The limitation of current anti inflammatory

The limitation of current anti-inflammatory therapies is widely acknowledged and evident in the continuous efforts in the pharmaceutical industry to develop drugs targeting specific steps in the inflammatory cascade. Natural products have the potential to fill this therapeutic gap addressing the complexity in the inflammatory cascade thereby reducing side effects and compensatory reactions requiring secondary treatment [8]. Orange peel is rich in flavonoids including methylated derivatives such as polymethoxyflavones (PMFs). PMFs have been shown to exhibit strong anti-inflammatory effects both at the level of gene adrenergic antagonist and enzyme activity [9–16]. In addition, induction of apoptosis by PMFs-mediated calcium-signaling may attenuate inflammation [17]. Flavonoids are typically found throughout the whole fruit whereas PMFs are found exclusively in the peels of Citrus genus, particularly in the peels of sweet oranges (Citrus sinensis) and mandarin oranges (Citrus reticulate). As the most abundant PMFs in orange peel extract (OPE), tangeretin and nobiletin have been demonstrated to have strong anti-inflammatory effects as indicated by inhibition of PLA, COX-2, iNOS, TNF-α, 15-LOX, IL-1β, IL-6, and NADPH oxidase in different cell-based and animal models [10,12–16,18,19]. Down-regulation of inflammatory genes by PMFs corresponded to suppression of NFκB, AP-1, and CREB [18]. Noteworthy, strong anti-inflammatory activities were found for 3,5,6,7,8,3′,4′-heptamethoxyflavone [20]. Strong anti-inflammatory activities were found also for OH-PMFs derived from OPE such as 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 3′-demethylnobiletin (3′-dNob), 4′-demethylnobiletin (4′-dNob), and 3′,4′-didemethylnobiletin (3′,4′-dNob) which attenuated iNOS, TNF-α, and COX-2 expression [10,15,21–23]. In view of the growing evidence of anti-inflammatory bioactives in orange peel extracts, we have prepared six different OPEs containing different concentrations of PMFs and OH-PMFs. Effects of different OPEs on cell viability by the MTT-method were evaluated and correlated to different chemical profiles. The nutrigenomic method [24,25] was used as measure for anti-inflammatory bioactivity using a subset of inflammatory surrogate genes in a human monocyte–macrophage differentiation model [8]. The OPE enriched with bioactive polymethoxyflavones showed strong anti-inflammatory effects as demonstrated in a cell-based human in vitro monocyte–macrophage differentiation model and a paw edema in vivo mouse model.
Materials and methods
Results
Discussion In this study, we prepared six different orange peel extracts and quantified major compounds by HPLC. By comparing the different OPEs, we looked for relationships between chemical profiles and cell viability profiles. Our analysis revealed cytoprotective effects of PMFs as indicated by an increase of IC50 values for OPEs with increasing amounts of total PMFs after long-term exposure for 5 d and 24h. On the other hand, OH-PMFs appear to have cytotoxic effects as indicated by a decrease of IC50 values for OPE with increasing concentration of OH-PMFs. OPE-5 containing the highest amount of total OH-PMFs (around 53%) showed the lowest IC50 value after long-term exposures of 24h and 5 d. Since OPEs are complex extracts, we can only speculate whether higher concentrations of OH-PMF are responsible for the differential effects on cell viability. But our results correlate to pro-apoptotic effects of OH-PMFs as observed earlier [17,40]. Despite of potential cytotoxic effects, OH-PMFs are reported to be strong agents against inflammation [10,15,21–23]. To gain insights in possible structure-activity relationships, we compared effects on cell viability by 3,5,6,7,3′,4′-hexamethoxyflavone and 3,5,6,7,8,3′,4′-heptamethoxyflavone. Intriguingly, HeptaMF showed less cytotoxic effects as compared to HexaMF. Previously, it has been reported that HeptaMF have strong anti-inflammatory activity [20]. In the light of HeptaMF, PMFs and OH-PMFs as anti-inflammatory bioactives, we selected OPE-4 for further evaluation of its anti-inflammatory effects since it contains high amounts of cytoprotective HeptaMF (14%), PMFs (52%) and OH-PMFs (21%) reported to have strong anti-inflammatory effects in cell-based in vivo and animal in vivo models [10,12–16,18–23].