Evaluation of the immunomodulatory potential of innovative herbal formulation (BVGL19/1) through in-silico, in-vitro, and in-vivo approaches

• Herbal formulation (BVGL19/1) screened for immunomodulatory potential using in-silico, in-vitro , and in-vivo methods • Bioactive compounds from 20 medicinal plants showed strong binding to key immune proteins. • Antioxidant assays revealed potent activity with IC 50 values of 22.81 µl/ml (ABTS), 10.63 µl/ml (DPPH), and 21 µl/ml (hydroxyl radical scavenging), respectively. • In-vivo rat model showed reduced antibody response and inflammation markers after treatment. • The finding highlights the potential of herbal formulation in modulating immune function and reducing inflammation. Herbal therapies have been historically employed to enhance health and well-being, with recent interest focusing on plant-based natural products for immunomodulation and inflammation mitigation. This study evaluates the immunomodulatory effects of a specific herbal formulation through in-silico, in-vitro , and in-vivo experimentation using sheep red blood cells (SRBC) immunized rat model. Bioactive compounds were selected from 20 medicinal plants based on their oral bioavailability and drug-likeness score. Molecular docking (AutoDock 4.2.6.) evaluate binding affinity of phytoconstituents with key target proteins involved in immune responses and inflammation. Molecular dynamics (MD) simulation was performed to validate the stability of the best ligand–protein complexes. The in-vitro antioxidant activity was assed using ABTS, DPPH, and hydroxyl radical scavenging assays, with ascorbic acid used as the positive control.. For the in-vivo study, 24 Wistar rats were divided into 4 groups, immunized via SRBC at a concentration of 0.5 × 10 9 cells/ml/100gm body weight. Groups III and IV received herbal formulation at doses of 2.06 and 4.12 ml/kg for 7 days. Haemagglutination antibody titer (HAT), delayed-type hypersensitivity reaction (DTH), serum IgG and Histopathology were evaluated. Computational analysis indicated strong binding energies of bioactive components with key proteins such as NLRP3, IL-1β, IL-23, TLR4, and TNF-α (surpassing Levamisole). MD simulation confirmed stable protein–ligand interactions, supporting the docking results. The IC 50 values from the in-vitro assays were recorded at 22.81 µl/ml (ABTS), 10.63 µl/ml (DPPH), and 21 µl/ml (hydroxyl radical scavenging). In-vivo results showed a significant reduction in HAT, DTH and serum IgG level of test groups as compared to control group. This study highlights the potential of herbal formulation in immune modulation and reducing inflammation, underscoring the need for clinical trials for further analysis.