Anti-Inflammatory Constituents of Antrodia camphorata on RAW 264.7 Cells Induced by Polyinosinic-Polycytidylic Acid

Antrodia camphorata is an endemic mushroom in Taiwan. This study was designed to screen anti-inflammatory compounds from the methanolic extract of the mycelium of A. camphorata on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA) known to be present in viral infection. A combination of bioactivity-guided isolation with an NMR-based identification led to the isolation of 4-acetylantroquinonol B (1), along with seven compounds. The structure of new compounds (4 and 5) was elucidated by spectroscopic experiments, including MS, IR, and NMR analysis. The anti-inflammatory activity of all isolated compounds was assessed at non-cytotoxic concentrations. 4-Acetylantroquinonol B (1) was the most potent compound against poly I:C-induced NO production in RAW 264.7 cells with an IC50 value of 0.57 ± 0.06 μM.


Introduction
Inflammation is part of the innate, or nonspecific, immune responses against invading pathogens. It is generally initiated when pathogen-associated molecular patterns (PAMPs), such as pathogen-specific carbohydrates, lipoproteins, and nucleic acids, are recognized by a large family of pattern-recognition receptors (PRRs), expressed by macrophages, monocytes, dendritic cells, and neutrophils [1]. Among these immune cells, macrophages play a crucial role in inflammatory reaction through producing inflammatory factors, such as reactive oxygen species (ROS), nitric oxide (NO), cytokines, chemokines, and prostaglandins [2,3].
Notable outbreaks in the last two decades mostly involved respiratory viruses-such as the 2003 severe acute respiratory syndrome (SARS) epidemic, 2009 A(H1N1) pandemic, Middle Eastern respiratory syndrome (MERS) epidemic, and ongoing coronavirus disease 2019 (COVID- 19) pandemic-that represent a considerable challenge to worldwide public health [4]. Novel respiratory viruses commonly lead to severe respiratory tract infections. Owing to the involvement of inflammatory signaling pathways in imbalanced host immune response, which cause acute lung injury in respiratory virus infection, mounting evidence points to the therapeutic potential of targeting viral induced-inflammation [5][6][7].
Plants are generally considered a rich source of natural products that can prevent the onset of diseases and reduce health care costs. A variety of natural products, including flavonoids, terpenoids, alkaloids, glycosides, quinones, and phenolic compounds, from plants traditionally used as antiviral agents have been reported to possess remarkable Molecules 2022, 27, 5320 2 of 9 potential against viral infection [8][9][10]. Among them, anti-inflammatory phytochemicals have attracted much attention to providing potential therapeutic interventions in combating viruses and related complications [11]. Antrodia camphorata is an edible and precious mushroom endemic to Taiwan, which has been used as traditional medicine. A. camphorata has been found to exhibit a broad range of pharmacological effects, which include anti-microbial, anti-oxidative, anti-inflammatory, anti-diabetic, anti-aging, anticarcinogenic, neuroprotective, hepatoprotective, cardioprotective, and immunomodulatory effects [12][13][14][15]. Phytochemical investigation of A. camphorata led to the isolation and characterization of diverse bioactive phytochemicals that include benzenoids, ubiquinones, maleic anhydride and maleimide derivatives, terpenoids, and steroids [12][13][14][15]. Antcins, unique steroid-like compounds isolated from A. camphorata, were reported to be novel anti-angiotensin-converting enzyme 2 (ACE2) agents for prophylaxis of COVID-19 [16]. Whether anti-inflammatory phytochemicals from A. camphorata show potency against inflammation in viral infection remains to be fully elucidated.
Recently, metabolomics can provide a rapid tool by allowing mixture analysis and simultaneously detecting diverse phytochemicals without the need for time-consuming chromatographic procedures. NMR-based identification is extremely powerful for rapid identification of natural products with relative concentration determination. Furthermore, relating metabolites with bioactivities is feasible by integrating metabolite profiling with bioassay results [17,18]. In this study, we combined an NMR-based identification approach with an anti-inflammatory assay to select natural products with inhibitory effects on NO production in RAW 264.7 cells induced by poly I:C, a synthetic analog of dsRNA known to be produced by viral replication and presented in viral infection.

Results and Discussion
Viral infections might provoke hyper-inflammation responses in macrophages. dsR-NAs are produced by viral replication during viral infections and recognized by PPR [19]. As a synthetic analog of dsRNA, poly I:C was used to mimic the viral infection model both in vivo and in vitro in previous studies [19][20][21].
The inhibitory effects of the mycelium of A. camphorata on viral infection-associated inflammation have not been studied previously. Here, poly I:C was applied to stimulate NO production in a RAW 264.7 mouse macrophage cell line as a virus-induced inflammation model for investigating the anti-inflammatory effects of A. camphorata. As shown in Figure S1, the methanolic extract of A. camphorata mycelium (ACM) showed significant inhibition activity against excessive NO production in poly I:C-induced RAW 264.7 cells. The percentages of inhibition of NO production treated with ACM at concentrations of 5, 10, 20, and 40 µg/mL were 53.39 ± 4.87, 67.83 ± 5.74, 84.35 ± 1.51, and 90.15 ± 1.33%, respectively. The methanolic extract of A. camphorata mycelium was then subjected to MPLC separation, and each of the collected fractions was assigned to one of ten main fractions based on TLC characterization. Fraction 5 potently inhibited NO production with percentages of inhibition of 42.00 ± 2.86, 51.46 ± 5.62, 69.05 ± 6.25, and 78.58 ± 3.68% at concentrations of 1, 2, 5, and 10 µg/mL, respectively. Thus, Fraction 5 was chosen for further analysis.
The 1 H NMR experiment offers metabolite profiling, achieved by chemical shifts, J-couplings, and signal area integration. Particularly, the intensity of the signal in 1 H NMR spectra is proportional to the relative number of (equivalent) protons that give rise to the signal and thus could provide quantitative information about the different compounds in the mixture. Relating putative bioactivities with these metabolites is feasible by integrating metabolite profiling with bioactivity results. According to the inspection of 1 H NMR spectra, the presence of 4-acetylantroquinonol B (1) in Fraction 5 was deduced from the characteristic signals as follows: A highly deshielded proton signal at δ H 5.72 (1H, d, J = 3.2 Hz) assigned as H-4; two olefinic proton signals at δ H 5.21 and 5.11 (1H each, t, J = 7.0 Hz); an oxygenated proton signal at δ H 4.62 (1H, m) assigned as H-15; two methoxy proton signals at δ H 4.00 and 3.67 (3H each, s); acetyl proton signal at δ H 2.09 (3H, s); two methyl proton singlets at δ H 1.65 and 1.55 (3H each, s); and one methyl proton doublet at δ H 1.19 (3H, d, J = 7.3 Hz). The remaining methyl proton doublet at δ H 1.26 was overlapped. The relative abundance of different compounds in Fraction 5 obtained by comparing the signal intensity in the 1 H NMR spectrum ( Figure 1) then indicated that the anti-inflammation activity might be contributed to by the presence of 4-acetylantroquinonol B (1), which is a natural ubiquinone analogue isolated from the mycelium of A. camphorata in 2009 [22]. As one of the bioactive compounds in A. camphorata, it has been reported to possess various bioactivities, which include anti-inflammatory, anti-carcinogenic, hepatoprotective, and immunomodulatory properties [22][23][24][25][26][27][28]. However, the role of 4-acetylantroquinonol B (1) in a viral-induced inflammation model has not been reported previously.
Evaluations for the inhibitory effect on NO production in RAW 264.7 macrophages induced by poly I:C (Table 1) were performed on all isolated compounds. In order to exclude the cytotoxic effect of tested compounds, those were also applied for cell viability assessment. No cytotoxicity was observed (cell viability >90%) when treated with the tested compounds under the half maximal inhibitory concentration. As expected, 4-acetylantroquinonol B (1) shows most potent inhibitory activity against poly I:C induced-NO production in RAW 264.7 cells with an IC 50 value of 0.57 ± 0.06 µM. Additionally, two antroquinonols (2 and 3), a benzenoid (4), and two maleimide anhydride derivatives (5 and 6) showed moderate inhibitory activity with the IC 50 values ranging from 2.96 to 27.14 µM. However, maleic anhydride derivatives (7 and 8), compared to maleimide derivatives, were found to be not effective in this study. According to the bioactivity results above, together with the relative abundance of different metabolites in the bioactive Fraction 5, 4-acetylantroquinonol B (1) was not only the most abundant compound but the most potent compound against poly I:C-induced NO production in RAW 264.7 cells. 67.10 ± 2.23 1 The IC 50 values were calculated based on the dose-response curves and expressed as mean ± SD of triplicates. 2 Indomethacin was used as a positive control.
Toll-like receptors (TLRs), a class of PPR, play a crucial role in regulating the innate immune system. TLR3 could recognize dsRNA and drive the inflammatory cytokine signaling, such as nuclear factor-κB (NF-κB) and mitogen-activated protein (MAP) kinase activation [19]. The preliminary activity of ACM, together with the anti-inflammatory compounds in this study, urges the need for further studies on the TLR3 signaling pathway.

General Experimental Procedures
Silica gel (25 µm) was used for medium-pressure column chromatography (MPLC). Silica gel 60 F254 plates (200 µm) was used for thin-layer chromatography (TLC). Highperformance liquid chromatography (HPLC) was carried out on a KNAUER HPLC system equipped with a refractive index (RI) detector. IR spectra were measured with a Shimadzu IRPrestige-21 Fourier transform infrared (FT-IR) spectrophotometer. High-resolution electrospray mass (HRESIMS) spectra were acquired on a Bruker Daltonics maXis impact ESI-Q-TOF mass spectrophotometer. UV spectra were obtained using a PerkinElmer Lambda 265 UV-Visible spectrophotometer. Nuclear magnetic resonance (NMR) spectra were measured with a Bruker AVANCE NEO 400 MHz FTNMR spectrometer and a Bruker AVANCE 500 MHz FTNMR spectrophotometer.

Extraction and Isolation
The mycelium of A. camphorata (4.1 kg) was extracted with methanol three times at room temperature, and the crude extract was concentrated and applied on MPLC with a linear gradient solvent system of n-hexane and EtOAc. Each collected fraction was assigned into ten main fractions based on TLC characterization.

Statistical Analysis
The results were expressed as mean ± SD of triplicates. Significant differences were examined using a two-tailed unpaired Student's t test. p values of less than 0.05 were considered statistically significant.

Conclusions
In this study, the inhibitory effects of the methanolic extract of the mycelium of A. camphorata were assessed on viral infection-associated inflammation using poly I:C-stimulated NO production in a RAW 264.7 mouse macrophage model. Our study provides a precise strategy for screening anti-inflammatory compounds from the mycelium of A. camphorata by a combination of bioactivity-guided isolation with an NMR-based identification approach, which led to isolation and identification of eight compounds, including two new compounds 4 and 5. Among them, 4-acetylantroquinonol B (1) was not only the most abundant compound, but the most potent compound in the bioactive Fraction 5 from the mycelium of A. camphorata. Thus, it could be a chemical marker for the potential anti-inflammatory agent A. camphorata mycelium during viral infection.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27165320/s1: Figure S1 The effects of the mycelium of Antrodia camphorata (ACM) on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C). Figure S2 1 H NMR spectrum of the major compound identified as 4-acetylantroquinonol B in the bioactive fraction 5. Figure S3 MS spectrum of compound 4. Figure S4 1 H NMR spectrum of compound 4. Figure S5 13 C NMR spectrum of compound 4. Figure  S6 HSQC NMR spectrum of compound 4. Figure S7 HMBC NMR spectrum of compound 4. Figure  S8 MS spectrum of compound 5. Figure S9 1 H NMR spectrum of compound 5. Figure S10 13 C NMR spectrum of compound 5. Figure S11 HSQC NMR spectrum of compound 5. Figure S12 HMBC NMR spectrum of compound 5. Figure