Nor-triterpenoids from the fruiting bodies of Ganoderma lucidum and their inhibitory activity against FAAH

Abstract Two new nor-triterpenoids ganodrenol A (1), B (2), and a new natural product ganodrenol C (3), along with three known nor-triterpenoids (4–6) were isolated from the fruiting bodies of Ganoderma lucidum. The chemical structures of these isolates were determined by 1 D and 2 D NMR, HRESIMS, and X-ray crystallography analysis. The inhibitory effects of isolated triterpenoids (1–6) against FAAH were evaluated by an in vitro assay, and compound 4 showed an inhibition rate of 70.27%. In addition, the cytotoxic effect of compounds (1–6) was evaluated against LOVO, MCF-7, and RAW264.7 cells, which displayed no significant cytotoxicity. Graphical Abstract


Introduction
Ganoderma lucidum (G. lucidum) is a kind of large medicinal fungus belonging to family Ganodermataceae, named 'Lingzhi', 'Reishi', and 'Yeongji' in China, Japan, and Korea, respectively according to the American Herbal Pharmacopoeia and Therapeutic Compendium (AHP 2006;Sanodiya et al. 2009;Nahata 2013;Wu et al. 2019). G. lucidum has been used for the treatment of chronic diseases, such as cancer, asthma, bronchitis, hepatitis, hypertension, leucopenia, neurasthenia, and hypercholesterolaemia (Wu et al. 2019). In addition, G. lucidum is also an inhibitor of testosterone-induced prostatic hyperplasia (Nahata and Dixit 2012). Studies have showed that G. lucidum contained various constituents including triterpenoids (Wei et al. 2017), meroterpenoids (Lu et al. 2019;Cai et al. 2021), steroids (Zhang et al. 2008), polysaccharides (Carrieri et al. 2017), peptides (Zhao et al. 2015). Triterpenoids are considered as the major constituents and possess extensive biological activities, such as cytotoxic (Guan et al. 2008;Cheng et al. 2010), anti-inflammatory (Su et al. 2020), hepatoprotective (Wu et al. 2016), FAAH, a-glucosidase, and P-glycoprotein inhibitory activities (Zhao et al. 2015;Lin et al. 2022). Fatty acid amide hydrolase (FAAH) is a vital target enzyme that modulates the intracellular arachidonoylethanolamide level and plays an important role in various neurodegenerative diseases, such as Parkinson's disease, depression, and Alzheimer's disease (Tian et al. 2021). As part of our work about bioactive substances investigation from natural products, the chemical constituents and bioactivity have been investigated for G. lucidum in the present work. The current chemical and bioactive studies were carried out on G. lucidum, which led to the identification of six nor-triterpenoids ( Figure 1). Considering the biological effects of G. lucidum, the FAAH inhibitory effect and cytotoxic activities of all compounds (1 À 6) were assessed. Detailed information on separation, structural elucidation, and bioactivity of 1 À 6 are reported here.
157.1 (C-8), 142.8 (C-9), and 198.2 (C-11)], and an unconjugated ketone carbon [d C 218.3 (C-15)] (Supporting Information Table S1). Analyses of the 1 H and 13 C NMR data of 1 indicated that it was a pentanorlanostane derivative and was similar to those of ganosineniol A (Liu et al. 2012). On the basis of the comparison about the 1 D NMR data of 1 and ganosineniol A, it was deduced that the hydroxyl group at C-15 in ganosineniol A was oxygenated to carbonyl in 1, which was confirmed by 2 D NMR data analysis (Supporting Information Figure S1). Analysis of the HMBC correlations of H-5, H 3 -28, and H 3 -29/C-3, H-7/C-8 and C-9, and H 2 -22/C-17 and C-21 indicated the positions of 3-OH, 7-OH, and 22-OH respectively (Supporting Information Figure S1). In addition, the location of a ketone moiety was assigned at C-15 by the HMBC correlations from H 3 -30 to C-15 (Supporting Information Figure S1). Meanwhile, the a,b-unsaturated ketone at C-8/C-9/C-11 was defined on the basis of the HMBC correlations from H 3 -19 to C-9, H-7 to C-8 and C-9, and H 2 -12 to C-11 (Supporting Information Figure S1). Additionally, the separated spin/spin systems of H 2 -1/H 2 -2/H-3, H-5/H 2 -6/H-7, H 2 -16/H-17/H-20/H 3 -21 and H-20/H 2 -22 were deduced using the 1 HÀ 1 H COSY spectrum (Supporting Information Figure S1). The relative configurations of the hydroxyl groups were elucidated as 3b, 7b-oriented based on the correlations of H-3/ H-5 and H-7/H-5 in the NOESY spectrum (Supporting Information Figure S2). Thus, 1 was elucidated as a pentanorlanostane triterpenoid and named ganodrenol A.
The 1 D NMR spectra and HRESIMS data of 2 indicated that it was an analogue of 1 with the molecular formula C 25 H 36 O 6. Comparison of the NMR data of 2 and 1 indicated that the 7-OH group in 1 was replaced by a carbonyl group (d C 199.0) in 2 and the presence of an additional hydroxyl group at C-12 in 2. This was confirmed by the 2 D NMR data of 2, especially by the HMBC correlations of H 3 -18/C-12, H-5 and H-6/C-7 (Supporting Information Figure S1). The relative configuration of the 3-OH and 12-OH were both elucidated as b-oriented based on the correlations of H-3/H-5 and H-12/H 3 -30 observed in the NOESY spectrum (Supporting Information Figure S2). Therefore, 2 was determined as shown in Figure 1 and named ganodrenol B.
Compound 3 was a hexanorcucurbitane triterpenoid, which was ever synthesized by Ren et al (Ren et al. 2016). However, the 1 H, 13 C NMR data of this compound was not attributed in the literature. It is the first report of an isolation of 3 as the new natural product and named ganodrenol C. Fortunately, the single-crystal of 3 was cultivated suitably for X-ray analysis (Supporting Information Figure S3), which determined the absolute configuration of 3 on the basis of Cu Ka radiation with Flack/Hooft parameter À0.07(13)/-0.09(6) (Supporting Information Table S2).

General experimental procedures
Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. HRESIMS data were acquired on an AB SciexX500r TOF mass spectrometer. The NMR spectra of isolated compounds were acquired with Bruker 600 MHz spectrometer. Analytical HPLC data were collected on an UltiMate 3000 instrument (Thermo Scientific Dionex) equipped with a diode array detector (DAD). MPLC separation was performed on an Agel liquid chromatography instrument with a UV detector. Preparative HPLC separation was performed on an SEP liquid chromatography instrument with a YMC (250 Â 10 mm i.d.) preparative C 18 (5 lm) column. Column chromatography (CC) was performed with silica gel (200 À 300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China). TLC was conducted on pre-coated silica gel GF254 plates (5 Â 10 cm, 2.5 Â 7.5 cm, Qingdao Marine Chemical Inc.). Chromatographic grade methanol was purchased from Sigma-Aldrich. All other solvents were of chemical grade (Kermel Chemical Co. Ltd, Tianjin, China). Spots were visualized under UV light or by spraying with 10% sulfuric acid in 95% aqueous EtOH followed by heating.

Plant material
The ethanolic extracts of the fruiting bodies of Ganoderma lucidum (20120411 G) were purchased from Kingsci Biotechnology Co., Ltd. (China) in July 2012, which were identified by Professor Jingming Jia (Shenyang Pharmaceutical University, p-20140511).

Cell lines and cell culture
All three cell lines were obtained from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China). They were cultured in DMEM (KeyGEN BioTECH Co., Ltd. Nanjing, China) containing 10% fetal bovine serum (FBS, ScienCell, USA) and incubated at a temperature of 37 C in a humidified atmosphere of 5% CO 2 .

Cell viability assay
The cytotoxicity of isolated nor-triterpenoids (100 lM) was evaluated using the cell viability assay for human colon cancer cell line (LOVO), and human breast cancer cell line (MCF-7), and RAW264.7 cell line by CCK-8 method.
In brief, 5 Â 10 3 cells were seeded into 96-well culture plates allowed to adhere for overnight, and then the cells were changed to fresh medium containing the isolated compounds (100 lM) dissolved in DMSO (final concentration, 0.1%). After incubation for 48 h, CCK-8 was added, and the absorbance was measured at 450 nm by Microplate reader (BioTek, USA) (Jin et al. 2021). Cell viability in vehicle control groups was considered 100%. Taxol (5 lM) was used as the positive control. The data points represented the mean value of triplicate experiments.

Bioassay about the inhibitory effects of nor-triterpenoids against FAAH
The inhibitory effects of isolated nor-triterpenoids (100 lM) against FAAH were evaluated using a fluorescent probe (Tian et al. 2021). In a standard 200 lL buffer containing 125 mM Tris-HCl, 1 mM EDTA, 0.1% EDTA (pH ¼ 8), FAAH (2.5 lg/mL) and nortriterpenoids (100 lM) were preincubated at 37 C for 3 min. Then 1 lL fluorescent probe (10 lM) was added to initiate reaction and incubated for another 30 min. Finally, 100 lL of ice acetonitrile was added to terminate the reaction. Then the vails were centrifuged at 4 C for 10 minutes at 20000 g to obtain supernatants for fluorescence intensity detection (k ex 600 nm, k em 660 nm). URB597 as a classic FAAH inhibitor was used a positive drug (50 nM). Values are means of triplicate experiments.

Conclusions
In summary, two new nor-triterpenoids ganodrenol A (1), B (2), and a new natural product ganodrenol C (3), along with three known nor-triterpenoids (4-6) were isolated from the ethanolic extracts of G. lucidum. The undoubted structures of isolated compounds were determined by various spectroscopic data analysis. The discovery of compounds (1-3) increased the chemical diversity of characteristic nor-triterpenoids in G. lucidum. Nor-triterpenoids 1-6 displayed no significant cytotoxicity. What's more, compound 4 was discovered to be a natural inhibitor for FAAH from G. lucidum, which could be used as potential candidate drug for the treatment of neurodegenerative diseases in the future.

Disclosure statement
No potential conflict of interest was reported by the authors.