Objective:

2

To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of

Polygala sibirica

L.

var megalopha

Fr. (EEP) on RAW264.7 mouse macrophages.

Methods:

2

RAW264.7 cells were pretreated with 0–200 μg/mL EEP or vehicle for 2 h prior to exposure to 1 μg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE

2

) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA)

respectively. The mRNA levels of inducible nitric oxide synthase (iNOS)

cyclooxygenase-2 (COX-2)

tumor necrosis factor α (TNF-α)

interleukin-1beta (IL-1β)

and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS

COX-2

phosphorylation of extracellular regulated protein kinases (ERK1/2)

c-Jun N-terminal kinase (JNK)

inhibitory subunit of nuclear factor Kappa B alpha (IκB-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κB p65 (NF-κB p65). Additionally

the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2

2-diphenyl-1-picrylhydrazyl (DPPH)

hydroxyl (OH)

superoxide anion (O

2

–

) radical and nitrite scavenging activity were also measured.

Results:

2

The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 μg/mL)

there was a notable decrease in NO and PGE

2

production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (

P

<

0.01 or

P

<

0.05). Furthermore

with EEP treatment (150 μg/mL)

there was a decrease in the mRNA expression levels of TNF-α

IL-1β and IL-6

as well as in the phosphorylation of ERK

JNK and p38 mitogen-activated protein kinase (MAPK

P

<

0.01 or

P

<

0.05)

by blocking the nuclear translocation of NF-κB p65 in LPS-stimulated cells. In addition

EEP (100 and 150 μg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT

with a concomitant decrease in ROS production (

P

<

0.01 or

P

<

0.05). EEP also indicated the DPPH

OH

O

2

–

radical and nitrite scavenging activity.

Conclusion:

2

EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κB pathway and protected against oxidative stress.