Nutr Res Pract.  2015 Dec;9(6):569-578. 10.4162/nrp.2015.9.6.569.

Antioxidant activity and anti-inflammatory activity of ethanol extract and fractions of Doenjang in LPS-stimulated RAW 264.7 macrophages

• Kwak CS
• Son D
• Chung YS
• Kwon YH

• KMID: 2313879
• DOI: http://doi.org/10.4162/nrp.2015.9.6.569

Abstract

BACKGROUND/OBJECTIVES
Fermentation can increase functional compounds in fermented soybean products, thereby improving antioxidant and/or anti-inflammatory activities. We investigated the changes in the contents of phenolics and isoflavones, antioxidant activity and anti-inflammatory activity of Doenjang during fermentation and aging.
MATERIALS/METHODS
Doenjang was made by inoculating Aspergillus oryzae and Bacillus licheniformis in soybeans, fermenting and aging for 1, 3, 6, 8, and 12 months (D1, D3, D6, D8, and D12). Doenjang was extracted using ethanol, and sequentially fractioned by hexane, dichloromethane (DM), ethylacetate (EA), n-butanol, and water. The contents of total phenolics, flavonoids and isoflavones, 2,2-diphenyl-1 picryl hydrazyl (DPPH) radical scavenging activity, and ferric reducing antioxidant power (FRAP) were measured. Anti-inflammatory effects in terms of nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokine production and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions were also measured using LPS-treated RAW 264.7 macrophages.
RESULTS
Total phenolic and flavonoid contents showed a gradual increase during fermentation and 6 months of aging and were sustained thereafter. DPPH radical scavenging activity and FRAP were increased by fermentation. FRAP was further increased by aging, but DPPH radical scavenging activity was not. Total isoflavone and glycoside contents decreased during fermentation and the aging process, while aglycone content and its proportion increased up to 3 or 6 months of aging and then showed a slow decrease. DM and EA fractions of Doenjang showed much higher total phenolic and flavonoid contents, and DPPH radical scavenging activity than the others. At 100 microg/mL, DM and EA fractions of D12 showed strongly suppressed NO production to 55.6% and 52.5% of control, respectively, and PGE2 production to 25.0% and 28.3% of control with inhibition of iNOS or COX-2 protein expression in macrophages.
CONCLUSIONS
Twelve month-aged Doenjang has potent antioxidant and anti-inflammatory activities with high levels of phenolics and isoflavone aglycones, and can be used as a beneficial food for human health.

Keyword

Doenjang; isoflavone aglycone; antioxidant; inflammation; RAW 264.7 macrophages

MeSH Terms

1-Butanol
Aging
Aspergillus oryzae
Bacillus
Dinoprostone
Ethanol*
Fermentation
Flavonoids
Humans
Inflammation
Isoflavones
Macrophages*
Methylene Chloride
Nitric Oxide
Nitric Oxide Synthase Type II
Phenol
Prostaglandin-Endoperoxide Synthases
Soybeans
Water
1-Butanol
Dinoprostone
Ethanol
Flavonoids
Isoflavones
Methylene Chloride
Nitric Oxide
Nitric Oxide Synthase Type II
Phenol
Prostaglandin-Endoperoxide Synthases
Water

Figure

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  Fig. 1 Manufacturing process of Doenjang

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  Fig. 2 Effect of dichloromethane (DM) and ethylacetate (EA) fractions of ethanol extract from steamed soybeans and Doenjang on the cell viability of RAW 264.7 macrophages. Each bar represents the mean ± SD (n = 3). Cells were treated with different concentrations of DM or EA fraction of sample extract for 24 h, and cell viability was determined by MTT assay. SB: Steamed soybeans, D0: Doenjang without aging, D6: 6 month-aged Doenjang, D12: 12 month-aged Doenjang

• 

  Fig. 3 Effect of dichloromethane (DM) and ethylacetate (EA) fractions of ethanol extract from steamed soybeans and Doenjang on NO and PGE2 synthesis in LPS-treated RAW 264.7 macrophages. Each bar represents the mean ± SD (n = 3). Cells were treated with different concentrations of DM or EA fraction of sample extract for 2 h, followed by addition LPS (1 µg/mL) and incubation for 22 h. NO (A) and PGE2 (B) concentrations in cultured medium were measured. SB: Steamed soybeans, D0: Doenjang without aging, D6: 6 month-aged Doenjang, D12: 12 month-aged Doenjang. NS: not significant. Means sharing the same alphabet in superscript within each sample are not significantly different at P < 0.05 by ANOVA and Duncan's multiple range test.

• 

  Fig. 4 Effect of dichloromethane (DM) and ethylacetate (EA) fractions of ethanol extract from steamed soybeans and Doenjang on IL-6 and TNF-α synthesis in LPS-treated RAW 264.7 macrophages. Each bar represents the mean ± SD (n = 3). Cells were treated with different concentrations of DM or EA fraction of sample extract for 2 h, followed by addition of LPS (1 µg/mL) and incubation for 22 h. IL-6 (A) and TNF-α (B) concentration in cultured medium were measured. SB: steamed soybeans, D0: Doenjang without aging, D6: 6 month-aged Doenjang, D12: 12 month-aged Doenjang, Means sharing the same alphabet in superscript within each sample are not significantly different at P < 0.05 by ANOVA and Duncan's multiple range test.

• 

  Fig. 5 Effects of dichloromethane (DM) and ethylacetate (EA) fractions of ethanol extract from steamed soybeans and Doenjang on iNOS and COX-2 synthesis in LPS-treated RAW 264.7 macrophage cells. (A) Cells were treated with 50 or 100 µg/mL of DM or EA fraction of sample extract for 2 h, followed by addition of LPS (1 µg/mL) and incubation for 22 h. Cells were lysed and equal amounts of cellular proteins (60 µg/well) were resolved by SDS-PAGE and subjected to Western blotting. The proteins were visualized using iNOS and COX-2 antibodies and ECL detection system. (B) The visualized bands were quantified by densitometry. Each bar represents the mean ± SD (n = 3). *significantly different from LPS-treated control at P < 0.05 by ANOVA and Duncan's multiple range test. SB: Steamed soybeans, D12:12 month-aged Doenjang

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