Terminal Cyclohexane-Type Meroterpenoids from the Fruiting Bodies of Ganoderma cochlear

Eleven new cyclohexane-type meroterpenoids (1, 3–5, 7, 8, 11–15) and four known similar meroterpenoids (2, 6, 9, and 10) were isolated from Ganoderma cochlear. Their structures and absolute configurations at stereogenic centers were elucidated by using HRESIMS, NMR spectroscopy and computational methods. In addition, the structure of the known meroterpenoid, cochlearol G (2), was revised, and the absolute configurations at the stereogenic centers of known meroterpenoids 9 and 10 were determined. All the isolated meroterpenoids were evaluated for their activities against renal fibrosis and triple negative breast cancer, and their insulin resistance. The results of the renal fibrosis study showed that meroterpenoid 11 inhibits over-expression of fibronectin, collagen I and α-SMA. Results of the wound healing study revealed that 4, 6 and 8 significantly inhibit migration of BT549 cells. Observations made in Western blotting experiments showed that 6 decreases the levels of TWIST1 and ZEB1, and increases the level of E-cadherin. Finally, meroterpenoids 7, 9, 11, and 15 significantly up-regulate p-AMPK protein expression in normal L6 myotubes cells.


INTRODUCTION
Ganoderma is not only a famous Chinese medicine, it is also used globally as a food, in the form of tea, coffee and other beverages, and in syrups and dietary supplements (Wang et al., 2020;Kumar, 2021). Polysaccharides and triterpenoids are representative of the important biologically active components of Ganoderma (Wang et al., 2020). Recent ongoing research studies exploring Ganoderma demonstrated that it contains meroterpenoid components that possess extensive biological activities, such as renal protection and neuroprotection, anti-inflammation, -tumor and -oxidation properties, and analgesic effects . These studies led to a deeper understanding of the components of Ganoderma and insight into active ingredients responsible for its traditional medical properties.
Ganoderma meroterpenoids comprise a class of substances with great potential, not only because they contain a variety of structural subtypes, but also because many members possess a host of biological activities. For instance, in 2009 ganomycin I was found to inhibit HIV-1 protease, and then in 2014 it was shown to have inhibitory effects on the production of monocyte chemotactic protein 1 (MCP-1) and fibronectin. Following these discoveries, ganomycin I was observed to inhibit NSC proliferation in 2015, and then in 2017 it was discovered to display hypoglycemic, hypolipidemic and insulin-sensitizing effects (Dine et al., 2009;Luo et al., 2015;Yan et al., 2015;Wang et al., 2017). As a result, we have been engaged in a program to isolate and identify new Ganoderma meroterpenoids and to assess their unique biological activities. In a previous effort, we found that these substances have inhibitory effects on renal fibrosis Meng et al., 2021). In the current investigation, we isolated fifteen terminal cyclohexane-type meroterpenoids (1-15) from Ganoderma cochlear and evaluated their biological activities against renal fibrosis and insulin resistance. Breast cancer is one of the malignant cancer, and the morbidity and mortality is highest in women, with triple negative breast cancer (TNBC) being. Based on the fact that Ganoderma has been used to treat cancer, we also investigated the activities of the meroterpenoids against cells of triple negative breast cancer, which is difficult to treat cancers and has an extremely high mortality rate (Collignon et al., 2016). The results of this study are described below.

Fungal Material
The dried fruiting bodies of G. cochlear were purchased from Tongkang Pharmaceutical Co. Ltd. Guangdong province, China, in July 2014. This fungus was authenticated by Prof. Zhu-Liang Yang at Kunming Institute of Botany, Chinese Academy of Sciences, China, and a voucher specimen (CHYX-0589) is deposited at Institute for Inheritance-Based Innovation of Chinese Medicine, Shenzhen University Health Science Center, China.

Extraction and Isolation
Powders of G. cochlear (200 kg) fruiting bodies were extracted with refluxing 80% EtOH (3 × 120 L, 4, 3, 3 h) and the extract was concentrated under reduced pressure to afford a crude residue. An aliquot (8 kg of the residue corresponding to 95 kg fungal material) was suspended in water and extracted three times with EtOAc, followed by concentration of the combined extracts to afford an EtOAc soluble residue (4 kg). The residue was subjected to silica gel column using an eluant comprised of increasing amounts of acetone in petroleum ether to provide four parts  .4 (0.3 g) was divided into two subfractions by using semi-preparative HPLC (MeOH:H 2 O containing 0.05% TFA, 55%, flow rate: 3 mL/ min), the first subfraction was subjected to chiral Daicel Chiralpak IC column chromatography (n-hexane/ethanol containing 0.05% TFA, 90:10) to yield 4 (18.2 mg, t R 19.7 min) and 5 (17.6 mg, t R 24. 1 min).

Renal Fibrosis Activity Assay
TGF-β1 induced rat renal proximal tubular cells (NRK-52E) were used to assess expression of the target gene. The cell culture method, and cell viability and western blotting assays were conducted following our previously reported protocols (Meng et al., 2021

Cell Viability Assay
Cell viability was evaluated by using a CCK-8 assay kit (meilunbio, Cat No. MA0218, China) according to the manufacturer's instructions. MDA-MB-231, BT549 and HCC1806 cells were seeded into 96-well plates with 3 × 10 3 cells per well. After 72 h exposure in the medium containing desired compounds (20 μM), 100 μL fresh medium including 10 μL CCK-8 reagent was added, and then the plates were incubated at 37°C for 2 h. The absorbances of CCK-8 in each well was measured at 450 and 600 nm by using a Cytation5 (BioTek, United States). DMSO was used as a control. Each sample was plated in triplicate.

Wound Healing Assays
Confluent MDA-MB-231, BT549, and HCC1806 cells were wounded by scratching using Wound Making Tool-Auto Scratch (BioTek, United States). After exposure for 24 h in the medium containing desired compounds (20 μM), the scratched gap area of each cell monolayer was photographed by using Cytation5 (BioTek, United States) and quantified by using Image-Pro Plus 6.0. (http://rsb. info.nih.gov/ij/download.html). The migration efficiency of each cell was then determined as average percentage of closure of the scratch area. Each sample was plated in triplicate. All product recommended protocols were followed.

Western Blot Analysis
The total proteins of the whole cell lysates were separated by using SDS-PAGE and transferred to a PVDF membrane (Millipore, Cat No. IPVH00010, United States). After being probed with primary antibodies overnight at 4°C, the proteins of interest were then detected using HRP-conjugated IgG (CST, Cat No.7074P2, United States) and visualized by using ECL substrate (4ABiotech, Cat No.4AW011, China) based imaging with a Minichemi ™ chemiluminescence imaging system (SAGECREATION, China).

Statistical Analysis
Analysis of statistical data, obtained from triplicate measurements, was performed by using the Student's t-test for two groups or by one-way ANOVA for multiple groups. #p < 0.05 was considered to be significant.

Biological Activity Assay on L6 Myotubes Cells
Cell Culture L6 myotubes cells were maintained in α-MEM culture medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin, and incubated at 37°C in an atmosphere of 5% CO 2 .

Western Blotting Analysis
Cells were washed with pre-cold PBS, lysed using RIPA buffer supplemented with Proteinase Inhibitors Cocktail (MCE), and PMSF (MCE). AMPK, p-AMPK, AKT, p-AKT and GAPDH antibody were incubated overnight and the secondary antibody was incubated for 1 h at room temperature. Signals were detected using a Western blotting Imaging System according to the manufacturer's specifications. The following primary antibodies were used for blotting: p-AKT (4060S, CST), AKT (9272S, CST), p-AMPK (2535S, CST), AMPK (2532, CST) and GAPDH (5174S, CST).

RESULTS AND DISCUSSION
Powders of the dried fruiting bodies of the fungus G. cochlear (200 kg) were first extracted with refluxing 80% EtOH and partitioned in water to obtain an ethyl acetate soluble fractions, an qliquote was then subjected to multiple chromatographic separation steps. This procedure led to isolation of fifteen the cyclohexane-type meroterpenoids 1-15 (Figure 1).
Cochlearol G (2) has been previously described as a component of fruiting bodies of G. cochlear by Wang and co-workers (Wang et al., 2019a). Although the ROESY experiment performed by Wang et al. led to assignment of the relative configurations of the two stereogenic centers in the terminal six-member ring of 2, the configuration of the Δ 3(4) double bond remained unresolved. As a result, we carefully analyzed the ROESY spectrum and found the existence of a correlation H 2 -2 (δ H 4.16)/H 2 -5 (δ H 2.65 and 2.42). This finding demonstrated that the Δ 3(4) double bond in 2 has E configuration, rather than the Z configuration depicted by Wang et al. (Wang et al., 2019a).
Ganodercin H (3) was isolated as a yellow gum. The HRESIMS of 3 gave a molecular ion m/z 375.1804 [M + H] + consistent with the molecular formula C 21 H 26 O 6 , indicating nine degrees of unsaturation. Detailed analysis of the 1D and 2D NMR data of 3 and ganotheaecoloid D shows that the E Δ 3(4) -double bond is present in 3 rather than Z-configuration found in ganotheaecoloid D (8). This conclusion is supported by the ROESY correlation in 3 of H-2 (δ H 4.09)/H-5 (δ H 2.45). The relative configurations of their stereogenic centers in the terminal cyclohexane ring in 3 were assigned as 6R*,10S* by using ROESY correlation of H-6 (δ H 1.94)/H-10 (δ H 3.38). Moreover, the absolute configurations at these centers were assigned as 6R,10S by comparing its CD spectrum to that of ganotheaecoloid D.
Two isolated meroterpenoids 7 (ganodercin K, yellow gum) and 8 (3-epi-ganodercin K, yellow gum) were found to have the same molecular formula (C 21 H 28 O 6 ) and nearly identical NMR data, suggesting that they have the same planar structure with both lacking unsaturation in chains connecting the aryl and cyclohexane moieties. Indeed, a detailed comparison of their 1D NMR data with those of 1 shows that 7 and 8 lack the Δ 3(4) double bond in 1, a conclusion supported by 1 H-1 H COSY correlations of H-2/H-3/H-4/H-5, and the HMBC correlations of H-2/C-1, C-3, C-4, C-15 and H-3/C-4, C-15. It was likely that 7 and 8 are diastereomers having 3S*,10S* and 3R*,10S* relative configurations at their stereogenic centers. Analysis of the CD spectra of 7 and 8 enabled the use of computational methods to elucidate the absolute configurations at their stereogenic centers. As seen by viewing Figure 4, the experimental CD spectrum of 7 well matches the calculated ECD curve of 3R,10S. In addition, the calculated ECD spectrum of 3S,10S matches the experimental CD spectrum of 8.
Compounds 9 and 10 were previously isolated from G. theaecolum by Luo et al. (Luo et al., 2018) and characterized the enantiomers of ganotheaecoloid C. However, the absolute configurations at the three stereogenic centers in these substances were not determined in the earlier effort. As a result, we used TDDFT-ECD calculations to determine their absolute configurations. It can be seen from viewing Figure 4, that the experimental CD spectrum of 9 agrees well with the calculated Frontiers in Chemistry | www.frontiersin.org December 2021 | Volume 9 | Article 783705 8 ECD curve of the 3S,6R,10S stereoisomer. Importantly, the experimental CD spectrum of 10 is in close agreement with the calculated ECD spectrum of the 3R,6R,10S stereoisomer, showing that 9 and 10 are actually epimers rather than enantiomers. By carefully analyzing the 1D NMR data reported by Luo, it was found that pairs of signals are present in the 13 C NMR spectrum, supporting our conclusion. The revised structures of 9 and 10 were renamed as ganodercin L for 9 and 3-epi-ganodercin L for 10. Ganodercin M (11) was obtained as a yellow gum and has the molecular formula C 22 H 30 O 6 (HRESIMS ion observed at m/z 391.2120 [M + H] + , calcd for 391.2115). The NMR data of 11 are similar to those of 9, except that the free C-15 carboxylic acid group in 9 is a methyl ester in 11. This conclusion is confirmed by observations of the HMBC correlation of OCH 3 /C-15. The ROESY correlation of H-6/H-10 showed that the relative configurations at the stereogenic centers in the terminal ring in 11 are 6R*,10S*. The computational methods applied to 9 and 10 were used to determine the absolute configurations at the stereogenic centers in 11. Matching experimental and calculated CD spectra showed that 11 is the 3S,6R,10S stereoisomer. The calculated ECD spectrum of (3S,10S)-7a at B3LYP/6-31G (d,p) level, σ 0.25 eV; shift +20 nm and calculated ECD spectrum of (3R,10S)-7b at B3LYP/6-31G (d,p) level, σ 0.23 eV; shift +20 nm. (E): The calculated ECD spectrum of (3S,6R,10S)-9a at CAM-B3LYP/def2SVP level, σ 0.28 eV; shift +13 nm and calculated ECD spectrum of (3R,6R,10S)-9b at B3LYP/6-31G (d,p) level, σ 0.20 eV; shift +16 nm. (F): The calculated ECD spectrum of (6S,6R,10S)-13 at CAM-B3LYP/def2SVP level, σ 0.20 eV; shift -2 nm.
The NMR spectroscopic data of ganodercin N (12) (yellow powder, C 21 H 26 O 6 ) are similar to those of (-)-ganotheaecoloid F (Luo et al., 2018). One difference is that a methine (δ C 57.1) and a nonprotonated carbon (δ C 88.6) are present in 12 instead of the double bond (δ C 136.7 and δ C 128.6) in (-)-ganotheaecoloid F. This proposal is supported by the HMBC correlations of H 3 -14 (δ H 1.28)/C-6 (δ C 57.1), C-7 (δ C 88.6), C-8 (δ C 39.7). Also, the downfield chemical shift of C-10 at 87.6 ppm, indicated that C-10 is the oxygenated. Moreover, the HMBC correlation of H-10/C-7 indicated that C-10 is linked to C-7 via an oxygen bridge. The relative configurations at the stereogenic centers in the terminal bicyclic ring in 12 were determined by using ROESY data. ROESY correlation of H-6 (δ H 1.26)/H 3 -12 (δ H 1.02) suggested that both H-6 and CH 3 -12 have a β-orientation. Accordingly, the ROESY correlation of H-10 (δ H 3.70)/H 3 -13 (δ H 0.97) revealed that H-10 and CH 3 -13 have a α-orientation. The C-14 methyl was determined to have a α-orientation, when consideration is given to the existence of the oxygen bridge. This proposal was further supported by the ROESY correlation of H 2 -4 (δ H 2.58 and 2.52)/H 3 -14. Thus, the relative configurations at the stereogenic centers in 12 were assigned as 6S*,7R*,10S*. Since no strong correlation of H-2/H-4 was observed, we hypothesized that the Δ 2(3) double bond in 12 is E. Density functional theory (DFT, B3LYP/6-311G(d,p) level) was used to verify this proposal. The experimental NMR data of 12 were compared with those calculated for 12a (E-isomer) and 12b (Z-isomer). It was found that the calculated 13 C NMR data for 12a possesses the highest R 2 value and a 100% probability in DP4+ analysis. Because no observable Cotton effects are present in the experimental CD spectrum of 12, it was not possible to use ECD calculations to assign stereochemistry. However, the absolute configuration at the stereogenic centers in 12 were assigned as 6S,7R,10S by comparing the calculated specific optical rotation +22.2 for (6S,7R,10S) with the experimental one of 12 ([α] D 20 +22.5). Ganodercin O (13) has the same molecular formula as 12 and similar NMR data, differing only by resonances associated with the location of the double bond. The position of double bond in 13 was demonstrated to be Δ 3(4) by the observation of 1 H-1 H COSY correlations of H-4/H-5/H-6, and the HMBC correlations of H-2/C-1, C-3, C-4, C-15 and H-4/C-15. The double bond Δ 7(8) was deduced to have the E-configuration by existence of the ROESY correlation of H-2/H-4. Similar to that in 12, H-6 and CH 3 -12 were determined to have a β-orientation by presence of the ROESY correlation of H-6/H 3 -12. Moreover, H-10 and CH 3 -14 were assigned to have a α-orientation by the ROESY correlations between H-10/H 3 -13, H 2 -5/H-13, H-14. As a result, the relative configurations of the stereogenic centers in 13 were determined to be 6S*,7R*,10S*. Finally, a match between the computed [B3LYP/6-31 g(d,p)] ECD spectrum of 6S,7R,10S-13 and the experimental one provided the absolute configurations at the stereogenic centers in 13 (Figure 1).
Ganodercin P (14) and 3-epi-ganodercin P (15) were found to have the same molecular formula of C 21 H 28 O 6 by using HRESIMS. Careful analysis revealed that their NMR data were similar, suggesting that they have the same planar structures. The observations of 1 H-1 H COSY correlations of H-2/H-3/H-4, and the HMBC correlations of H-2/C-1, C-15 and H-3/C-15 clearly revealed that one methylene (C-2) and one methine (C-3) in both 14 and 15 replace the E-double bond in 12. Because 14 and 15 contain the same terminal bicyclic ring that is present in 12 and 13, the relative configurations at the stereogenic centers were determined to be 6S*,7R*,10S* by the presence of ROESY correlations H-6/H 3 -12, H-10/H 3 -13, H 2 -5/  Frontiers in Chemistry | www.frontiersin.org December 2021 | Volume 9 | Article 783705 11 the terminal oxygen bridge doe not contribute to the Cotton effects ( Figure 5). As a result, the absolute configuration of C-3 in 14 is R and that of compound 15 is S.
X-ray crustallographic analysis of 15 was carried out, which was generated by crystallization from Kappa single diffractometer in CuKα with a Flack parameter of -0.08 (7). Analysis of the crystal data leads to assignment of the absolute configurations at the stereogenic centers in 15 to be 3S,6S,7R,10S. Since 14 and 15 are C-3 epimers, the absolute configurations at the centers in 14 are 3R,6S,7R,10S.
The final substance isolated was (−)-ganotheaecoloid F (6), which was identified by comparison its spectroscopic data with those reported in the literature (Luo et al., 2018).
In this study, we have determined the structures and absolute configurations at the stereogenic centers in 15 meroterpenoids isolated from dried fruiting bodies of G. cochlear. It is worthy noting that all of these substances contain a terminal cyclohexane ring and all are enantiomerically pure. Meroterpenoids of this type isolated from Ganoderma have been also found to exist in enantiomerically pure (Luo et al., 2018;Wang et al., 2019a;Wang et al., 2019b). Among them, the absolute configuration of four structures was successfully determined. The absolute configurations at the centers in these substances as well as the fact that C-10 in all have the S-configuration might be related to their biosynthetic origin, and might aid subsequent structural identification of analogues.
The protective activity of the meroterpenoids isolated in this effort were assessed by observing the expression of renal fibroblast biomarkers in TGF-β1-induced NRK-52E, which plays an important role in stimulation of renal fibroblasts (Zhang et al., 2012;Meng et al., 2015). The results showed that 11 displays selective inhibitory activity in that it significantly inhibits over-expression of fibronectin, collagen I and α-SMA Frontiers in Chemistry | www.frontiersin.org December 2021 | Volume 9 | Article 783705 at the protein level at a concentration of 40 μM ( Figures  6A,B). In addition, western blot analysis, carried out in a dose-concentration dependent manner, demonstrated that the optimum activity of 11 is 40 μM ( Figure 6D). To explore the mechanism underlying the antifibrotic effect of, Smad2/3 phosphorylation was investigated. We found that 11 has no effect on phosphorylation of Smad2 or Smad3 in TGF-β1-induced NRK-52E cells at concentrations of 10 μM, 20 and 40 μM ( Figure 6C), suggesting that its effects could play potential roles in renal fibrosis through a non-Smad pathway. We investigated the cellular phenotype promoted by the isolated substances in breast cancer cells by using a cell viability and a wound healing assay in three TNBC cell lines including MDA-MB-231, BT549 and HCC1806. Inspection of the plots given in Figure 7A shows that treatment with 7 and 9 results in significant decreases in cell viability of 29.2 and 26.3% in MDA-MB-231 and HCC1806 cells, respectively. The other meroterpenoids have negligible inhibitory effects on cell viability even at concentrations as high as 20 μM ( Figure 7A). Interestingly, although all the fifteen substances have rather low cytotoxicities toward these three breast cancer cell lines, three of the isolates including 4, 6 and 8 significant inhibit the migration ability of BT549 cells. All of the other meroterpenoids display no significant effects on MDA-MB-231 and HCC1806 cells ( Figures 7B,C). Moreover, we observed that treatment with 4 and 8 markedly decreases the protein level of TWIST1 and ZEB1 without noticeably affecting the E-cadherin level in BT549 cell lysates. Meroterpenoid 6 not only decreases the protein level of TWIST1 and ZEB1, it also increases the protein level of E-cadherin ( Figure 7D). ZEB1, E-cadherin and TWIST1 are generally acknowledged to be transcriptional factors driving the Epithelial-Mesenchymal Transition (EMT), one of the most important pathogenic events occurring in the initiation of cancer metastasis. Thus, the data indicate that 4, 6 and 8 suppress the metastatic potential of TNBC cells through down regulation of EMT, and consequently they are promising lead compounds for the development of the anti-cancer drugs against metastasis of TNBC.
All the isolated meroterpenoids at a concentration of 20 μM were exposed to normal L6 myotubes cells for 24 h. The control group was treated with insulin (INS, 100 nM, 15 min) and Berberine (BBR, 10 μM, 24 h). Notably, INS and BBR treatment led to significant increases in the phosphorylation AKT and AMPK, respectively. Compared with the control group, the meroterpenoids have no significant impact on the AKT pathway. Meanwhile, 7, 9, 11 and 15 significantly upregulate p-AMPK protein expression (Figure 8). This finding suggests that these substances might enhance insulin sensitivity by activating AMP-activated protein kinase (AMPK) in normal L6 myotubes cells.

CONCLUSION
In conclusion, the study described above resulted in the isolation of eleven new meroterpenoids, and four known meroterpenoids. The structure of cochlearol G (2) was revised, and the absolute configurations at the stereogenic centers in 9 and 10 were determined by using ECD calculations. Biological studies related to renal fibrosis showed that (1) 11 inhibits over-expression of fibronectin, collagen I and α-SMA, (2) 4, 6 and 8 significantly inhibitof the migration ability of BT549 cells, (3) 6 decreases the protein level of TWIST1 and ZEB1 and increases the protein level of E-cadherin, and (4) 7, 9, 11 and 15 significantly up-regulate p-AMPK protein expression in normal L6 myotubes cells.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
YC designed the research. FQ conducted chemical experiments. TX, YL, and DC conducted biological experiments in vitro. FQ, TX, HZ, LL, and YC analyzed data. FQ and YC wrote and revised the manuscript. All authors discussed the results and commented on the manuscript at all stages.