In this study, the leaves of Lycopus lucidus were extracted with 70% ethanol. The ethanol extracts were successively fractionated using n-hexane, methylene chloride (MC), ethyl acetate(EtOAc), n-butanol, and water, and the antioxidative and anti-inflammatory activities of the solvent fractions were investigated. The results are below.

The yield of n­hexane, MC, EtOAc, n­butanol and water fraction from 70% ethanol extract of L. lucidus were 4.25%, 2.40%, 2.20%, 5.90% and 3.25%, respectively.

The total polyphenol and flavonoid contents in the EtOAc fraction of L. lucidus were 747.65±35.65 mg/g and 113.46±0.99 mg/g, respectively, and higher than the other solvent fractions.

At 50 μg/mL concentration, the DPPH, ABTS, and hydroxyl radical scavenging activities of the EtOAc fraction of L. lucidus were 88.68 ±0.06%, 94.91±0.16% and 16.05±1.25%, respectively

The Fe2+ chelating ability of the water fraction of L. lucidus was 51.77±2.19%, which was higher than that of the control deferoxamine (DFRXM ; 31.32±1.76%).

The nitrite scavenging ability of the EtOAc fraction of L. lucidus was 75.88±0.15% (at pH 1.2) and 66.65±0.19% (at pH 3.0), indicating high scavenging ability comparable to that of the control BHT.

Effects of the solvent fractions of L. lucidus were assessed on the viability of the macrophage cell line RAW264.7. Cell viability decreased with increasing concentration of the solvent fractions. The survival rate was >80% at concentrations below 50 μg/mL.

When solvent fractions of L. lucidus were used at a concentration of 50 μg/mL, the EtOAc fraction showed the lowest nitric oxide (NO) production, demonstrating similar activity to the untreated control.

The 70% ethanol extract, EtOAc fraction, and water fraction were non-toxic to RAW 264.7 cells at concentrations of 10, 50, and 100 μg/mL(indicated by ≥90% cell viability). However, at 100 μg/mL, hexane, MC, and BuOH fractions exhibited cytotoxicity (68.50%, 66.17%, and 79.16% cell viability, respectively)

NO production in RAW264.7 cells decreased in the order of EtOAc>Water>BuOH>MC>Hexane>70% ethanol extract of L. lucidus. The NO production of the EtOAc fraction (10.98 μM) was similar to that of untreated control (9.90 μM). At concentrations of 2, 10, and 50 μg/mL, the EtOAc fraction showed concentration -dependent reduction in NO production compared with the untreated controll.

At concentrations of 2, 10, and 50 μg/mL, the EtOAc fraction of L. lucidus showed concentration-dependent reduction in PGE2 production compared with the LPS-treated control group.

TNF-α, IL-1β, IL-6, and GM-CSF production decreased with an increase in the concentration of EtOAc fraction of L. lucidus compared with the LPS-treated group. IL-10 production increased with an increase in the concentration of EtOAc fraction when compared with the LPS-treated group.

The expression of iNOS and COX-2 proteins decreased with an increase in the concentration of EtOAc fraction of L. lucidus. In addition, phosphorylation of ERK, p38 and JNK in LPS-stimulated RAW264.7 cells decreased with an increase in the concentration of the EtOAc fraction.

IκB-α expression increased in a concentration-dependent manner in LPS-stimulated RAW264.7 cells compared with that in the control. However, the expression of phosphorylated IκB-α decreased in a concentration-dependent manner compared with the control. The expression of p65, a subunit of NF-κB, was concentration­dependent.

In conclusion, the EtOAc fraction of L. lucidus exhibited excellent antioxidative and anti-inflammatory activities, demonstrating its potential to be developed as an antioxidant and anti-inflammatory agent.