This study demonstrates that Echinacea alcohol extracts in vitro inhibit LPS-induced generation of NO and TNF-α in macrophages. Although NO and TNF-α are needed for clearing and containing bacterial infection, excessive NO and TNF-α are implicated in a pathological role in inflammatory responses. Such an inhibitory property may endow Echinacea alcohol extracts as an effective anti-inflammatory remedy. In the present study, we found evidence that Echinacea can decrease inflammatory mediators in vitro, but that the effect in vivo was observed only in comparison with the vehicle control group, which exhibited an excessive production of NO during Salmonella infection. Interestingly enough the excessive NO production and suppressed NO production did not correlate with any biological effects of bacterial killing in the PECs.
Echinacea alcohol extracts consist of both hydrophilic and lipophilic fractions. There is evidence to believe that polar caffeic acid derivatives have anti-inflammatory activities (Speroni et al. 2002
). There are several types of caffeic acid derivatives in Echinacea (). Echinacoside was predominately present in EPA and EA, whereas cichoric acid was the principal caffeic acid derivative of EP. In recent years, much attention has been paid to the antioxidant activities and free radical scavenging abilities of Echinacea-derived caffeic acid derivatives (Facino et al. 1995
; Hu & Kitts 2000
; Pellati et al. 2004
; Dalby-Brown et al. 2005
). The alkamide mixture of Echinacea alone shows weak or no antioxidant activity, however, the alkamides have been found to significantly increase the antioxidant activity of the caffeic acid derivatives, possibly due to the lipophilic nature of the alkamides (Dalby-Brown et al. 2005
). Moreover, individual alkamides and mixtures of alkamides have been reported to have anti-inflammatory effect by inhibiting NO production in LPS-stimulated macrophages in vitro
(Chen et al. 2005
). The observed inhibition of inflammatory mediators by Echinacea alcohol extracts in vitro
in the present study might be due to the additive or synergistic action of hydrophilic fractions (caffeic acid derivatives) and lipophilic fractions (alkamides).
The NO and TNF-α inhibitory potential of the three alcohol extracts in vitro is generally EPA > EP > EA. The basis of this difference in the modulation of NO and TNF-α production may rest on the distinct amounts and types of phytochemicals between the three herbal preparations. HPLC data showed that EPA and EP had higher levels of echinacoside or cichoric acid, respectively, and they share high levels of some individual alkamides, such as alkamide 2 that occurs in relatively small amount in EA. These herbal components may be quantitatively important as modulators of NO and TNF-α production. Further studies are needed to address the respective role of individual phytochemicals and their interaction in the modulation of inflammatory mediators.
NO generated by LPS-activated macrophages is a strong inducer of cell damage and apoptosis (Brune et al. 1997
; Chang et al. 2000
). The downregulation of NO production by Echinacea alcohol extracts possibly protects macrophages from NO damage. Since production of TNF-α and NO by macrophages are precisely controlled by several ubiquitous transcription factors including NF-κB (Baeuerle & Baltimore 1996
), Echinacea-mediated reduction of both inflammatory mediators probably results from the perturbance of a common upstream signaling pathway.
Interestingly, Sharma et al. (2006)
recently reported that an alcohol tincture from EP roots increased the nuclear expression of multiple pro-inflammatory transcription factors (e.g. NF-κB and STATs) in non-activated human bronchial epithelial cell line BEAS-2B, but inhibited the expression of these transcription factors when the cells were infected with rhinovirus, thus providing strong mechanistic evidence to explain the observed phenomena in this study; that is, Echinacea alcohol extracts have different influences on the non-activated and activated macrophages, and the effects of Echinacea on the inflammatory mediators are associated with the modulation of transcription factor expression.
The opposite effects of Echinacea alcohol extracts on the non-activated and activated macrophages were also seen in cell numbers with the use of the MTS assay (). In comparison to the vehicle, Echinacea resulted in decreased cell number in the presence of LPS. While macrophages are in a resting state, Echinacea stimulated, to some extent, an increase in cell number. Echinacea alcohol extracts might stimulate the non-activated macrophages (e.g. increase cell number and/or function), but reduce macrophage proliferation when they are activated.
Macrophages constitute one of the first lines of host defense against microbial infections based on their abilities to produce NO and reactive oxygen species. NO is believed to represent an important effector molecule in the killing of a variety of pathogens including Salmonella
(MacMicking et al. 1997
; Burgner et al. 1999
; Alam et al. 2002
; Babu et al. 2006
). Since Echinacea preparations, mainly from fresh-pressed juice or the high molecular weight polysaccharide fraction, have been shown to upregulate production and secretion of proinflammatory cytokines and oxygen radicals, consistent with an immune activated antimicrobial effect (Stimpel et al. 1984
; Steinmuller et al. 1993
; Burger et al. 1997
), it is interesting to know the possible consequence upon inhibition of NO production by Echinacea alcohol extracts. We attempted to investigate and correlate the phagocytic activity and bacterial killing of macrophages with NO production following exposure to Echinacea alcohol extracts. Although EA enhanced phagocytic activity, treatment with EA or EP clearly showed an inhibitory effect on intracellular bacterial killing. This inhibition may be a secondary effect of the downregulation of NO production. Macrophages infected with bacteria produce high levels of NO that can be blocked by alcohol extracts of Echinacea, especially at high concentrations. Nonetheless, EPA at 200 μg/ml simultaneously increased the potential of bacterial killing but inhibited NO production by macrophages. A reasonable explanation is that macrophages may depend on multiple mediators (i.e. reactive oxygen intermediates (Cherayil & Antos 2001
)), not just NO, to provide them with Salmonella
RAW 264.7 cells closely resemble the murine peritoneal macrophages in their response to inflammatory stimuli and pathogenic microbes. We expected that Echinacea alcohol extracts could exert a similar, although not identical, effect on PECs as they did on the macrophage-like RAW 264.7 cells. Unfortunately, a weak in vivo
effect on inflammatory PECs was seen. The only notable change in PECs was an EP-mediated reduction in NO production at the 4-h time point after Salmonella
engulfment. Interestingly, the reduction in NO production did not result in a significant decrease in bacterial killing. The in vivo
effect in this regard might be largely influenced by several factors, such as the metabolism of the phytochemicals, the activation state of PECs and the complex in vivo
environment (e.g. constitutive expression of cytokines, growth factors and hormones (Pruett et al. 2005
)). Although inflammatory agents, such as proteose peptone and thioglycollate, are commonly applied to induce an inflammatory response resulting in an influx of strongly activated macrophages into the peritoneum and thus an increased peritoneal macrophage yield, it is unclear as to how and to what extent these inflammatory agents affect the functional state of macrophages (Fortier & Falk 1994
). On the other hand, lack of an inhibitory effect of orally administered Echinacea alcohol extracts on bacterial phagocytosis and killing by PECs may indicate that Echinacea will not adversely affect these important innate immune functions.
To summarize, Echinacea alcohol extracts have a potential anti-inflammatory activity, but this effect was mainly observed in vitro, especially at relatively high concentrations of the extracts. Increased knowledge of the biological properties and the mode of action of the physiologically relevant concentrations of Echinacea alcohol extracts are necessary.