AH is a wild herb cultivated in India, Myanmar, Nepal, and China. AH is mainly used as food supplement and medicinal food. Since, AH is introduced in South Korea, the beneficial effects of AH has been extensively investigated including, anti-oxidant [1416], anti-inflammatory [17], anti-microbial [18], anti-obesity [20] and anti-diabetic [2123]. Its beneficial effect is attributed to the sulfur compounds, phenolic compounds, phytosterols and vitamin C of AH [11]. Allicin and ten alkyl thiosulfinates from AH root extract were characterized by HPLC-ESI-MS [26]. Non-volatile organosulfur compounds, methiin and cycloalliin were detected as major compounds and volatile compounds such as allyl methyl sulphides and dimethyl sulphides were identified from AH [27]. Some of chemicals in AH have showed biological activities. Ferulic acid-4-O-?-D-glucopyranoside isolated from AH root has antioxidant activity [14]. The hot-water extract of AH containing alliin, sinapic acid, and ferulic acid exhibited beneficial effects on bone health [19].

We previously demonstrated that water extracts of AH root ameliorated oxidative stress-induced inflammatory responses and ?-cell damage in pancreas of STZ-induced diabetic rats [23]. In addition, supplement of AH root lowered blood glucose and increased insulin immunoreactives cells in the type 2 diabetic db/db mice [21]. In this study, we found that AHE effectively suppressed LPS-induced inflammation. AHE treatment inhibited increased NO, ROS, proinflammatory cytokines in RAW264.7 cells stimulated with LPS. AHE also significantly decreased the expression of iNOS and COX-2 through inhibiting NF-?B activation.

Macrophage activation by LPS, a component of the outer membrane of Gram-negative bacteria, promotes the synthesis and release of large amounts of mediators involved in the inflammatory onset such as cytokines, NO, pro-inflammatory enzymes and ROS [28]. Accumulating evidence has indicated that NO is well known for its involvement in the development of inflammation. NO has important functions as signaling molecules in diverse physiological systems, such as cardiovascular, nervous and immunological systems [29]. High concentration of NO synthesized by iNOS, can mediate inflammation and cause cell death by inducing apoptosis [30]. Therefore, identifying new agents capable of lowering the production of this proinflammatory agent is regarded as an essential requirement for the alleviation of a number of inflammation-related disorders attributed to macrophage activation [10]. ROS have also been reported to be involved in the activation of NF-?B by pro-inflammatory cytokines such as TNF-? [31]. NF-?B has been reported to play a pivotal role in inflammatory response through the induction of inflammation-related cytokines (i.e. IL-6, IL-1?, TNF-?) and enzymes such as COX-2 and iNOS [32, 33]. In this study, we demonstrate that AHE treatment significantly inhibited NO production and iNOS expression at a concentration 100–300 ?g/mL in a dose-dependent manner in RAW264.7 cells. In particular, the profound inhibition of LPS-induced NO and ROS was observed at a concentration of 100 ?g/mL of AHE. In addition, our previous study also demonstrated that water extract of AH root (100 mg/kg body weight) significantly inhibited ROS production and protein expression of cytokines in the pancreas of STZ-induced diabetic rats [23].

In this study, the anti-inflammatory effect of AHE may be mediated by its inhibitory effect on the pro-inflammatory cytokines, including IL-6 and TNF-?, as well as the hallmarks of inflammation, NO, and activation of NF-?B at least in part. NF-?B is composed mainly of two proteins, p50 and p65. In resting cells, the NF-?B heterodimer is held in the cytosol through interaction with I?B inhibitory proteins [34, 35]. According to the pro-inflammatory stimuli, I?B becomes phosphorylated, ubiquitinated, and then degraded. Thus, the liberated NF-?B dimers are translocated to nucleus, where the transcription of target gene is induced [10]. We observed that AHE decreased the LPS-induced nuclear accumulation of the p65 subunit of NF-kB, but not that of I?B?, in LPS-activated RAW264.7 macrophages. Other studies have showed that the inhibitory effects of sulfur-containing compounds from garlic towards LPS-activated NF-?B dependent pathway [36]. The major water-soluble sulfur compound, SAC, in garlic extract seems to have direct inhibitory effect on NF-?B and indirect inhibitory effect on LPS-induced IL-1 and TNF-? in human whole blood [36]. The lipid-soluble sulfur compounds allicin and diallyl disulfide (DADS) also inhibit NF-?B and reduce the expression of iNOS in LPS-stimulated macrophages [36]. Dially trisulfide (DATS), an organic polysulfide compound found in garlic, attenuates the initiation of LPS-mediated intracellular signaling cascades by suppressing activation of NF-?B and by inhibiting binding of LPS to toll like receptor4 on macrophages [37]. DADS also reduced the airway inflammation via regulation of nuclear factor E2-related factor 2/heme oxygenase (HO)-1 and NF-?B [38]. Accumulated data demonstrated that the NF-?B pathway mediates the expression of iNOS, COX-2 and various pro-inflammatory cytokines [35]. DATS effectively suppressed phosphorylation and the degradation of I?B in RAW264.7 cells, and downregulated serine/threonine protein kinase B/transforming growth factors-?-activated kinase-mediated mitogen-activated protein kinase (MAPK) and NF-?B signaling pathways [39]. Several lines of study have demonstrated that DADS suppressed LPS-induced MAPKs signaling to attenuate inflammation responses [40]. Ethanol extracts of AH root exerts anti-inflammatory through up-regulation of HO-1 and deactivation of p38 in the LPS-stimulated RAW264.7 cells [17]. In agreement with our study, Kim et al. reported similar results. The anti-inflammatory activities of aged black garlic water extract were similar to those of raw garlic extract at nontoxic concentrations up to 250 ?g/ml. Both garlic extracts inhibited cytokine production and activation of MAPK and NF-?B signal transduction pathway induced by LPS stimulated RAW264.7 cells and LPS-induced lethal shock in mice [41]. The findings from our study reveal, for the first time, a protective effect of AHE in the LPS-induced RAW264.7 cells. This effect, in part, may have been contributed by the anti-inflammatory effects of sulfur compounds in AHE. Therefore, we believe that AHE could potentially be used in the treatment of inflammation related diseases.