Protective Role of Phenolic Compounds from Whole Cardamom (Elettaria cardamomum (L.) Maton) against LPS-Induced Inflammation in Colon and Macrophage Cells

Abstract

The chemical profiling of phenolic and terpenoid compounds in whole cardamom, skin, and seeds (Elettaria cardamomum (L.) Maton) showed 11 phenolics and 16 terpenoids, many of which are reported for the first time. Herein, we report the anti-inflammatory properties of a methanolic extract of whole cardamom in colon and macrophage cells stimulated with an inflammatory bacteria lipopolysaccharide (LPS). The results show that cardamom extracts lowered the expression of pro-inflammatory genes NFkβ, TNFα, IL-6, and COX2 in colon cells by reducing reactive oxygen species (ROS) while not affecting LXRα. In macrophages, cardamom extracts lowered the expression of pro-inflammatory genes NFkβ, TNFα, IL-6, and COX2 and decreased NO levels through a reduction in ROS and enhanced gene expression of nuclear receptors LXRα and PPARγ. The cardamom extracts in a range of 200-800 μg/mL did not show toxicity effects in colon or macrophage cells. The whole-cardamom methanolic extracts contained high levels of phenolics compounds (e.g., protocatechuic acid, caffeic acid, syringic acid, and 5-O-caffeoylquinic acid, among others) and are likely responsible for the anti-inflammatory and multifunctional effects observed in this study. The generated information suggests that cardamom may play a protective role against low-grade inflammation that can be the basis of future in vivo studies using mice models of inflammation and associated chronic diseases.

Keywords: Elettaria cardamomum (L.) Maton; cardamom; colon and macrophage cells; inflammation; mode of action; oxidative stress; polyphenols; pro-inflammatory genes and nuclear receptors.

Figure 1

LC chromatograms of methanolic extracts of cardamom seed, cardamom skin, and whole cardamom. Individual peaks of phenolic compounds (peak numbers 1–11 in blue) were identified by LC-MS analysis and are reported in Table 2.

Figure 1

LC chromatograms of methanolic extracts of cardamom seed, cardamom skin, and whole cardamom. Individual peaks of phenolic compounds (peak numbers 1–11 in blue) were identified by LC-MS analysis and are reported in Table 2.

Figure 2

GC-MS analysis of cardamom seed, cardamom skin, and whole cardamom essential oils obtained by steam distillation. Individual peaks of volatile compounds (peak numbers 1–20 in blue) are reported in Table 3.

Figure 2

GC-MS analysis of cardamom seed, cardamom skin, and whole cardamom essential oils obtained by steam distillation. Individual peaks of volatile compounds (peak numbers 1–20 in blue) are reported in Table 3.

Figure 3

Cell viability (MTS test), reactive oxygen species (ROS), and nitric oxide (NO) production in colon and macrophage cells exposed to whole-cardamom methanolic extracts. Cell viability was assayed in colon Caco-2 cells (8  ×  10³ cells/well) and macrophage Raw 264.7 cells (5 ×  10⁴ cells/well) pretreated with 0–1000 µg/mL of whole-cardamom extract for 24 h. ROS production was assayed in Caco-2 cells (8  ×  10³ cells/well) and Raw 264.7 cells (5  ×  10⁴ cells/well), while NO was assayed in Raw 264.7 cells by pretreatment with 0–800 µg/mL of whole-cardamon extract for 5 h and then co-incubated for 19 h with 50 and 1 µg/mL LPS for Caco2 and Raw 264.7, respectively.

Figure 4

Anti-inflammatory properties of whole-cardamom methanolic extract in colon Caco-2 cells. Cardamom extract (200–800 µg/mL) effects on expression of pro-inflammatory genes (NFkβ, TNFα, IL-6, COX-2) and nuclear receptor LXRα were tested in colon cells (0.5 × 10⁶ cells/well) pre-incubated 5 h with cardamom extract and co-incubated afterwards 19 h with 50 µg/mL LPS. Cardamom extracts were dissolved in the growth medium including 0.5% DMSO and used as a control in all experiments. Different letters indicate significant differences among treatments at p < 0.05 by ANOVA and Tukey analysis.

Figure 5

Anti-inflammatory dual mode of action of whole-cardamom methanolic extract in colon cells and macrophages. Bacteria lipopolysaccharide (LPS) binds to receptor TLR4 and triggers inflammation in colon and macrophage cells by activating NOX-enzyme-producing reactive oxygen species (ROS) that activate transcription factor NFk-β, which in turn upregulates pro-inflammatory genes. (A) In colon cells, cardamom extract decreases ROS and attenuates NFk-β, which in turn downregulate the pro-inflammatory genes of cytokines TNF-α and IL-6 and enzyme COX-2. (B) In macrophages, cardamom extracts show a dual mode of action by decreasing ROS and activating nuclear receptors LXR-α and PPARγ, which in turn attenuate NFk-β levels and downregulate the pro-inflammatory genes of cytokines TNF-α and IL-6 and enzymes COX-2 and iNOS, associated to NO production.

Figure 6

Anti-inflammatory properties of cardamom extract in Raw 264.7 macrophage cells. Cardamom extract (200–800 µg/mL) effects on expression of pro-inflammatory genes (NFkβ, TNFα, IL-6, COX-2) and nuclear receptors LXRα and PPARγ were tested in macrophage cells (0.5 × 10⁶ cells/well) pre-incubated 5 h with cardamom extract and co-incubated afterwards 19 h with 1 µg/mL LPS. Cardamom extracts were dissolved in the growth medium including 0.5% DMSO and used as a control in all experiments. Different letters indicate significant differences among treatments at p ≤ 0.05 by ANOVA and Tukey analyses.

Figure 6

Anti-inflammatory properties of cardamom extract in Raw 264.7 macrophage cells. Cardamom extract (200–800 µg/mL) effects on expression of pro-inflammatory genes (NFkβ, TNFα, IL-6, COX-2) and nuclear receptors LXRα and PPARγ were tested in macrophage cells (0.5 × 10⁶ cells/well) pre-incubated 5 h with cardamom extract and co-incubated afterwards 19 h with 1 µg/mL LPS. Cardamom extracts were dissolved in the growth medium including 0.5% DMSO and used as a control in all experiments. Different letters indicate significant differences among treatments at p ≤ 0.05 by ANOVA and Tukey analyses.

Figure 7

Hypothetical model of multifunctional effects of cardamom phenolic bioactives against low-grade inflammation. Cardamom may exert multifunctional effects against inflammation in three different fronts, including a possible prebiotic effect (to be confirmed) as well as by reducing LPS-induced inflammation in the intestinal epithelium due to diet-induced gut microbiota dysbiosis. In addition, cardamom may reduce LPS-induced inflammation in macrophages due to a leaky intestinal epithelium that allows LPS to reach the blood capillaries and induce inflammation in circulating macrophages and possibly initiate a low-grade inflammatory state.