In order to compare the anti-inflammatory effects of five selected cereal grains-proso millet, hwanggeumchal sorghum, foxtail millet, barnyard millet, and adlay-the inhibitory activities of 80% ethanol (EtOH) extracts obtained from the individual grains on lipopolysaccharide (LPS)-induced nitric oxide (NO) generation were investigated in RAW264.7 cells. The EtOH extract of barnyard millet (
Echinochloa crus-galli
var.
frumentacea
) grains exhibited more potent anti-inflammatory activity than that of the other grains. When the EtOH extract of barnyard millet grains was sequentially fractionated with n-hexane, methylene chloride (MC), ethyl acetate (EtOAc), and n-butanol, the majority of the anti-inflammatory activity was detected in the MC fraction, followed by the EtOAc fraction. Pretreatment with the MC fraction caused downregulation of the expression levels of iNOS- and COX-2-specific transcripts and proteins, as well as proinflammatory cytokine gene transcripts (IL-1β, IL-6, and TNF-α) in LPS-stimulated RAW264.7 cells. Additionally, the MC fraction could suppress not only the LPS-induced nuclear translocation of cytosolic NF-kB, but also the LPS-induced activation of MAPKs, such as ERK, JNK, and p38MAPK. Further analysis of the MC fraction by HPLC identified kaempferol, biochanin A, and formononetin as the major phenolic components. Both kaempferol and biochanin A, but not formononetin, could exert anti-inflammatory effect at the same concentrations as those of the MC fraction. Consequently, these results indicate that kaempferol and biochanin A are among the most effective anti-inflammatory phenolic components in barnyard millet grains. This finding suggests that barnyard millet grains and the MC extract enriched in kaempferol and biochanin A could be beneficial functional food sources that have an anti-inflammatory effect.
Introduction
Lipopolysaccharide (LPS) is an endotoxic component of the outer membranes of gram-negative bacteria and stimulates several pathological inflammatory responses, such as systemic inflammatory response syndrome, septic shock, disseminated intravascular coagulation and multiple organ dysfunctions
[5]
. These LPS-induced inflammatory diseases are known to be mediated primarily via deregulated overproduction of pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E
2
(PGE
2
), interleukin-1β (IL-1β), interleukin- 6 (IL-6), and tumor necrosis factor-α (TNF-α), by activated macrophages
[21
,
38]
. The LPS-induced production of these pro-inflammatory mediators by macrophages is tightly regulated by two principal transcription factors, NF-κ B and AP-1. These factors are activated by the transmembrane signaling pathway, which is triggered by the interaction of the cell surface CD14/toll-like receptor 4 (TLR4) complex with the hydrophobic lipid A portion of LPS, and then relayed by sequential activation of protein kinases
[14
,
22]
. Among the critical protein kinases required for the signaling pathway are IL-1 receptor-associated kinase 4 (IRAK4) and IRAK1, TGF-β-activated kinase 1 (TAK1), IκB kinase (IKK), and MAPKs (p38MAPK, JNK and ERK)
[2
,
11]
.
In relation to LPS-induced TLR4-dependent activation of NF-κB in macrophages, the redox signaling mediated by reactive oxygen species (ROS) has also been implicated
[9
,
17
,
20]
. Supplementation of antioxidants in early inflammation stage has been shown to attenuate the oxidative stress by acting as ROS scavenger, and could prevent developing chronic inflammation
[3]
. In this context, it is likely that inhibition of oxidative stress might be one of the reliable strategies, which is beneficial for ameliorating metabolic diseases being accompanied by inflammation.
Much attention has been paid to the physiological functionality of foods, due to the increasing interest in human health, and research into the health benefits of foods has been increasing last years. Traditionally, miscellaneous cereal grains have been considered as a beneficial functional food source, which can improve the metabolic diseases
[7
,
8
,
30]
. Barnyard millet is one of the hardy crops that have adapted to sterile environments throughout the world
[28]
. The cultivation area of barnyard millet was gradually reduced due to various changes in current diet and cereal breeding. However, in recent years, barnyard millet grains are re-evaluated because of their high nutritional value in preventing metabolic diseases. The nutritive and biological studies about barnyard millet grains have demonstrated that the grains are rich in protein, lipid, vitamin B complex and nicotinic acid, when compared with common cereal grains. Previously, it has been reported that the extract of barnyard millet grains possess anti-oxidant activity
[12
,
13]
, a beneficial influence on metabolism of cholesterol and lipid
[27]
, immunosuppressive activity
[15]
, anti-microbial activity
[29]
and lowering glycemic index and anti-diabetes effects
[23
,
27
,
36]
. Several bioactive components have been found from barnyard millet grains including serotonin, luteolin, tricin, deoxynojirimycin and coumaric acid derivatives
[33
,
37]
, but studies on the health benefits of barnyard millet grains with respect to anti-inflammatory activity are not well-established.
In the present study, as an attempt to compare anti-inflammatory activities of five selected miscellaneous cereal grains, such as proso millet (
Panicum miliaceum
), hwanggeumchal sorghum (
Sorghum bicolor
(L.) Moench var.
hwanggeumchal
), yellow glutinous foxtail millet (
Setaria italica
), adlay (
Coix lacryma-jobi
), and barnyard millet (
Echinochloa crus-galli
var.
frumentacea
) harvested in Korea, we investigated the anti-NO production activity of the 80% ethanol (EtOH) extracts from the individual grains in LPS-stimulated murine macrophage RAW264.7 cells. The EtOH extract of barnyard millet grains, which exhibited more potent anti-inflammatory effect compared with other grain extracts, were sequentially fractionated by n-hexane, MC, EtOAc, and BuOH. Because the inhibitory effect on LPS-induced NO production in RAW264.7 cells were mainly detected in the MC fraction followed by the EtOAc fraction, the anti-inflammatory activity of the MC fraction was further examined by investigating its inhibitory action against LPS-induced inflammatory events and by detecting the active phenolic components in the MC fraction.
Materials and Methods
- Reagents, chemicals, antibodies and culture medium
The ECL Western blotting kit was purchased from PerkinElmer (Boston, MA, USA), and Immobilon-P membrane was obtained from Millipore Corporation (Bedford, MA, USA). The anti-COX-2, anti-β-actin, anti-NF-κB p65, anti- p-JNK, anti-JNK, anti-Sp1 and anti-p38 MAPK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the anti-p-p38 MAPK, anti-IκBα, anti-p-IκBα and p-c-jun antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The anti-p-ERK was obtained from Millipore Corporation, and anti-ERK antibody was obtained from Zymed Laboratories (South San Francisco, CA, USA). Anti-iNOS was purchased from BD Biosciences (Chicago, IL, USA). Horse radish peroxide (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were obtained from Cell Signaling, and HRP-conjugated anti-goat IgG was obtained from Santa Cruz Biotechnology. The murine macrophage cell line RAW264.7 was purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco
’
s modified Eagle
’
s medium (DMEM) which was supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml gentamycin at 37℃ in a humidified 5% CO
2
atmosphere. For the experiments, the cells were grown to 80-90% confluences, and were subjected to no more than 15 cell passages.
- Sample extraction
Barnyard millet grains were provided by National Institute of Crop Science of Miryang, Korea, and a voucher specimen has been deposited in Laboratory of Immunology, College of Natural Sciences, Kyungpook National University, Daegu, Korea. The dried grains (250 g) were milled on a Blender 7012 (Dynamics Corporation, USA) for 10 min, and then extracted with 80% ethanol (EtOH) for 3 hr at 80℃. The ethanol extract was evaporated, dissolved in water, and then sequentially extracted with n-hexane, methylene chloride (MC), ethyl acetate (EtOAc) and n-butanol (BuOH). Each organic solvent fractionation was repeated three times. Each organic solvent fraction as well as the remnant aqueous fraction was centrifuged at 7,500 rpm for 15 min to remove insoluble substances. The recovered supernatant of each fraction was then concentrated by rotary vacuum evaporator (Heidolph LR 4000, Germany). The yields of hexane fraction, MC fraction, EtOAc fraction, BuOH fraction, remnant aqueous fraction were 9.4 g, 2.5 g, 1.1 g, 0.7 g, 2.3 g and 3.1 g, respectively.
- Cell viability assay
The Cytotoxic effect of samples on RAW264.7 cells was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells (0.5×10
5
/well) were cultured with serial dilutions of samples in 96-well plates. After incubation for 18 hr, 50 μl of MTT solution (1.1 mg/ml) was added to each well and incubated for an additional 2 hr. The colored formazan crystal produced from MTT was dissolved in DMSO. The absorbance was measured at 540 nm by a plate reader (Molecular Devices, Thermo Max, USA) to determine the formazan concentration, which reflects the cell viability.
- Nitric oxide assay
As an indicator of NO production, the concentration of nitrite, a stable metabolite of NO, in the culture medium was assessed by Griess reagent
[10]
. Briefly, RAW264.7 cells (2×10
5
cells/well) were cultured overnight in 96-well plates, and then treated with LPS (0.1 μg/ml) in the absence or in the presence of various concentrations of sample for 16 hr. The culture supernatant (100 μl) was mixed with an equal volume of Griess reagent for 15 min at room temperature in dark condition, and then the absorbance of the chromophoric azo-derivative molecule was measured using a microplate reader at 540 nm. To ensure the validity of the results, experiments were done in three independent experiments with three replicates per independent experiment.
- Total RNA isolation and RT-PCR
Cells were washed twice in PBS, then total RNA was isolated using the Trizol reagent from Invitrogen (Carlsbad, CA, USA) according to the manufacturer
’
s instructions and DNase I treatment. After RNA quantification by GE NanoVue Spectrophotometer (GE healthcare, Buckinghamshire, UK), 1 μg RNA was reversely transcribed using First strand cDNA synthesis kit (Thermo scientific, Logan, UT, USA) for cDNA synthesis. Gene expression values were normalized to housekeeping GAPDH gene. GAPDH was amplified with forward (5
’
-ATCCTGCGTCTGGACCTGGCT-3
’
) and reverse (5
’
-CTGATCCACATCTGCTGGAAG-3
’
) primers. PCR amplification was done using AccuPower™ PCR PreMix (Bioneer, Seoul, Korea) and specific primers. The following primers were used for PCR: iNOS-forward, 5
’
- ATGTCCGAAGCAAACATCAC-3
’
; iNOS-reverse, 5
’
-TAATGTCCAGGAAGTAGGTG- 3
’
; COX-2-forward, 5-CAGCAAATCCTTGCTGTTCC- 3; COX-2-reverse, 5
’
-TGGGCAAAGAATGCAAACATC- 3
’
; TNF-α-forward, 5
’
-TACTGAACTTCGGGGTGATCGGTCC- 3
’
; TNF-α-reverse, 5
’
-CAGCCTTGTCCCTTGAAGAGAACC- 3
’
; IL-6-forward, 5
’
-GAAATGATGGATGCTTCCAAACTGG- 3
’
; IL-1β-forward, 5
’
-CAAGGAGAACCAAGCAAC- 3
’
; IL-1β-reverse, 5
’
-GGGGAAGGCAATAGAAAC- 3
’
. To ensure that the same amount of RNA was being used, the concentration of the total RNA for each sample was confirmed by spectrophotometry and normalized with GAPDH as the message of a housekeeping gene. The PCR products were electrophoresed using 1.2% agarose gel and visualized under UV light after ethidium bromide staining.
- Preparation of cell lysate and western blot analysis
Cellular lysates were prepared by suspending cells (5×10
6
) in 300 μl of lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl
2
, 25 mM MOPS, 1 mM PMSF, and 5.0 μg/ml proteinase inhibitor E-64, 0.1% Triton X-100, pH 7.2). The cells were disrupted by sonication and extracted at 4℃ for 30 min. An equivalent amount of protein lysate (25 μg) was electrophorersed on 4~12% NuPAGE gradient gel (Invitrogen/Novex, Carlsbad, CA, USA) with MOPS buffer and then electrotransferred to Immobilon-P membranes. Detection of each protein was performed utilizing the ECL Western blotting kit following the manufacturer
’
s instructions. Densitometry was performed using ImageQuant TL software (Amersham, Arlington Heights, IL, USA). Arbitrary densitometric units for the protein of interest were normalized to the densitometric units of β-actin.
- High-performance liquid chromatography (HPLC) analysis of 80% ethanol extract and its organic solvent fractions of barnyard millet grains
The contents of phenolic compounds were analyzed as previously described
[32]
. Samples were filtered through a 0.45 μm syringe filter (Millipore, Billerica, MA, USA) and analyzed by HPLC (Agilent 1200, Agilent Technologies, Waldbronn, Germany). The analytical column was a ZORBAX ODS (4.6×250 mm, Agilent Technologies) with a guard column (Phenomenex, Torrance, CA, USA). The detection wavelength was set at 280 nm and the solvent flow rate was held constant at 1.0 ml/min. The mobile phase used for the separation consisted of solvent A (distilled water included 0.1% acetic acid) and solvent B (acetonitrile included 0.1% acetic acid). A gradient elution procedure was used as 0 min 92% A, 2-27 min 90% A, 27-50 min 70% A, 50-51 min 10% A, 51-60 min 0% A, and 60-62 min 92% A. The injection volume was 20 μl for analysis. The standards used were biochanin A, caffeic acid, (±)-catechin hydrate, chlorogenic acid,
trans
-cinnamic acid, formononetin, gallic acid, hesperidin, homogentisic acid, isoorientin, kaempferol, naringin, orientin, protocatechuic acid, pyrogallol, quercetin, resveratrol, rutin hydrate, syringic acid, vanillic acid, vanillin, veratric acid and all samples were analyzed in triplicate.
- Statistical analysis
Unless indicated otherwise, each result in this paper is representative of at least three separate experiments. Values represent the mean standard deviation (SD) of these experiments. The statistical significance was calculated with Student
’
s t-test.
P
values less than 0.05 were considered significant.
Results and Discussion
- Anti-NO production activity of the 80% EtOH extract and its organic solvent fractions obtained from barn yard millet grains in LPS-stimulated RAW264.7 cells
In order to compare the anti-inflammatory effects of five selected miscellaneous cereal grains, including proso millet (
Panicum miliaceum
), hwanggeumchal sorghum (
Sorghum bicolor
(L.) Moench var.
hwanggeumchal
), yellow glutinous foxtail millet (
Setaria italica
), adlay (
Coix lacryma-jobi
), and barnyard millet (
Echinochloa crus-galli
var.
frumentacea
) harvested in Korea, the inhibitory activities of the 80% ethanol extracts of the individual cereal grains on LPS-induced NO proproduction were investigated in RAW264.7 cells. As shown in
Fig. 1A
, the EtOH extracts of barnyard millet grains, hwanggeumchal sorghum grains, and proso millet grains appeared to exhibit more potent anti-NO production activities compared with other cereal grains tested. The LPS-induced NO productions in the presence of the EtOH extracts of barnyard millet grains and hwanggeumchal sorghum grains at concentrations of 100 μg/ml were reduced to the levels of 56.4% and 52.3%, respectively, whereas those at concentrations of 200 μg/ml were reduced to the levels of 3.4% and 10.8%, respectively. However, under the same conditions, none of these EtOH extracts could affect the cell viability of RAW264.7 cells (
Fig. 1B
). These results indicated that anti-inflammatory effect of barnyard millet grains was the most potent among the five selected miscellaneous cereal grains tested, and that the EtOH extract of barnyard millet grains at concentrations of 100-200 μg/ml could significantly reduce the LPS-induced production of NO without affecting cell viability in RAW264.7 cells.
Effect of the 80% EtOH extracts of five selected different miscellaneous cereal grains on LPS-induced NO production (A) and viability (B) in RAW264.7 cells. After RAW 264.7 cells (2×105 cells/well) were incubated in 96 well plates for 16 hr, the cells were treated with the individual 80% ethanol extracts (50, 100, and 200 μg/ml) for 1 hr and then continuously incubated with LPS (0.1 μg/ml) for 20 hr. The nitrite concentration as an indicator of NO production in culture medium was measured using Griess reagent. The cell viability was determined by the MTT assay as described in Materials and Methods. Each value is expressed as mean ± SD (n=3 with six replicates per independent experiment). *p<0.05, significant compared with vehicle- treated control.
In order to examine further the anti-NO production property of barnyard millet grains, the 80% EtOH extract of barnyard millet grains was sequentially fractionated with n-hexane, MC, EtOAc, and BuOH, and then individual organic solvent fractions at concentrations ranging from 25-100 μg/ ml were tested for the anti-NO production activity. As shown in
Fig. 2A
, the LPS-induced NO production was the most significantly suppressed in the presence of the MC fraction, followed by the EtOAc fraction. In addition, the LPS-induced NO productions in the presence of the MC fraction at concentrations of 25 μg/ml, 50 μg/ml and 100 μg/ml were reduced to the levels of 81.7%, 35.2% and 1.4%, respectively. At the same time, the MC fraction at concentrations of up to 100 μg/ml did not affect the cell viability (
Fig. 2B
).
Effect of the 80% EtOH extract and its organic solvent fraction of barnyard millet grains on LPS-induced NO production (A) and viability (B) in RAW264.7 cells. After RAW 264.7 cells (2×105 cells/well) were incubated in 96 well plates for 16 hr, the cells were treated with the individual 80% ethanol extracts (25, 50, and 100 μg/ml) for 1 hr and then continuously incubated with LPS (0.1 μg/ml) for 20 hr. The nitrite concentration as an indicator of NO production in culture medium was measured using Griess reagent. The cell viability was determined by the MTT assay as described in Materials and Methods. Each value is expressed as mean ± SD (n=3 with six replicates per independent experiment). *p<0.05, significant compared with vehicle-treated control.
Consequently, these results indicate that the MC fraction of barnyard millet grains at concentrations of 25-100 μg/ml could suppress the LPS-induced NO production in a dose-dependent manner, and that the IC
50
value of the MC fraction was 44.8 μg/ml.
- Inhibitory effect of the MC fraction of barnyard millet grains on LPS-induced expression of iNOS, COX-2, and pro-inflammatory cytokines in RAW264.7 cells
Because the production of pro-inflammatory mediators, such as NO and prostaglandin E
2
(PGE
2
), in RAW64.7 cells stimulated with LPS is governed by the enzymes iNOS and COX-2, RT-PCR and western blot analysis were performed to examine whether the MC fraction of barnyard millet grains could suppress the expression levels of iNOS and COX-2 in RAW264.7 cells stimulated with LPS. As shown in
Fig. 3A
, RT-PCR data revealed that the expression level of mRNAs specific for iNOS and COX-2, which were not detected in unstimulated RAW264.7 cells, were significantly enhanced following LPS-stimulation; however, the presence of the MC fraction at concentration of 25-100 μg/ml down-regulated the levels of iNOS and COX-2 mRNAs. Under these conditions, western blot analysis data also revealed that although the proteins specific for iNOS and COX-2 were not detected in continuously growing RAW 264.7 cells, the levels of both proteins were enhanced by 5.8 folds and 5.2 folds, respectively, in RAW264.7 cells stimulated with LPS (
Fig. 3B
). However, the LPS-induced increase in the levels of iNOS and COX-2 proteins was markedly reduced by the MC fraction in a dose-dependent manner. In particular, LPS-induced expression of both iNOS and COX-2 proteins was not detected in the presence of the MC fraction at a concentration of 100 μg/ml. These results demonstrated that the MC fraction of barnyard millet grains at concentrations of 25‒100 μg/ml could reduce the expression levels of the pro-inflammatory proteins (iNOS and COX-2) in RAW264.7 cells stimulated with LPS via the down-regulation of mRNA levels.
Effect of the MC fraction of barnyard millet grains on the expression levels of iNOS, COX-2 and pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in RAW264.7 cells stimulated with LPS. The cells were pretreated with the MC fraction at indicated concentrations, prior to stimulation with LPS (0.1 μg/ml) for 20 hr. RT-PCR analysis of transcripts of iNOS, COX-2 and GAPDH (A) and IL-6, IL-1β, TNF-α and GAPDH (C), and western blot analysis of iNOS, COX-2 and β-actin proteins (B) were performed as described in the Materials and Methods. The expression level of GAPDH mRNA or β-actin protein was used as control. Arbitrary densitometric units for the individual transcripts and proteins of interest were normalized to the densitometric units of GAPDH transcript and β-actin protein, respectively. A representative study is shown and two additional experiments yielded similar results.
To examine whether the MC fraction could suppress the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, RAW264.7 cells were stimulated with LPS for 4 hr following pretreatment with the MC fraction (25-100 μg/ml) for 1 hr. The levels of mRNAs specific for IL-1β, IL-6, and TNF-α were assessed by RT-PCR. As shown in
Fig. 3C
, the mRNAs specific for IL-1β, IL-6, and TNF-α were not detected in unstimulated RAW264.7 cells, but the expression levels of these cytokine-specific mRNAs were markedly up-regulated in RAW264.7 cells following stimulation with LPS. Under the same conditions, the LPS-induced expression of IL-1β, IL-6, and TNF-α mRNA was reduced in the presence of the MC fraction, more efficiently at a concentration of 100 μg/ml. These results suggested that the MC fraction at concentrations of 25-100 μg/ml could inhibit the LPS-induced up-regulation of the expression levels of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α.
- Inhibitory effect of the MC fraction of barnyard millet grains on the nuclear translocation of NF-κB complex in RAW264.7 cells
The LPS-induced expression of pro-inflammatory mediators including iNOS, COX-2, IL-1β, IL-6 and TNF-α in macrophages is known to be tightly regulated by two principal transcription factors, NF-κB and AP-1
[21
,
34
,
38]
. Nuclear translocation of NF-κB and proteasomal degradation of IκBα have been considered as prominent markers that exhibit molecular inflammation initiation for the expression of pro-inflammatory mediators in activated macrophages.
To determine whether the MC fraction of barnyard millet grains could inhibit LPS-induced activation of NF-κB in RAW264.7 cells, LPS-induced phosphorylation of IκBα, alterations in the level of IκBα, and nuclear translocation of NF-κB (p65), all of which are known to be critical for the activation of NF-kB, were compared by western blot analysis in RAW264.7 cells stimulated with LPS with and without the MC fraction (25-100 μg/ml). As shown in
Fig. 4
, LPS-stimulation resulted in an increase in the phosphorylation level of IκBα and a decrease in the protein level of IκBα in RAW264.7 cells. At the same time, LPS-stimulation caused nuclear translocation of cytosolic NF-κB (p65) so that the level of nuclear NF-κB could be enhanced by approximately 7 folds compared with that of untreated RAW264.7 cells. However, the LPS-induced phosphorylation of IκBα, down-regulation of IκBα protein levels, and nuclear translocation of cytosolic NF-κB (p65) appeared to be suppressed by the MC fraction in a dose-dependent manner. These results suggested that the MC fraction of barnyard millet grains could inhibit LPS-induced activation of NF-κB by suppressing the LPS-induced phosphorylation and degradation of IκBα, and subsequent nuclear translocation of NF-kB in RAW264.7 cells.
Inhibition of the MC fraction of barnyard millet grains on IκBα phosphorylation, IκBα protein levels, and nuclear translocation of cytosolic NF-κB (p65) in RAW264.7 cells following LPS stimulation. The cells were treated by the MC fraction at the indicated concentrations for 1 hr, and then stimulated with LPS (0.1 μg/ml) for 4 hr to detect IκBα and its phosphorylation by western analysis. A representative study is shown and two additional experiments yielded similar results.
- Inhibitory effect of the MC fraction of barnyard millet grains on LPS-induced phosphorylation of MAPKs in RAW264.7 cells
Several studies have reported that MAPKs (p38MAPK, JNK and ERK) are closely associated with the TLR4-mediated proximal signaling events that lead to the activation of transcription factors NF-kB and AP-1 in LPS-stimulated macrophages
[2
,
11]
. Because the MC fraction of barnyard millet grains appeared to suppress the LPS-induced phosphorylation of IκBα, alterations in the level of IκBα, and nuclear translocation of NF-κB (p65), all of which are critical for activation of the transcription factor NF-kB, we decided to examine whether the LPS-induced activation of p38MAPK, JNK, and ERK could be targeted by the inhibitory action of the MC fraction of barnyard millet grains by western blot analysis using individual antibodies specific for the phosphosphorylated active forms of these MAPKs. Although the total protein levels of p38MAPK, JNK, and ERK all of which were easily detected in unstimulated RAW264.7 cells, were not changed regardless of the presence of the MC fractions, the active phosphorylated forms of p38MAPK, JNK, and ERK were significantly enhanced after LPS treatment (
Fig. 5
). Under these conditions, however, the LPS-induced phosphorylation of p38MAPK, JNK and ERK was commonly reduced by the MC fraction. In accordance with the observed reduction in the level of LPS-induced phosphorylation of JNK, the phosphorylation of c-Jun protein at Ser-63, which is catalyzed by JNK
[6]
, was markedly reduced in the presence of the MC fractions. This finding indicated that the phosphorylated JNK that was detected in RAW264.7 cells after treatment with LPS possessed enough enzymatic activity to phosphorylate c-Jun. Consequently, these results indicated that the LPS-induced activation of p38MAPK, JNK and ERK was influenced by the inhibitory action of the MC fraction. In addition, these results also suggested that inhibitory action of the MC fraction against the LPS-induced nuclear translocation of NF-kB in RAW264.7 cells might be due to the decline in the level of activation of p38MAPK, JNK and ERK.
Effect of the MC fraction of barnyard millet grains on LPS-induced activation of MAPKs (p38MAPK, JNK and ERK) in RAW264.7 cells. The cells were pre-treated with indicated concentrations of the MC fraction for 1 hr, and then with LPS (0.1 μg/ml) for 20 hr. The levels of the active phosphorylated forms of p38MAPK, c-Jun, JNK, and ERK were assessed by western blot analysis using antibodies specific for the phosphorylated forms of individual kinases. A representative study is shown and two additional experiments yielded similar results.
- Identification of the major anti-inflammatory phenolic compounds in the MC fraction of barnyard millet grains
Among phytochemicals, phenolic compounds have been reported to have various ameliorating effects on neurodegenerative diseases, multiple sclerosis, cardiovascular diseases, and metabolic syndrome from oxidative stress
[16
,
35]
. In order to identify the major anti-inflammatory ingredient( s) of the MC fraction of barnyard millet grains, we decided to analyze phenolic compounds contained in the MC fraction by HPLC. As the major phenolic components, kaempferol (9.17 μg/mg), biochanin A (2.22 μg/mg), and formononectin (1.52 μg/mg), which accounted for 85% of the total phenolic compounds, were detected in the MC fraction.
Because it has previously been reported that kaempferol
[4
,
18]
, biochanin A
[19]
, and formononectin
[26]
possess anti- inflammatory activity, the anti-NO production activities of these phenolic compounds were examined in LPS-stimulated RAW264.7 cells. As shown in
Fig. 6A
, when the inhibitory activities of kaempferol, biochanin A, and formononectin against LPS-induced NO production were examined at concentrations of 10 μM, 25 μM, and 50 μM in RAW264.7 cells, kaempferol reduced the LPS-induced NO production to the levels of 85.3%, 69.1%, and 45.7%, respectively, whereas biochanin A reduced the NO production to the levels of 79.6%, 60.9%, and 39.7%, respectively. Under these conditions, both kaempferol and biochanin A did not show a significant cytotoxic effect on RAW264.7 cells. Although formononetin has previously been reported to inhibit the inflammation in mouse lung injury model
[26]
, it failed to inhibit the LPS-induced NO production to a remarkable level. It is noteworthy that hesperidin (0.33 μg/ mg)
[1
,
31]
, naringin (0.56 μg/mg)
[25]
, and protocatechuic acid (0.34 μg/mg)
[24]
as the minor components, which were reported to possess anti-inflammatory activity, were detected in the MC fraction. Consequently, these results indicated that kaempferol and biochanin A, but not formononetin, could inhibit the LPS-induced NO production in a dose-dependent manner, and that both kaempferol and biochanin A were among the most effective anti-inflammatory phenolic components in barnyard millet grains.
Comparison of the anti-NO production activities (A) and cytotoxicity (B) of phenolic compounds (kaempferol, biochanin A, and formononetin) in LPS-stimulated RAW 264.7 cells. The cells were pre-incubated for 1 hr with the MC fraction (12.5 μg/ml, 20 μg/ml and 50 μg/ml), kaempferol (10 μM, 25 μM, and 50 μM), biochanin A (10 μM, 25 μM, and 50 μM), or formononetin (10 μM, 25 μM, and 50 μM) in triplicate and then treated with LPS (0.1 μg/ml) for 4 hr. The culture supernatants were saved and used to determine NO production. MTS were employed to check the cell viability. Each value is expressed as mean ± SD (n=3 with six replicates per independent experiment). *p<0.05, significant compared with vehicle- treated control.
In conclusion, this study describes an anti-inflammatory activity of barnyard millet grains against LPS-induced inflammatory events in mouse macrophage cell line RAW 264.7, and demonstrates that this anti-inflammatory action is attributable to suppression of LPS-induced up-regulation of pro-inflammatory modulators including iNOS, COX-2, IL-1β, IL-6, and TNF-α, via inhibition of nuclear translocation of cytosolic NF-kB as well as inactivation of MAPKs. As the active phenolic ingredient in the MC fraction responsible for the inflammatory activities, kaempferol and biochanin A, which are detected as the major phenolic compounds in the MC fraction of barnyard millet grains, are identified. Current results also suggest that barnyard millet grains and the MC extract enriched in kaempferol and biochanin A could be beneficial functional food sources applicable to improving inflammatory conditions.
Acknowledgements
This work was carried out with the support of“Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009865)”Rural Development Administration, Republic of Korea.
Abuelsaad A. S.
,
Allam G.
,
Al-Solumani A. A.
2014
Hesperidin inhibits inflammatory response induced by Aeromonas hydrophila infection and alters CD4+/CD8+ T cell ratio
Mediators Inflamm
2014
393217 -
Basu S.
,
Ghosh A.
,
Hazra B.
2005
Evaluation of the antibacterial activity of Ventilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn.: isolation of emodin and physcion as active antibacterial agents
Phytother Res
19
888 -
894
DOI : 10.1002/ptr.1752
Choi I. S.
,
Choi E. Y.
,
Jin J. Y.
,
Park H. R.
,
Choi J. I.
,
Kim S. J.
2013
Kaempferol inhibits P. intermedia lipopolysaccharide- induced production of nitric oxide through translational regulation in murine macrophages: critical role of heme oxygenase-1-mediated ROS reduction
J Periodontol
84
545 -
555
DOI : 10.1902/jop.2012.120180
Corriveau C. C.
,
Danner R. L.
1993
Endotoxin as a therapeutic target in septic shock
Infect Agents Dis
2
35 -
43
Esmaillzadeh A.
,
Mirmiran P.
,
Azizi F.
2005
Wholegrain intake and the prevalence of hypertriglyceridemic waist phenotype in Tehranian adults
Am J Clin Nutr
81
55 -
63
Fardet A.
2010
New hypotheses for the health-protective mechanisms of whole-grain cereals: what is beyond fibre?
Nutr Res Rev
23
65 -
134
Flohe L.
,
Brigelius-Flohe R.
,
Saliou C.
,
Traber M. G.
,
Packer L.
1997
Redox regulation of NF-κB activation
Free Radic Biol Med
22
1156 -
1126
Green L. C.
,
Wanger D. A.
,
Glogowski J.
1982
Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids
Anal Biochem
126
131 -
138
DOI : 10.1016/0003-2697(82)90118-X
Hegde P. S.
,
Chandra T. S.
2005
ESR spectroscopic study reveals higher free radical quenching potential in kodo millet (Paspalum scrobiculatum) compared to other millets
Food Chem
92
177 -
182
DOI : 10.1016/j.foodchem.2004.08.002
Higashi-Okai K.
,
Ishida E.
,
Nakamura Y.
,
Fujiwara S.
,
Okai Y.
2008
Potent antioxidant and radical-scavenging activities of traditional Japanese cereal grains
J UOEH
30
375 -
389
Hoebe K.
,
Janssen E.
,
Beutler B.
2004
The interface between innate and adaptive immunity
Nat Immunol
5
971 -
974
Hosoda A.
,
Okai Y.
,
Kasahara E.
,
Inoue M.
,
Snhimizu M.
,
Usui Y.
,
Sekiyama A.
,
Higashi-Okai K.
2012
Potent immunomodulating effects of bran extracts of traditional Japanese millets on nitric oxide and cytokine production of macrophages (RAW264.7) induced by lipopolysaccharide
J UOEH
34
285 -
296
DOI : 10.7888/juoeh.34.285
Hur S. J
,
Kang S. H.
,
Jung H. S.
,
Kim S. C.
,
Jeon H. S.
,
Kim I. H.
,
Lee J. D.
2012
Review of natural products actions on cytokines in inflammatory bowel disease
Nutr Res
32
801 -
816
DOI : 10.1016/j.nutres.2012.09.013
Janessen-Heininger Y. M.
,
Poynter M. E.
,
Baeuerle P. A.
2000
Recent advances towards understanding redox mechanisms in the activation of nuclear factor kappa B
Free Radic Biol Med
28
1317 -
1327
DOI : 10.1016/S0891-5849(00)00218-5
Kim H. K.
,
Park A. H.
,
Lee J. S.
,
Chung T. S.
,
Chung H. Y.
,
Chung A. J.
2007
Down-regulation of iNOS and TNF-a expression by kaempferol via NF-κB inactivation in aged rat gingival tissues
Biogerontology
8
399 -
408
DOI : 10.1007/s10522-007-9083-9
Kole L.
,
Giri B.
,
Manna S. K.
,
Pal B.
,
Ghosh S.
2011
Biochanin-A, an isoflavone, showed anti-proliferative and anti-inflammatory activities through the inhibition of iNOS expression, p38-MAPK and ATF-2 phosphorylation and blocking NF-κB nuclear translocation
Eur J Pharmacol
653
8 -
15
DOI : 10.1016/j.ejphar.2010.11.026
Kong X.
,
Thimmulappa R.
,
Kombairaju P.
,
Biswal S.
2010
NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice
J Immunol
185
569 -
577
DOI : 10.4049/jimmunol.0902315
Kurt-Jones E. A.
,
Popova L.
,
Kwinn L.
,
Haynes L. M.
,
Jones L. P.
,
Tripp R. A.
,
Walsh E. E.
,
Freeman M. W.
,
Golenbock D. T.
,
Anderson L. J.
,
Finberg R. W.
2000
Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus
Nat Immunol
1
398 -
401
DOI : 10.1038/80833
Lee S. H.
,
Chung I. M.
,
Cha Y. S.
,
Park Y.
2010
Millet consumption decreased serum concentration of triglyceride and C-reactive protein but not oxidative status in hyperlipidemic rats
Nutr Res
30
290 -
296
DOI : 10.1016/j.nutres.2010.04.007
Lende A. B.
,
Kshirsagar A. D.
,
Deshpande A. D.
,
Muley M. M.
,
Patil R. R.
,
Bafna P. A.
,
Naik S. R.
2011
Anti-inflammatory and analgesic activity of protocatechuic acid in rats and mice
Inflammopharmacology
19
255 -
263
DOI : 10.1007/s10787-011-0086-4
Liu Y.
,
Wu H.
,
Nie Y. C.
,
Chen J. L.
,
Su W. W.
,
Li P. B
2011
Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-κB pathway
Int Immunopharmacol
11
1606 -
1612
DOI : 10.1016/j.intimp.2011.05.022
Ma Z.
,
Ji W.
,
Fu Q.
,
Ma S
2013
Formononetin inhibited the inflammation of LPS-induced acute lung injury in mice associated with induction of PPAR gamma expression
Inflammation
36
1560 -
1566
DOI : 10.1007/s10753-013-9700-5
Nishizawa N.
,
Togawa T.
,
Park K. O.
,
Sato D.
,
Miyakoshi Y.
,
Inagaki K.
,
Ohmori N.
,
Ito Y.
,
Nagasawa T.
2009
Dietary Japanese millet protein ameliorates plasma levels of adiponectin, glucose, and lipids in type 2 diabetic mice
Biosci Biotechnol Biochem
73
351 -
360
DOI : 10.1271/bbb.80589
Odintsova T.
,
Rogozhin E. A.
,
Baranov Y.
,
Musolyamov A. Kh.
,
Yalpani N.
,
Egorov T. A.
,
Grishin E. V.
2008
Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv
Biochimie
90
1667 -
1673
DOI : 10.1016/j.biochi.2008.06.007
Ryazantsev D. Y.
,
Rogozhin E. A.
,
Dimitrieva T. V.
,
Drobyazina P. E.
,
Khadeeva N. V.
,
Egorov T. A.
,
Grishin E. V.
,
Zavriev S. K.
2014
A novel hairpin-like antimicrobial peptide from barnyard grass (Echinochloa crusgalli L.) seeds: Structure functional and molecular-genetics characterization
Biochimie
99
63 -
70
DOI : 10.1016/j.biochi.2013.11.005
Sahyoun N. R.
,
Jacques P. F.
,
Zhang X. L.
,
Juan W.
,
McKeown N. M.
2006
Whole-grain intake is inversely associated with the metabolic syndrome and mortality in older adults
Am J Clin Nutr
83
124 -
131
Saiprasad G.
,
Chitra P.
,
Manikandan R.
,
Sudhandiran G.
2013
Hesperidin alleviates oxidative stress and downregulates the expressions of proliferative and inflammatory markers in azoxymethane-induced experimental colon carcinogenesis in mice
Inflamm Res
62
425 -
440
DOI : 10.1007/s00011-013-0595-2
Seo M. C.
,
Ko J. Y.
,
Song S. B.
,
Lee J. S.
,
Kang J. R.
,
Kwak D. Y.
,
Oh B. G.
,
Yoon Y. N.
,
Nam M. H.
,
Jeong H. S.
,
Woo K. S.
2011
Antioxidant compounds and activities of foxtail millet, proso millet and sorghum with different pulverizing methods
J Korean Soc Food Sci Nutr
40
790 -
797
DOI : 10.3746/jkfn.2011.40.6.790
Seo W. D.
,
Kim J. Y.
,
Jang K. C.
,
Han S. I.
,
Ra J. E.
,
Oh S. H.
,
Lee J. H.
,
Kim Y. G.
,
Kang H. J.
,
Kim B. J.
,
Nam M. H.
2012
Anti-pigmentation effect of serotonin alkaloid isolated from Korean barnyard millet (Echinochola utilis)
J Korean Soc Appl Biol Chem
55
579 -
586
DOI : 10.1007/s13765-012-2112-7
Surh Y. J.
,
Chun K. S.
,
Cha H. H.
,
Han S. S.
,
Keum Y. S.
,
Park K. K.
,
Lee S. S
2001
Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-κB activation
Mutat Res
480-481
243 -
268
DOI : 10.1016/S0027-5107(01)00183-X
Talero E.
,
Ávila-Roman J.
,
Motilva V
2012
Chemoprevention with phytonutrients and microalgae products in chronic inflammation and colon cancer
Curr Pharm Des
18
3939 -
3965
DOI : 10.2174/138161212802083725
Ugare R.
,
Chimmad B.
,
Naik R.
,
Bharati P.
,
Itagi S.
2014
Glycemic index and significance of barnyard millet (Echinochloa frumentacae) in type II diabetics
J Food Sci Technol
51
392 -
395
DOI : 10.1007/s13197-011-0516-8
Watanabe M.
1999
Antioxidative phenolic compounds from Japanese barnyard millet (Echinochloa utilis) grains
J Agric Food Chem
47
4500 -
4505
DOI : 10.1021/jf990498s
Watson W. H.
,
Zhao Y.
,
Chawla R. K.
1999
S-adenosylmethionine attenuates the lipopolysaccharide-induced expression of the gene for tumor necrosis factor alpha
Biochem J
342
21 -
25
DOI : 10.1042/0264-6021:3420021