Immunomodulatory Effects of Diterpene Quinone Derivatives from the Roots of Horminum pyrenaicum in Human PBMC

Abstract

Several phytochemicals were shown to interfere with redox biology in the human system. Moreover, redox biochemistry is crucially involved in the orchestration of immunological cascades. When screening for immunomodulatory compounds, the two interferon gamma- (IFN-γ-) dependent immunometabolic pathways of tryptophan breakdown via indoleamine 2,3-dioxygenase-1 (IDO-1) and neopterin formation by GTP-cyclohydrolase 1 (GTP-CH-I) represent prominent targets, as IFN-γ-related signaling is strongly sensitive to oxidative triggers. Herein, the analysis of these pathway activities in human peripheral mononuclear cells was successfully applied in a bioactivity-guided fractionation strategy to screen for anti-inflammatory substances contained in the root of Horminum (H.) pyrenaicum L. (syn. Dragon's mouth), the only representative of the monophyletic genus Horminum. Four abietane diterpene quinone derivatives (horminone, 7-O-acetylhorminone, inuroyleanol and its 15,16-dehydro-derivative, a novel natural product), two nor-abietane diterpene quinones (agastaquinone and 3-deoxyagastaquinone) and two abeo 18 (4 → 3) abietane diterpene quinones (agastol and its 15,16-dehydro-derivative) could be identified. These compounds were able to dose-dependently suppress the above mentioned pathways with different potency. Beside the description of new active compounds, this study demonstrates the feasibility of integrating IDO-1 and GTP-CH-I activity in the search for novel anti-inflammatory compounds, which can then be directed towards a more detailed mode of action analysis.

Figure 1

Concentrations of neopterin (Neo), tryptophan (Trp), kynurenine (Kyn) as well as the Kyn/Trp, a measure of IDO-1 activity, in the supernatant of unstimulated PBMC and cells stimulated with 10 μg/mL concanavalin A (Con A) or phytohaemagglutinin (PHA) for 48 h. Results shown are the mean values ± SEM of seven independent experiments run in duplicates (^∗ p < 0.05, compared to unstimulated cells; please note log-scale).

Figure 2

Bioactivity of the Horminum pyrenaicum root extract: Unstimulated (grey squares) and phytohemagglutinin (PHA)-stimulated peripheral mononuclear cells (PBMC; black squares) were treated with increasing extract concentrations for 48 h. Neopterin formation (a) and tryptophan breakdown to kynurenine, indicated by the Kyn/Trp (b), was measured in the cell supernatants. Results shown are the mean values ± SEM of three independent experiments run in duplicates. ^∗ p < 0.05 indicates significant differences compared to the respective unstimulated or stimulated control cells (set as 100%).

Figure 3

Chromatogram of the HPLC analysis (280 nm) of the bioactive fraction HOR-a3 (1.0 mg/mL) with identified compounds (see Figure 4).

Figure 4

Identified constituents of the roots of Horminum pyrenaicum. Black, blue and violet colors represent abietane-diterpene quinones, nor-abietane diterpene quinones, and abeo 18 (4 → 3) abietane diterpene quinones, respectively.

Figure 5

Inhibition of indoleamine 2,3-dioxygenase, as indicated by Kyn/Trp, in mitogen-stimulated PBMC by the identified compounds. PBMC treated with PHA alone were used as a control for each experiment (set as 100%). (A) (1) horminone (n = 1), (2) 7-O-acetylhorminone (n = 3), (3) agastaqiunone (n = 2) and HP-r4, which is an equimolar mixture of the compounds (4) 15,16-dehydroinuroyleanol and (5) 15,16-dehydroagastol (the average molecular weight was used for calculations; n = 3) and (B) (6) inuroyleanol (n = 3), (7) agastol (n = 3) and (8) 3-deoxyagastaquinone (n = 3). The number of replicates is indicated in brackets behind each compound. It was not possible to repeat the experiments in all cases due to the low amount of substance yield.