Individual Botanicals Perform Better than AYUSH-64 Formulation in Suppressing TLR4-Driven Inflammatory Cytokines

Dysregulated activation of macrophages through the Toll-like receptor 4 (TLR4) pathway plays a central role in the pathophysiology of numerous inflammatory diseases. The immunomodulatory potential of AYUSH-64, a polyherbal formulation, has recently gained renewed interest. However, the individual contributions of its constituent botanicals, Alstonia scholaris (AS), Picrorhiza kurroa (PK), Swertia chirata (SC), and Caesalpinia crista (CC), remain poorly characterized.

To evaluate and compare the prophylactic immunomodulatory effects of AYUSH-64 and its individual plant extracts on lipopolysaccharide (LPS)-induced inflammation in THP-1-derived macrophage-like cells.

THP-1 monocytes were differentiated using phorbol 12-myristate 13-acetate (PMA) and subsequently stimulated with LPS (100 ng/mL) to activate TLR4 signaling. Before stimulation, cells were pre-treated with AYUSH-64 or its individual extracts (AS, PK, SC, and CC) at concentrations of 30, 100, and 300 µg/mL. Dexamethasone (30 nM) was used as a positive control. Gene expression of pro-inflammatory and regulatory cytokines (TNF-α, IL-1β, and IL-10) was quantified using qRT-PCR, and cytokine secretion levels via ELISA.

All treatments, including AYUSH-64 and the individual botanical extracts, were non-cytotoxic to PMA-differentiated THP-1 cells. AYUSH-64 elicited a moderate suppressive effect on TNF-α and IL-10 at both the transcriptional and protein levels, with minimal impact on IL-1β expression. In contrast, individual extracts specifically SC, PK, and CC, demonstrated more pronounced and selective inhibition of inflammatory markers. Notably, SC and PK significantly reduced IL-10 secretion, achieving effects superior to those of dexamethasone. AS showed strong downregulation of TNF-α and IL-10 gene expression, while CC induced a dose-dependent reduction in IL-10 expression and secretion.

Individual components of AYUSH-64, especially SC, PK, and CC, demonstrate stronger and more selective anti-inflammatory effects in vitro. They outperformed AYUSH-64 in reducing IL-10 secretion. Thus, constituent-level evaluation in polyherbal formulations is warranted, though further in vivo and clinical studies are needed to confirm their therapeutic potential.

AYUSH-64, Alstonia scholaris, Picrorhiza kurroa, Swertia chirata, Caesalpinia crista, toll-like receptor 4, inflammation, macrophage.