Background:
Cistanche tubulosa is a tonic in traditional Chinese medicines and has a broad spectrum of biological activity, including
anti-inflammatory. However, its anti-inflammatory major constituents of C. tubulosa and their underlying mechanisms are still unknown.
Objective:
The aim of the current study was to explore the separation and structural characterization of lignan glycosides from C. tubulosa
(Schenk) Wight., their anti-inflammatory activity and underlying mechanism.
Materials and Methods:
Fractionation and isolation of the 85% EtOH extract of C. tubulosa (Schenk) Wight. were carried out and the primary
ingredients lignan glycosides (1-6) were structurally characterized. CCK8 methods were used to evaluate the cytotoxic effect of lignan
glycosides (1-6). Effects of lignan glycosides (1-6) on NO production in LPS/IFN-γ-induced RAW264.7 macrophages cells were measured
using Griess reagent by reaction with nitrite. The mRNA expression levels of iNOS, COX-2, IL-1β, IL-6, TNF-a, and TGF-β treated
RAW264.7 cells with various concentrations (0, 25 and 50 μg/ml) of lignan glycosides (1, 4) in the presence of LPS (10 ng/ml) and IFN-γ
(20 ng/ml) for 24 h were analyzed by quantitative RT-PCR. Also the protein expressions of iNOS, COX-2, PI3K, AKT, p-AKT and β-actin
were determined using Western blot analysis. A molecular docking study was performed to investigate the interactions between the lignan
glycosides and the PI3K using Autodock vina 1.1.2 package.
Results:
Six lignan glycosides (1-6) were isolated from stems of C. tubulosa. Among them, (+)-pinoresinol-4-O-β-D-glucopyranosyl-
(1→6)-β-D- glucopyranoside (5) and eleutheroside E (6) were firstly isolated from C. tubulosa. Of these lignans, 1 and 4 exhibited pronounced
inhibitions on NO production with the values of 33.63 ± 4.78 and 39.28 ± 5.52 % at 50 μg/ml, respectively. Additionally,
LPS/IFN-γ-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-6, and tumor
necrosis factor-a (TNF-a) was significantly suppressed by pre-treatment of 1 and 4 in a dose-dependent manner. While 1 and 4 increased
the mRNA levels of anti-inflammatory cytokines (TGF-β). Furthermore, 1 and 4 significantly inhibited the protein levels of PI3K
and p-AKT in a dose-dependent manner.
Conclusion:
Taken together, these results suggest that 1 and 4 play an important role in the attenuation of LPS/IFN-γ-induced inflammatory
responses in RAW264.7 cells and that the mechanisms involve down-regulation of the PI3K/AKT pathway.