Lanostane Triterpenoids and Ergostane Steroids from Ganoderma luteomarginatum and Their Cytotoxicity

Macrofungus Ganoderma luteomarginatum is one of the main species of Ganoderma fungi distributed in Hainan province of China, the fruiting bodies of which have been widely used in folk as a healthy food to prevent tumors. To explore the potential cytotoxic constituents from G. luteomarginatum, the phytochemical investigation on the ethyl acetate soluble fraction of 95% ethanolic extract from the fruiting bodies of this fungus led to the isolation of twenty-six lanostane triterpenoids (1–26), including three undescribed ones (1–3), together with eight ergostane steroids (27–34). The structures of three new lanostane triterpenoids were elucidated as lanosta-7,9(11)-dien-3β-acetyloxy-24,25-diol (1), lanosta-7,9(11)-dien-3-oxo-24,26-diol-25-methoxy (2), and lanosta-8,20(22)-dien-3,11,23-trioxo-7β,15β-diol-26-oic acid methyl ester (3) by the analysis of 1D, 2D NMR, and HRESIMS spectroscopic data. All isolates were assayed for their cytotoxic activities using three human cancer cell lines (K562, BEL-7402, and SGC-7901) and seven lanostane triterpenoids (1, 2, 7, 13, 18, 22, and 24), and one ergostane steroid (34) showed definite cytotoxicity with IC50 values that ranged from 6.64 to 47.63 μg/mL. Among these cytotoxic lanostane triterpenoids, compounds 2 and 13 showed general cytotoxicity against three human cancer cell lines, while compounds 1 and 18 exhibited significant selective cytotoxicity against K562 cells with IC50 values of 8.59 and 8.82 μg/mL, respectively. Furthermore, the preliminary structure–cytotoxicity relationships was proposed.


Introduction
The genus Ganoderma, belonging to the family Ganodermataceae and known as "Lingzhi" in Chinese, has been widely used as traditional Chinese medicine and functional foods for health in China and Southeast Asia for thousands of years [1]. There are more than 100 species in this family growing on cut or rotten trees in China, and 78 wild ones were found in Hainan Province [2]. As the major genus in this family, Ganoderma is a prolific producer of novel natural products responsible for its health benefits, mainly containing polysaccharides with an immunostimulative effect and triterpenes with a cytotoxic action [3]. Two main species, G. lucidum and G. sinensis, are recorded in Pharmacopoeia of the People's Republic of China and used as an addition to conventional therapy in a clinical treatment of chronic bronchitis, bronchial asthma, leukopenia, coronary heart disease, arrhythmia, and acute infectious hepatitis. Recent research on chemical constituents of Ganoderma species showed that lanostane-type triterpenoids are the main characteristic natural products [4], and more than 400 lanostanoids have been isolated from the fungi of Ganoderma. These small molecule compounds have attracted considerable attention due to their extensive biological and pharmacological activities [5,6], including cytotoxic [7][8][9], hepatoprotective [10,11], anti-inflammatory [12][13][14], antidiabetic [15,16], neuroprotective [17], antiviral [18], antiaging [19], and antioxidant [20][21][22] effects. The genus Ganoderma is used as a healthy food and has been traditionally used for the prevention of numerous diseases or various pathological conditions, including complementary cancer therapy, especially a broad-spectrum application for the treatment of cancer.
Cancer has been considered as a huge threat to human health, and most governments are committed to diminishing this threat. The prevention and treatment of cancer becomes a key health goal. Finding antitumor drugs with high efficiency and low toxicity has become the urgent task, and countless researchers are dedicated to discovering bioactive ingredients from nature resources. Ganoderma is a promising anticancer immunotherapy agent owing to its low toxicology and efficacy as a combination therapy through the regulation of the immune system [23]. Polysaccharides and triterpenes from Ganoderma have been known to possess chemopreventive and antitumor activity. Many studies indicate that lanostane-type triterpenoids act as an inhibitor on different cancer cell lines, including the lung, liver, colon, pancreas, breast, skin, and prostate [6]. Among the reported active lanostanoids, the ganoderic acids are the main types of triterpene that play key roles in the biological activity. Lanostane-type triterpenoids can cause cell cycle arrest by the downregulation of cyclin D1 in the G1 phase of cell growth and inhibition of PKC activity in the G2 growth phase. Moreover, lanostane-type triterpenoids also prevent tumor metastasis by modulating MMPs and IL-8 and inhibit the excretion of inflammatory cytokines [24].
Ganoderma has been used as a healthy food and medicinal purposes for centuries particularly in China, Japan, and Korea. A great deal of work has been carried out on over thirty species of Ganoderma. Two types of natural products, lanostane triterpenoids (1-26) and ergostane steroids (27)(28)(29)(30)(31)(32)(33)(34), were discovered from the fruiting bodies of G. luteomarginatum collected in Hainan Province, China. These two types of compounds are widely found in genus Ganoderma [1], and lanostanoids (Ganoderma triterpenoids) was the characteristic active metabolites in species of Ganoderma, which are a class of compounds with various chemical structures. Here, the isolated lanostane triterpenoids were divided into two groups according to the conjugated system at C-7, C-8, C-9, and C-11. The first group possessed the conjugated double bond ∆ 7,9(11) as shown in 1, 2, 6-18, 23, and 24. The second group had (∆ 8 )α,β-unsaturated ketone at C-7 or C-9. The C-26 in lanostanoid structures is often oxidized to alcohols, aldehydes, and acids [5]. Among the structures of identified lanostanoids (1-26), ganoderiol derivative was the main type. In addition, three ganoderic aldehydes (5, 23, 24) and one ganoderic acid (3) were also found. The β-configuration of OH-15 in new ganoderic acid (3) from G. luteomarginatum was consistent with the previously discovered ganoderic acid derivatives from this fungus [27], which was opposite of that shown in the corresponding compounds from other Ganoderma species [7,12,50]. Moreover, norlanostanoids with 24 carbon atoms often occur in Ganoderma. In present study, two hexanorlanostanoids (25 and 26) were isolated. Some lanostane triterpenoids besides the three new ones in our study are structurally different from those previously reported lanostanoids [26,27] from G. luteomarginatum collected in Guangxi Province, China. This may be related to the different growth environment of this fungus or different growth period for collection, which needs further comparative analysis in our subsequent studies.

Cytotoxic Activities of Compounds
The cytotoxic activities of all the isolates were evaluated by MTT method toward three human cancer cell lines (K562, BEL-7402, and SGC-7901). The results were presented in Table 2. Of the compounds tested, seven lanostane triterpenoids (1, 2, 7, 13, 18, 22, and 24) showed definite cytotoxicity against K562 with IC 50 values range from 6.64 to 17.38 µg/mL, among which compounds 1, 13 and 18 showed the IC 50 values of 8.59, 6.64, and 8.82 µg/mL, respectively. Compounds 2 and 13 also showed moderate cytotoxicity against two human cancer cell lines (BEL-7402 and SGC-7901). Moreover, compound 7 showed moderate cytotoxicity against human cancer cell lines BEL-7402 with IC 50 value of 20.05 µg/mL . Compounds 1, 18, 22, and 24 had no obvious cytotoxicity on BEL-7402 and SGC-7901 cell lines (IC 50 > 50 µg/mL). Among these cytotoxic lanostanoids, compounds 2 and 13 showed general cytotoxicity against three human cancer cell lines, while compounds 1 and 18 exhibited significant selective cytotoxicity against K562 cell lines. One ergostane steroid (34) showed general cytotoxicity against three human cancer cell lines. Lanostane-type triterpenoids of Ganoderma are considered to be the major pharmacologically active compounds that contribute to its antitumor efficacy. The lanostane-type triterpenoids were extensively evaluated for cytotoxic activities against a series of tumor cell lines [5] related to lung, liver, colon, pancreas, breast, skin, and prostate [6]. The lanostanoids with structural complexity and functional group variety may be specific to different cell lines and the structure-cytotoxicity relationships could be raised. Compounds 1, 18, 22, and 24 exhibited selective cytotoxicity against K562 cell lines may be due to their unique structures. From the results of isolated lanostanoids against three human cancer cell lines (K562, BEL-7402, and SGC-7901), the conjugated double-bond ∆ 7,9(11) system in tetracyclic skeleton (1, 2, 7, 13, 18, and 24) seemed to be more important than (∆ 8 )α,β-unsaturated ketone system for potent cytotoxic activity. Comparing the cytotoxicity between 7/9, 18/17, and 24/5 with only difference at C-3, it suggested that acetylation may be the negative factor for cytotoxic activity. In addition, compound 13 exhibited significant cytotoxicity, while their keto-3 analog 12 was inactive, assumed that reduction of the keto-3 group to OH-3 in lanostane triterpenoids would improve the cytotoxicity against K562, BEL-7402, and SGC-7901 significantly. The above preliminary structure-cytotoxicity relationships provide an approach to understanding the structural requirements of lanostane-type triterpenoids.

General Experimental Procedures
The NMR spectra were recorded with a Bruker AV-500 spectrometer (Bruker, Bremen, Germany) with TMS as an internal standard. HRESIMS data were determined on a mass spectrometer API QSTAR Pulsar (Bruker, Bremen, Germany). Optical rotations were measured on a Rudolph Autopol III polarimeter (USA). UV spectra were obtained on a Shimadzu UV-2550 spectrometer. IR spectra were obtained on a Nicolet 380 FT-IR spectrometer with KBr pellets. Silica gel (60-80 and 200-300 mesh, Marine Chemical Industry Factory, Qingdao, China), Rp-C18 (20-45 mL; Fuji Silysia Chemical Ltd., Aichi, Japan), and Sephadex LH-20 (Merck, Germany) were used for column chromatography. Fractions were monitored by TLC and spots were visualized by heating after spraying with 5% H 2 SO 4 in ethanol.

Bioassay of Cytotoxic Activity
All the compounds was assayed for their cytotoxic activity against three human tumor cell lines: K562 (leukemic cell line), BEL7402 (hepatoma cell line), and SGC7901 (gastric cancer cell line) using MTT methods reported previously [7]. Briefly, each tested compound was dissolved with DMSO at concentration of 10 mM and then diluted to the required concentrations with the medium. Cells were cultured in 96-well plates with initial density of 5000 cells/well 12 h before treatment and exposed to different concentrations (40, 8, 1.6, 0.32, and 0.064 µM, respectively) of compounds, with paclitaxel (Sigma, Livonia, MI, USA) as the positive control. After the culturing period, 20 µL of MTT (5 mg/mL) was added per well and incubated for 4 h at 37 • C. Finally, absorbance was measured at 570 nm using a microplate reader. Each assay was replicated three times. The effect of the compounds on cell viability was calculated and expressed as the IC 50 .