By systematic analysis of the pharmacological activities of the
All NMR spectra were measured on an Agilent VNS-600 spectrometer or on a Bruker DPX 300 spectrometer operating at 600 or 300 MHz for 1H-NMR and 150 or 75 MHz for 13C-NMR in DMSO-
Powdered water extract of fruit bodies of
Metabolic profiling was performed on an Agilent 1200 serious HPLC coupled to an Agilent DAD detector. A Luna C18 column (250 mm×4.6 mm i.d., 5 μm; Phenomenex, Torrance, CA, USA) was used and the mobile phase consisted of water (A) and acetonitrile (B) at a flow rate of 0.9 ml/min, as reported previously (Vo
HEK293 cells (1×106 cells/ml) were transfected in 12-well plates with NF-κB-Luc or AP-1-Luc (each 1 μg/ml), as well as β-galactosidase (0.25 μg/ml), using the PEI method, as reported previously (Chen
To determine cytokine mRNA expression levels, total RNA was isolated from LPS-treated RAW264.7 cells with TRIzol Reagent (GIBCO BRL), according to the manufacturer’s instructions (Moon
All data are presented as the mean ± SEM of 3 different experiments performed with 4 samples (Fig. 1B, 3A, 3B, 3D) for
Since the total water extract of
Continuously, we evaluated the anti-inflammatory activities of 8 compounds using the same reporter gene assay and RT-PCR analysis of inflammatory genes. As Fig. 3 shows that compound 1 blocked luciferase activity induced by NF-κB activation, whilst compounds 1, 3, 4, 7, and 8 significantly suppressed AP-1-mediated luciferase activity, suggesting that these compounds may have an anti-inflammatory function. To confirm these activities, we next examined whether these compounds are able to modulate the expression of TNF-α and COX-2. At 200 μM, compound 1 clearly suppressed the mRNA levels of COX-2, whereas compounds 1, 3, 4, and 8 at 400 μM strongly suppressed COX-2 expression and compounds 1, 4, and 8 diminished the mRNA expression of TNF-α.
After establishing a technology to artificially cultivate the fruit bodies of
We next investigated the anti-inflammatory activities of the isolated constituents at the transcriptional levels. As Fig. 3 indicates, compound 1 was found to suppress NF-κB-mediated luciferase activity, whereas others [compounds 1, 3, 4, 7, and 8] targeted to block AP-1-mediated luciferase activity, implying that these compounds may have an anti-inflammatory function. This possibility was also confirmed by testing the levels of TNF-α and COX-2 genes, representative inflammatory genes mediated by the activation of NF-κB and AP-1 (Yu
Conclusively, we have isolated 8 compounds, 1,9-dimethylguanine (1), adenosine (2), uridine (3), nicotinamide (4), 3-methyluracil (5), 1,7-dimethylxanthine (6), nudifloric acid (7), and mannitol (8) from the water extract of
This work was supported by the BK21 plus program through the National Research Foundation (NRF) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. NRF-2016R1A6A1A03007648), Korea.