Isolation, Structural Elucidation, and α-Glucosidase Inhibitory Activities of Triterpenoid Lactones and Their Relevant Biogenetic Constituents from Ganoderma resinaceum

Ganoderma resinaceum has been used as an ethnomedicine for lowering blood sugar. To clarify the bioactive chemical constituents contributing to lower blood sugar, chemical investigation on the fruiting bodies of Ganoderma resinaceum was conducted by chromatographic techniques, and led to the isolation of 14 compounds. Their structures were elucidated as triterpenoid lactones (1–4 and 8) and ganoderma acids (5–7 and 9–14) based on the analysis of extensive spectroscopy (mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared (IR), and ultraviolet (UV)) and comparison with literature data. Compounds 3, 5, 6, and 9–14 were evaluated for α-glucosidase inhibitory activity. Compounds 1–7 are new compounds. Compounds 1–4 and 8 were characteristic of an oxaspirolactone moiety, consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon. It is rare for natural products that such an oxaspirolactone moiety occurred in the lanostane-type triterpenoids. Compounds 5–7 and 9–14 may be important intermediates of the biosynthetic pathways of 1–4 and 8. Compounds 1 and 2 showed more potent inhibitory activity against α-glucosidase compared with the positive control drug acarbose with IC50 value of 0.75 ± 0.018 mM and 1.64 ± 0.022 mM, respectively.


Structural Elucidation
Compound 1 was obtained as a white powder. Its molecular formula was defined as C 30 H 44 O 6 by high resolution electrospray ionization mass spectrometry (HRESIMS) at m/z 499.3033 [M−H] − (calcd for C 30 H 43 O 6 , 499.3065) and 1D NMR data (Table 1). Its IR spectrum showed the presence of hydroxy (3471 cm −1 ), carbonyl (1767 cm −1 ), and α,β-unsaturated carbonyl (1635 cm −1 ) groups. The UV spectrum showed an absorption band to 258 nm due to the presence of α,β-unsaturated carbonyl group. The 1 H NMR spectrum displayed seven methyl signals [δ H 1.15 (3H, s), 1.42 (3H, s), 0.91 (3H, d), 1.23 (3H, d), 1.27 (3H, s), 1.13 (3H, s), and 1.68 (3H, s)] and two oxygenated methine signals (δ H 3.52 and 4.72). The 13 C NMR and HSQC spectra revealed the presence of 30 carbon signals consisting of seven methyls, eight methylenes, five methines including two oxygenated methines at δ C 77.8 and 72.9, six sp 3 quaternary carbons (two oxygenated carbon signals at δ C 95.6 and 113.3), one ketone group (δ C 198.6), one carboxylic group (δ C 178.7), and two olefinic carbons (δ C 165.2 and 139.9). The above spectroscopic data suggested 1 to be lanostane-type triterpenoid. The rings A-D were established by the heteronuclear multiple bond correlation (HMBC) and 1 H-1 H correlation spectroscopy (COSY) experiment ( Figure 2 . Apart from seven degrees of unsaturation ascribing to the rings A-D, two carbonyls, and one double bond, the remaining two degrees of unsaturation indicated that 1 should have two rings in the side chain. According to a carboxylic carbon (δ C 178.7), an acetal secondary carbon (δ C 113.3), and a downfield oxygenated sp 3 tertiary carbon (δ C 95.6), it was assumed that the structure of 1 possessed a novel oxaspirolactone moiety consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon. The hypothesis of oxaspirolactone moiety was supported by the HMBC correlations from H-21 (δ H 0.91) to C-17 (δ C 95.6), C-20 (δ C 43.7), and C- 22 (8), except for the disappearance of hydroxy group attached to C-7 in 1.

Bioassay
In an in vitro α-glucosidase inhibitory assay, compounds 1-3, 5, 6, and 9-14 were evaluated for α-glucosidase inhibitory activity. Compounds 1 and 2 exhibited more potent inhibitory activity against α-glucosidase compared with the positive control drug acarbose (IC 50 value 2.76 mM) with IC 50 value of 0.75 ± 0.018 mM and 1.64 ± 0.022 mM, respectively. The inhibition rate of other compounds was less than 50% at the concentration of 3 mM (Table S1 in Supplementary Material), suggesting that they showed no significant inhibitory activity against α-glucosidase compared with acarbose; therefore, their IC 50 value were not measured. The bioassay results are in line with the structure-activity relationships of α-glucosidase inhibitory activity of triterpenoids, which was summarized in our previous report [3].

Discussion
Fourteen triterpenoids including seven new triterpenoids (1-7) and seven known analogues (8)(9)(10)(11)(12)(13)(14) were isolated from the fruiting bodies of G. resinaceum. The IC 50 values of 1 and 2 are lower than that of the positive control drug acarbose, suggesting that 1 and 2 are strong α-glucosidase inhibitors. Compounds 1-4 and 8 were determined to be triterpenoid lactones which possessed an oxaspirolactone moiety in the side chain, consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon. It is rare that such an oxaspirolactone moiety ring occurred in the lanostane-type triterpenoids. At present, no more than 40 lanostane-type triterpenoids isolated from natural products possessed such an oxaspirolactone moiety [12,13,21]. We proposed that compounds 1-4 and 8 were biogenetically derived from lanosterol derived by the mevalonic acid pathway. In the biosynthetic pathway (Figure 3 The hydroxy at C-17 attacks ketone via nucleophilic addition reaction, inducing tandem cyclization reaction to generate 1-4 and 8. Of note, Me-27 is α-or β-orientation in 1-4 and 8, this phenomenon allows us to tentatively hypothesize that the carboxylic group at C-26 was randomly attacked via nucleophilic addition reaction in the process of free rotation of side chain. Notably, compounds 1-4 and 8 provide unique insights into the biosynthetic pathway of lanostane-type triterpenoids in Ganoderma genus, for the formation of oxaspirolactone moiety involved the tandem cyclization reaction. nucleophilic addition reaction, inducing tandem cyclization reaction to generate 1-4 and 8. Of note, Me-27 is α-or β-orientation in 1-4 and 8, this phenomenon allows us to tentatively hypothesize that the carboxylic group at C-26 was randomly attacked via nucleophilic addition reaction in the process of free rotation of side chain. Notably, compounds 1-4 and 8 provide unique insights into the biosynthetic pathway of lanostane-type triterpenoids in Ganoderma genus, for the formation of oxaspirolactone moiety involved the tandem cyclization reaction.

Conclusions
Phytochemical investigation of G. resinaceum led to the isolation of 14 triterpenoids, including seven new triterpenoids (1-7) and seven known analogues (8)(9)(10)(11)(12)(13)(14) from the fruiting bodies of G. resinaceum. Compounds 1-4 and 8 were determined to be triterpenoid lactones which possessed an oxaspirolactone moiety in the side chain, consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon. It is rare that such an oxaspirolactone moiety ring occurred in the lanostane-type triterpenoids. Based on the analysis of plausible biogenetic pathway, compounds 5-7 and 9-14 may be important intermediates in the biosynthesis of 1-4 and 8. Compounds 1 and 2 showed stronger α-glucosidase inhibitory activity than the positive control drug acarbose, which are strong α-glucosidase inhibitors with IC 50 value of 0.75 ± 0.018 mM and 1.64 ± 0.022 mM, respectively.
Supplementary Materials: The following are available online, Spectrum copies of Mass, NMR, and IR, Table S1: Inhibition rate of compounds 3, 5, 6, 9, and 11-13 at the concentration of 3 mM.