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. 2016 Mar 30;21(4):430.
doi: 10.3390/molecules21040430.

Comparison of Anti-Oxidant and Anti-Inflammatory Effects between Fresh and Aged Black Garlic Extracts

Yi Yeong Jeong  1 , Ji Hyeon Ryu  2 , Jung-Hye Shin  3 , Min Jung Kang  4 , Jae Ran Kang  5 , Jaehee Han  6   7 , Dawon Kang  8   9
Affiliations

Comparison of Anti-Oxidant and Anti-Inflammatory Effects between Fresh and Aged Black Garlic Extracts

Yi Yeong Jeong et al. Molecules. .

Abstract

Numerous studies have demonstrated that aged black garlic (ABG) has strong anti-oxidant activity. Little is known however regarding the anti-inflammatory activity of ABG. This study was performed to identify and compare the anti-oxidant and anti-inflammatory effects of ABG extract (ABGE) with those of fresh raw garlic (FRG) extract (FRGE). In addition, we investigated which components are responsible for the observed effects. Hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) were used as a pro-oxidant and pro-inflammatory stressor, respectively. ABGE showed high ABTS and DPPH radical scavenging activities and low ROS generation in RAW264.7 cells compared with FRGE. However, inhibition of cyclooxygenase-2 and 5-lipooxygenase activities by FRGE was stronger than that by ABGE. FRGE reduced PGE₂, NO, IL-6, IL-1β, LTD₄, and LTE₄ production in LPS-activated RAW264.7 cells more than did ABGE. The combination of FRGE and sugar (galactose, glucose, fructose, or sucrose), which is more abundant in ABGE than in FRGE, decreased the anti-inflammatory activity compared with FRGE. FRGE-induced inhibition of NF-κB activation and pro-inflammatory gene expression was blocked by combination with sugars. The lower anti-inflammatory activity in ABGE than FRGE could result from the presence of sugars. Our results suggest that ABGE might be helpful for the treatment of diseases mediated predominantly by ROS.

Keywords: NF-kappa B; anti-inflammatory agents; antioxidants; garlic; sugar.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Antioxidant activity of garlic extracts. (A) ABTS and (B) DPPH radical scavenging activities of garlic extracts. Each bar represents the half maximal inhibitory concentration (IC50). * p < 0.05 compared with the FRGE treatment. † p < 0.05 compared with the HRGE treatment. # p < 0.05 compared with the ABGE treatment; (C) Effect of garlic extracts on viability of RAW264.7 cells. Different concentrations of garlic extracts were treated to cells for 24 h; (D,E) Effect of garlic extracts on H2O2-induced ROS generation in RAW264.7 cells. Cells were treated with 100 μM H2O2, 1000 μg/mL garlic extracts, or 3 mM NAC and then stained with H2DCFDA to evaluate ROS generation. The ROS levels in the cells were quantified using fluorescence microscopy after 3 h of treatment with the extract. The plus sign (+) represents conditions co-treated with H2O2. The scale bar represents 50 μm. * p < 0.05 compared with the control (CTL). † p < 0.05 compared with the H2O2 treatment. # p < 0.05 compared with the FRGE treatment.
Figure 2
Figure 2
Anti-inflammatory activity of garlic extracts. (A) Inhibition of COX-2 and 5-LO activities by garlic extracts. The activities of garlic extracts were measured by calculating absorbance. * p < 0.05 compared with the FRGE treatment. † p < 0.05 compared with the HRGE treatment; (B) Inhibitory effects of garlic extracts on the production of NO and the release of pro-inflammatory cytokines (IL-6 and IL-1β) in LPS-activated RAW264.7 cells. The cells were pretreated for 2 h with garlic extract (1000 μg/mL) before stimulation with LPS (1 μg/mL) for 24 h; (C,D) Inhibitory effect of garlic extracts on the LPS-induced PGE2 secretion and COX-2 expression (C) and on the release of LTD4 and LTE4 (D). The concentrations of nitrite, IL-6, IL-1β, PGE2, LTD4, and LTE4 were measured in the collected media. Total protein isolated from the cells was subjected to western blot analysis for COX-2. The numbers below the blot represent the normalized ratio of the expression levels of COX-2 to those of β-actin for each lane. Equal quantities (30 μg) of total protein were loaded in each lane. β-actin was used as a loading control. * p < 0.05 compared with the control (CTL). † p < 0.05 compared with the LPS treatment. # p < 0.05 compared with the FRGE treatment.
Figure 3
Figure 3
High anti-oxidant and low anti-inflammatory effects of ABGE compared with FRGE. (A) High anti-oxidant effect of ABGE. Pyruvate, not sugar, is responsible for the anti-oxidant effect of ABGE. RAW264.7 cells were incubated with H2DCFDA to evaluate ROS generation. The ROS levels were quantified using fluorescence microscopy after 3 h of treatment with 100 μM pyruvate, 1 μM allicin, 100 μM sugars, 1000 μg/mL garlic extracts, or 1 mM NAC in the H2O2 (100 μM)-treated cells. The scale bar represents 50 μm. * p < 0.05 compared with the control (CTL). † p < 0.05 compared with the H2O2 treatment. # p < 0.05 compared with the ABGE treatment; (B) Reduction of the anti-inflammatory effect of FRGE by pretreatment with sugars. Production of NO and PGE2 was measured in media collected from LPS-stimulated RAW264.7 cells. * p < 0.05 compared with the control (CTL). † p < 0.05 compared with the LPS treatment. # p < 0.05 compared with the FRGE treatment. Each bar represents the mean ± SD of three independent experiments. The plus (+) sign represents conditions with treatment. Pyru, pyruvate; Galac, galactose; Glu, glucose; Fruc, fructose; Suc, sucrose.
Figure 4
Figure 4
Sugars block the anti-inflammatory effect of FRGE in LPS-stimulated RAW264.7 cells through NF-κB activation. (A) Nuclear translocation of NF-κB upon treatment with FRGE and either glucose or fructose. RAW264.7 cells were pretreated with Bay 11-7085, FRGE, and/or glucose or fructose for 12 h and then exposed to LPS for 1 h. Fluorescent cells are labeled with the NF-κB-specific antibody and Cy3-conjugated anti-rabbit IgG. The nucleus was stained with DAPI. The primary antibody was omitted in the negative control (NC). No red fluorescence was observed in the NC; only the DAPI stain was observed (blue color). The scale bar represents 50 μm; (B) FRGE-induced NF-κB suppression was decreased by co-treatment with glucose or sucrose in LPS-stimulated RAW264.7 cells. Total and phosphorylated NF-κB p65 levels were analyzed by ELISA using total cell lysate. Each bar represents the mean ± SD of three independent experiments. The plus (+) sign represents conditions with treatment. * p < 0.05 compared with the control (CTL). † p < 0.05 compared with the LPS treatment; (C) FRGE-induce reduction in the expression of iNOS, COX, and IL-1 mRNA was decreased by treatment with glucose or fructose in LPS-activated RAW264.7 cells. Total cell lysates were obtained using lysis buffer and subjected to RT-PCR. First-strand cDNAs were synthesized from 1 μg of total RNA isolated from the RAW264.7 cells, and the same concentration was used as the template for PCR. GAPDH was used as a loading control for the mRNA expression levels. The numbers below the PCR band represent the normalized ratio of the expression levels of iNOS, COX-2, and IL-1 to those of GAPDH for each lane. Bay, Bay 11-7085; Glu, glucose; Fruc, fructose.
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