Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (5): 939-948.doi: 10.3864/j.issn.0578-1752.2019.05.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Antioxidative and Anti-inflammatory Activities of Ethanol Extract of Geopropolis from Stingless Bees

WANG Bei1,2,CHANG HuaSong2,SU SongKun1,SUN LiPing2,WANG Kai2()   

  1. 1 College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002
    2 Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093
  • Received:2018-09-28 Accepted:2018-11-30 Online:2019-03-01 Published:2019-03-12
  • Contact: Kai WANG E-mail:kaiwang628@gmail.com

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;

Table 1

The primer sequence of related genes in RT-qPCR"

基因Gene 上游引物序列Upstream primer sequence 下游引物序列Downstream primer sequence
INOS 5′-TTTCCAGAAGCAGAATGTGACC-3′ 5′-AACACCACTTTCACCAAGACTC-3′
HO-1 5′-ACATTGAGCTGTTTGAGGAG-3′ 5′-TACATGGCATAAATTCCCACTG-3′
IL-1β 5′-CCAACAAGTGATATTCTCCATGAG-3′ 5′-ACTCTGCAGACTCAAACTCCA-3′
IL-10 5′-CTATGCTGCCTGCTCTTACTG-3′ 5′-CAACCCAAGTAACCCTTAAAGTC-3′
GAPDH 5′-GAGAAACCTGCCAAGTATGATGAC-3′ 5′-TAGCCGTATTCATTGTCATACCAG-3′

Table 2

Total phenolic acid, total flavonoids contents and radical-scavenging activity of EEHI"

总酚酸含量
Total phenolic acid content
(mg GAE·g-1)
总黄酮含量
Total flavonoids content
(mg QE·g-1)
DPPH自由基清除力IC50
DPPH radical-scavenging activity IC50 (μg·mL-1)
ABTS自由基清除力IC50
ABTSradical-scavenging activity IC50 (μg·mL-1)
54.70±0.65 116.20±0.12 275.60±0.22 284.00±0.01

Fig. 1

Effects of different concentrations of EEHI on the viability of RAW 264.7 cells **: P <0.01"

Fig. 2

Effects of different concentrations of EEHI on the NO production of RAW 264.7 cells ***: P <0.001"

Fig. 3

Effects of different concentrations of EEHI on the related inflammatory genes expression in RAW 264.7 cells"

Fig. 4

Inhibitory effects of different concentrations of EEHI on the activation of IκΒ α induced by LPS"

Fig. 5

Effects of EEHI on NF-κB-p65 activation"

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