NF-κB-targeted anti-inflammatory activity of Prunella vulgaris var. lilacina in macrophages RAW 264.7

Abstract

Prunella vulgaris var. lilacina, a herbal medicine, has long been used in Korea for the treatment of sore throat, and to alleviate fever and accelerate wound healing. Although the therapeutic effect of P. vulgaris var. lilacina is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here, we sought to elucidate the possible mechanisms of the anti-inflammatory activity. We have investigated the anti-inflammatory activity of the various solvent fractions (hexane, butanol, chloroform and water) from the ethanol extract of P. vulgaris var. lilacina in activated macrophages. The hexane fraction exhibited higher anti-inflammatory activities, inducing inhibition of nitric oxide and prostaglandin E2 production as well as inducible nitric oxide synthase, cyclooxygenase-2, and tumor necrosis factor-α mRNA expression in response to lipopolysaccharide stimulation. Moreover, the hexane fraction from P. vulgaris var. lilacina significantly inhibited the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the nuclear translocation of the NF-κB p50 and p65 subunits. These results indicate that P. vulgaris var. lilacina has an anti-inflammatory capacity in vitro, suggesting that it could be a potential source of natural anti-inflammatory agents.

Figure 1

Effects of solvent fractions from Prunella vulgaris var. lilacina on the viability of RAW 264.7 cells. (A) Lipopolysaccharide (LPS) untreated; (B) LPS treated. Bars represent the mean and standard deviations from three different experiments performed in triplicate. *p < 0.05 significantly different from the LPS group.

Figure 2

Effects of the solvent fractions from Prunella vulgaris var. lilacina on LPS-induced nitric oxide (NO) (A) and prostaglandin E2 (PGE2); (B) production in RAW 264.7 cells. Values show the means and standard deviations of three different experiments performed in triplicate. *p < 0.05 significantly different from the LPS-treated PBS group.

Figure 3

Effects of solvent fractions from Prunella vulgaris var. lilacina on LPS-induced pro-inflammatory mRNA expression and protein levels in RAW 264.7 cells. Cells were treated with fractions (50 μg/mL) and stimulated with LPS (1 μg/mL) for 12 h. (A,B) After incubation, cells were harvested for real-time RT-PCR to determine mRNA expression for (A) iNOS and (B) COX-2. (C) After incubation, cell lysates were used to determine iNOS and COX-2 protein levels via Western blot. Values show the means and standard deviations of three different experiments performed in triplicate. *p < 0.05 significantly different from the LPS-treated PBS group.

Figure 4

Effects of solvent fractions from Prunella vulgaris var. lilacina on LPS-induced TNFα levels (A) and TNFα mRNA expression (B) in RAW 264.7 cells. Cells were treated with fractions (50 μg/mL) and stimulated with LPS (1 μg/mL) for 12 h. Values show the means and standard deviations of three different experiments performed in triplicate. *p < 0.05 significantly different from the LPS-treated PBS group.

Figure 5

Effect of Prunella vulgaris var. lilacina on NF-κB activation as measured by the luciferase assay (A), and on nuclear translocation of the p65 and p50 subunits (B) in LPS-stimulated RAW 264.7 cells. Cells were treated with fractions (50 μg/mL) and stimulated with LPS (1 μg/mL) for 12 h. *p < 0.05 significantly different from the LPS-stimulated PBS group.

Figure 6

Gas chromatogram and MS Spectra of hexane fractions from Prunella vulgaris var. lilacina. (A) Gas chromatogram of hexane fractions from Prunella vulgaris var. lilacina; (B) hexadecanoic acid; (C) ethyl palmitate; (D) phytol; (E) ethyl linileate; (F) (Z,Z,Z)-9,12,15-octadecatrien-1-ol; (G) linoleic acid ethyl ester; (H) (Z,Z,Z)-ethy lester-9,12,15-octadecatrienoic acid, (I) 3-oxo-8,beta, H-eudesma-1,4,7(11)-trien-8,12-olide; (J) 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol; (K) nerol; (L) linalyl formate, (M) 3-ethylenetricyclo[3.3.1.1(3,7)]decane.

Figure 6

Gas chromatogram and MS Spectra of hexane fractions from Prunella vulgaris var. lilacina. (A) Gas chromatogram of hexane fractions from Prunella vulgaris var. lilacina; (B) hexadecanoic acid; (C) ethyl palmitate; (D) phytol; (E) ethyl linileate; (F) (Z,Z,Z)-9,12,15-octadecatrien-1-ol; (G) linoleic acid ethyl ester; (H) (Z,Z,Z)-ethy lester-9,12,15-octadecatrienoic acid, (I) 3-oxo-8,beta, H-eudesma-1,4,7(11)-trien-8,12-olide; (J) 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol; (K) nerol; (L) linalyl formate, (M) 3-ethylenetricyclo[3.3.1.1(3,7)]decane.

Figure 6

Gas chromatogram and MS Spectra of hexane fractions from Prunella vulgaris var. lilacina. (A) Gas chromatogram of hexane fractions from Prunella vulgaris var. lilacina; (B) hexadecanoic acid; (C) ethyl palmitate; (D) phytol; (E) ethyl linileate; (F) (Z,Z,Z)-9,12,15-octadecatrien-1-ol; (G) linoleic acid ethyl ester; (H) (Z,Z,Z)-ethy lester-9,12,15-octadecatrienoic acid, (I) 3-oxo-8,beta, H-eudesma-1,4,7(11)-trien-8,12-olide; (J) 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol; (K) nerol; (L) linalyl formate, (M) 3-ethylenetricyclo[3.3.1.1(3,7)]decane.