Investigation into the Modulatory Effects of Kawakawa Extract on Macrophage Functions

Abstract

Macrophage activation is essential for innate immune defence; however, excessive or prolonged activation contributes to chronic inflammation and tissue damage. Kawakawa (Piper excelsum), a medicinal plant traditionally used in rongoā Māori in Aotearoa New Zealand, contains bioactive compounds with reported anti-inflammatory properties, although its immunomodulatory effects on macrophages remain incompletely understood.

This study investigated the concentration-, time-, stimulus-, and treatment-timing-dependent effects of a hydroethanolic kawakawa leaf extract in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS), interferon-gamma (IFN-γ), or a combination of LPS and IFN-γ. Cell viability was assessed using trypan blue exclusion and the MTT assay, while nitrite accumulation, used as an indirect indicator of nitric oxide (NO) production, was quantified via the Griess assay.

Kawakawa extract did not significantly affect macrophage viability across the concentrations tested in most conditions, indicating that the observed biological effects were generally not attributable to overt cytotoxicity. In the LPS-induced inflammation model, significant reductions in NO production were observed following pre-treatment and co-treatment with 100 μg/mL kawakawa extract (p < 0.001). Under the IFN-γ stimulation, the inhibitory effects of the extract were observed under both pre-treatment and co-treatment conditions after 48 hours (p < 0.01), suggesting a delayed anti-inflammatory response compared with the LPS model. For LPS + IFN-γ-stimulated macrophages, kawakawa extract produced the greatest inhibition under co-treatment conditions (p < 0.001). Across all treatment strategies, pre-treatment and co-treatment generally produced stronger inhibitory effects than post-treatment, although the magnitude and statistical significance of these responses varied according to inflammatory stimulus, incubation time, and experimental endpoint. MTT-reducing activity was not consistently affected by kawakawa treatment, with significant reductions observed under selected conditions at the highest concentration tested.

In conclusion, hydroethanolic kawakawa leaf extract modulated inflammatory nitrite accumulation in activated RAW 264.7 macrophages while generally maintaining cell viability across most experimental conditions. The findings indicate that its biological activity depends on extract concentration, inflammatory stimulus, treatment timing, and incubation period, with greater inhibitory effects observed when the extract was administered before or concurrently with inflammatory stimulation. These results provide experimental evidence supporting the immunomodulatory potential of kawakawa and establish a foundation for future studies using chemically standardised extracts to identify the active constituents, uncover the molecular mechanisms, and evaluate its potential therapeutic applications.