Three New Lanostanoids from the Mushroom Ganoderma tropicum

Three new lanostanoid triterpenes—ganotropic acid (1), 3β,7β,15α,24-tetra-hydroxy-11,23-dioxo-lanost-8-en-26-oic acid (2) and 3β,7β,15α,28-tetrahydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (3)—were isolated from the n-BuOH extract of the fruiting bodies of the mushroom Ganoderma tropicum. Their structures were elucidated by 1D and 2D NMR spectroscopy, as well as HR-EI-MS data.


Introduction
Ganoderma, the major genus in the family Ganodermataceae, are widely used to cure various chronic diseases such as hypertension, diabetes, hepatitis and cancers [1][2][3]. Among them some species are used as valuable Traditional Chinese Medicines (TCM). Phytochemical investigations on some Ganoderma OPEN ACCESS species showed that ganoderma triterpenes (GTs) are mainly lanostanoid-type triterpenes with extensive biological and pharmacological activities, including cytotoxic [4,5], hepatoprotective [6,7], anti-inflammatory [8,9], and antioxidant [10] properties. Ganoderma tropicum is a main wild Ganoderma mushroom species found distributed in tropical areas of China. It is used as a health supplement and folk medicine alternative to Ganoderma lucidum and Ganoderma sinensis which are recorded in the Chinese Pharmacopeia to treat coronary heart disease and chronic hepatitis [11].

Results and Discussion
Compound 1 was obtained as a white power, and its molecular formula was assigned to be C30H44O7, with nine degrees of unsaturation, from its HREIMS (m/z 516.3095 [M] + ). The IR spectrum revealed the presence of hydroxyl (3743 and 3442 cm −1 ), carbonyl (1768 cm −1 ) and double bond (1652 cm −1 ) absorptions. The 1 H-NMR spectrum (Table 1) Table 2) showed 30 carbon resonances, including seven methyls, seven methylenes, six methines (three oxygenated), and ten quaternary carbons (two olefinic, two carbonyl, and two oxygenated), suggesting a triterpenoid skeleton. Further comprehensive analysis of the 1D and 2D NMR spectra indicated that compound 1 had the lanostane skeleton as ganoderic acid C2 [17]. However, the 13 C-NMR data for compound 1 had two oxygenated quaternary carbons (C-23 and C-17) instead of the corresponding carbonyl (C-23) and methyl (C-17) in ganoderic acid C2. Apart from seven degrees of unsaturation (four rings, two carbonyls and one double bond), the remaining elements of the unsaturation in compound 1 were assumed to be two rings in the side chain. These were reminiscent of the presence of two oxygenic five-membered rings in compound 1 with the characteristic C-23 spiro carbon (δC 113.4) similar to that of abietospiran [18]. This partial structure was supported by HMBC correlations of H-27 with C-24 (δC 44.8) and C-26 (δC 179.2), H-24 and H-22 with C-23 as well as H-21 with C-17 (δC 94.9) and C-22 (δC 44.7) (Figure 2). The stereo-configuration of the lanostane skeleton for compound 1 was determined based on ROESY spectroscopic data and comparison of the NMR data with those similar structures. The NOE correlations of H-3/H-5, H-3/Me-28, H-7/H-5, H-7/Me-30 and H-15/Me-18 in the ROESY experiment indicated that 3-OH and 7-OH were β-oriented and 15-OH was α-oriented. Table 1. 1 H-NMR (500 MHz) spectroscopic data (δH in ppm, J in Hz) of compounds 1 (in CDCl3) and 2, 3 (in CD3OD).  The stereoconfigurations of chiral carbons C-17, C-20 and C-23 in the oxygenated five-membered ring in the side chain were determined to be the same as in abietospiran from their similar NMR data. The α-orientation of Me-27 was deduced based on analysis of ROESY correlations of H-24α (δH 2.48 dd (J = 8.1, 12.8)) with Me-21 and Me-27. Therefore, compound 1 was identified as (23S,25R)-3β,7β,15α-trihydroxy-11-oxo-17,23-epoxy lanost-8-en-26,23-olide, named ganotropic acid.
The inhibitory activities of compounds 1-3 against AChE were tested using a spectrophotometric method [19,20]. The results showed that these compounds possessed low percentage inhibition (<10%) at the concentration of 100 μM, compared to the tacrine control, indicating no significant inhibitory activities against AChE. In addition, these isolates were evaluated for antibacterial activity against Staphylococcus aureus, as well as cytotoxic activity against six tumour cells (K-562/HL-60/SMMC-7721/A-549/MCF-7/SW-480) according to the methods described previously [4,21], but they also showed no significant bioactivities.

Fungal Material
Fruiting bodies of G. tropicum were collected in Lingshui County, Hainan Province, China (May, 2011), and identified by Prof. Xing-Liang Wu of Hainan University. A voucher specimen (No. 2011LZ01) is deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

Bioassay of AChE Inhibitory Activity
AChE inhibitory activity of the three compounds was assayed by the spectrophotometric method developed by Ellman [19,20]. Acetylthiocholine iodide was used as substrate in the assay. Na2HPO4 (94.7 mL, 0.1 M) and NaH2PO4 (5.3 mL, 0.1 M) were mixed to prepare phosphate buffer (PB, pH 8.0). Compounds were dissolved in DMSO (2% in PB). The reaction mixture contained PB (110 μL), test compound solution (10 μL, 2000 μM) and acetyl cholinesterase solution (40 μL, 0.1 U/mL), which were mixed and incubated for 20 min (30 °C). The reaction was initiated by the addition of DTNB (20 μL, 6.25 mM) and acetylthiocholine iodide (20 μL, 6.25 mM). The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s. Tacrine was used as positive control. All reactions were performed in triplicate. The percentage inhibition was calculated as follows: % age inhibition = (E − S)/E × 100 (E is activity of the enzyme without test compound and S is the activity of enzyme with test compound).