Immunostimulant activity of Centella asiatica (ECa 233) on RAW 264.7 macrophages

The innate immune system serves as the body’s first line of defense, with macrophages playing a central role in pathogen recognition and elimination. Although Centella asiatica has long been used in traditional medicine, its immunostimulatory effects are not yet fully elucidated. This study investigated the immunostimulant activity of a standardized C. asiatica extract (ECa 233) on RAW 264.7 macrophages. ECa 233 was first characterized using FT-IR and NMR spectroscopy. FT-IR identified key functional groups, while NMR provided detailed insights into molecular structure, together ensuring precise extract characterization for subsequent pharmacological evaluation. RAW 264.7 cells were treated with ECa 233 at concentrations of 1.5625–200 μg/mL. The production of nitric oxide (NO), hydrogen peroxide (H2O2), and reactive oxygen species (ROS) was assessed, along with macrophage functional activity using zymosan-NBT reduction and neutral red uptake assays. Inflammatory gene expression was analyzed via Reverse transcriptase polymerase chain reaction (RT-PCR). ECa 233 significantly increased NO, H2O2, and ROS production without compromising cell viability and enhanced macrophage functional activity. RT-PCR results revealed upregulation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), regulatory cytokines (TGF-β, IL-10, IL-12), surface markers (CXCR-1, CXCR-2), and inflammatory mediators (iNOS, COX-2). These findings suggest that ECa 233 activates macrophages and promotes immune responses, supporting its potential as an immunostimulant. Further in vivo studies are warranted to confirm its efficacy and therapeutic relevance.