Cytotoxic lanostane-type triterpenes from the fruiting bodies of Ganoderma lucidum

Abstract A new lanostane-type triterpene, namely 3-oxo-5α-lanosta-7,9(11)-dien-24-oic acid methyl ester (2), and three known compounds including ganoderal A (1), ganoderiol B (3) and ganodermenonol (4) were isolated from the fruiting bodies of Ganoderma lucidum by silica gel column chromatography and Sephadex LH-20 column chromatography. Their structures were determined by extensive NMR data and mass spectral analysis. The in vitro cytotoxic activity of the isolated compounds against SK-Hep-1, HepG2, Hela and Hela/VCR cancer cell lines was assessed by using MTT assay. The IC50 values of compound 1 were 43.09 ± 2.86, 42.31 ± 1.78 and 46.51 ± 1.95 μM in SK-Hep-1, HepG2 and Hela cells, respectively, after 48 h. The IC50 values of compound 4 were 44.70 ± 2.32 and 41.33 ± 2.15 μM in Hela and Hela/VCR cells, respectively, after 48 h. Graphical Abstract


Introduction
Ganoderma lucidum (G. lucidum), commonly known as Lingzhi in Chinese, has been used as an herbal remedy in China for more than 2000 years . G. lucidum contains various compounds such as triterpenes, polysaccharides, alkaloids, flavonoids, steroids, proteins, amino acids, enzymes, vitamins and minerals (Ahmad 2018). Lanostane-type triterpenes are typical constituents of G. lucidum which attract considerable attention due to their significant pharmacological effects such as antitumor, anti-inflammation, liver protection, anti-virus, bacteriostasis, anti-oxidation, anti-radiation and reversal of Cancer Multidrug Resistance (MDR) (Satria et al. 2019;Wang et al. 2020;Wu et al. 2022). Mycochemical investigation has led to the isolation of more than 300 different types of triterpenes from the fruiting bodies, mycelia and spores of G. lucidum over the previous 4 decades (Ahmad 2020;Zhang et al. 2022). We reported two new lanostane-type triterpenes isolated from the fruiting bodies of G. lucidum in the previous mycochemical investigation of Ganoderma lucidum (Li et al. 2013(Li et al. , 2016. This article describes the isolation and structure elucidation of a new lanostane-type triterpene, namely 3-oxo-5a-lanosta-7,9(11)-dien-24-oic acid methyl ester (2), and three known compounds from the fruiting bodies of G. lucidum (Figure 1). We also describe the cytotoxic effect of the isolated compounds on SK-Hep-1, HepG2, Hela and Hela/ VCR cancer cells.
All compounds were tested for cytotoxicity against human liver cancer (SK-Hep-1 and HepG2) and human cervical carcinoma (Hela and Hela/VCR) cell lines by using MTT assay. The IC 50 values were compiled in the Table S3. It could be observed from cytotoxicity assays that compound 1 showed cytotoxic activity against SK-Hep-1, HepG2 and Hela cells with IC 50 values of 43.09 ± 2.86, 42.31 ± 1.78 and 46.51 ± 1.95 lM, respectively. Compound 4 showed cytotoxic activity against Hela and Hela/VCR cell lines with IC 50 values of 44.70 ± 2.32 and 41.33 ± 2.15 lM, respectively. Compound 2 exhibited cytotoxic activity against Hela/VCR cell lines with IC 50 values of 87.13 ± 3.66 lM. In addition, Compound 3 showed weak cytotoxicity against these tumor cells lines (IC 50 >100 lM). Compared with compounds 1-4, adriamycin, as a positive control, showed significant cytotoxicity against these tumor cells lines within 0.66-5.89 lM range of IC 50 value. Several antitumor triterpenes had been obtained from G. lucidum such as ganodermanondiol, ganodermanontriol, ganoderiol A and lucialdehyde C (Gao et al. 2002;Chen et al. 2017). The cytotoxicity test results indicated that ganoderal A (1) and ganodermenonol (4) might be antitumor triterpenes of G. lucidum.

General
UV spectra were measured using a Varian Cary 50 Bio UV-visible spectrophotometer. IR spectra were measured using a Thermo Scientific Nicolet iS50 FT-IR spectrometer. Nuclear magnetic resonance (NMR) spectra were obtained with a Bruker Avance III 500 MHz spectrometer. HR-EI-MS spectra were recorded on a Thermo Q-Extractive mass spectrometer. Column chromatography was performed with a silica gel (200 À 300 mesh and 300 À 400 mesh, Qingdao Marine Chemical Plant, China). TLC analyses were carried out using precoated silica gel GF 254 (Qingdao Marine Chemical Plant, China). Spots were detected on TLC under UV light or by heating after spraying with phospho-molybdic acid reagent. Adriamycin, dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all purchased from Sigma-Aldrich (St. Louis, MO, USA).
Human liver cancer cell lines SK-Hep-1 and HepG2 and human cervical carcinoma cell lines Hela/VCR and Hela were obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China), and cultured in RPMI1640 medium (Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 IU/mL penicillin, and 100 mg/mL streptomycin (Sigma, St. Louis, MO, USA) in a humidified incubator containing 5% CO 2 at 37 C.

Mushroom material
The cultivated Ganoderma lucidum (Leyss. ex Fr.) Karst. was purchased from Fujian Xianzhilou Biological Science and Technology Co., Ltd, Fuzhou, China in 2018, and identified by research fellow Xiaoqing Zhang, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. A voucher specimen (No. HMAS 247932) had been deposited in the Fungarium of Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

Cytotoxicity assay
The cytotoxicity of compounds 1-4 and adriamycin was analysed following the procedure described (Abaci et al. 2022) in a 96-well plate. 135 lL of cells suspension at a density of 2 Â 10 4 cells/mL were seeded in the growth medium into 96-well microtiter plates and incubated at 37 C in a 5% CO 2 incubator. The compounds were dissolved in DMSO and adjusted to different concentrations by diluting with the growth medium. The final DMSO concentration was adjusted to <0.2%. After standing for 24 h, the cells were treated with the medium containing different concentrations of compounds respectively. The same volume of medium with 0.2% DMSO was added to the negative control wells. After the test sample was added for 48 h, MTT reagent (20 lL, 5 mg/mL) was added to each well and incubated for 4 h. After careful removal of the MTT solution, 180 lL of DMSO was added to each well. Absorbance was measured on a microplate reader (Thermo Scientific, Model Multiskan FC, USA) with a test wavelength of 570 nm. Drug concentrations producing 50% inhibitory concentration (IC 50 ) were determined by curve fitting analysis using Prism software (GraphPad Software 6.0, San Diego, CA).

Statistics
Student's t test was used for the statistical analysis through SPSS statistical software (SPSS 16.0). p<0.05 or p<0.01 was considered statistically significant. Data were expressed as mean ± standard deviation (SD). All the experiments were repeated at least three times.

Conclusion
A new lanostane-type triterpene, namely 3-oxo-5a-lanosta-7,9(11)-dien-24-oic acid methyl ester, and three known compounds including ganoderal A, ganoderiol B and ganodermenonol were isolated from the fruiting bodies of G. lucidum. Ganoderal A and ganodermenonol showed anti-cancer activity and might be the active components of G. lucidum.

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