Phytochemical Profiling, Antioxidant, Antibacterial and Biomarkers Enzyme Activity of Aristolochia Indica: Host Plant Effects on Immune Response Across Development of Specialized Caterpillar (Pachliopta Hector)

Abstract

Introduction: The medicinal plant Aristolochia indica is the exclusive host for the larvae of the crimson rose swallowtail butterfly, Pachliopta hector, which sequester the plant's toxic secondary metabolites for defense.

Objectives: This study presents a comprehensive phytochemical and bioactivity profile of A. indica leaf extracts and investigates its impact on the immune response of its specialized herbivore across larval development.

Methods: Preliminary phytochemical screening confirmed the presence of various compound classes. Gas Chromatography-Mass Spectrometry (GC-MS) analysis further identified and quantified specific bioactive compounds. The extract's antioxidant potential was assessed using multiple in vitro assays (DPPH, ABTS⁺, nitric oxide, superoxide anion scavenging, FRAP), and its antibacterial activity was tested against Gram-positive and Gram-negative pathogens. To assess host plant effects, P. hector caterpillars were reared on A. indica leaves, and hemolymph was collected across five larval instars to analyze immune parameters (phenoloxidase activity, total hemocyte count) and key antioxidant enzyme activity (GST, GPx, CAT).

Results: The extract exhibited significant in vitro antioxidant potential and broad-spectrum antibacterial activity. A notable, instar-specific modulation of immune parameters was observed in the caterpillars. Later instars, corresponding with increased consumption of the toxic foliage, exhibited a significantly heightened immune response, including increased phenoloxidase activity and total hemocyte count, compared to early instars and controls fed on a non-toxic host. The activity of key antioxidant enzymes in the hemolymph provided a direct biochemical measure of the response to oxidative stress.

Conclusions: The findings conclusively establish A. indica as a rich source of bioactive compounds with potent antioxidant and antibacterial properties. More importantly, they demonstrate that the plant's chemical arsenal acts as a powerful selective pressure, driving the evolution of a robust and inducible immune system in its specialized herbivore, P. hector, to facilitate successful sequestration and ensure fitness.