Immunostimulatory activity of complex extracts containing Codonopsis lanceolata radix

The immune system plays a crucial role in protecting the body against pathogens. However, immune function declines with age, making the use of immunomodulators essential for enhancing immune responses. Codonopsis lanceolata Radix (CL), the root of Codonopsis lanceolata (Siebold and Zucc.) Benth. and Hook.f. ex Trautv., contains bioactive compounds such as polyphenols and saponins that exhibit immune activity and have well-documented immunomodulatory effects. However, its efficacy when used alone may be limited, as it may not sufficiently counteract the decline in immune function associated with aging. To overcome this limitation, combining CL with other herbal medicines is essential to enhance its immunostimulatory effects through synergistic interactions and by targeting additional immune pathways. Therefore, this study aimed to prepare six herbal medicine mixtures (CDGLS) by combining CL with Dioscoreae Rhizoma, Glycine Semen Nigra, Lycii Fructus, and Schisandrae Fructus in various ratios to identify the most effective mixture in terms of immunostimulatory effects. The immunostimulatory activity of the six mixtures was evaluated in interferon-gamma (IFN-γ)-primed RAW 264.7 macrophages, and statistical significance was assessed using one-way analysis of variance followed by Dunnett's post-hoc test (p < 0.05), with IFN-γ-primed RAW 264.7 macrophages used as the control group. The antioxidant activities of six CDGLS candidates were evaluated through DPPH and superoxide radical scavenging assays, demonstrating higher activity than CL alone. To measure nitric oxide (NO) production by individual herbal medicines and the six CDGLS candidates, treatments were applied at concentrations of 125, 250, and 500 μg/ml, which were selected based on cell viability assays to ensure non-cytotoxic conditions. Among these candidates, CDGLS-1 demonstrated significantly higher NO production at all tested concentrations (p < 0.001), leading to its selection as the final mixing ratio. The production and mRNA expression of cytokines, including tumor necrosis factor-α, interleukin (IL)-6, and IL-1β, were measured through quantitative real-time PCR and enzyme immunoassay, and all showed significantly increased levels after CDGLS-1 treatment (p < 0.01 to p < 0.001). Western blot analysis showed that CDGLS-1 activated the phosphorylation of inhibitory κB-α, promoted the nuclear translocation of nuclear factor-κB p65, and increased the expression of inducible NO synthase and cyclooxygenase-2, with statistical significance ranging from p < 0.05 to p < 0.001. These results suggest that CDGLS-1 has potential as an immunostimulatory agent for use in dietary supplements or adjunct therapies. However, further studies, including in vivo studies, are needed to validate its efficacy and clarify its mechanism of action.