Synergistic effect of garlic and andrographis extracts on digestive enzyme activity, immune modulation, and anti- inflammation in nile tilapia (Oreochromis niloticus)

• This study was designed to evaluate the biological effects of Allum sativum (GE), Andrographis paniculata (AE), and a combined 1:1 formulation (GAF) on digestive enzyme activities and immune responses in Nile tilapia at organismal level. • The effects of these extracts were assessed in LPS-induced RAW 264.7 cells by analyzing cell viability and inflammatory cytokine gene expression, to provide mechanism of action on immune response at cellular levels. • The GAF formulation exhibited a well-balanced effect on both the digestive and immune systems. This research investigated the effects and their mechanisms of garlic extract (GE), Andrographis extract (AE), and their combined formulation (GAF) on digestion, immune response, and inflammation in Nile tilapia (Oreochromis niloticus), a freshwater species commonly raised in high-density aquaculture systems prone to stress and disease. Following 14 days of fish dietary supplementation, the GAF group showed significantly enhanced digestive enzyme activities, including amylase, lipase, and trypsin while chymotrypsin was reduced, yielding a higher trypsin/chymotrypsin ratio indicative of improved protein digestion. Lysozyme activity, a key innate immune marker, was also significantly elevated in the GAF group, followed by AE and GE. In vitro tests using RAW 264.7 macrophages confirmed no cytotoxicity at extract concentrations of 250–4000 µg/mL, and increased cell viability at 1000–2000 µg/mL. Gene expression analysis demonstrated that AE strongly suppressed proinflammatory markers (IL-1β, TNF-α, iNOS, IL-6), with GAF and GE showing moderate effects. All treated groups significantly lowered the IL-6/IL-10 ratio, reflecting improved immune balance. Collectively, AE, GE, and GAF enhance digestive efficiency, boost immune function, and anti- inflammation. The balanced response from GAF supports its use as a natural feed additive to reduce antibiotic reliance and promote sustainable aquaculture.