Anti-Oxidation and Anti-Inflammatory Potency Evaluation of Ferulic Acid Derivatives Obtained through Virtual Screening

Abstract

Various factors such as ultraviolet rays can cause a continuous threat to our skin, resulting in inflammation or oxidation problems. Ferulic acid (FA), with certain antioxidant and anti-inflammatory properties, is widely used in many cosmetics, even used to treat various diseases in the clinic. In this study, the FA structural skeleton was used to search for FA derivatives. Then, molecular docking, the rule of five, and Veber rules were performed to virtually screen compounds that can bind to proteins with a good drug likeness. DPPH and ABTS were used to evaluate their antioxidant potency and an MTT assay was employed to investigate the toxicities of the compounds, while Griess Reaction System and ELISA were used to judge the concentration variations of NO and different inflammatory factors (TNF-α, IL-1β, and IL-6). Western blotting featured nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression levels. The trend of the intracellular changes of reactive oxygen species (ROS) was detected by the DCFH-DA method and fluorescence staining. As a result, we found that the ferulic acid derivative S-52372 not only had certain scavenging effects on free radicals in biochemical experiments, but also prevented inflammation and oxidative stress in LPS-stimulated RAW264.7 cells in the cellular environment; intracellular ROS and inflammatory mediators, including iNOS, COX-2, TNF-α, and IL-6, were also suppressed. In a computer prediction, S-52372 owned better water solubility and lower toxicity than FA. This compound deserves further research to find an ideal FA derivative.

Keywords: anti-inflammation; antioxidant; ferulic acid; free radical scavenging; virtual screening.

Figure 1

The whole process in this study.

Figure 2

The docking results of FA with the active site of 2I3Y and the determined skeleton structure (green means Pi–Pi stacking, pink means H-bond, and the transition color means salt bridge).

Figure 3

A floor plan view of the docking results, including (A) S-11593, (B) S-25207, (C) S-69904, and (D) S-52372.

Figure 4

The graph of free radical scavenging ability. (A) The DPPH scavenging abilities of FA and its skeleton derivative, S-52372. (B) The ABTS scavenging ability of FA and its skeleton derivative S-52372. * p < 0.05 means FA compared with corresponding concentration of S-52372 group.

Figure 5

Cytotoxicity of ferulic acid and its derivative, S-52372, on RAW264.7 cells. RAW264.7 cells were treated with ferulic acid and S-52372 for 24 h, and an MTT assay was carried to detect the cell viability. (A) Cytotoxicity of ferulic acid on RAW264.7 cells. (B) Cytotoxicity of S-52372 on RAW264.7 cells. Each bar illustrates the average ± standard deviation (SD) counted from three experiments.

Figure 6

Inhibition of the FA and S-52372 on LPS-induced nitric oxide (NO) in RAW 264.7 macrophages. Each bar illustrates the average ± standard deviation (SD) counted from three experiments. * p < 0.05 compared to the only LPS−treated group; ^# p < 0.05 compared to the control group.

Figure 7

Effects of FA and S-52372 on TNF-α, IL-1β, and IL-6 induced by LPS. In addition to an untreated control sample, RAW264.7 cells were pretreated with 1.6 μmol/L of FA as a positive control to compare to RAW264.7 cells pretreated with with S-52372 at 1.6, 0.32, and 0.064 µM for 1.5 h, and then treated all groups with 1 µg/mL of LPS, except the control group, at 37 °C for 24 h. The levels of (A) TNF-α, (B) IL-1β, and (C) IL-6 in the culture supernatants of RAW264.7 macrophages were then detected by ELISA. Each bar represents the mean ± standard deviation (SD) calculated from three independent experiments. * p < 0.05 compared to only LPS−treated group; ^# p < 0.05 compared to the control group.

Figure 8

Ferulic acid and S-52372 decreased LPS−induced protein expressions of iNOS and COX-2. The protein expression of iNOS and COX-2 was detected by Western blot analysis. (A) iNOS and COX-2 protein expression levels. (B) Relative ration analysis. In addition to an untreated control sample, RAW264.7 cells were pretreated with 1.6 μmol/L of FA as a positive control to compare with RAW264.7 cells pretreated with with S-52372 at 1.6, 0.32, and 0.064 µM for 1.5 h, and then all groups were treated with 1 µg/mL of LPS, except the control group, at 37 °C for 24 h. Each bar represents the mean ± standard deviation (SD) calculated from three independent experiments. * p < 0.05 compared to the only LPS−treated group; ^# p < 0.05 compared to the control group; ^& p < 0.05 compared to the FA group.

Figure 9

Ferulic acid and its derivative, S-52372, inhibited LPS−induced intracellular ROS production. In addition to an untreated control sample, RAW264.7 cells were pretreated with 1.6 μmol/L of FA as a positive control to compare with RAW264.7 cells pretreated with with S-52372 at 1.6, 0.32, and 0.064 µM for 1.5 h, and then all groups were treated with 1 µg/mL of LPS, except the control group, at 37 °C for 24 h. (A) ROS fluorescence images of FA (1.6 µM) and S-52372 (1.6, 0.32, and 0.064 µM). (B) ROS production at 0.064 µM of S-52372 to compare with FA at 1.6 µM. (C) ROS production at 0.32 µM of S-52372 to compare with FA at 1.6 µM. (D) ROS production at 1.6 µM of S-52372 to compare with FA at 1.6 µM. The results were presented in the above superposition of the fluorescence spectra of all groups. (E) Relative fluorescence intensity analysis. * p < 0.05 compared to the only LPS-treated group; ^# p < 0.05 compared to the control group.