Feucht, Germany) according to manufacturers’ instructions. The expression of genes that are involved in the regulation of inflammation and antioxidant was examined using the Fast Start Essential DNA Green Light Master kit (Roche, Indianapolis, IN, USA) in a LightCycler 96 (Roche) [18
]. Primer sequences were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/
) and confirmed using a Primer-Blast (NCBI database). Primer sequences were listed in Table 1
. Ribosomal protein L 32 (RPL32) was used as the housekeeping gene.
2.5. Enzyme-Linked Immunosorbent Assay (ELISA) for TNFα
RAW 264.7 cells were plated into 12-well plates at a density of 0.5 × 106/well and incubated for 24 h. Cells were then treated with 0, 5, 10, and 20 μg/mL of RAC for 12 h and then treated with the same samples and 1 μg/mL of LPS for 3 h. TNFα concentrations in the culture supernatant were measured using an ELISA kit (eBioscience, San Diego, CA, USA) following the manufacturer’s instruction.
2.6. Western Blot Analysis
Total protein extraction and a Western blot analysis were performed as previously described [18
]. To examine the inhibitory effects of RCA on translocation of NF-κB between nuclear and cytoplasm, cells had the same pretreatments with RCA described above and then were treated with RCA and 0.5 μg/mL LPS for 1 h [19
]. Nuclear and cytoplasmic fractions were obtained using a nuclear extraction kit (Cayman Chemical, Ann Harbor, MI, USA) according to the manufacturer’s instruction. To find each incubation time that produces the highest amount of iNOS, COX2, and p47phox
proteins, cells were treated with 0.5 μg/mL LPS during different hours. Based on this result, cells were treated with RCA and LPS for 12 h and for 3 h for determining effects of RCA on iNOS, COX2, and p47phox
proteins, respectively after RCA pretreatment. Primary antibodies were used as follows: rabbit anti-mouse COX2, iNOS, and NADPH oxidase 1 (NOX1 (1:1000, ABclonal, Woburn, MA, USA), anti-phospho-p47phox
, and NF-κB p65 (1:1000, Thermo Fisher Scientific), TATA-binding protein (1:3000, Thermo Fisher Scientific), and β-actin (1:3000, Sigma-Aldrich, St. Louis, MO, USA). Horseradish-peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG, 1:5000, Invitrogen, Carlsbad, CA, USA) were used.
2.7. Identification and Quantification of Red Clover Polyphenols Using HPLC/ESI-MS Analysis
Separation, detection, and identification of polyphenols from RC were performed using an HPLC/ESI-MS (Waters) according to the method of Cho et al. [20
]. The identified polyphenols were quantified by an HPLC (Waters) [20
]. Polyphenols were detected at 330 nm and quantified as daidzein equivalents. Total polyphenols were calculated as the sum of individual polyphenols. Anthocyanins were quantified as delphinidin, cyanidin, petunidin, peonidin, and malvidin glucoside equivalents at 510 nm. Total anthocyanins were calculated as the sum of individual anthocyanin monoglucosides.
2.8. Statistical Analysis
Data were analyzed using a one-way analysis of variance with Tukey’s post hoc analysis (Prism 7.0, Graphpad Software Inc., San Diego, CA, USA). P values less than 0.05 were considered significant. Data were presented as mean ± standard deviation.
This study determined anti-inflammatory and antioxidant effects of anthocyanins of red clover using LPS-stimulated macrophage cells and identified and quantified anthocyanins and other dietary compounds of red clover. RC that was extracted using DMSO inhibited the secretion of TNFα and IL6 and suppressed iNOS, COX2, and NF-κB proteins in LPS-stimulated RAW 264.7 cells [17
]. Muller et al. showed that isoflavones such as biochanin A, genistein, and daidzein were responsible for the anti-inflammatory effect of red clover [17
]. In this study, both RC and RCA suppressed LPS-induced IL1β
, and COX2
genes. This indicates that anthocyanins of red clover may be critical for the anti-inflammatory effects of red clover. RC reduced LPS-induced TNFα
gene expression in this study, but this gene was not changed by RCA. Interestingly, without a change in TNFα
gene expression, the secretion of TNFα was suppressed by RCA. Chemokines, e.g., TNFα are regulated by post-transcriptional regulation by controlling RNA stability and/or translational regulation [21
]. Mice deleting TNF AU-rich elements (ARE) in the 3´-untranslated region of a transcript encoding TNFα exhibited increased secretion of TNFα by decreasing a rate of TNFα decay and translational repression in hemopoietic and stromal cells [22
]. Therefore, RCA may suppress LPS-induced TNFα secretion by controlling translational inhibition without a change in RNA stability. Consistent with RCA-suppressed genes and proteins related to cytokines and inflammatory enzymes, translocation of p65 subunit of NF-κB into the nucleus was inhibited by RCA in this study. This indicates that the anti-inflammatory effects of anthocyanins of red clover may be mediated by regulating the NF-κB signaling pathway.
In response to LPS-induced cytokines, iNOS and COX2 catalyze the production of NO and prostaglandin E2, respectively, which are potent pro-inflammatory mediators [23
]. In this study, LPS-induced both iNOS and COX2 genes and proteins were suppressed by RCA. The iNOS positively regulates NOX which produces superoxide by transferring electrons from NADPH to oxygen [24
]. Under stimulated conditions, e.g., LPS, NOX enzymes are activated by protein–protein interactions via its cytosolic subunits (p47phox
, and p40phox
) and membrane subunits (small-G-protein, Rac1, or Rac 2) and by phosphorylation of p47phox
]. Moreover, p47phox
is a critical organizer to bring these subunits to complexes by its localization to the membrane and its phosphorylation-induced conformational changes on the protein [25
]. In this study, LPS-induced NOX1 gene and phosphorylation on p47phox
were completely abolished by RCA treatment at 5 and 20 µg/mL, respectively, which supports reduced intracellular ROS production. Because LPS-induced NOX1 is NF-κB dependent [27
], our findings indicate that RCA modulates cellular oxidative stress by suppressing NOX1 through reduced NF-κB.
When ROS increases in the body, the antioxidant system is promoted to remove free radicals. NRF2 is a major transcriptional factor to regulate genes and proteins that are involved in the antioxidant system. LPS-stimulated RAW 264.7 cells that were treated with berry anthocyanins showed a significant decrease in NRF2 and its downstream genes including catalase and superoxide dismutase [28
]. Extracts from red clover showed high antioxidant activities using ABTS radical, DPPH radical, hydrogen peroxide, and superoxide radical scavenging assays [29
], which may be effective at cellular levels. In this study, LPS-induced ROS production led to an increase in NRF2
gene expression, which activates cellular antioxidant systems to remove ROS. NRF2
gene expression was reduced by RCA, due to possibly either less ROS production by decreased NOX1 or anthocyanins’ antioxidant activities. Consistent with this, NOX1 gene and phosphorylation of p47phox
were reduced to the basal level of cells untreated with LPS and samples, but LPS-stimulated cells with RC or RCA exhibited lower ROS levels than the basal level, suggesting that further ROS reduction beyond the NOX1 and its regulation observed in this study may be caused by the antioxidant activity of red clover, especially its anthocyanins [29
]. Therefore, its anthocyanins may save NRF2 activity by directly removing ROS using its strong antioxidant capacity.
Some dietary compounds that are previously found in red clover [30
], were also identified in this study, but additional dietary compounds were identified, which may be due to detection at a different wavelength. Cyanidin-3-O-sophoroside and cyanidin-3-O-sambubioside were identified as major anthocyanins from red clover using thin-layer chromatography [31
]. However, these compounds were not identified in this study using LC-MS. This discrepancy results from different methodologies and approaches for extraction and identification. In this study, malvidin-3-O-galactoside was the most abundant anthocyanin of red clover. Although malvidin-3-glucose had a higher anti-inflammatory effect than malvidin-3-O-galactoside, malvidin-3-O-galactoside also suppressed the expression of MCP1, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 genes and proteins by inhibiting IkB degradation and translocation of p65 protein of NF-kB in TNFα-stimulated human umbilical vein endothelial cells [12
]. Other anthocyanins detected in the red clover including peonidin-3-O-monogalactoside, cyanidin-3-O-monogalactoside, cyanidin-3-O-monoglucoside, petunidin-3-O-monogalactoside, delphinidin-3,5-O-diglucoside, and petunidin-3-O-rutinoside are also found in blueberry, concord grape, and myrtle berry, which have strong antioxidant and anti-inflammatory effects [28