Chemical investigations and cytotoxic effects of metabolites from Antrodia camphorata against human hepatocellular carcinoma cells

Abstract Antrodia camphorata is used as a medicinal fungus in Taiwan to treat fatigue, food intoxication, and enhance liver function. Here we identified fermented metabolic components from the mycelium of A. camphorata KH37 and explored their anti-hepatoma potentials with study models of human hepatoblastoma cell lines. Bioassay-guided fractionation of the solid fermentation powder of A. camphorata KH37 led to the isolation of one new quinonol, antroquinonol Z (1), and nine known compounds (2–10). Treatment with 10 μM antrocamols LT1 (2) or LT3 (3) reduced cell viability of HepG2 and Huh-7 cells to about 60% in 48 hours. Antroquinonol Z (1) exhibited mild cytotoxicity against Huh-7 cells in 48 and 72 hours. Interestingly, two fractions showed cytotoxicity in HepG2 and Huh-7 cells, even better than compounds isolated from these fractions. The significant cytotoxicity of partially purified samples from A. camphorata KH37 exhibited a potential for developing alternative or complementary therapeutics against hepatoma. Graphical Abstract


Introduction
Natural products have been shown to possess significant pharmacological activities to regulate various body functions for thousands of years. Nowadays, many natural products are developed and processed as pharmaceutical agents alleviating human diseases, such as infection, inflammation, cancer, or metabolic disorders (Ramana et al. 2014). Recent studies indicated that fungi occupy an important position in natural product research because of their plethora of active compounds (Wu et al. 2019). The chemical diversity and biodiversity of fungal metabolites have driven more investigations to explore novel bioactive compounds produced by fungi (Hoeksma et al. 2019 Wu, Yu, Dai & Su), belonging to the Polyporaceae family, is an endemic medicinal fungus to Taiwan . Wild A. camphorata grows tightly in the brown, rotting empty heartwood of Formosan endemic plant Cinnamomum kanehirai (Lauraceous family) (Lu et al. 2013;Chen et al. 2017;Yen et al. 2020). The fruiting body of A. camphorata has long been used as indigenous medicine to relieve food intoxication, abdominal pain, and fatigue and enhance liver function (Lu et al. 2013;Chen et al. 2017;Yen et al. 2020). However, there are still many challenges for the large-scale cultivation and commercialization of A. camphorata, including the mushroom-host specificity, slow growth rate, and scarcity in nature, leading to the high price of wild fruiting bodies for $15,000 to $25,000 per kg (Lu et al. 2013;Kuang et al. 2021). Besides, it is not easy to induce the growth and formation of A. camphorata fruiting-body even under the controlled laboratory culture systems (Lin et al. 2015;Chung et al. 2016). Therefore, the mycelium-based products of A. camphorata are widely used to investigate bioactive compounds because they can quickly produce a large amount of mycelium accompanied by bioactive metabolites (Lin et al. 2015). The previous investigation also indicated that the ethanolic extracts of the mycelium-based products of A. camphorata contain similar important bioactive compounds as their fruiting bodies (Wu et al. 2018).
The characteristic phytochemicals of A. camphorata include triterpenoid, antroquinonol-meroterpenoids, succinic and maleic acid derivatives, steroids, polysaccharides, lignans, benzoquinones, benzenoids, and lactones (Shao et al. 2008;Geethangili and Tzeng 2011;Lu et al. 2013;Chen et al. 2017;Zhang et al. 2017;Senthil Kumar et al. 2020). Triterpenoids are bitter resources mainly observed in the fruiting body of A. camphorata Angamuthu et al. 2019). Some triterpenes showed anti-inflammatory, wound healing, and anticancer activities in previous studies (Ganesan et al. 2019;Wang et al. 2019;Kuang et al. 2021). Antroquinonol-meroterpenoids (ubiquinones) are the representative skeleton compounds in the mycelia of A. camphorata . They possessed bioactive components with cytotoxic (Geethangili and Tzeng 2011;Tong et al. 2017;Zhang et al. 2017), anti-inflammatory (Yang et al. 2009;Lu et al. 2013), and hepato-protective activities (Lu et al. 2011;Lu et al. 2013), as well as the therapeutic potential to alleviate Alzheimer's disease  in previous studies. Succinic and maleic acid derivatives are only found in the fruiting body or mycelium of A. camphorata (Angamuthu et al. 2019). They also possessed anti-inflammatory, cytotoxic, and hepatitis C virus protease inhibition activities in previous studies (Ganesan et al. 2019;Wang et al. 2019). Antrodin C is one of the most famous maleic acids in A. camphorata and has been investigated extensively . Sterols from A. camphorata exhibited significant antioxidant, anti-inflammation, and anticancer activities (Wang et al. 2019;Chao et al. 2021). Polysaccharides could be found in the fruiting body or mycelia of A. camphorata (Wu et al. 2019;Zhang et al. 2019). They are composed of high molecular weight substances and have shown hepatitis B virus (HBV) inhibition and anti-inflammatory activity in previous studies (Wu et al. 2019;Zhang et al. 2019).
Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related death in 2020 (Sung et al. 2021). Hepatocellular carcinoma (HCC) is the most common form of liver cancer and comprises $90% of liver cancer cases (Ogunwobi et al. 2019). The primary cause of HCC in Asia is chronic HBV infection. In contrast, chronic hepatitis C virus (HCV), alcoholic cirrhosis, and non-alcoholic steatohepatitis (NASH) are the main risk factors of HCC in Western countries (Ogunwobi et al. 2019). Other established causes of HCC include alcohol abuse, non-alcoholic fatty liver disease, obesity, type 2 diabetes, and tobacco smoking (Ogunwobi et al. 2019). The treatment options for HCC patients include surgical resection or liver transplantation in early-stage, chemotherapy, targeted cancer therapy, and immunotherapy. However, despite the advancement in the HCC therapies, the survival rate of advanced or metastatic HCC is still far from acceptable (Severi et al. 2010;Ogunwobi et al. 2019;Chen Z et al. 2020). Thus, discovering newer or more potent agents as the combined or alternative therapies for HCC is urgent.
A. camphorata has long been used as a folk remedy for ameliorating liver disorders in Taiwan. Additionally, many studies have suggested the therapeutic potentials of A. camphorata in various liver diseases. Therefore, we were motivated to investigate the bioactive constituents with anti-HCC activities from A. camphorata KH37. This study aimed to isolate secondary metabolites from the solid-state fermentation extracts of A. camphorata KH37 and evaluate their anti-HCC activity with the study models of human hepatoblastoma cell lines HepG2 and Huh-7.

Results and discussion
With the bioactivity-guided fractionation of the solid-state fermentation extracts of A. camphorata KH37, one new quinonol, antroquinonol Z (1), and nine known compounds (2-10) were successfully isolated ( Figure 1). The phytochemical spectra of compound 1 were shown in the Supplementary Materials. The 1D and 2D NMR spectroscopy techniques were used for structural elucidation and the identification of compounds. Moreover, the anti-HCC activities of isolates were evaluated on two different human hepatoblastoma cell lines, HepG2 and Huh-7. The structural identification of the new compound and anti-HCC activity results were as follows.
Unlike the ubiquinone compounds isolated from A. camphorata (Kuang et al. 2021), antroquinonol Z (1) is a ubiquinone compound without the isoprenoid side chain at C-5. Besides, the relative configuration of OH-6/H-7 in 1 was on the same side different from known compounds were on the opposite phase. The skeleton of ubiquinones Figure 2. Effects of Fr. 14, compounds 1-3 and 6 of A. camphorata on the cell viability of human hepatoblastoma HepG2 and Huh-7 cells. Cells were plated for 24 hours and then treated with various samples for another 72 hours. Data were represented as the cell viability relative to the control group (0.1% DMSO). Values were mean ± SEM from three independent experiments. Ã p < 0.01, compared with the control group by one-way ANOVA with a Dunnett's post hoc test.
In an attempt to discover bioactive compounds by the bioassay-guided fractionation method, the inhibitory effects of fractions and isolates from A. camphorata on the cell viability of human hepatoblastoma HepG2 and Huh-7 cells were evaluated. As shown in Supplementary Figure S2 and Table S1, treatment of Fr.1, Fr.3, Fr.10-Fr.16 at the concentration of 100 lg/mL exhibited the potent inhibition of the cell viability of HepG2 cells at 48 and 72 hours. Besides, Fr.3, Fr.10, Fr.14, and Fr.15 showed the significant cytotoxicity toward Huh-7 cells at the concentration of 100 lg/mL at 48 and 72 hours, with the most potent effects found in Fr.10 and Fr.14. Regarding the isolated compounds, treatment of antrocamols LT1 (2) or LT3 (3) at the concentration of 10 lM reduced the cell viability of HepG2 and Huh-7 cells to about 60% at 48 and 72 hours (Figure 2 and Figure S3). In contrast, antroquinonol Z (1) exhibited weak cytotoxic activity against Huh-7 cells, but not HepG2, at 48 and 72 hours (Figure 2 and Figure  S3). The moderated cytotoxicity of antroquinonol Z (1) in Huh-7 cells supported the isoprenoid side chain's attachment at C-5 of cyclohexanone moiety necessary for cytotoxicity enhancement. Antrodin C (6) did not show significant cytotoxicity for HepG2 and Huh-7 cells (Figure 2). These results suggested the anti-HCC potentials of the fractions and isolates from A. camphorata.

Cultivation and preparation of antrodia camphorata KH37
Antrodia camphorata KH37 was provided by KWANG HSIN Technology Co., Ltd, New Taipei City, Taiwan, and identified by the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan. The voucher specimen (2013AG036) was kept in BCRC in Taiwan. The mycelium of this strain was inoculated into 250 mL flasks, each containing 0.2 g of Difco potato dextrose broth (Becton, Dickinson and Company, Sparks, USA), 20 g of barley, and 15 mL of deionized water. The fermentation was conducted at 24 C for four months. After four months of cultivation, the A. camphorata KH37 was harvested and lyophilized for the extraction of metabolites.

Cell culture
Human hepatoblastoma cell lines HepG2 and Huh-7 were kindly provided by Professor Chia-Hung Yen in the Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan (Chen et al. 2021). Briefly, HepG2 and Huh-7 cells were cultured in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), and Penicillin-Streptomycin-Amphotericin B Solution and maintained at 37 C in a humidified incubator with 5% CO 2 and 95% air. The cell culture medium was replaced every 2-3 days, and confluent cells were passed every 3-5 days with trypsinization.

Cell viability assay
The cell viability of HepG2 and Huh-7 cells was determined by the resazurin-based viability assay, in which the blue non-fluorescent dye resazurin can be reduced by viable cells to the highly red-fluorescent product resorufin (Czekanska 2011;Gong et al. 2020). Briefly, 5,000 cells were plated onto 96-well plates for 24 hours, and the treatments were applied with indicated concentrations and incubation time. After treatment, ten lL of 0.2 mg/mL resazurin (Cayman Chemical, USA) was added into the culture medium, and then the plates were incubated for two hours at 37 C in a cell culture incubator. Fluorescence with an excitation wavelength of 560 nm and emission of 590 nm recorded using a fluorescent microplate reader (Bio-Tek Synergy HT, Winooski, VT, USA) was used to calculate cell viability.

Statistic analyses
All data are presented as the mean ± standard error of the mean (SEM), derived from at least three independent experiments in triplicate for each treatment group. Statistical significance was analyzed using the unpaired Student's t-test or one-way analysis of variance (ANOVA) test (SPSS 13 Inc., Chicago, IL, USA). A value of p < 0.01 was considered statistically significant.

Conclusions
In this study, one new compound and nine known compounds were isolated from the solid-fermentation extracts of Antrodia camphorata KH37. The moderated cytotoxicity of antroquinonol Z (1) in Huh-7 cells supported the isoprenoid side chain's attachment at C-5 of cyclohexanone moiety necessary for cytotoxicity enhancement. Our cytotoxicity results for two hepatocellular carcinoma cell lines revealed the significant cytotoxic effects of fractions. This study provides evidence for developing alternative or complementary anti-HCC therapeutics from A. camphorata in a partially purified formula.