Histological Evidence of Enhanced Humoral Immunity by a Polyherbal Formulation in an Animal Immunogenicity Model

Abstract

Background: Polyherbal formulations, which combine multiple medicinal plants, may provide enhanced immunomodulatory effects through synergistic interactions among their bioactive constituents. Objective: This study aimed to evaluate the immunomodulatory effects of a polyherbal formulation on splenic white pulp structure and splenic index in mice immunized with tetanus toxoid. Methods: Twenty-five male BALB/c mice were randomly divided into five groups: a normal control group, a vaccine control group, and three treatment groups receiving low, medium, or high doses of the polyherbal formulation for 28 days. Tetanus toxoid was administered to the vaccine control and treatment groups on days 7 and 14. The splenic index was calculated as the ratio of spleen weight (mg) to body weight (g). Splenic white pulp diameter was assessed histologically in hematoxylin and eosin-stained spleen sections at 100× magnification. Data were analyzed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) post hoc test. Results: The polyherbal formulation did not significantly affect the splenic index among the experimental groups (p = 0.103), indicating no significant splenic enlargement associated with treatment. In contrast, splenic white pulp diameter increased significantly in all polyherbal treatment groups compared with the vaccine control group (p < 0.001). The mean white pulp diameters were 354.2 μm, 436.4 μm, and 518.0 μm in the low-, medium-, and high-dose groups, respectively, demonstrating a dose-related increase in white pulp development. Conclusion: The polyherbal formulation significantly increased splenic white pulp diameter without significantly altering the splenic index. These findings suggest that the formulation may enhance local adaptive immune responses without inducing significant systemic splenic enlargement in tetanus-toxoid-immunized mice.