Commiphora leptophloeos leaf and bark extracts modulate OxInflammation through TLR4/ NF-κB/ Nrf2 pathways

Abstract

Ethnopharmacological relevance: Commiphora leptophloeos (Imburana or Umburuna) represents a significant botanical resource within the Burseraceae family. Indigenous populations across Brazil have historically utilized this medicinal plant for addressing various pathological conditions, including respiratory diseases, gastrointestinal disorders, ulcerative lesions, and diverse inflammatory manifestations. Despite its ethnomedicinal significance, comprehensive investigations into the molecular mechanisms governing its anti-inflammatory properties remain inadequately explored, limiting our understanding of its potential therapeutic uses.

Aim of the study: This research evaluated the capacity of leaf and bark extracts derived from C. leptophloeos to modulate oxidative stress-induced inflammation (OxInflammation) in vitro, while also investigating the molecular signalling cascades that mediate these effects.

Materials and methods: Phytochemical profiling of C. leptophloeos leaf and bark extracts was performed using flow injection analysis coupled with electrospray ionization ion trap tandem mass spectrometry (FIA-ESI-IT-MS/MS). Total phenolic and total flavonoid contents were determined through the Folin-Ciocalteu methodology, and the aluminum chloride colorimetric technique, respectively. Antioxidant capacity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The anti-inflammatory potential was assessed through multiple complementary methodologies, including: (i) inhibition of protein denaturation via bovine serum albumin (BSA) assay; (ii) erythrocyte membrane stabilization against hypotonicity-induced lysis; and (iii) trypsin proteolytic activity inhibition. Cellular viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Catalase (CAT) enzymatic activity was measured following hydrogen peroxide (H₂O₂) exposure. Nitric oxide (NO) production in RAW 264.7 macrophages was quantified using the Griess reaction. Cyclooxygenase (COX) activity was assessed via commercial ELISA methodology. Transcriptional regulation of pro-inflammatory markers (TLR4, NF-κB, BAX, COX-2, TNF-α, IL-6) and anti-inflammatory/antioxidant mediators (Nrf2, HO-1, HIF-1, IL-10) was quantified through real-time quantitative polymerase chain reaction (RT-qPCR).

Results: Chromatographic analysis of C. leptophloeos leaf extracts (HECL-LE) revealed a complex phytochemical profile comprising one phenolic acid and fourteen glycosylated flavonoids derived from apigenin, luteolin, and quercetin. Conversely, bark extracts (HECL-BA) exhibited a distinctive chemical signature characterized by seven major constituents, including quinic acid and six oligomeric B-type procyanidins. Both extracts demonstrated strong antioxidant properties, inhibiting more than 80 % of DPPH radicals. Additionally, neither extract exhibited hemolytic activity while providing approximately 20 % protection against protein denaturation. Bark extracts enhanced cellular viability and migration capacity, whereas leaf extracts enhanced catalase enzymatic activity. Both extracts exhibited significant anti-inflammatory properties, as evidenced by reduced nitric oxide production and downregulation of key inflammatory mediators including TLR4, NF-κB, IL-6, TNF-α, BAX, and COX-2. Notably, the extracts upregulated Nrf2 expression, a critical transcription factor orchestrating cellular antioxidant responses and oxidative stress regulation.

Conclusions: The leaf extracts of C. leptophloeos, characterized by their rich flavonoid content-including two novel compounds (luteolin-O-feruloyl-O-hexose and apigenin-8-C-hexose-6-O-4-hydroxybenzoic acid)-and bark extracts containing oligomeric proanthocyanidins and phenolic compounds, demonstrated substantial antioxidant and anti-inflammatory properties with minimal cytotoxicity. The primary molecular mechanisms underlying OxInflammation modulation involve attenuation of the TLR4 and NF-κB pathways, decreased levels of pro-inflammatory mediators (BAX, IL-6, TNF-α, COX-2), and reduction of nitric oxide production. Notably, Nrf2 expression was significantly upregulated, particularly in response to bark extract treatment, underscoring its key contribution to oxidative stress modulation and facilitating the resolution of acute inflammation. These results collectively demonstrate the pharmacological efficacy of C. leptophloeos extracts in managing inflammatory conditions linked to oxidative stress, thereby providing a scientific basis for their ethnomedicinal applications.

Keywords: Antioxidant; Commiphora leptophloeos; Flavonoids; Inflammation; OxInflammation; Proanthocyanidins.