Differential Modulation of IRF1, IRF3 and IRF7 Gene Expression by an n-Hexane Fraction of a Polyherbal Formulation in Human Peripheral Blood Mononuclear Cells: An In-Vitro Study

Abstract

Background: Interferon regulatory factors (IRFs) coordinate antiviral gene programmes, but the effects of non-polar polyherbal fractions on individual IRF pathways in primary human immune cells remain poorly defined. This exploratory study evaluated the effect of an n-hexane fraction of a polyherbal formulation on IRF1, IRF3 and IRF7 expression in peripheral blood mononuclear cells (PBMCs) from HIV-negative donors. Methods: PHA-stimulated PBMCs were cultured without treatment or with 50 µg/mL of the n-hexane fraction. Total RNA was extracted, reverse-transcribed and analysed by quantitative reverse-transcription PCR, using GAPDH as the reference gene and the 2^-ΔΔCt method for relative expression. The available dataset comprised four observations per group. Independent-samples t-tests were used, and Welch’s result was prioritised when variance homogeneity was violated. Results: IRF1 expression decreased from 1.06 ± 0.05-fold in untreated cells to 0.50 ± 0.09-fold after treatment (52.7% reduction; p < 0.001). IRF3 increased from 1.06 ± 0.05-fold to 2.05 ± 0.63-fold (93.5% increase). The pooled-variance test yielded p = 0.021, but the variance-robust Welch test yielded p = 0.052. IRF7 changed from 1.06 ± 0.05-fold to 0.96 ± 0.62-fold and was not significant (Welch p = 0.763). Conclusion: The n-hexane fraction produced a gene-specific transcriptional pattern characterised by marked IRF1 suppression, a large but statistically uncertain increase in IRF3 and no clear change in IRF7. These findings identify candidate immunomodulatory activity but do not establish antiviral efficacy. Larger donor-matched, dose-response and time-course studies with MIQE-compliant qPCR reporting, protein-level validation, cytotoxicity assessment and functional viral challenge are required.