Ethanolic extract of Parkia speciosa pods exhibits antioxidant and anti-inflammatory properties in lipopolysaccharide-induced murine macrophages by inhibiting the p38 MAPK pathway

Abstract

Background: Parkia speciosa (PS) is commonly used in Southeast Asian cuisine and traditional medicine to treat diabetes, hypertension, dermatitis, and kidney diseases. PS has emerged as a subject of interest because of its potential antioxidation and anti-inflammatory properties. However, despite its historically long and wide usage, a comprehensive investigation of these properties in PS pods (PSp) have not been conducted.

Aims of this study: This study aimed to identify the phytochemical compounds in the ethanolic extract of PSp collected from Southern Thailand and assess whether PSp exhibit antioxidant properties and mitigate inflammation in a lipopolysaccharide (LPS)-induced RAW264.7 model.

Materials and methods: The ethanolic extract of PSp was comprehensively analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC/MS) to identify its phytochemical constituents. To assess the antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS) assays were performed, and cytotoxicity was evaluated using the MTT assay. The effect of PSp on reactive nitrogen and oxygen species (RNS and ROS) was determined using a nitric oxide (NO) assay, and its effect on pro-inflammatory cytokines was assessed using enzyme-linked immunosorbent assay (ELISA) and real-time quatitvative polymerase chain reaction (qPCR). Morphological changes following treatment were observed using a microscope. Western blot analysis was performed to quantify MAPK pathway expression.

Results: PSp contain polyphenols, phytosterols, triterpenes, oxaloacetic acid, and unsaturated fatty acids. PSp demonstrated high antioxidant potential in scavenging free radicals and exhibited no cytotoxic effects on macrophages. Moreover, PSp effectively reduced NO release and inhibited pro-inflammatory cytokines such as IL1-β, TNF-α, and IL-6. PSp treatment induced notable morphological changes in macrophages, characterized by an increase in cell size and the presence of intracellular vacuoles. In addition, Western blot analysis showed the selective suppressive effect of PSp on the p38-MAPK pathway.

Conclusion: PSp possess strong antioxidant and anti-inflammatory properties, making it a potential therapeutic agent for the treatment of inflammatory disorders.

Keywords: Anti-Inflammation; Antioxidation; Parkia speciosa pod; Pro-inflammatory cytokine; p38-MAPK.

Graphical abstract

Fig. 1

GC-MS chromatogram of empty PSp extract.

Fig. 2

Antioxidant activities of PSp extract. A) DPPH % radical scavenging activity of PSp extract. B) ABTS % radical scavenging activity of PSp extract. Each value in the graph is expressed as mean ± standard deviation (SD) (n = 3).

Fig. 3

Effect of PSp extract on viability of murine macrophages; RAW264.7 detection was performed using the MTT assay. (A) Cells were treated with extract (0–200 μg/mL) for 24 h. (B) Cells were treated with 0.1 μg/mL LPS and PSp (concentrations ranging 0–200 μg/mL) for 24 h. Values in the graphs are expressed as mean ± SD (n = 3). Statistical significant, denoted as ∗p < 0.05, was assessed among the specified groups.

Fig. 4

Effect of PSp extract on NO production. (A) Murine macrophages were categorized into the negative control (not induced by LPS) and experimental groups treated with 0.1 μg/mL LPS in combination with varying concentrations of PSp extract (0–200 μg/mL). (B) Real-time PCR was performed to detect iNOS expression. Each data point is presented as mean ± SD (n = 3). Statistical significant, denoted as ∗p < 0.05, was assessed among the specified groups.

Fig. 5

Effects of PS on proinflammatory cytokine expression at transcriptional and translational levels. (A–C) ELISA were performed to examine IL1-β, IL-6, and TNF-α concentrations in cell supernatants. (D, E) Real-time PCR was performed to examine expression levels of mRNAs encoding IL1-β, IL-6, and TNF-α. GADPH mRNA expression was used as an internal reference. Each data point is presented as mean ± SD (n = 3). Statistical significant, denoted as ∗p < 0.05, was assessed among the specified groups.

Fig. 6

Effect of PSp extract on the morphology of LPS-induced macrophages. Cells were treated with the designated conditions for 24 h. Cell morphology was observed using phase contrast microscopy (magnification, 40 × ). Scale bar, 100 μm.

Fig. 7

Effect of PSp extract on MAPK induced by LPS. (A) Western blot analysis was performed to assess p-p38, p-JNK, and p-ERK protein levels. Actin was used as an internal reference. Densitometric data represent the means of three independent experiments assessing (B) p-p38, (C) p-JNK, and (D) p-ERK levels. Statistical significant, denoted as ∗p < 0.05, was assessed among the specified groups.