New Diterpenes from Cultures of the Fungus Engleromyces goetzii and Their CETP Inhibitory Activity

One new cleistanthane-type diterpene named engleromycenolic acid A (1), one new rosane-type diterpene named engleromycenolic acid B (2) and one new natural rosane-type diterpene, engleromycenol (3), along with three known rosane-type diterpenes, rosololactone (4), rosenonolactone (5) and 7-deoxyrosenonolactone (6) were isolated from cultures of the fungus Engleromyces goetzii, where it naturally grows on Alpine bamboo culms. The new compounds were elucidated based on their spectroscopic data. In addition, compounds 1–6 were evaluated for their cholesterol ester transfer protein (CETP) inhibition activity. This paper reports the isolation, structural elucidation, and CETP inhibition activity of these compounds.


Introduction
The fungus Engleromyces goetzii is widely distributed in Tibet, Sichuan and Yunnan province. It grows on Alpine bamboo culms, and ripens during rainy season from July to August. Local residents usually boil the fruiting bodies in water to treat infection, inflammation and cancer [1][2][3]. Previous investigation on the fruiting bodies of E. goetzii has led to the isolation of neoengleromycin, cytochalasin D and 19,20-epoxycytochalasin D [4][5][6]. There are no reports about the chemical constituents of the cultures of this fungus.
In order to search for more novel and potentially bioactive secondary metabolites, the chemical constituents of E. goetzii cultures were investigated by altering the culture conditions of the fungus and enlarging the fermentation scale. This investigation led to the isolation and identification of one new cleistanthane-type diterpene named engleromycenolic acid A (1), one new rosane-type diterpene named engleromycenolic acid B (2) and one new natural rosane-type diterpene named engleromycenol (3), along with three known rosane-type diterpenes: rosololactone (4), rosenonolactone (5) and 7-deoxyrosenonolactone (6) [7]. Their structures (Fig. 1) were elucidated based on the spectroscopic data analyses. The cholesterol ester transfer protein (CETP) inhibition activities of compounds 1-6 were examined and engleromycenolic acid A (1) showed CETP inhibition activity with IC 50 value at 7.55 lM.
There are two kinds of lipoproteinsin plasma: one is low-density lipoprotein (LDL) and another is high-density lipoprotein (HDL) [8,9]. They are in charge of carrying cholesteryl esters in plasma. HDL is responsible for transferring cholesteryl esters from periphery plasma to the liver to metabolize, and LDL mediates the transport of cholesteryl esters from the liver to periphery plasma [8,10,11]. CETP promotes the transfer of cholesteryl esters from HDL to LDL. Inhibiting the activity of CETP can promotes the metabolism of cholesteryl esters in the liver and avoids accumulation of cholesterol esters in plasma that may cause atherosclerosis [9,[11][12][13][14].  13 C NMR spectrum showed 20 carbon resonances, including one carboxyl, four sp 2 olefinic carbons, one oxygen-bearing methine, two tertiary methyls, six methylenes, four methines as well as two quaternary carbons. These data suggested compound 1 might be a tricyclic diterpene. The 1 H and 13 C NMR spectral data of 1 were similar to those of auricularic acid [15], which indicated that 1 was a cleistanthane-type diterpene. However, there are certain differences as follows: the resonance of C-2 at d C 19.5 in auricularic acid is down shifted to d C 65.3 in 1, suggesting that the methylene at C-2 was replaced by an oxygen-bearing methine. Furthermore, the HMBC correlations from d H 2.41, 2.13, 1.00, 0.93 to carbon at d C 65.3, and the 1 H-1 H COSY correlations from d H 4.14 to d H 2.41, 2.13, 1.00 and 0.93 in 1, indicating that the oxygenbearing methane was placed at C-2. According to the molecular formula, we can conclude the oxygen-bearing methine at C-2 in 1 was substituted by a hydroxyl. In the ROSEY spectrum, the observed cross peaks of CH 3 -20/H- The 13 C NMR spectrum showed 20 carbon resonances including one carbonyl, four olefinic carbons, one oxygenbearing methine, three methyls, seven methylenes, one methine and three quaternary carbons. Comparison of NMR data of 2 with those of 7-deoxyrosenonolactone (6) [16], revealed the presence of the characteristic signals of a rosane-type diterpene. The resonances at d C 129.5 and 138.8 in the 13  indicated that the oxygen-bearing methine at C-2 in 2 was replaced by a methylene in 3. The configuration of C-8, C-9 and C-13 were established by comparing the NMR data of 3 with 2. The H-3 signal at d H 1.78 (1H, ddd, J = 13.5, 5.0, 3.5 Hz) suggested it to be equatorial b-oriented. In the ROSEY spectrum, the observed cross peak of d H 1.78 and 3.32 indicated the hydroxymethyl group in C-4 was located in axial b-oriented. In addition, there are no cross peak between H-18 (3.58, 3.32) and H-3a (1.27) in ROSEY spectrum, which further approved the conclusion above (Fig. 4). According to this analysis, compound 3 was confirmed as engleromycenol.
All the isolates were assayed for their CETP inhibition activity with the CETP Inhibitor Drug Screening Kit. The result showed that compounds 2-6 exhibited weak

Fungal Material and Cultivation Conditions
Fruiting bodies of E. goetzii were collected from Shangri-La county in Yunnan Province, China. A voucher specimen has been deposited in the Herbarium of the Kunming Institute of Botany of the Chinese Academy of Sciences. The mycelia cultures were derived from the tissue plugs. The culture PDA medium consisted of glucose (5 %), peptone from porcine meat (0.15 %), yeast powder (0.5 %), KH 2 PO 4 (0.05 %) and MgSO 4 (0.05 %). The inoculums of E. goetzii were prepared in a 15 L-fermentation tank for 6 days under the following conditions: culture temperature, 24°C; initial pH, 6.0; agitation speed, 250 r/min; inoculation volume, 10 % (by volume); and aeration rate, 1.0 volume/culture volume/min. Subsequently, the liquid seed was transferred into a 100 L-fermentation tank for cultivation under the same conditions for 20 days to afford an 80 L culture broth.

CETP Inhibition Activity Assay
Cholesterol ester transfer protein inhibition activity assay were carried out using a CETP Inhibitor Screening Kit (BioVision Incorporated., Milpitas, USA). CETP is a member of the lipid transfer/lipopolysaccharide binding protein gene family. CETP transfers neutral lipids from HDL to LDL and is present in normal human plasma and serum. The CETP Drug Screening Kit uses a donor molecule containing a fluorescent self-quenched neutral lipid that is transferred to an acceptor molecule in the presence of CETP (rabbit serum). CETP-mediated transfer of the fluorescent neutral lipid to the acceptor molecule results in an increase in fluorescence (ExEm = 465/535 nm). Inhibitor of CETP will inhibit the lipid transfer and subsequently decrease fluorescence intensity. The assay was carried out in a microtiter plate. Reagents were kept on ice prior to setting up the assay. The reaction mixture, containing test sample in 160 lL dH 2 O or control vehicle (160 lL dH 2 O); 20 lL CETP assay buffer; 10 lL of donor molecule and 10 lL of acceptor molecule was mixed well. The reaction was initiated by the addition of 3 lL of rabbit serum. After 60 min of incubation at 37°C, transfer was measured by the fluorescence intensity with BioTek Instrument (Gene Company Limited., USA). Background values were obtained from a blank with 160 lL dH 2 O. Percent inhibition of CETP activity was calculated by subtracting the background values from both control and test sample values. The IC 50 value was calculated by Reed and Muench's method.