A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX

Abstract

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Figure 1

3D representation of the binding of N-feruloylserotonin to the chemokine CCL22.

Figure 2

Chromatographic profile of a typical cornflower extract obtained from raw material after cold pressure in UV at 313 nm (concentration 0.5 mg/mL).

Figure 3

Molecular structure of N-feruloylserotonin (C₂₀H₂₀N₂O₄; MW = 352.4 g/mol).

Figure 4

Molecular structure of N-p-coumaroylserotonin (C₁₉H₁₈N₂O₃; MW = 322.13 g/mol).

Figure 5

Molecular structure of p-coumaroylquinic acid (C₁₆H₁₈O₈; MW = 338.31 g/mol).

Figure 6

Effect of the pure cornflower extract (CFE, 1 mg/mL) on CCL5, MMP-2, and IL-8 expression after inflammation induction by CCL17. Relative gene expression was calculated using the delta CT method after normalization to ribosomal 18S expression. Experiment was performed in triplicate. One-way ANOVA: ^∗∗∗∗p < 0.0001; ^∗∗∗p < 0.005.

Figure 7

Effect of the pure cornflower extract (CFE, 1 mg/mL) on MMP-2, IL-8, and CCL26 expression after inflammation induction with CCL22. Relative gene expression was calculated using the delta-delta CT method after normalization to ribosomal 18S expression. Experiment was performed in triplicate. One-way ANOVA: ns: nonsignificant; ^∗p < 0.05; ^∗∗∗p < 0.005.

Figure 8

(a) Human COX-2 (PDB 5KIR) with docked N-feruloylserotonin in the active site shown as green surface. The heme group is depicted as red stick model. The three-pocket entry-restricting residues Arg120, Glu524, and Tyr355 surround the docked ligand as stick models. (b) Binding hypothesis of N-feruloylserotonin to COX-2 (docking score -11.41 kcal/mol). Protein-ligand interactions are indicated as blue dashed lines for aromatic interactions and yellow dashed lines for hydrogen bonds. Tyr385 has been shown to be actively involved in electron transfer between natural substrate arachidonic acid and heme [60].

Figure 9

COX-2 enzymatic inhibition: endogenous COX-2 enzymatic activity in THP-1 cells differentiated in macrophages (PMA). Macrophages pretreated with CFEM or N-FS (N-feruloylserotonin) for 1 hour. COX-2 induction (LPS) with the test substance for 24 hours. One-way ANOVA: ^∗∗∗∗p < 0.0001.

Figure 10

5-LOX enzymatic inhibition: endogenous 5-LOX enzymatic activity in THP-1 cells differentiated in macrophages (PMA). Macrophages pretreated with CFEM or N-FS (N-feruloylserotonin) for 1 hour. 5-LOX induction (IL-3) with the test substance for 24 hours. One-way ANOVA: ^∗∗∗p < 0.005; ^∗∗∗∗p < 0.0001.

Figure 11

Mode of action of the cornflower extract (in red). The biological activities of N-feruloylserotonin are highlighted in red boxes.