Two lanostane triterpenoids with α-glucosidase inhibitory activity from the fruiting bodies of Ganoderma weberianum

Abstract A new oxygenated lanostane-type triterpenoid, 20S,24S-epoxy-lanosta-7,9(11)-dien-3β,15α,25R,26-tetraol (1), together with three known compounds (2–4) were isolated from the fruiting bodies of Ganoderma weberianum. Extensive NMR spectrometry and HRESIMS analysis, as well as NMR and ECD calculations elucidated the structure of the new compound. 27-nor-3β-hydroxylanosta-7,9(11),23E-trien-25-one (2) showed superior α-glucosidase inhibitory activity with IC50 value of 122.1 μM to that of positive control acarbose (304.6 μM). Graphical Abstract

Ganoderma weberianum belongs to the genus Ganoderma and mainly distributed in Guizhou, Guangxi and Hainan provinces in China. G. weberianum can produce laccase to promote the biodegradation and detoxification of dyes and industrial wastewater (Dwivedi et al. 2011;Torres-Farrad a et al. 2018). The chemical constituents and pharmacological effects of the fruiting bodies of G. weberianum were rarely reported (Isaka, Chinthanom, et al. 2020). Over the course of our search for bioactive constituents from Ganoderma fungus, we performed a chemical study on the G. weberianum, leading to isolation of a new lanostane-type triterpenoid (1) and three known compounds (2-4) ( Figure 1). Previous studies revealed that many of lanostane-type triterpenoids are potential inhibitors of a-glucosidase Guo et al. 2021), which inspired us to screen the a-glucosidase inhibitory activity for compounds 1 and 2. Herein, we described the isolation, structural characterisation and a-glucosidase inhibitory activity of compounds 1-4 obtained from G. weberianum.

Results and discussion
Compound 1 was obtained as white amorphous powder and its molecular formula was determined to be C 30 (Table S1) spectra presented 30 carbon signals for seven methyls, eight methylenes (one oxygenated), seven methines (three oxygenated and two olefinic) and eight quaternary carbons (two olefinic and two oxygenated). Above-mentioned NMR data suggested that 1 was a lanostane triterpenoid and structurally similar to that of ganoderiol A . The main differences between them were that the methylene group at C-15 and the methine group at C-20 in ganoderiol A were replaced by an oxygenated methine (d C 74.4) and an oxygenated quaternary carbon (d C 85.2), respectively, which was confirmed by HMBC correlations ( Figure S2) from H 3 -30 (d H 0.96) to C-15 (d C 74.4), C-14 (d C 52.3), C-13 (d C 45.0), and C-8 (d C 140.7), and from H 3 -21 (d H 1.21) to C-20 (d C 85.2), C-17 (d C 52.0) and C-22 (d C 38.8). The presence of a tetracyclic unit and two double bonds, together with its molecular formula from HRESIMS data, indicated that one more ring was required to fulfill the seven doublebond equivalents. Detailed comparison of the chemical shifts of 1 and 26-hydroxypinnatasterone, an ecdysteroid (Santos et al. 2001) with four hydroxylated carbons (C-20 and C-24-C-26) on its side chain, revealed that the 13 C NMR chemical shifts of C-20 and C-24 of 1 were downfield shifted. Therefore, we inferred an ether bond formed between C-20 and C-24 in 1, which was supported by the similar NMR chemical shifts of C-20-C-27 of 1 with those of (20R,24R)-ocotillone (Nuanyai et al. 2011) containing a tetrahydrofuran ring on its side chain and the ROESY correlation of H 3 -21/H 2 -26 ( Figure S2). The relative configuration of the tetracyclic core structure of 1 was determined by the ROESY data ( Figure S2  tetrahydrofuran ring. However, owing to the flexibility of the single bond between C-17 and C-20, and C-24 and C-25, it was difficult to unambiguously define the relative configuration of stereocenters C-20, C-24 and C-25 only based on the ROESY correlations. Therefore, we performed theoretical NMR calculations of the four possible diastereomers (1a: 20R,24R,25R-1, 1 b: 20R,24R,25S-1, 1c: 20S,24S,25R-1 and 1d: 20S,24S,25S-1) of 1 ( Figure S13) at the PCM/mPW1PW91/6-311G(d,p) level in methanol with the GIAO method. The computed chemical shifts of 1c showed best agreement with the experimental values. Furthermore, DP4þ analysis also predicted that 1c was the most likely candidates with 100% probability.
Compound 2 showed stronger inhibitory activity against a-glucosidase with IC 50 value of 122.1 ± 5.5 lM, than that of acarbose (IC 50 304.6 ± 15.9 lM), but compound 1 only showed 13.5% inhibition at the highest tested concentration of 200 lM.

Fungal material
The fruiting bodies of G. weberianum were collected in August 2013 from Leye county, Guangxi province and identified as G. weberianum (Bres. and Henn.) by professor Niankai Zeng of Hainan Medical University. The voucher specimen (No. 2013WBLZ01) was deposited in the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

a-Glucosidase inhibitory activity
Compounds 1 and 2 were evaluated for their inhibitory effects against a-glucosidase using p-NPG as the substrate referring to the previous method (Ye et al. 2017) with acarbose as the positive control.

Conclusion
In summary, the chemical study on the ethanol extract of the G. weberianum fruiting bodies lead to isolation of four compounds including one new oxygenated lanostanetype triterpenoid, 20S,24S-epoxy-lanosta-7,9(11)-dien-3b,15a,25R,26-tetraol (1) and three known compounds. Compound 2 showed stronger inhibitory activities against a-glucosidase than that of positive control acarbose with IC 50 value of 122.1 lM. Our study enriched the chemical constituents in G. weberianum and provided new insights for the usage of G. weberianum.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Natural Science Foundation of China (81973568)