Comparative pharmacokinetic study of the five anti-inflammatory active ingredients of Inula cappa in a normal and an LPS-induced inflammatory cell model

Abstract

Inula cappa is a commonly used medicine in the Miao area of Guizhou Province in China. We established an in vitro inflammatory model of mouse macrophage RAW264.7 cells to study the different pharmacokinetics of five anti-inflammatory active ingredients in the I. cappa extract namely luteolin (LUT), chlorogenic acid (CA), cryptochlorogenic acid (CCA), 3,4-dicaffeoylquinic acid (3,4-DCQA) and 4,5-dicaffeoylquinic acid (4,5-DCQA), in a normal and an inflammatory cell model. First, RAW264.7 cells were treated in vitro with l μg/mL lipopolysaccharide (LPS) for 24 h to establish an inflammatory cell model. Then, the pharmacokinetic characteristics of the five ingredients were compared in normal and inflammatory cells after treatment with 200 μg/ml and 800 μg/ml of I. cappa extracts. After treatment with 1 μg/ml LPS for 24 h, the volume of RAW264.7 cells was increased, the morphology was changed, the antennae were obvious, and the secretion of inflammatory factors nitric oxide and TNF-α was increased. The pharmacokinetics results showed that the five ingredients in normal and inflammatory cells exhibited an increase in C_max and AUC values with increasing doses, and the C_max and AUC values of five ingredients were positively correlated with the extract concentration. Each of these five ingredients presented nonlinear pharmacokinetic characteristics. After treatment with 200 μg/ml of I. cappa extract, the uptake of five ingredients increased in inflammatory cells, T_max was prolonged, MRT and t_1/2 were prolonged, and CL_F and Vz_F were decreased, while after treatment with 800 μg/ml of I. cappa extract, the uptake of five ingredients decreased, T_max was prolonged, absorption was faster, and MRT and t_1/2 were prolonged. The five analyzed components in I. cappa extract exerted different effects on normal cells and LPS-induced inflammatory cells. Compared to normal cells, the uptake of five ingredients in inflammatory cells was faster and the AUC and C_max values increased with increasing doses, showing a dose-dependent nonlinear pharmacokinetic profile. These results indicate that the pharmacokinetic effects of the five analyzed ingredients in I. cappa extract are changed in the inflammatory state.

Keywords: Inula cappa; RAW264.7 cells; UPLC-MS/MS; cellular pharmacokinetics; inflammatory model.

FIGURE 1

Chemical structures of luteolin (A), chlorogenic acid (B), cryptochlorogenic acid (C), 3,4-dicaffeoylquinic acid (D), and 4,5-dicaffeoylquinic acid (E).

FIGURE 2

Cell morphology of LPS-induced RAW264.7 cells at different times (A): normal cells, (B) 1 μg/ml LPS-induced cells at 6 h, (C) 1 μg/ml LPS-induced cells at 12 h, (D) 1 μg/ml LPS-induced cells at 24 h) (100×).

FIGURE 3

Survival rate of LPS-induced RAW264.7 cells at LPS different concentrations for 24 h (A), the accumulation of NO (B) and TNF-α (C) in LPS-induced RAW264.7 cells at different concentrations for 6, 12 and 24 h ( $\overline{x}$ ± SD, n = 3, nmol/mL), and survival rate of RAW264.7 cells after the addition of I. cappa extract (D). Compared with the blank group, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

The mean concentration (ng/mg) of luteolin (A), chlorogenic acid (B), cryptochlorogenic acid (C), 3,4-dicaffeoylquinic acid (D), and 4,5-dicaffeoylquinic acid (E) versus time profiles in RAW264.7 cells after treatment with 200 μg/ml and 800 μg/ml of I. cappa extracts ( $\overline{x}$ ± SD, n = 6).