Lanostane Triterpenoids from the Fruiting Bodies of Fomes officinalis and Their Anti-Inflammatory Activities

In the current study, further chemical investigation of the fruiting bodies of Fomes officinalis led to isolate seven new 24-methyl-lanostane triterpenoids, named officimalonic acids I−O (1–7). Their structures were elucidated based on the analysis of spectroscopic data (HR-MS, 1D and 2D NMR, UV, IR). Compounds 1−3 possessed an unusual C-23 spirostructure moiety, while compounds 4−7 had 23,26-lactone unit. Anti-inflammatory assay revealed that compounds 3 and 5 exhibited significant inhibitory activities against NO production in LPS-induced RAW 264.7 cells and cyclooxygenase (COX-2).


Introduction
24-methyl-lanostane triterpenoids are a class of compounds present in most fungal species of the family Polyporaceae, which has attracted wide attention among the natural product chemistry community due to their diversity structure and bioactivities [1]. Fomes officinalis (Vill. Ex Fr.) Ames (Polyporaceae) is a perennial wood-decay fungus, distributed in the Northern regions of China and in the Pacific Northwest United States, Canada, and Europe [2,3]. F. officinalis was traditionally used to treat cough and asthma in the Chinese Uygur Medicine prescription [2]. Previous phytochemical investigation on this species led to isolate a variety of lanostane triterpenoids, which exhibited broad biological activity such as cytotoxicity, anti-inflammation, antibacterial, and anti-leukaemia [4][5][6][7]. In our search for bioactive compounds from Xinjiang medicinal materials, eight new 24-methyl-lanostane triterpenoids, officimalonic acids A−H, were previously isolated and identified from the fruiting bodies of F. officinalis by our group [8]. In the current study, further chemical investigation on this species led to isolate another seven undescribed lanostane triterpenoids, named officimalonic acids I−O (1-7) (Figure 1). The inhibitory activity of the isolates against NO production in LPS-induced RAW264.7 cells were also evaluated. Herein, the isolation, structural elucidation, and the evaluation of anti-inflammatory activity, were present.

Results and Discussion
Compound 1 was isolated as colorless amorphous solid; its molecular formula was identified as C 34 4.21 (t,J = 7.1 Hz,, as well as a oxygenated methylene protons at 3.91 (1H, d, J = 14.3 Hz) and 3.88 (1H,d,J = 14.3 Hz). The 13 C-NMR (Table 2) spectra indicated the presence of 33 carbons sorted to seven methyls, nine methylenes, seven methines and 10 quaternary carbons (three of which are carbonyls). It is worth to mention that the NMR data of CH 2 -2 were not observed when they were acquired in CD 3 OD, which is consistent with that of officimalonic acid A [8].  Further analysis of 2D NMR data ( 1 H-1 H COSY, HSQC, HMBC) revealed that the structure of 1 was similar with that of officimalonic acid C, previously isolated from this species by our group [8]. The only difference between them is the absence of C-7 carbonyl in 1. The 1 H-1 H COSY correlations (Figure 2a) of H-5/H-6/H-7, and the HMBC correlations ( Figure 2a) from H-6 to C-8 and from H-7 to C-9, confirmed the assignment. The relative configurations of 1 were determined by the NOESY correlations ( Figure 2b) and coupling constant. The H-3 was established as β-configuration by its broad singlet-like signal. The NOESY correlation between H-12 and H 3 -30 indicated they were α-configuration. Considering the biogenetic relationship, the absolute configurations of 1 were tentatively assigned as those of officimalonic acid C. Thus, the structure of compound 1 was elucidated as depicted, given the trivial name officimalonic acid I. was that instead of a methylene signal, the signals of oxygenated methine (CH-12) were missed in 2, suggesting that compound 2 is one less hydroxyl than 1. The 1 H-1 H COSY correlations of H-11/H-12 and the HMBC correlation from H 2 -18 to C-12, supported the assignment. The structure of 2 was thus elucidated as depicted, named officimalonic acid J.
Compound 3 had a molecular formula C 34 H 48 O 8 , which was determined by the HR-ESI-MS at m/z 607.3218 [M + Na] + (calculated for C 34 H 48 O 8 Na, 607.3247). Analysis of 1 H and 13 C-NMR (Tables 1 and 2) data of 3 indicated that it had a similar structure with 1. The obvious differences between them were the presence of two double bonds (∆ 7 and ∆ 9(11) ) in 3 instead of one double bond (∆ 8 ) present in 1. The two olefinic protons signals at δ H 5.57 (brs, H-7) and 5.40 (brs, H-11) supported the assignment. The 1 H-1 H COSY correlations of H-5/H-6/H-7 and H-11/H-12, together with the HMBC correlations from H-7 to C-9, and from H-11 to C-8, confirmed the above assignment. Therefore, the structure of 3 was elucidated as depicted, named officimalonic acid K.
Compound 4 was obtained as a colorless amorphous solid; its HR-ESI-MS displayed a quasi-molecular ion peak at m/z 615.3512 [M + HCOO] − , corresponding to the molecular formula C 34 H 50 O 7 . Analysis of 1 H and 13 C-NMR (Tables 1 and 2) data of 4 revealed it shared the close structure similarity with that of officimalonic acid B, previously isolated from the same species [8]. The difference between these two compounds is the absence of a carbonyl group signal and the presence of a methylene in 4. The 1 H-1 H COSY correlations (Figure 3) of H-5/H-6/H-7, and the HMBC correlations ( Figure 3) from H-6 to C-8 and from H-7 to C-9, indicated that compound 4 possess methylene at C-7 in 4 instead of a carbonyl group in officimalonic acid B. The structure of 3 was thus elucidated as depicted, gave a trivial name officimalonic acid L.   (Tables 1 and 2) data of 5 showed almost the same as those of officimalonic acid B, the only difference being the presence of one more methoxyl signal in 5. The methoxy group was attached to C-3 of malonate half-ester by the HMBC correlation from δ H 3.68 (3H, s) to C-3 (δ C 167.4). Therefore, the structure of 5 was determined as depicted, named officimalonic acid M.
Compound 6 had a molecular formula of C 31 H 48 O 4 as determined on the basis of HR-ESI-MS at m/z 507.3427 [M + Na] + (calculated for C 35 H 50 O 8 Na,507.3450). Analysis of 1 H and 13 C-NMR (Tables 1 and 2) data of 6 revealed it had a similar structure with that of 4. The difference is that the signals for malonate half-ester at C-3 are not observed in 6, correspondingly, its proton signal of H-3 is up-field shifted to δ H 3.36 (brs), indicated that compound 6 had hydroxy group at C-3, rather than carboxyacetyloxy group at C-3. The HMBC correlations from H 3 -28 (or H 3 -29) to C-3, C-4, and C-5, supported the assignment. The structure of 6 was thus elucidated as depicted, given the trivial name officimalonic acid N.
All of the new compounds had their inhibitory activities tested against NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells. Their cell viabilities were firstly examined by the CCK-8 method. As compounds 2 and 4 showed strong cytotoxic effects against RAW 264.7 cells at the concentration of 60 µM, they would not be conducted, following anti-inflammation evaluation. Except compounds 2 and 4, other tested compounds showed NO inhibition lower than 60 µM. The results indicated that compounds 3 and 5 exhibited significant inhibitory activities against NO production in LPS-induced RAW 264.7 cells with IC 50 values at 33.0 and 25.4 µM, compared to that of positive control dexamethasone (IC 50 = 20.35 µM).
In addition, the cyclooxygenase (COX-2) inhibitory activities of these new compounds were evaluated using the in vitro assay kit [10]. Interestingly, the similar results were observed as those of NO production inhibition assay, only compounds 3 and 5 showed COX-2 inhibitory activities with IC 50 values at 30.1 and 42.3 µM, compared to that of positive control naproxen (IC 50 = 8.2 µM).

General Experimental Procedures
IR spectra were recorded on a Nicolet 380 FT-IR spectrometer. The optical rotations were measured on an Auto Pol IV automatic polarimeter (Rudolph Research, Flanders, NJ, USA) at room temperature. 1D and 2D NMR data were recorded on a Varian 400 MHz instrument with TMS as internal standard. HR-ESI-MS data were acquired using a Triple TOF 6600 mass spectrometer (AB Sciex, Framingham, MA, USA). Semi-preparative HPLC separations were performed on a Hitachi Chromaster system consisting of a 5110 pump, 5210 autosampler, 5310 column oven, 5430 diode array detector, and a Phenomenex Luna C18 column (250 × 10 mm, S-5 µm), all operated using EZChrom Elite software. All solvents were of ACS or HPLC grade, and were obtained from Tansoole (Shanghai, China), Sigma-Aldrich (St. Louis, MO, USA), respectively. Silica gel (300−400 mesh), C18 reverse-phased silica gel Merck,city,country),and MCI gel (CHP20P,Mitsubishi Chemical Industries Ltd.,Tokyo,Japan) were used for column chromatography (CC), and pre-coated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, China) were used for TLC.