Ganoderma lucidum-Derived Meroterpenoids Show Anti-Inflammatory Activity In Vitro

Ganoderma lucidum, known as the “herb of spiritual potency”, is used for the treatment and prevention of various diseases, but the responsible constituents for its therapeutic effects are largely unknown. For the purpose of obtaining insight into the chemical and biological profiling of meroterpenoids in G. lucidum, various chromatographic approaches were utilized for the title fungus. As a result, six undescribed meroterpenoids, chizhienes A–F (1–6), containing two pairs of enantiomers (4 and 5), were isolated. Their structures were identified using spectroscopic and computational methods. In addition, the anti-inflammatory activities of all the isolates were evaluated by Western blot analysis in LPS-induced macrophage cells (RAW264.7), showing that 1 and 3 could dose dependently inhibit iNOS but not COX-2 expression. Further, 1 and 3 were found to inhibit nitric oxide (NO) production using the Greiss reagent test. The current study will aid in enriching the structural and biological diversity of Ganoderma-derived meroterpenoids.

Chizhiene B (2) was isolated as a yellow oil.Comparing the 1D and 2D NMR da (Table 1) of 2 and petchiene B [34] suggested that 2 is a structural analog of petchiene The difference between 2 and petchiene B is that the hydroxyl group at C-9′ in petchie B is replaced by an ethoxy moiety in 2. The 1 H-1 H COSY correlation of H3-11′/H2-10′ a the HMBC correlations of H2-10′/C-9′ and H2-6′/C-7′, C-8′, and C-9′ (Figure 2) confirm the aforementioned conclusion.Thus, the structure of 2 was identified.
Of note, compounds 1-3 and 6 were found to bear an ethyl group in the structure, forming an ethoxy group.Since the structure for 2 without the ethyl group was characterized, ref. [34] and ethanol was used for extraction under heat, we highly speculate that all these isolates with the ethyl group should be artifacts during extraction procedures, although no further efforts were made to detect whether they are natural products or artifacts due to the extremely low content in the material.Further literature search found that compounds 1, 3 and 6 and their ethyl products are undescribed, meaning that the structures of 1, 3 and 6 with a terminal "OH" group are new natural products which will add structure diversity for GMs family.Despite the possible artifact nature for these compounds, the following biological potency for 1 and 3 may arise from the presence of the additional ethyl group, although further comparison between "-OH" and "CH 3 CH 2 -" forms was not conducted due to the unavailable amounts of the samples.

Biological Activity toward Inflammation
Inflammation is an essential process that allows our bodies to fight against various pathogenic bacteria, viruses, and parasites [38].The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins are tightly associated with inflammation, indicating its occurrence to a certain extent.Ganoderma fungi have been reported to have anti-inflammatory effects [39].Therefore, the anti-inflammatory activities of all the isolates were evaluated.Initially, the cytotoxic effects of compounds were assessed using the cell proliferation and toxicity detection kit (CCK8) assay.As shown in Figure 5, there was no cytotoxicity of compounds observed in RAW264.7 cells at 20 µM for 24 h.
Inflammation is an essential process that allows our bodies to fight against various pathogenic bacteria, viruses, and parasites [38].The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins are tightly associated with inflammation, indicating its occurrence to a certain extent.Ganoderma fungi have been reported to have anti-inflammatory effects [39].Therefore, the anti-inflammatory activities of all the isolates were evaluated.Initially, the cytotoxic effects of compounds were assessed using the cell proliferation and toxicity detection kit (CCK8) assay.As shown in Figure 5, there was no cytotoxicity of compounds observed in RAW264.7 cells at 20 μM for 24 h.
Following this, the protein expression of iNOS and COX-2 was detected by the Western blotting assay in LPS-stimulated macrophage RAW264.7 cells.The results revealed that all compounds down regulated iNOS protein, particularly compounds 1 and 3 (Figure 5).Hence, a dose-response curve for compounds 1 and 3 was further performed.Similarly, the cytotoxic effects of compounds 1 and 3 were firstly detected.The results showed that no cytotoxicity for compound 1 and faint cytotoxicity for compound 3 at 40 μM (Figure S53).Then, the Western blotting assay revealed that the protein level of iNOS was down regulated by compounds 1 and 3 dose dependently in LPS-induced RAW264.7 cells (Figure 6).Meanwhile, the NO production of compounds 1 and 3 was also examined.It was found that compounds 1 and 3 both could inhibit NO release in LPS-stimulated RAW264.7 cells (Figure 7).Interestingly, compounds 1 and 3, rather than 2 and 4, are active toward inflammation inhibition.Upon inspecting their structures, we could conclude that the  2′(3′) double bond might have an influence on the biological activity.In detail, the presence of the  2′(3′) double bond is not advantageous for keeping the anti-inflammatory property.These findings may provide inspirations for structure optimization using these meroterpenoids as lead compounds against inflammation.Following this, the protein expression of iNOS and COX-2 was detected by the Western blotting assay in LPS-stimulated macrophage RAW264.7 cells.The results revealed that all compounds down regulated iNOS protein, particularly compounds 1 and 3 (Figure 5).Hence, a dose-response curve for compounds 1 and 3 was further performed.Similarly, the cytotoxic effects of compounds 1 and 3 were firstly detected.The results showed that no cytotoxicity for compound 1 and faint cytotoxicity for compound 3 at 40 µM (Figure S53).Then, the Western blotting assay revealed that the protein level of iNOS was down regulated by compounds 1 and 3 dose dependently in LPS-induced RAW264.7 cells (Figure 6).Meanwhile, the NO production of compounds 1 and 3 was also examined.It was found that compounds 1 and 3 both could inhibit NO release in LPS-stimulated RAW264.7 cells (Figure 7).Interestingly, compounds 1 and 3, rather than 2 and 4, are active toward inflammation inhibition.Upon inspecting their structures, we could conclude that the ∆ 2 ′ (3 ′ ) double bond might have an influence on the biological activity.In detail, the presence of the ∆ 2 ′ (3 ′ ) double bond is not advantageous for keeping the anti-inflammatory property.These findings may provide inspirations for structure optimization using these meroterpenoids as lead compounds against inflammation.

Fungal Material
The source and authentication of G. lucidum fruiting bodies were identical with our previous study [35] and the voucher specimen (CHYX-0619) of G. lucidum has been deposited in Inheritance-Based Innovation of Chinese Medicine, School of Pharmacy, Shenzhen University Medical School, Shenzhen University.

Extraction and Isolation
The initial extraction process of the dried fruiting bodies of G. lucidum (500.0 kg) and fractionation of the extract to yield 17 fractions (Fr.1-Fr.17)refers to a previous report [35].

Measurement of NO Production
RAW 264.7 cells were seeded in a 24-well plate at 1 × 10 5 cells/well overnight and treated with compounds 1 and 3 (10 µM, 20 µM, and 40 µM) and LPS for 24 h.Cell culture medium was collected and mixed with equal volumes of Griess reagent (Nitric Oxide Assay Kit, S0021M, 042723230918 Beyotime, Shanghai, China) at room temperature in the dark [41].The OD values were measured using Cytation1 (BioTek, Winooski, VT, USA) at 540 nm and NO production was detected using a sodium nitrite standard calibration curve.

Statistical Analysis
All the experimental data were performed in three replicates.The results are represented as the mean ± SEM.Statistical analyses were carried out using GraphPad Prism 8 with Student's t-test, and one-way ANOVA.Differences were considered significant with * p < 0.05 or # p < 0.05.

Conclusions
To conclude, the current study led to the characterization of six new meroterpenoids (1-6) from G. lucidum.The possible artificial nature of compounds 1-3 and 6 and their contribution to biological potential were briefly discussed.Biological evaluations revealed that compounds 1 and 3 could significantly attenuate the protein expression level of iNOS and NO production in LPS-stimulated RAW264.7 cells, indicating their potential in inflammatory disease.In addition, the present findings are also beneficial for insights into GMs structure alterations in the context of trace content in the material.Last but not least, the hydroxy group on the benzene ring is more readily reacted with ethanol than a primary alcohol, contrary to our present observations.Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules29051149/s1.

Figure 4 .
Figure 4.The calculated and experimental ECD spectra of 4 and 5.

Figure 4 .
Figure 4.The calculated and experimental ECD spectra of 4 and 5.

Figure S53 :
The cytotoxic effects of compounds 1 and 3 in RAW264.7 cells.TableS1: Extracted heats and weighting factors of the optimized conformers of 4 and 5. TableS2: The Cartesian coordinates of the lowest-energy conformers for 4 and 5. Author Contributions: Y.-X.C. conceived, designed the experiments the paper, D.C. carried out biological experiments.Y.-Y.L. and X.-P.T. performed chemical experiments.All authors have read and agreed to the published version of the manuscript.