Potential of Coffee Cherry Pulp Extract against Polycyclic Aromatic Hydrocarbons in Air Pollution Induced Inflammation and Oxidative Stress for Topical Applications

Abstract

Airborne particulate matter (PM) contains polycyclic aromatic hydrocarbons (PAHs) as primary toxic components, causing oxidative damage and being associated with various inflammatory skin pathologies such as premature aging, atopic dermatitis, and psoriasis. Coffee cherry pulp (CCS) extract, rich in chlorogenic acid, caffeine, and theophylline, has demonstrated strong antioxidant properties. However, its specific anti-inflammatory effects and ability to protect macrophages against PAH-induced inflammation remain unexplored. Thus, this study aimed to evaluate the anti-inflammatory properties of CCS extract on RAW 264.7 macrophage cells exposed to atmospheric PAHs, compared to chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP) standards. The CCS extract was assessed for its impact on the production of nitric oxide (NO) and expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Results showed that CCS extract exhibited significant antioxidant activities and effectively inhibited protease and lipoxygenase (LOX) activities. The PAH induced the increase in intracellular reactive oxygen species, NO, TNF-α, IL-6, iNOS, and COX-2, which were markedly suppressed by CCS extract in a dose-dependent manner, comparable to the effects of chlorogenic acid, caffeine, and theophylline. In conclusion, CCS extract inhibits PAH-induced inflammation by reducing pro-inflammatory cytokines and reactive oxygen species (ROS) production in RAW 264.7 cells. This effect is likely due to the synergistic effects of its bioactive compounds. Chlorogenic acid showed strong antioxidant and anti-inflammatory activities, while caffeine and theophylline enhanced anti-inflammatory activity. CCS extract did not irritate the hen's egg chorioallantoic membrane. Therefore, CCS extract shows its potential as a promising cosmeceutical ingredient for safely alleviating inflammatory skin diseases caused by air pollution.

Keywords: anti-inflammation; caffeine; chlorogenic acid; coffee cherry pulp; cyclooxygenase-2; inducible nitric oxide synthase; interleukin-6; pollution; polycyclic aromatic hydrocarbons; theophylline; tumor necrosis factor-alpha.

Figure 1

The ability of coffee cherry pulp (CCS) extract to inhibit (a) lipoxygenases (LOX) and (b) protease enzymes compared to a positive control (diclofenac sodium) and standard compounds chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP), all tested at the same concentration of 1000 µg/mL. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–d) are not significantly different, with a significance level of p < 0.05.

Figure 2

Effects of PAHs on the viability of RAW 264.7 cells using the MTT assay; the bars represent the percent cell viability of the cells treated with different concentrations of the PAHs (6.3–100 µg/mL) for 24 h. Data are expressed as mean ± S.D. (n = 3). An asterisk (*) indicates a significant difference compared to the untreated cells (0 µg/mL) at p < 0.05.

Figure 3

Effects of (a) coffee cherry pulp (CCS) extract, (b) chlorogenic acid (CGA), (c) caffeine (CAF), and (d) theophylline (THP) on the viability of RAW 264.7 cells using the MTT assay; the bars represent the percent cell viability of the cells treated with tested compounds in different concentrations (62.5–1000 µg/mL) then exposed to 12.5 µg/mL of PAHs for 24 h. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–e) are not significantly different, with a significance level of p < 0.05.

Figure 4

(a) Effect of coffee cherry pulp (CCS) extract, chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP) at the same concentration of 100 μg/mL on ROS production in RAW 264.7 cells exposed to PAHs. The cells were pre-treated with tested compounds and PAHs (12.5 μg/mL) for 2 h to determine ROS production. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–c) are not significantly different, with a significance level of p < 0.05. (b) Histograms of the RAW 264.7 cell counts versus fluorescence intensity are shown with a mark to define fluorescing cells of the tested compounds (100 μg/mL) alone or in combination with PAHs.

Figure 5

Effects of PAHs on NO production in RAW 264.7 cells; the bars represent the nitric oxide production of the cells treated with different concentrations of the PAHs (1.6–12.5 µg/mL) for 24 h. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–d) are not significantly different, with a significance level of p < 0.05.

Figure 6

Inhibitory effects of coffee cherry pulp (CCS) extract, chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP) on PAH-induced production of nitric oxide. RAW 264.7 cells were pre-treated with various concentrations (10, 50, and 100 μg/mL) of the tested compounds for 2 h and then incubated with PAHs (12.5 μg/mL) for 22 h. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–g) are not significantly different, with a significance level of p < 0.05.

Figure 7

Effects of PAHs on TNF-α and IL-6 secretion in RAW 264.7 cells; the bars represent (a) the TNF-α and (b) IL-6 production of the cells treated with different concentrations of the PAHs (1.6–12.5 µg/mL) for 24 h. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–e) are not significantly different, with a significance level of p < 0.05.

Figure 8

Inhibitory effects of coffee cherry pulp (CCS) extract, chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP) on PAH-induced production of (a) TNF-α and (b) IL-6 in RAW 264.7 cells at 24 h. The cells were pre-treated with various concentrations (10, 50 and 100 μg/mL) of the tested compounds for 2 h and then incubated with PAHs (12.5 μg/mL) for 22 h. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–g) are not significantly different, with a significance level of p < 0.05.

Figure 9

Inhibitory effects of coffee cherry pulp (CCS) extract, chlorogenic acid (CGA), caffeine (CAF), and theophylline (THP) on PAH-induced mRNA expression of (a) COX-2 and (b) iNOS. RAW 264.7 cells were pre-treated with various concentrations (10, 50, and 100 μg/mL) of the tested compounds for 2 h and then incubated with PAHs (12.5 μg/mL) for 22 h. Data are presented as the mean ± S.D. (n = 3). Tukey’s HSD test was conducted to compare all group means. Groups sharing the same letters (a–d) are not significantly different, with a significance level of p < 0.05.

Figure 10

CAM was exposed to a positive control (1% w/v SLS), a negative control (0.9% w/v NaCl), and coffee cherry pulp (CCS) extract (10 mg/mL) for 5 and 60 min, respectively.