Mechanistic insights into the immunomodulatory and antibacterial activity of the Japanese Kampo Formula Hainosankyuto

Abstract

Purpose This study aimed to quantify the in vitro antibacterial activity of the Japanese herbal formula Hainosankyuto (HNST) against Gram-positive and Gram-negative bacteria and to elucidate its immunomodulatory effects on human monocytes. Methods The antibacterial activity of HNST was assessed against Escherichia ( E .) coli DH5α and Streptococcus ( S .) pneumoniae D39 using bacterial growth curve analysis. The immunomodulatory effects were examined in THP-1 monocytes by quantifying phagocytosis via a gentamicin protection assay, cytokine release by enzyme immunoassay and NF-κB activation using the THP1-Blue™ NF-κB reporter cell line. Cell viability was evaluated by microscopy and lactate dehydrogenase (LDH) release assays. The potential for lipopolysaccharide (LPS) contamination was assessed using a Limulus amoebocyte lysate (LAL) assay and validated with the TLR4-deficient THP1-Dual™ KO-TLR4 reporter cell line. Results HNST exhibited significant antibacterial activity against the Gram-positive strain S. pneumoniae , but not against E. coli . It enhanced phagocytic activity and induced NF-κB activation in THP-1 monocytes in a concentration-dependent manner. Compared to an unstimulated control, HNST moderately increased intracellular killing of bacteria. These effects were independent of its LPS content. No significant cytokine release was detected at non-cytotoxic concentrations. Cytotoxicity was observed at higher concentrations. Conclusion HNST demonstrated immunomodulatory and selective antibacterial activity, which supports its traditional use as an immune-enhancing agent. However, these preliminary findings require further pharmacological and in vivo validation. The data highlight the potential of HNST as a possible complementary therapeutic approach in the era of increasing antibiotic resistance. Further studies are necessary to elucidate its active components and mechanisms of action.