Nortriterpenoids from the Fruiting Bodies of the Mushroom Ganoderma resinaceum

Ganoderma resinaceum is usually used as ethnomedicine for immune-regulation, hyperglycemia, and liver disease. To date, only a few chemical constituents have been reported from G. resinaceum. In this study, fifteen nortriterpenoids including six new nortriterpenoids (1–6) and nine known analogs (7–15), were separated and purified from the fruiting bodies of G. resinaceum. New compounds were identified as lucidone I (1), lucidone J (2), lucidone K (3), lucidone I (4), ganosineniol B (5), and ganosineniol C (6), based on analysis of extensive spectroscopic data (high resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR), infrared (IR), and ultraviolet (UV)). The known compounds were assigned as lucidone A (7), lucidone B (8), lucidone H (9), lucidone E (10), lucidone F (11), lucidone D (12), lucidone C (13), ganoderense F (14), and ganosineniol A (15), by comparing their spectroscopic data with those reported in the literature. Compounds 3, 4, and 7–13 were examined for α-glucosidase inhibitory activity and display no significant activity, but the finding may support that the side chain of ganoderma triterpenoids played an important role in α-glucosidase inhibitory activity.


Introduction
Nortriterpenoids, derived from lanostane-type triterpenoids due to degradation of side chain, are a class of secondary metabolites in Ganoderma [1,2]. Although substantial triterpenoids have been reported from Ganoderma, nortriterpenoid is rare. The common nortriterpenoids possess 24 or 27 carbons skeleton in Ganoderma, for example, lucidones A-H, and lucidenic acids A-N. However, two novel nortriterpenoids, methyl ganosinensate A and ganosinensic acid A, contain an unusual four-membered ring skeleton produced by a bond across C-1 to C-11 [3]. Nortriterpenoids isolated from Ganoderma showed a wide range of biological activities, such as anti-tumor [4][5][6], anti-inflammatory [7], neurotrophic [8], hepatoprotective [9], and anti-HIV-1 protease activities [10]. Hence, nortriterpenoids deserve our close attention due to its structure diversity and good model for the pharmaceutical field.
As a member of the genus Ganoderma, G. resinaceum has been used for immune-regulation, hyperglycemia, and liver disease [11]. The extract of G. resinaceum exhibited antimicrobial, antioxidant, and inhibitory activities against acetyl cholinesterase, tyrosinase, α-amylase and α-glucosidase [12]. At present, only 17 triterpenoids had been obtained from the fruiting bodies of G. resinaceum, whose biological properties included cytotoxicity [13] and hepatoprotective activities [9]. Obviously, the chemical constituents isolated from G. resinaceum and their biological activities have not been
Compound 1 was obtained as white amorphous powder. Its molecular formula was established as C24H36O5 by electrospray ionization (ESI)-HRMS at m/z 403.2484 [M − H] − (calcd. for C24H35O5, 403.2484). Its IR spectrum showed the presence of hydroxy group (3429 cm −1 ) and carbonyl group (1717 cm −1 ). 1 H-NMR spectrum showed the presences of six singlet methyl signals [δH 0.77, 0.79, 0.90, 1.08, 1.21, and 2.13 (each 3H, s)]. Heteronuclear single quantum coherence (HSQC) and 13 C-NMR spectra displayed 24 carbon resonances, including six methyls, five methylenes, five methines including three oxygenated carbon resonances at δC 79.5, 66.9 and 65.3, and eight quaternary carbons including a tetrasubstituted olefinic carbon δC 136.8 and 146.2, and two ketone carbons δC 220.2 and 209.3. The above spectroscopic data suggested 1 to be nortriterpenoid similar to lucidone A (7). However, a comparison of their 13 C-NMR data showed that 1 possesses an additional oxygenated methine at δC 65.3 but absent of one ketone group signal at δC 196.7 in 7. Moreover, the chemical sifts of tetrasubstituted olefinic carbons of 1 in 13 C-NMR spectrum were significantly different from those of 7 due to the disappearance of α,β-unsaturated ketone conjugated system at C-8/C-9/C-11. Accordingly, it was assumed that 1 was the 11-OH derivative of 7. The 11-OH was further confirmed
Compound 1 was obtained as white amorphous powder. The above spectroscopic data suggested 1 to be nortriterpenoid similar to lucidone A (7). However, a comparison of their 13 C-NMR data showed that 1 possesses an additional oxygenated methine at δ C 65.3 but absent of one ketone group signal at δ C 196.7 in 7. Moreover, the chemical sifts of tetrasubstituted olefinic carbons of 1 in 13 C-NMR spectrum were significantly different from those of 7 due to the disappearance of α,β-unsaturated ketone conjugated system at C-8/C-9/C-11. Accordingly, it was assumed that 1 was the 11-OH derivative of 7. The 11-OH was further confirmed on the basis of the heteronuclear multiple bond correlation (HMBC) correlations from H-11 (δ H 4.48) to C-8 (δ H 136.8), C-9 (δ C 146.2) and C-13 (δ C 43.8), and H-12β (δ H 2.00) to C-11 (δ H 65.3), and the hydrogen-hydrogen correlation spectroscopy ( 1 H-1 H COSY) correlation of H-11 (δ H 4.48) with H-12α (δ H 2.51). Finally, the planar structure of 1 was established by 1D and 2D NMR spectra.
Triterpenoids, biogenetically derived via mevalonic acid pathway, are the main secondary metabolites of Ganoderma. The above-mentioned nortriterpenoids are considered to originate from triterpenoid due to the degradation of its side chain. A possible biogenetic pathway for C24 nortriterpenoids is proposed as shown in Figure 4. The precursor, Ganoderenic acid and its esterified derivatives, undergoes oxidation to yield intermediate A (1, and 7-13). Compounds 7 and 12 were further transformed into 2 and 3, respectively, through an addition reaction. Finally, compound 3 (intermediate B) generated 4 (D) via elimination reaction and addition reaction. Triterpenoids, biogenetically derived via mevalonic acid pathway, are the main secondary metabolites of Ganoderma. The above-mentioned nortriterpenoids are considered to originate from triterpenoid due to the degradation of its side chain. A possible biogenetic pathway for C24 nortriterpenoids is proposed as shown in Figure 4. The precursor, Ganoderenic acid and its esterified derivatives, undergoes oxidation to yield intermediate A (1, and 7-13). Compounds 7 and 12 were further transformed into 2 and 3, respectively, through an addition reaction. Finally, compound 3 (intermediate B) generated 4 (D) via elimination reaction and addition reaction. G. resinaceum has been used as ethnomedicine for lowering blood sugar in Nigeria [19]. However, it is unknown whether chemical constituents from G. resinaceum contribute to the traditional medicinal efficacy. Therefore, we considered carrying out α-glucosidase inhibitory assay of isolates from G. resinaceum. Triterpenoids from Ganoderma exhibited significant α-glucosidase inhibitory activity [20][21][22]. Nortriterpenoids from G. resinaceum are considered to originate from triterpenoids due to the degradation of its side chain on basis of analysis of possible biogenetic pathway. Aiming to explore biological active constituents and structure-activity relationship of terpenoids from Ganoderma, compounds 3, 4, and 7-13 were evaluated for α-glucosidase inhibitory activity. Their inhibitions were less than 50% at the concentration of 3 mM (see Supplementary Materials, Table S1). All isolates measured showed lower inhibitory activity compared with positive drug acarbose (IC50 value 2.76 mM), hence, their IC50 value were not measured. Our bioactivity studies may support previous research results that the side chain played an important role in α-glucosidase inhibitory activity of ganoderma triterpenoids, especially the presence of carboxyl acid group [22]. For example, the only difference between ganoderic acid A and 11 was that ganoderic acid A had aliphatic side chain, whereas 11 did not. Ganoderic acid A displayed the same inhibitory activity as positive drug acarbose [22], however, Compound 11 was almost inactive (16% inhibition at the concentration of 3 mM).

General Experimental Procedures
NMR spectra data were recorded on a Bruker ascend 600 spectrometer (Bruker, Karlsruhe, Germany) with TMS used as a reference. Optical rotations were measured on PerkinElmer Model 341 polarimeter (PerkinElmer, Waltham, MA, USA). UV spectrum data were acquired using HACH DR6000 UV-visible spectrophotometer (Hach, Loveland, CO, USA). IR spectra were recorded as KBr disks on PerkinElmer Spectrum 100 Series FT-IR spectrometers (PerkinElmer, Waltham, MA, USA). HRESIMS data were obtained on a LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap FT-MS spectrometer (Thermo, Waltham, MA, USA). TLC was carried out on silica gel GF254 plates (Yantai Institute of Chemical industry, Yantai, China) and spots were visualized by UV light (254 and/or 365 nm) and spraying with 10% H2SO4 followed by heating. Column chromatography was carried out using silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), MCI gel (CHP-20P, 75-150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), ODS (35-70 μm, Grace, Maryland, MD, USA), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) as packing materials. Semi-preparative HPLC was performed on a Shimadzu instrument (Shimadzu, Tokyo, Japan) coupled to CBM-20A system G. resinaceum has been used as ethnomedicine for lowering blood sugar in Nigeria [19]. However, it is unknown whether chemical constituents from G. resinaceum contribute to the traditional medicinal efficacy. Therefore, we considered carrying out α-glucosidase inhibitory assay of isolates from G. resinaceum. Triterpenoids from Ganoderma exhibited significant α-glucosidase inhibitory activity [20][21][22]. Nortriterpenoids from G. resinaceum are considered to originate from triterpenoids due to the degradation of its side chain on basis of analysis of possible biogenetic pathway. Aiming to explore biological active constituents and structure-activity relationship of terpenoids from Ganoderma, compounds 3, 4, and 7-13 were evaluated for α-glucosidase inhibitory activity. Their inhibitions were less than 50% at the concentration of 3 mM (see Supplementary Materials, Table S1). All isolates measured showed lower inhibitory activity compared with positive drug acarbose (IC 50 value 2.76 mM), hence, their IC 50 value were not measured. Our bioactivity studies may support previous research results that the side chain played an important role in α-glucosidase inhibitory activity of ganoderma triterpenoids, especially the presence of carboxyl acid group [22]. For example, the only difference between ganoderic acid A and 11 was that ganoderic acid A had aliphatic side chain, whereas 11 did not. Ganoderic acid A displayed the same inhibitory activity as positive drug acarbose [22], however, Compound 11 was almost inactive (16% inhibition at the concentration of 3 mM).

Activity Assay
α-Glucosidase inhibitory activity was examined by the method described by Dengqiang Li et al. [23]. Acarbose, a definite α-glucosidase inhibition, was used as positive drug.

Conclusions
In summary, six new nortriterpenoids (1-6), together with nine known analogs (7-15), were isolated and identified from the fruiting bodies of Ganoderma resinaceum. The possible biogenetic pathway for C24 nortriterpenoids was proposed Compounds 3, 4, and 7-15 displayed no significant α-glucosidase inhibitory activity, however, this finding may support that the side chain of ganoderma triterpenoids is critical for α-glucosidase inhibitory activity, especially the presence of carboxyl acid group.
Supplementary Materials: The following are available online: Spectrum copies of Mass, NMR, and IR.