The study protocol was approved by the Ethical Committee of The First Hospital of Xiamen Univesity (Fujian, China) and written informed consent was obtained from all participants.

Water was prepared from distilled water using a Milli-Q system (Millipore Laboratory, Bedford, MA, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and 12-o-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 was supplied by Gibco (Thermo Fisher Scientific Inc., Rockville, MD, USA). A TRAP-polymerase chain reaction (PCR)-ELISA kit was purchased from Roche Diagnostics (Basel, Switzerland). The other solvents used in this study were of analytical purity grade. Sephadex LH-20 was purchased from GE Healthcare Life Sciences (Shanghai, China). Silica gel was obtained from Qingdao Oceanic Chemical Co., Ltd. (Qingdao, China).

Fruit bodies of G. lucidum were purchased from Fujian Xianzhilou Biotechnology Co., Ltd. (Fujian, China) in 2013 and identified by Mr Feng Li at the same institute. A sample of fruiting bodies of G. lucidum was deposited in our institution as voucher specimen (M20130606). EBV early antigen (EA) positive serum and EBV capsid antigen (CA) positive serum were collected from 5 patients (male:femal, 2:3) with NPC at our institution between July and December 2015 according to the diagnostic guidelines from the Chinese Medical Association (21). Patients without positive EA or CA were excluded.

Air dried fruit bodies of G. lucidum (3.0 kg) were ground and extracted with 95% EtOH (6.0 l) three times under reflux for 3 h each time. The solvent was evaporated under reduced pressure and the residue was suspended in water (1.5 l) and partitioned successively with dichloromethane (DCM; 1.5 l) three times. Following this, the solvent was evaporated to yield the DCM extract (65.0 g).

DCM extract was subjected to common chromatography (CC) on silica gel eluted with gradient petroleum ether (PE)/ethyl acetate (EA) (from 100:0 to 10:90; v/v) and gave 7 fractions according to the thin-layer chromatography assay. Fraction 2 was separated on silica gel and crystalized in DCM to give compounds 3 (18.5 mg) and 5 (26.0 mg). Fraction 3 was subjected to CC over Sephadex LH-20 eluted with DCM to yield three subfractions. Subfraction 3 was chromatographed over silica gel with gradient PE/EA (from 100:0 to 50:50; v/v) to obtain compound 4 (21.0 mg). Fraction 5 was handled with silica gel CC eluted with gradient PE/acetone and DCM/EA repeatedly to afford compounds 1 (22.0 mg) and 2 (10.5 mg). Fraction 6 was separated on silica gel CC with gradient DCM/acetone (from 100:0 to 70:30; v/v) and further purified by Sephadex LH-20 CC with isocratic DCM/MeOH (3:1; v/v) to yield compound 1 (27.0 mg).

The compounds obtained were dissolved in CDCl₃ and the nuclear magnetic resonance spectra were recorded on a Bruker DRX400 NMR spectrometer (Bruker, Billerica, MA, USA). Their chemical structures were identified on the basis of the spectra analysis.

B_95-8 cells and Raji cells were purchased from Cell Bank of Shanghai Institute of Biological Sciences (Shanghai, China). Human nasopharyngeal carcinoma 5–8 F cells (NPC 5–8 F cells) were obtained from Nanjing Haeckel Biotechnology Co., Ltd. (Nanjing, China). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin, and incubated in a humid atmosphere containing 5% CO₂ at 37°C.

To determine if there are toxic effects induced by natural compounds on Raji cells and B95-8 cells as well as the inhibitory effect on NPC 5–8 F cells, an MTT assay was performed. Cells in the flask were adjusted to a density of 1×10⁶/ml and 100 µl suspension was added into 96-well microplates for each well. Following this, the cells were divided into control group and test groups. Test groups were administered with compounds at a concentration of 1 mM. Following incubation for 48 h, MTT was added into each well at 0.5 mg/ml further incubated for 4 h at 37°C. The medium was subsequently removed and DMSO was used to dissolve the formazan. After 10 min, the absorbance was measured at 550 nm on a microplate reader. Cell viability was expressed as a relative percentage of optical density (OD) values compared with the control group.

Inhibiton of EBV EA activation was measured as previously described (22,23). Raji cells, the EBV genome carrying lymphoblastoid cells derived from Burkitt's lymphoma, were adjusted to 1×10⁶/ml and cultured in 1 ml RPMI-1640 for 48 h at 37°C, with the medium containing 4 µmol n-butyric acid and 32 pmol TPA as co-inducers, as well as 16 or 3.2 nmol compounds in DMSO. Smears were prepared from the cell suspension. Raji cells expressing EBV EA were stained with EBV EA-positive serum from patients with NPC and detected through an indirect immunofluorescence technique (24). The number of stained cells (positive cells) among 500 cells was recorded. Each assay was carried out three times. Inhibition of EBV EA activation was represented as a relative ratio compared with the positive control group (100%), which was exposed to 4 µmol n-butyric acid and 32 pmol TPA. In the experiments, the ratio of EBV EA activation was typically ~35%.

To further evaluate the inhibitory effects of compounds obtained on EBV antigens, B_95-8 cells, the EBV-transformed tamarin cells were employed. According to a previously published protocol (22), the experimental procedure is similar to the assay above. Cells were diluted to 1×10⁶/ml and cultivated in 1 ml medium containing 4 µmol n-butyric acid and 16 or 3.2 nmol of the compounds obtained. Following incubation at 37°C for 48 h, smears were made and the cells were stained with EBV CA positive serum from patients with NPC. In total, 500 cells were counted and the number of stained cells was recorded. The inhibitory effect on EBV CA activation was expressed as a relative ratio compared with the positive control group (100%) treated with 4 µmol n-butyric acid. The ratio of EBV CA activation was ~30%.

The inhibitory effects of these compounds on telomerase were determined based on the telomeric repeat amplification protocol (TRAP) (25). Herein, the TRAP-PCR-ELISA kit was employed according to the manufacturer's instructions, as previously described (26). Briefly, the NPC 5–6 F cells (5×10⁴ cells/ml) in logarithmic growth were incubated with the compounds at 10 µM for 24 h. Subsequently, cells were washed with PBS and lysed with lysis buffer. Subsequently, the lysate was centrifuged at 15,000 × g for 20 min at 4°C to isolate the supernatant for analysis. In total, 2 µl cellular extract was added into the reaction mixture. PCR was performed at 94°C for 120 sec for initial denaturation, followed by 35 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 90 sec. Subsequently, PCR products (20 µl) were hybridized with a digoxigenin-labeled telomeric repeat specific detection probe (Roche Diagnostics). PCR products were immobilized to a streptavidin-coated microplate via a biotin-labeled primer (5′-biotin-ACGACGTCCATAAGCA ACT-3′; Roche Diagnostics). Immobilized DNA fragments were detected with a peroxidase-conjugated anti-digoxigenin antibody (cat. no. 1185466; 1:1,000; Roche Diagnostics) and visualized in the presence of the stop regent. The absorbance was recorded on a microplate reader at 490 nm. Results were presented as the inhibition ratio compared with the control group.

According to the results of telomerase inhibition, compound 1, a typical triterpenoid in G. lucidum, was chosen as the ligand to reveal the binding mode with telomerase through molecular docking. The 3D structure of compound 1 was generated by SYBYL sketch software X2.0 (Tripos, Inc., St. Louis, MO, USA). The protein crystal structure was retrieved from Protein Data Bank (http://www.rcsb.org/pdb) (PDB code, 3DU6); hydrogen atoms and charges were added, and water molecules were removed. The docking process was implemented using Surflex-Dock software X2.0 (Tripos, Inc.). In total, 20 ligand conformations were obtained.

Lipophilicity/hydrophilicity has a strong influence on the ADME properties of drugs in vivo. Hydrophobic drugs are inclined to bind to hydrophobic sites while hydrophilic ones favor hydrophilic positions. Hence, the lipophilicity/hydrophilicity expressed as logp was theoretically calculated to elucidate the physicochemical properties of these compounds. Typically, the values of logp for drugs were between 2 and 5, which can be considered to indicate that the drugs are easily delivered to the binding sites (27).

Polar surface area (PSA) is defined as the sum of the surface over all polar atoms, primarily oxygen and nitrogen atoms, and their attached hydrogen atoms. PSA is typically used to optimize the drug's ability to permeate cells (28). Molecules with a PSA of >140 angstroms² tend to be poor at permeating cell membranes.

All data were expressed as the mean ± standard deviation. GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was employed for data analyses. Statistical differences of the data were analyzed by one way analysis of variance followed by Dunnetts test for multiple comparisons, and Student's t-test was employed for single comparisons. P<0.05 was considered to indicate a statistically significant difference.

Phytochemical studies on the DCM extract of G. lucidum led to the isolation of five natural compounds. All the compounds showed positive results via the Liebermann-Burchard reaction. Their structures were identified as ganoderic acid A (compound 1), ganoderic acid B (compound 2), ganoderol B (compound 3), ganodermanontriol (compound 4), and ganodermanondiol (compound 5) (Fig. 1) on the analysis of nuclear magnetic resonance spectra and comparison of the data with existing literature (29–31).

To evaluate whether these triterpenoids had toxic effects on Raji cells, B_95-8 cells and NPC 5–8 F cells. MTT assay was employed. The results demonstrated that the triterpenoids did not affect the survival of these cell lines and had no toxic effects against Raji cells and B_95-8 cells, even when treated with a concentration of 1 mM (Fig. 2). These results indicated the inhibitory effects of these compounds on EBV EA and CA activation should be further investigated. Conversely, the viability of NPC 5–8 F cells was significantly reduced by all five triterpenoids at 1 mM. The minimum viability was 81.87±5.10% after treatment with compound 1, and the maximum viability was 89.67±2.12% induced by compound 5.

The inhibitory effects of triterpenoids on EBV EA and CA activation were assessed via an indirect immunofluorescence assay. The results indicated that all five compounds contributed to the inhibition of EBV EA and CA activation (Fig. 3). When administered at a high concentration (16 nmol), compounds 1 and 2 exhibited the most potent inhibitory effects on EBV EA activation (18.97±1.76 and 17.40±2.10%), as compared with the others; all compounds were able to significantly inhibit the activation of EBV CA. At the low concentration of 3.2 nmol, all the compounds exhibited significant, yet moderate, inhibitory effects on EBV EA and CA. Of all the compounds, compounds 1 and 2 exhibited slightly greater inhibitiory effects (62.43±2.30 and 63.53±2.11%) than the others three compounds at 3.2 nmol.

Activity of telomerase in NPC 5–8 F cells was detected via the TRAP-PCR-ELISA method. The triterpenoids obtained exhibited significant inhibitory effects on telomerase. The relative inhibition ratio ranged from 78.62±6.41 to 82.79±3.12% (Fig. 4). There was no notable difference in the inhibitory effects among these compounds.

Molecular docking provides additional information about the interaction of compound 1 with telomerase. As shown in Fig. 5, the entire molecule of compound 1 is able to completely enter the binding pocket. The privileged conformation of compound 1 can bind to amino acid residues through hydrogen bonds, van der Waals force, electrostatic interaction and hydrophobic interaction. The 7-hydroxyl group of compound 1 forms a hydrogen bond with ASN-142 (2.2 Å) as a hydrogen donor. At the same time, 15-hydroxyl, 23-carbonyl and 26-carboxyl groups interacts as hydrogen acceptors with ASN-446 (1.9 Å) and LYS-416 (2.1 and 2.1 Å) to form hydrogen bonds (Fig. 5A). Compound 1 also interacts with LYS-406 and LYS-416 through electrostatic interaction. LEU-141, LEU-404, PHE-443 and ILE-444 contribute to the hydrophobic interaction. Meanwhile, van der Waals forces exist between compound 1 and several residues, including ASN-142, GLY-143, GLY-391, THR-403, TYR-405, LYS-406, LYS-416, CYS-445, ASN-446, SER-447 and CYS-473 (Fig. 5B). The total score was 6.67, which suggests that there is moderate interaction between the ligand and telomerase. The docking results indicate that compound 1 is able to inhibit telomerase as a ligand.

As shown in Table I, the logp values for the identified compounds 1 and 2 fall in the range of 2 to 5, which indicates that they shall be well delivered to the binding sites. Compounds 3–5 exhibited higher logp values when compared with compounds 1 and 2. On the contrary, the trend for PSA was the opposite; compounds 3–5 exhibited better permeability than compounds 1 and 2, which may attributed to the carboxyl groups of the two compounds. The oxygen atoms of carboxyl groups increase the PSA through the elevation of polarity.