H-NMR metabolomics for evaluating the protective effect of Clinacanthus nutans (Burm. f) Lindau water extract against nitric oxide production in LPS-IFN-γ activated RAW 264.7 macrophages.

Introduction: Clinacanthus nutans, a small shrub that is native to Southeast Asia, is commonly used in traditional herbal medicine and as a food source. Its anti-inflammation properties is influenced by the metabolites composition, which can be determined by different binary extraction solvent ratio and extraction methods used during plant post-harvesting stage.

Objective: Evaluate the relationship between the chemical composition of C. nutans and its anti-inflammatory properties using nuclear magnetic resonance (NMR) metabolomics approach.

Methodology: The anti-inflammatory effect of C. nutans air-dried leaves extracted using five different binary extraction solvent ratio and two extraction methods was determined based on their nitric oxide (NO) inhibition effect in lipopolysaccharide-interferon-gamma (LPS-IFN-γ) activated RAW 264.7 macrophages. The relationship between extract bioactivity and metabolite profiles and quantifications were established using H-NMR metabolomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The possible metabolite biosynthesis pathway was constructed to further strengthen the findings.

Results: Water and sonication prepared air-dried leaves possessed the highest NO inhibition activity (IC  = 190.43 ± 12.26 μg/mL, P < 0.05). A total of 56 metabolites were tentatively identified using H-NMR metabolomics. A partial least square (PLS) biplot suggested that sulphur containing glucoside, sulphur containing compounds, phytosterols, triterpenoids, flavones and some organic and amino acids were among the potential NO inhibitors. LC-MS/MS targeted quantification further supported sonicated water extract was among the extract that possessed the most abundant C-glycosyl flavones.

Conclusion: The present study may serve as a preliminary reference for the selection of optimum extract in further C. nutans in vivo anti-inflammatory study.