Abstract:

【Objective】Stingless bees are one of the important pollinators in tropic and subtropical area, differ from Apis mellifera ligustica with typical stingless characteristic. Stingless bee collected more propolis than that of A. m. ligustica . Nevertheless, the study on the geopropolis activity was relatively scarce. The objective of this study is to evaluate the in vitro antioxidant and anti-inflammatory activities of the ethanol extract of geopropolis collected from stingless bee, Heterotrigona itama , which is an indigenous stingless bee species in Malaysia. 【Method】The content of total phenolic acid and total flavonoids in the EEHI (ethanol extract of H. itama propolis) was determined by Folin-phenol method and AlNO₃ colorimetry, respectively. The antioxidant activity was investigated using DPPH and ABTS ^+·free radical scavenging assays. Moreover, the inflammatory model of murine macrophage RAW 264.7 was induced by bacterial endotoxin lipopolysaccharide (LPS) and the effect of EEHI on the relative cell viability was detected via CCK-8 method. On the basis of ensuring that EEHI has no cytotoxic effect on cells, Griess method and RT-qPCR technique were applied to evaluate the effect of EEHI on the release of inflammatory mediator NO, and on the expression of inflammatory factors ( IL-1β , IL-10 and INOS ) as well as antioxidant gene ( HO-1 ) in LPS-activated macrophages, respectively. In order to explore the potential anti-inflammatory mechanisms of EEHI, the effects of EEHI on the expression of p-IκB α and IκΒ α in macrophages induced by LPS and the translocation of NF-κB-p65 protein were further studied by Western blot and immunofluorescence methods. 【Result】The content of total phenolic acid and total flavonoids in the EEHI was 54.70 mg GAE·g ^-1 and 116.20 mg QE·g ^-1, and the IC₅₀ value of the EEHI for scavenging of DPPH and ABTS ^+· free radicals was 275.60 and 284.00 μg·mL ^-1, respectively. The safe concentration of EEHI to RAW 264.7 cells was 0-40 μg·mL ^-1. In LPS-challenged macrophages, EEHI at 0-40 μg·mL ^-1 significantly inhibited the release of NO as well as the expression of pro-inflammatory cytokine genes ( IL-1β , IL-10 and INOS ), and enhanced the expression of antioxidant gene HO-1 in a dose-dependent manner, compared with the LPS-treated control. Furthermore, it was noticed that EEHI at 0-40 μg·mL ^-1 significantly inhibited the LPS-stimulated phosphorylation of IκΒ α protein in a dose-dependent manner, and EEHI at 40 μg·mL ^-1 significantly reduced the nuclear migration of NF-κB-p65 protein. It was suggested that EEHI had potential anti-inflammatory effects by inhibiting the activation of NF-κB inflammatory signaling pathway induced by LPS.【Conclusion】The ethanol extract of geopropolis collected from H. itama contains a large number of polyphenols, which has good antioxidant and anti-inflammatory effects, and is of great value for exploitation and utilization in the future.

Key words: stingless bee, geopropolis, antioxidant, anti-inflammatory, NF-κB;