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

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.