Development of Niosome‐Encapsulated Standardized Water Extract of Eurycoma longifolia for Potential Immunomodulatory Activity In Vitro

Eurycoma longifolia is a well‐known medicinal plant recognized for its anticancer activity and immunomodulatory properties. Despite its therapeutic potential, the extract has demonstrated low bioavailability, particularly due to the limited permeability of eurycomanone in rodent studies. The objectives of the present study were to develop a niosome formulation incorporating a standardized water extract of E. longifolia and to evaluate its immunomodulatory activity. Three niosome formulations containing E. longifolia extract with different stabilizers were prepared using the heating method and were abbreviated based on the stabilizing agent used: Nio1, containing sodium dodecyl sulfate (SDS); Nio2, containing α‐tocopherol; and Nio3, containing phenonip. The prepared niosomes were characterized for their physicochemical properties, including particle size, polydispersity index (PDI), zeta potential, pH, stability, entrapment efficiency (EE), and in vitro drug release. All niosome formulations exhibited desirable particle size (195–493 nm), low PDI values (0.05–0.13), and negative zeta potentials (−30.63 to −44.90 mV) with pH values ranging from 7.05 to 7.55. The formulations remained physically stable at room temperature over the 3‐month storage period. EE ranged from 59.50% to 66.90%. In vitro drug release at pH 1.2, 6.8, and 7.4 demonstrated a diffusion‐controlled release mechanism. An in vitro cell viability and an immunomodulatory assay of the niosome on the lipopolysaccharides (LPS)‐induced murine macrophage (RAW264.7) cell line was evaluated. Cytotoxicity data showed that the Nio2 formulation was less toxic towards the Vero, THP‐1, and RAW 264.7 cells with IC 50 values of 19.90, 22.78, and 23.87 μg/mL, respectively. Immunomodulatory assays demonstrated that Nio2 treatment significantly suppressed nitric oxide release in LPS‐stimulated murine macrophages. The Nio2 formulation also enhanced the phagocytic ability of THP‐1 cells towards E. coli , similar to the nonformulated E. longifolia extract. These results indicate that Nio2 could serve as effective nanocarriers for enhancing immunomodulatory activity by activating the THP‐1 cells and potentially benefiting macrophage cells. In conclusion, this study demonstrates that the niosome formulation incorporating a standardized water extract of E. longifolia was successfully developed and exhibits potential immunomodulatory activity, supporting its further evaluation in in vivo immunomodulatory study.