Abstract
Guided by the MS/MS metabolome-based molecular networking, 50 compounds, including 18 new triterpenoid metabolites (ganoleucocontins A–R, 1–18), were obtained from Ganoderma leucocontextum. The chemical structures of these novel metabolites were determined based on comprehensive spectroscopic methods and chemical derivation. Compounds 12–15, 17, and 18 represent a new class of triterpenoid dimers linked by 3-hydroxy-3-methylglutaryl (HMG), which were reported for the first time. The cytotoxicity was assessed in Huh7 cells, revealing IC50 values of 24.39–47.29 μM for new compounds 10, 13, 15, 16, and 18. Further studies on the anticancer properties demonstrated that compounds 10 and 15 inhibited tumor cell growth by promoting apoptosis and disrupting the cell cycle. Meanwhile, the mass spectrometry imaging experiments showed that triterpenoids were predominantly distributed at the edge of the upper shell layer in both the cap and stipe, supporting the peels of G. leucocontexum as a main distribution of Ganoderma triterpenoids.
Introduction
Bioactive components form the chemical foundation for the health benefits associated with functional foods. To efficiently excavate the bioactive substances from the edible and medicinal materials, approaches based on genome, metabolome, or artificial intelligence have been developed. − The LC-MS/MS metabolome-based molecular networking (MN) screening is one of the successfully applied methods for variation analysis, target isolation, as well as the discovery of new constituents in food materials. , For example, the MN method was applied in the analysis of Rosa roxburghii Tratt, resulting in the discovery of 17 new ascorbic acid derivatives. By integrating an untargeted metabolomics approach with MN analysis, four peptides were identified for the first time in the wild Morchella. In another study, seven steroid compounds and eight fatty acid derivatives were discovered in the MeOH extracts of six Pleurotus with the aid of MN.
Mushrooms in the genus Ganoderma are reported to contain diverse natural products, including polysaccharides, triterpenoids, meroterpenoids, and steroids. − A search in the Reaxys database (accessed on December 01, 2024) revealed approximately 2000 secondary metabolites from Ganoderma species, with over 1300 of them classified as lanostane triterpenoids. The Ganoderma triterpenoids exhibited various biological properties, including antitumor, antiviral, anti-inflammatory, and hepatoprotective effects. Ganoderma leucocontextum is exclusively distributed in the region of the Qinghai-Tibet Plateau. In our earlier research on G. leucocontextum, we found that triterpenoids hybridized with the farnesyl hydroquinone or HMG moiety showed significant cytotoxicity and inhibitory effects on α-glucosidase and HMG-CoA reductase. −
In this study, an MS/MS metabolome-based molecular networking analysis was conducted on the EtOAc extract from the fruiting body of cultivated G. leucocontextum (Figure ). The visualized MN, comprising 25 clusters and 1681 connected nodes, revealed nodes containing potential new triterpenoid analogs with ganoleucoins A (27), K (28), and N (40) serving as probing molecules. Subsequently, guided by the MN analysis, 50 compounds including 18 new lanostane-type triterpenoids, ganoleucocontins A–R (1–18) were obtained (Figures and , Table S1). Herein, we report the MN-guided isolation, structural elucidation, biological evaluation on the human hepatocellular carcinoma Huh7 cell line, and spatial distribution of these triterpenes in the fruiting body of G. leucocontextum using MALDI-MSI technology.
1.
New compounds obtained under the guidance of the molecular network. (A) Molecular network of the EtOAc extract from G. leucocontextum using the GNPS platform. The molecular cluster including the novel triterpenoid analogues is highlighted, and the parent m/z values of the nodes are displayed. (B) New compounds (1–18) isolated from G. leucocontextum.
2.
Known compounds (19–50) obtained in this study.
Materials and Methods
General Experimental Procedures
The high-performance liquid chromatography (HPLC) separation and thin-layer chromatography (TLC) analysis were performed in the same manner as described in our early work. UV, CD, NMR, high-resolution time-of-flight mass spectrometry with Electrospray Ionization (HRESIMS) data, and optical rotation values were also obtained with the same instruments as previously reported. Electronic circular dichroism (ECD) calculation was conducted using the same software and protocol as previously reported.
Fungal Material
The fruiting bodies of G. leucocontexum were provided by Tibet Lingzhi Biotech Co. Ltd. and authenticated by Jingzu Sun on the basis of morphological characteristics consistent with published descriptions of G. leucocontexum.
Extraction and Isolation
The ethanol extracts (600.2 g) of G. leucocontexum (7 kg) were dried under vacuum and further partitioned between water and ethyl acetate (EtOAc). The obtained EtOAc extract (209.1 g) was separated on a silica gel chromatographic column (CC) eluted with hexane-ethyl acetate and dichloromethane-methanol to give 19 fractions (Fr. 1 to Fr. 19) based on TLC analysis.
Fr. Thirteen (35.2 g) and Fr. Fifteen (50.1 g) that contain potential new triterpenoid metabolites were separated on an ODS CC, eluting with MeOH-H2O from 30 to 100% to give ten (Fr. 15–1 to Fr.15–10) and 12 subfractions (Fr. 13–1 to Fr. 13–12), respectively.
Fr. 13–3 was subjected to Sephadex LH-20 CC to give six subfractions (Fr. 13–3–1 to Fr. 13–3–6). Next, Fr. 13–3–6 was further separated using an ODS CC to give five subfractions (Fr. 13–3–6–1 to Fr. 13–3–6–5). Compounds 45 (4.2 mg) and 46 (3.8 mg) were purified from Fr. 13–3–6–2 by RP-HPLC using 45% MeCN-H2O. Compounds 47 (3.9 mg), 48 (3.2 mg), 49 (4.5 mg min), and 50 (5.2 mg) were obtained from Fr. 13–3–6–4 by RP-HPLC using 60% MeCN-H2O. Fr. 13–4 was subjected to Sephadex LH-20 CC to give five subfractions (Fr. 13–4–1 to Fr. 13–4–5). Fr. 13–4–1 was purified by repeated RP-HPLC using 75% MeCN-H2O to yield compounds 13 (3.6 mg), 14 (3.9 mg), 16 (5.3 mg), 15 (11.9 mg), 17 (5.1 mg), and 18 (3.2 mg). Fr. 13–4–3 was further separated by ODS CC to give six subfractions (Fr. 13–4–3–1 to Fr. 13–4–3–6). Fr. 13–4–3–6 was separated by RP-HPLC using 80% MeCN-H2O to yield compounds 35 (5.3 mg), 36 (3.9 mg), and 37 (6.7 mg). Six subfractions (Fr. 13–5–1 to Fr. 13–5–6) were obtained from Fr. 13–5 by Sephadex LH-20 CC. Fraction 13–5–6 was further separated by repeated RP-HPLC (85% MeCN/H2O) to yield compounds 11 (4.6 mg), 12 (3.9 mg), 40 (6.3 mg), 41 (5.9 mg), 42 (5.1 mg), 43 (3.2 mg), and 44 (3.2 mg).
Fr. 15–2 was subjected to Sephadex LH-20 CC to afford five subfractions (Fr. 15–2–1 to Fr. 15–5–5). Compounds 31 (4.5 mg) and 32 (5.1 mg) were purified from the fraction Fr. 15–2–3 by RP-HPLC (27% MeCN/H2O). Fr. 15–3 was processed through Sephadex LH-20 CC to afford six subfractions (Fr. 15–3–1 to Fr. 15–3–6). Fr. 15–3–2 was further purified by RP-HPLC (42% MeCN/H2O), yielding compounds 7 (10.3 mg), 8 (4.2 mg), and compounds 9 (3.6 mg), 28 (35.7 mg), and 29 (24.0 mg). Fr. 15–3–4 was next separated on an ODS CC to give five subfractions (Fr. 15–3–4–1 to Fr. 15–3–4–5). Fr. 15–3–4–2 was separated by RP-HPLC (30% MeCN/H2O), resulting in purification of compounds 20 (5.8 mg), 21 (5.6 mg), and 23 (4.8 mg). Compounds 1 (5.2 mg), 2 (58.6 mg), 3 (7.5 mg), 4 (4.5 mg), 5 (6.1 mg), 27 (5.5 mg), 24 (56.8 mg), 22 (4.3 mg), 25 (58.4 mg), and 26 (4.4 mg) were obtained from Fr. 15–3–4–3 by repeated RP-HPLC (40% MeCN/H2O). Fr. 15–3–4–5 was purified by RP-HPLC (45% MeCN/H2O) to yield compounds 6 (3.9 mg), 10 (14.4 mg), and 19 (6.7 mg). Fr. 15–4 was subjected to Sephadex LH-20 CC, giving seven subfractions (Fr. 15–4–1 to Fr. 15–4–7). Fr. 15–4–3 was purified by RP-HPLC (50% MeCN/H2O) to yield 30 (30.2 mg). Fr. 15–5 was first subjected to Sephadex LH-20 CC to afford six subfractions (Fr. 15–5–1 to Fr. 15–5–6). Compounds 33 (9.5 mg), 34 (4.7 mg), and 38 (22.8 mg) were purified from Fr. 15–5–4 by RP-HPLC (55% MeCN/H2O).
Ganoleucocontin A (1): amorphous powder; UV (MeOH) λmax (log ε) 225 (4.01), 272 (2.00) nm; [α]25 D + 53.7 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 228 (+6.2), 280 (+3.4), 306 (−3.0) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 515.3009 (calcd. for C30H43O7, 515.3003); its 1H and 13C NMR (CDCl3) data; see Table ; the calculated ECD spectrum of its stereoisomer 1a was obtained by using an established method.
1. NMR Data of Compounds 1–6 (δ in ppm).
| 1
|
2
|
3
|
4
|
5
|
6
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| no. | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) |
| 1 | 35.7 | 2.91 m | 35.9 | 2.92 m | 34.7 | 2.89 m | 33.8 | 2.83 m | 34.9 | 2.99 m | 34.3 | 2.86 m |
| 1.55 m | 1.86 m | 1.51 m | 1.72 m | 1.85 m | 1.79 m | |||||||
| 2 | 34.7 | 2.58 m | 34.5 | 2.65 m | 34.5 | 2.59 m | 34.2 | 2.69 m | 34.5 | 2.71 m | 37.5 | 2.62 m |
| 2.42 m | 2.42 m | 2.47 m | 2.48 m | 2.42 m | 2.49 m | |||||||
| 3 | 220.3 | 220.6 | 219.1 | 218.1 | 218.4 | 217.6 | ||||||
| 4 | 50.2 | 50.1 | 50.4 | 50.3 | 50.0 | 53.4 | ||||||
| 5 | 49.3 | 1.80 dd (14.0, 1.9) | 48.9 | 1.94 dd (13.9, 2.0) | 49.3 | 1.81 dd (14.2, 1.9) | 51.6 | 2.45 dd (14.1, 1.9) | 49.8 | 2.42 dd (15.2, 3.1) | 45.2 | 2.89 dd (12.9, 2.4) |
| 6 | 27.0 | 2.18 m | 27.1 | 2.13 m | 27.2 | 2.24 m | 36.9 | 2.57 m | 36.4 | 2.60 m | 37.9 | 2.77 m |
| 1.56 m | 1.53 m | 1.66 m | 2.72 dd (14.1, 8.9) | 2.55 dd (14.8, 3.1) | 2.38 dd (13.1, 2.4) | |||||||
| 7 | 66.3 | 4.83 dd (9.6, 7.5) | 66.2 | 4.84 dd (9.4, 7.7) | 65.9 | 4.94 dd (9.4, 7.7) | 198.2 | 4.83 dd (9.6, 7.5) | 204.1 | 201.6 | ||
| 8 | 158.0 | 160.8 | 161.3 | 147.6 | 151.1 | 148.1 | ||||||
| 9 | 140.8 | 138.8 | 142.2 | 148.6 | 152.5 | 151.5 | ||||||
| 10 | 38.0 | 38.1 | 37.9 | 38.9 | 38.9 | 40.5 | ||||||
| 11 | 197.8 | 201.1 | 189.3 | 201.9 | 201.3 | 201.3 | ||||||
| 12 | 50.3 | 2.75 s | 78.9 | 3.82 s | 197.6 | 77.9 | 4.52 s | 52.0 | 2.81 d (16.8) | 50.0 | 3.10 d (15.9) | |
| 2.59 d (16.8) | 2.76 d (15.9) | |||||||||||
| 13 | 45.0 | 46.7 | 61.8 | 49.4 | 47.7 | 45.1 | ||||||
| 14 | 59.4 | 58.7 | 61.9 | 58.0 | 52.8 | 58.7 | ||||||
| 15 | 218.1 | 218.4 | 215.2 | 206.5 | 72.3 | 4.28 dd (9.2, 6.1) | 210.4 | |||||
| 16 | 41.4 | 2.75 dd (19.4, 8.1) | 41.5 | 2.79 dd (19.4, 8.6) | 41.2 | 2.85 dd (19.8, 8.6) | 37.5 | 2.72 dd (18.3, 8.3) | 36.4 | 2.58 m | 41.0 | 2.91 dd (18.3, 9.7) |
| 2.08 dd (19.4, 9.7) | 2.11 dd (19.4, 9.8) | 2.15 dd (19.8, 10.0) | 2.01 dd (18.3, 8.1) | 1.92 m | 1.87 dd (18.3, 8.1) | |||||||
| 17 | 46.4 | 1.99 m | 39.2 | 2.69 m | 39.6 | 2.66 m | 45.8 | 2.59 m | 48.4 | 1.82 m | 46.1 | 2.28 m |
| 18 | 18.0 | 1.00 s | 18.5 | 0.98 s | 14.2 | 1.29 s | 10.9 | 0.66 s | 17.6 | 0.86 s | 16.4 | 0.89 s |
| 19 | 19.3 | 1.21 s | 19.9 | 1.12 s | 18.8 | 1.33 s | 19.5 | 1.38 s | 18.2 | 1.27 s | 18.8 | 1.34 s |
| 20 | 35.7 | 2.01 m | 35.9 | 1.58 m | 36.3 | 1.46 m | 33.0 | 1.70 m | 36.0 | 1.39 m | 36.9 | 1.58 m |
| 21 | 18.3 | 1.02 d (6.5) | 17.2 | 1.11 d (6.5) | 18.3 | 1.01 d (6.5) | 20.4 | 1.16 d (6.5) | 18.6 | 0.89 d (6.5) | 18.9 | 1.05 d (6.5) |
| 22 | 34.6 | 1.65 m | 34.6 | 1.53 m | 34.3 | 1.51 m | 34.0 | 1.54 m | 34.5 | 1.55 m | 35.6 | 1.55 m |
| 1.45 m | 1.21 m | 1.34 m | 1.18 m | 1.21 m | 1.29 m | |||||||
| 23 | 25.8 | 2.28 m | 25.9 | 2.31 m | 26.1 | 2.33 m | 26.7 | 2.28 m | 25.8 | 2.34 m | 26.4 | 2.32 m |
| 2.15 m | 2.16 m | 2.16 m | 2.15 m | 2.11 m | 2.20 m | |||||||
| 24 | 144.2 | 6.84 t (7.3) | 144.7 | 6.87 t (7.3) | 144.2 | 6.84 t (7.3) | 144.6 | 6.85 t (7.3) | 144.7 | 6.85 t (7.3) | 143.8 | 6.79 t (7.3) |
| 25 | 127.5 | 127.3 | 127.5 | 127.2 | 127.3 | 129.1 | ||||||
| 26 | 172.1 | 172.1 | 171.5 | 172.0 | 172.4 | 171.6 | ||||||
| 27 | 12.3 | 1.85 s | 12.3 | 1.86 s | 12.3 | 1.86 s | 12.2 | 1.83 s | 12.2 | 1.83 s | 12.4 | 1.84 s |
| 28 | 22.2 | 1.33 s | 22.3 | 1.34 s | 22.2 | 1.32 s | 22.7 | 1.31 s | 22.7 | 1.31 s | 17.2 | 1.01 s |
| 29 | 65.7 | 4.08 d (11.3) | 65.6 | 4.07 d (11.3) | 65.5 | 4.05 d (11.3) | 65.5 | 3.99 d (11.3) | 65.4 | 3.98 d (11.3) | 68.5 | 3.50 d (10.8) |
| 3.44 d (11.3) | 3.44 d (11.3) | 3.49 d (11.3) | 3.47 d (11.3) | 3.47 d (11.3) | 3.39 d (10.8) | |||||||
| 30 | 24.9 | 1.34 s | 27.1 | 1.35 s | 27.5 | 1.27 s | 20.1 | 1.75 s | 20.6 | 1.19 s | 21.1 | 1.70 s |
1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data were measured in CDCl3.
1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data were measured in CD3OD.
Ganoleucocontin B (2): amorphous powder; UV (MeOH) λmax (log ε) 216 (4.09), 272 (2.10) nm; [α]25 D + 31.2 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 224 (+8.1), 281 (+4.9), 310 (−4.6) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 531.2952 (calcd. for C30H43O8, 531.2952); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin C (3): amorphous powder; UV (MeOH) λmax (log ε) 224 (4.04), 270 (2.00) nm; [α]25 D + 85.4 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 227 (+3.7), 278 (+2.8), 307 (−0.6) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 529.2794 (calcd. for C30H41O8, 529.2796); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin D (4): amorphous powder; UV (MeOH) λmax (log ε) 216 (4.10), 272 (2.01) nm; [α]25 D + 62.5 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 227 (+8.2), 278 (+4.1), 307 (−3.3) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 529.2796 (calcd. for C30H41O8, 529.2796); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin E (5): amorphous powder; UV (MeOH) λmax (log ε) 218 (4.16), 268 (2.00) nm; [α]25 D + 71.5 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 224 (+4.2), 281 (+2.1), 317 (−3.3) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 515.2997 (calcd. for C30H43O7, 515.3003); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin F (6): amorphous powder; UV (MeOH) λmax (log ε) 218 (4.04), 270 (1.98) nm; [α]25 D + 28.0 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 225 (+4.0), 279 (+2.5), 310 (−3.1) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 513.2854 (calcd. for C30H41O7, 513.2847); its 1H and 13C NMR (CD3OD) data, see Table .
Ganoleucocontin G (7): yellow powder; UV (MeOH) λmax (log ε) 224 (4.01), 282 (2.00) nm; [α]25 D + 19.2 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 237 (+8.2), 281 (+4.9), 317 (−5.1) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 715.3322 (calcd. for C38H51O13, 715.3324); its 1H and 13C NMR (CDCl3) data, see Table .
2. NMR Data of Compounds 7–9 (δ in ppm).
| 7
|
8
|
9
|
||||
|---|---|---|---|---|---|---|
| no. | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) |
| 1 | 33.8 | 2.88 m | 28.5 | 2.59 m | 28.8 | 2.67 m |
| 1.67 m | 1.57 m | 1.41 m | ||||
| 2 | 34.3 | 2.65 m | 25.8 | 1.94 m | 23.1 | 1.91 m |
| 2.61 m | 1.69 m | 1.74 m | ||||
| 3 | 211.4 | 69.9 | 3.72 t (3.2) | 72.1 | 4.91 t (2.8) | |
| 4 | 50.4 | 41.4 | 40.7 | |||
| 5 | 51.7 | 2.34 dd (14.1, 2.6) | 45.2 | 2.25 dd (14.9, 2.4) | 46.5 | 2.18 dd (14.1, 2.7) |
| 6 | 37.3 | 2.64 m | 36.8 | 2.65 m | 36.5 | 2.67 m |
| 2.76 dd (15.1, 2.6) | 2.54 dd (14.5, 2.4) | 2.57 dd (15.1, 2.7) | ||||
| 7 | 198.0 | 199.4 | 199.3 | |||
| 8 | 146.3 | 146.4 | 146.7 | |||
| 9 | 149.9 | 152.1 | 151.6 | |||
| 10 | 39.5 | 40.2 | 40.4 | |||
| 11 | 194.1 | 199.9 | 199.8 | |||
| 12 | 79.3 | 5.66 s | 49.5 | 2.89 d (15.7) | 49.5 | 2.91 d (15.7) |
| 2.72 d (15.7) | 2.57 d (15.7) | |||||
| 13 | 47.8 | 44.4 | 44.4 | |||
| 14 | 58.7 | 57.3 | 57.2 | |||
| 15 | 206.6 | 208.3 | 208.3 | |||
| 16 | 37.6 | 2.80 dd (18.5, 8.1) | 40.4 | 2.78 dd (18.3, 9.1) | 40.7 | 2.78 dd (18.5, 8.1) |
| 2.08 dd (18.5, 8.3) | 1.83 dd (18.3, 8.1) | 1.93 dd (18.5, 8.3) | ||||
| 17 | 45.3 | 2.49 m | 45.5 | 2.12 m | 45.6 | 2.13 m |
| 18 | 12.3 | 0.84 s | 16.2 | 0.82 s | 16.3 | 0.84 s |
| 19 | 18.5 | 1.43 s | 18.6 | 1.31 s | 18.0 | 1.30 s |
| 20 | 33.2 | 1.64 m | 35.7 | 1.52 m | 35.8 | 1.51 m |
| 21 | 21.1 | 1.03 d (6.5) | 18.2 | 0.99 d (6.7) | 18.5 | 0.99 d (6.5) |
| 22 | 33.6 | 1.56 m | 34.5 | 1.52 m | 34.5 | 1.51 m |
| 1.19 m | 1.25 m | 1.21 m | ||||
| 23 | 26.5 | 2.28 m | 25.8 | 2.29 m | 25.8 | 2.27 m |
| 2.16 m | 2.15 m | 2.13 m | ||||
| 24 | 144.2 | 6.85 t (7.3) | 144.7 | 6.86 t (7.3) | 144.4 | 6.84 t (7.3) |
| 25 | 127.7 | 127.3 | 127.5 | |||
| 26 | 172.8 | 172.3 | 172.5 | |||
| 27 | 12.2 | 1.83 s | 12.2 | 1.84 s | 12.2 | 1.83 s |
| 28 | 22.6 | 1.21 s | 22.1 | 1.09 s | 22.1 | 1.01 s |
| 29 | 65.7 | 4.51 d (11.7) | 68.3 | 4.26 d (11.4) | 66.9 | 4.51 d (11.4) |
| 4.15 d (11.7) | 4.07 d (11.4) | 4.15 d (11.4) | ||||
| 30 | 21.0 | 1.77 s | 21.6 | 1.57 s | 21.3 | 1.60 s |
| 1′ | 171.6 | 172.1 | 172.0 | |||
| 2′ | 44.7 | 2.71 d (15.8) | 44.8 | 2.72 d (15.6) | 44.8 | 2.74 d (15.6) |
| 2.60 d (15.8) | 2.63 d (15.6) | 2.64 d (15.6) | ||||
| 3′ | 69.9 | 69.9 | 69.9 | |||
| 4′ | 44.8 | 2.71 d (14.5) | 44.9 | 2.74 d (14.3) | 44.9 | 2.71 d (14.3) |
| 2.64 d (14.5) | 2.65 d (14.3) | 2.64 d (14.3) | ||||
| 5′ | 175.0 | 174.8 | 174.7 | |||
| 6′ | 27.4 | 1.37 s | 27.7 | 1.39 s | 27.7 | 1.39 s |
| –COCH3 | 170.3 | 170.7 | ||||
| –COCH 3 | 20.5 | 2.22 s | 21.7 | 2.06 s | ||
1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data were measured in CDCl3.
Ganoleucocontin H (8): yellow powder; UV (MeOH) λmax (log ε) 216 (4.10), 280 (2.03) nm; [α]25 D + 70.5 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 225 (+8.2), 281 (+4.9), 317 (−5.3) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 659.3476 (calcd. for C36H51O11, 659.3477); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin I (9): yellow powder; UV (MeOH) λmax (log ε) 218 (4.04), 280 (2.00) nm; [α]25 D + 34.8 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 237 (+3.1), 281 (+2.9), 317 (−3.3) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 701.3529 (calcd. for C38H53O12, 701.3532); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin J (10): amorphous powder; UV (MeOH) λmax (log ε) 302 (4.10) nm; [α]25 D + 28.0 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 292 (−9.2), 342 (4.6) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 487.3418 (calcd. for C30H47O5, 487.3419); its 1H and 13C NMR (CDCl3) data, see Table .
3. NMR Data of Compounds 10–12 (δ in ppm).
| 10
|
11
|
12
|
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|---|---|---|---|---|---|---|
| no. | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) |
| 1 | 36.6 | 2.29 m | 35.7 | 2.01 m | 35.7 | 2.01 m |
| 1.98 m | 1.47 m | 1.47 m | ||||
| 2 | 34.2 | 2.91 m | 27.8 | 1.73 m | 27.8 | 1.73 m |
| 2.38 m | 1.62 m | 1.62 m | ||||
| 3 | 214.8 | 79.1 | 3.26 dd (11.4, 4.4) | 79.1 | 3.26 dd (11.4, 4.4) | |
| 4 | 47.0 | 38.7 | 38.7 | |||
| 5 | 60.1 | 2.60 s | 49.1 | 1.09 dd (11.0, 4.5) | 49.1 | 1.09 dd (11.0, 4.5) |
| 6 | 198.9 | 22.7 | 2.16 m | 22.7 | 2.16 m | |
| 2.11 m | 2.11 m | |||||
| 7 | 122.1 | 5.70 s | 120.3 | 5.46 d (6.1) | 120.3 | 5.46 d (6.1) |
| 8 | 159.0 | 145.9 | 145.9 | |||
| 9 | 143.7 | 142.5 | 142.5 | |||
| 10 | 39.3 | 37.4 | 37.4 | |||
| 11 | 127.5 | 5.89 d (6.8) | 116.2 | 5.31 d (6.2) | 116.2 | 5.31 d (6.2) |
| 12 | 38.1 | 2.41 d (11.7) | 37.8 | 2.21 d (17.4) | 37.8 | 2.21 d (17.4) |
| 2.31 d (11.7) | 2.08 d (17.4) | 2.08 d (17.4) | ||||
| 13 | 44.2 | 43.8 | 43.8 | |||
| 14 | 51.4 | 50.3 | 50.3 | |||
| 15 | 31.0 | 1.73 m | 31.5 | 1.62 m | 31.5 | 1.59 m |
| 1.49 m | 1.39 m | 1.37 m | ||||
| 16 | 27.5 | 2.05 m | 27.9 | 1.72 m | 27.9 | 1.72 m |
| 1.42 m | 1.27 m | 1.26 m | ||||
| 17 | 50.9 | 1.68 m | 50.9 | 1.55 m | 50.9 | 1.58 m |
| 18 | 16.0 | 0.70 s | 15.8 | 0.57 s | 15.8 | 0.55 s |
| 19 | 24.8 | 1.43 s | 23.0 | 0.98 s | 23.0 | 0.98 s |
| 20 | 36.4 | 1.45 m | 35.7 | 2.01 m | 36.8 | 1.37 m |
| 21 | 18.8 | 0.93 d (6.2) | 18.4 | 0.91 d (6.3) | 18.6 | 0.92 d (6.3) |
| 22 | 33.3 | 1.83 m | 32.8 | 1.47 m | 33.2 | 1.49 m |
| 1.02 m | 1.06 m | 1.06 m | ||||
| 23 | 29.1 | 1.64 m | 24.8 | 2.02 m | 25.3 | 2.03 m |
| 1.25 m | 1.74 m | 1.49 m | ||||
| 24 | 79.7 | 3.52 d (10.7) | 76.9 | 4.98 dd (10.7, 1.8) | 77.7 | 4.94 dd (10.7, 1.8) |
| 25 | 74.7 | 73.3 | 73.4 | |||
| 26 | 67.2 | 3.87 d (11.1) | 66.8 | 3.49 d (12.1) | 66.8 | 3.48 d (12.1) |
| 3.53 d (11.1) | 3.32 d (12.1) | 3.31 d (12.1) | ||||
| 27 | 21.6 | 1.13 s | 18.2 | 1.13 s | 18.2 | 1.12 s |
| 28 | 22.3 | 1.48 s | 16.2 | 0.88 s | 16.2 | 0.88 s |
| 29 | 25.1 | 1.29 s | 28.1 | 1.01 s | 28.1 | 1.01 s |
| 30 | 25.5 | 1.01 s | 25.6 | 0.87 s | 25.6 | 0.87 s |
| 1′ | 31.7 | 3.65 dd (16.1, 8.5) | 31.7 | 3.65 dd (16.1, 8.5) | ||
| 3.59 dd (16.1, 8.5) | 3.62 dd (16.1, 8.5) | |||||
| 2′ | 141.0 | 5.99 t (8.6) | 140.8 | 5.99 t (8.6) | ||
| 3′ | 123.9 | 123.9 | ||||
| 4′ | 34.2 | 2.32 m | 34.2 | 2.32 m | ||
| 2.28 m | 2.28 m | |||||
| 5′ | 27.8 | 2.15 m | 27.8 | 2.15 m | ||
| 1.94 m | 1.94 m | |||||
| 6′ | 122.4 | 5.04 m | 122.4 | 5.04 m | ||
| 7′ | 136.6 | 136.6 | ||||
| 8′ | 39.6 | 2.01 m | 39.6 | 2.01 m | ||
| 1.95 m | 1.93 m | |||||
| 9′ | 26.6 | 2.02 m | 26.6 | 2.02 m | ||
| 1.63 m | 1.63 m | |||||
| 10′ | 124.2 | 5.06 m | 124.2 | 5.06 m | ||
| 11′ | 131.4 | 131.4 | ||||
| 12′ | 25.7 | 1.67 s | 25.7 | 1.67 s | ||
| 13′ | 17.7 | 1.59 s | 17.7 | 1.59 s | ||
| 14′ | 15.6 | 1.55 s | 15.6 | 1.55 s | ||
| 15′ | 171.2 | 171.2 | ||||
| 16′ | 130.9 | 130.9 | ||||
| 17′ | 117.0 | 6.63 d (1.5) | 117.0 | 6.63 d (1.5) | ||
| 18′ | 148.8 | 148.8 | ||||
| 19′ | 115.0 | 6.60 dd | 115.0 | 6.60 dd | ||
| (8.2, 1.5) | (8.2, 1.5) | |||||
| 20′ | 117.0 | 6.73 d (8.2) | 117.0 | 6.73 d (8.2) | ||
| 21′ | 149.3 | 149.3 | ||||
1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data were measured in CDCl3.
Ganoleucocontin K (11): yellow powder; UV (MeOH) λmax (log ε) 230 (4.16), 298 (2.00) nm; [α]25 D + 19.1 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 242 (+3.0), 305 (−1.2) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 801.5671 (calcd. for C51H77O7, 801.5664); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin L (12): yellow powder; UV (MeOH) λmax (log ε) 235 (4.01), 298 (2.10) nm; [α]25 D + 16.2 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 235 (+2.9), 307 (−1.3) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 801.5655 (calcd. for C51H77O7, 801.5664); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin M (13): amorphous powder; UV (MeOH) λmax (log ε) 238 (4.10) nm; [α]25 D + 35.8 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 225 (+4.1), 274 (+3.2), 309 (−2.5) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 1111.6718 (calcd. for C66H95O14, 1111.6716); its 1H and 13C NMR (CDCl3) data, see Table .
4. NMR Data of Compounds 13 and 18 (δ in ppm).
| 13
|
14
|
15
|
16
|
17
|
18
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| no. | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) |
| 1 | 36.6 | 2.27 m | 36.7 | 2.30 m | 36.6 | 2.27 m | 36.6 | 2.28 m | 36.8 | 2.29 m | 36.7 | 2.29 m |
| 1.75 m | 1.76 m | 1.75 m | 1.75 m | 1.77 m | 1.75 m | |||||||
| 2 | 34.9 | 2.77 m | 35.0 | 2.78 m | 34.9 | 2.79 m | 34.9 | 2.77 m | 35.0 | 2.78 m | 34.9 | 2.78 m |
| 2.34 m | 2.35 m | 2.36 m | 2.36 m | 2.34 m | 2.37 m | |||||||
| 3 | 216.9 | 217.0 | 217.1 | 216.9 | 217.0 | 217.0 | ||||||
| 4 | 47.5 | 47.6 | 47.6 | 47.6 | 47.6 | 47.6 | ||||||
| 5 | 50.7 | 1.53 dd (11.8, 3.8) | 50.7 | 1.54 dd (11.8, 3.8) | 50.7 | 1.59 dd (12.1, 3.7) | 50.7 | 1.56 dd (12.1, 3.7) | 50.7 | 1.55 dd (11.8, 3.8) | 50.7 | 1.58 dd (12.1, 3.7) |
| 6 | 23.6 | 2.20 m | 23.8 | 2.19 m | 23.8 | 2.19 m | 23.7 | 2.21 m | 23.8 | 2.18 m | 23.7 | 2.19 m |
| 2.05 m | 2.03 m | 2.04 m | 2.05 m | 2.04 m | 2.05 m | |||||||
| 7 | 119.9 | 5.49 d (6.6) | 119.9 | 5.49 d (6.6) | 119.9 | 5.49 d (6.6) | 119.9 | 5.49 d (6.6) | 119.9 | 5.49 d (6.6) | 119.9 | 5.50 d (6.6) |
| 8 | 142.8 | 142.9 | 142.8 | 142.8 | 142.9 | 142.9 | ||||||
| 9 | 144.5 | 144.5 | 144.5 | 144.5 | 144.5 | 144.6 | ||||||
| 10 | 37.2 | 37.3 | 37.2 | 37.2 | 37.3 | 37.2 | ||||||
| 11 | 117.2 | 5.38 d (6.1) | 117.3 | 5.38 d (6.1) | 117.4 | 5.38 d (6.1) | 117.2 | 5.39 d (6.1) | 117.3 | 5.38 d (6.1) | 117.3 | 5.39 d (6.1) |
| 12 | 37.8 | 2.21 m | 37.8 | 2.22 m | 37.7 | 2.23 m | 37.8 | 2.22 m | 37.9 | 2.23 m | 37.8 | 2.24 m |
| 2.08 m | 2.11 m | 2.11 m | 2.10 m | 2.11 m | 2.10 m | |||||||
| 13 | 43.6 | 43.8 | 43.7 | 1.63 m | 43.6 | 1.62 m | 43.8 | 43.7 | ||||
| 1.39 m | 1.39 m | |||||||||||
| 14 | 50.7 | 50.7 | 50.5 | 1.93 m | 50.6 | 1.95 m | 50.7 | 50.5 | ||||
| 1.28 m | 1.29 m | |||||||||||
| 15 | 31.4 | 1.62 m | 31.5 | 1.63 m | 31.6 | 1.56 m | 31.5 | 1.55 m | 31.4 | 1.64 m | 31.5 | 1.64 m |
| 1.38 m | 1.39 m | 1.38 m | 1.40 m | |||||||||
| 16 | 27.8 | 1.92 m | 27.8 | 1.94 m | 27.8 | 0.58 s | 27.8 | 0.58 s | 27.8 | 1.95 m | 27.8 | 1.93 m |
| 1.28 m | 1.29 m | 1.29 m | 1.28 m | |||||||||
| 17 | 50.6 | 1.56 m | 50.8 | 1.55 m | 50.7 | 1.19 s | 50.8 | 1.20 s | 50.8 | 1.56 m | 50.9 | 1.58 m |
| 18 | 15.7 | 0.58 s | 15.8 | 0.57 s | 15.8 | 1.42 m | 15.7 | 1.41 m | 15.8 | 0.58 s | 15.8 | 0.58 s |
| 19 | 22.3 | 1.20 s | 22.4 | 1.19 s | 22.2 | 0.90 d (6.5) | 22.3 | 0.91 d (6.5) | 22.4 | 1.19 s | 22.1 | 1.20 s |
| 20 | 36.4 | 1.37 m | 36.5 | 1.38 m | 36.5 | 1.80 m | 36.5 | 1.82 m | 35.8 | 1.44 m | 36.5 | 1.41 m |
| 1.01 m | 1.02 m | |||||||||||
| 21 | 18.4 | 0.91 d (6.4) | 18.7 | 0.90 d (6.5) | 18.7 | 1.70 m | 18.6 | 1.69 m | 18.6 | 0.89 d (6.5) | 18.6 | 0.91 d (6.5) |
| 1.21 m | 1.20 m | |||||||||||
| 22 | 32.9 | 1.39 m | 32.8 | 1.39 m | 33.4 | 3.41 d (10.2) | 33.5 | 3.41 d (10.2) | 32.7 | 1.42 m | 33.5 | 1.81 m |
| 0.98 m | 1.00 m | 1.05 m | 1.03 m | |||||||||
| 23 | 25.4 | 1.89 m | 25.3 | 1.91 m | 28.0 | 5.38 d (6.1) | 27.9 | 5.39 d (6.1) | 25.5 | 1.71 m | 28.0 | 1.68 m |
| 1.40 m | 1.41 m | 1.63 m | 1.20 m | |||||||||
| 24 | 77.7 | 4.82 dd (10.3, 2.1) | 77.7 | 4.82 dd (10.3, 2.1) | 77.5 | 2.23 m | 77.5 | 2.22 m | 77.2 | 4.88 dd | 77.1 | 3.38 d (10.2) |
| 2.11 m | 2.10 m | (10.3, 2.1) | ||||||||||
| 25 | 73.6 | 73.7 | 73.6 | 73.5 | 73.6 | 73.6 | ||||||
| 26 | 66.7 | 3.57 d (11.9) | 66.8 | 3.56 d (11.9) | 68.8 | 4.36 d (11.8) | 68.7 | 4.36 d (11.8) | 66.7 | 3.57 d (11.9) | 68.7 | 4.36 d (11.8) |
| 3.32 d (11.9) | 3.31 d (11.9) | 4.01 d (11.8) | 4.01 d (11.8) | 3.31 d (11.9) | 4.09 d (11.8) | |||||||
| 27 | 18.8 | 1.12 s | 18.8 | 1.12 s | 20.9 | 1.18 s | 20.7 | 1.19 s | 18.8 | 1.12 s | 20.9 | 1.18 s |
| 28 | 22.4 | 1.13 s | 22.5 | 1.13 s | 22.6 | 1.12 s | 22.5 | 1.13 s | 22.5 | 1.13 s | 22.6 | 1.12 s |
| 29 | 25.7 | 1.08 s | 25.7 | 1.08 s | 25.6 | 1.08 s | 25.4 | 1.09 s | 25.7 | 1.09 s | 25.6 | 1.08 s |
| 30 | 25.6 | 0.86 s | 25.6 | 0.86 s | 25.7 | 0.87 s | 25.7 | 0.86 s | 25.6 | 0.86 s | 25.7 | 0.87 s |
| 1′ | 34.2 | 3.04 m | 33.8 | 2.95 m | 33.8 | 2.95 m | 34.3 | 3.05 m | 33.9 | 2.96 m | 33.9 | 2.94 m |
| 1.68 m | 1.64 m | 1.62 m | 1.67 m | 1.67 m | 1.59 m | |||||||
| 2′ | 34.4 | 2.65 m | 34.5 | 2.72 m | 34.4 | 2.71 m | 34.5 | 2.65 m | 34.5 | 2.72 m | 34.5 | 2.69 m |
| 2.58 m | 2.59 m | 2.55 m | 2.56 m | 2.61 m | 2.56 m | |||||||
| 3′ | 211.6 | 211.1 | 211.3 | 211.7 | 211.1 | 211.4 | ||||||
| 4′ | 50.3 | 50.5 | 50.5 | 50.3 | 50.6 | 50.6 | ||||||
| 5′ | 51.5 | 2.32 m | 51.6 | 2.32 m | 51.9 | 2.31 m | 51.5 | 2.32 m | 51.8 | 2.32 m | 51.9 | 2.32 m |
| 6′ | 37.0 | 2.75 m | 37.3 | 2.84 m | 37.3 | 2.82 m | 37.0 | 2.75 m | 37.4 | 2.84 m | 37.3 | 2.81 m |
| 2.64 m | 2.65 m | 2.66 m | 2.65 m | 2.66 m | 2.64 m | |||||||
| 7′ | 198.6 | 197.8 | 197.7 | 198.5 | 197.7 | 197.6 | ||||||
| 8′ | 146.9 | 147.6 | 147.8 | 147.0 | 147.5 | 147.7 | ||||||
| 9′ | 149.8 | 148.8 | 148.8 | 149.8 | 148.8 | 148.8 | ||||||
| 10′ | 39.4 | 39.4 | 39.4 | 39.4 | 39.4 | 39.4 | ||||||
| 11′ | 199.4 | 201.8 | 201.8 | 199.4 | 201.7 | 201.7 | ||||||
| 12′ | 49.1 | 2.89 d (16.2) | 77.9 | 4.54 s | 77.8 | 4.53 s | 49.1 | 2.89 d (16.1) | 77.9 | 4.54 s | 77.9 | 4.53 s |
| 2.78 d (16.2) | 2.79 d (16.1) | |||||||||||
| 13′ | 43.7 | 49.6 | 49.6 | 43.8 | 49.6 | 49.6 | ||||||
| 14′ | 57.3 | 57.9 | 58.0 | 57.3 | 58.0 | 58.0 | ||||||
| 15′ | 207.7 | 206.8 | 206.7 | 207.6 | 206.7 | 206.6 | ||||||
| 16′ | 40.1 | 2.79 m | 37.5 | 2.75 m | 37.4 | 2.75 m | 40.1 | 2.79 m | 37.4 | 2.76 m | 37.4 | 2.74 m |
| 1.90 m | 2.00 m | 2.01 m | 1.88 m | 2.01 m | 1.98 m | |||||||
| 17′ | 45.2 | 2.13 m | 45.7 | 2.61 m | 45.6 | 2.60 m | 45.2 | 2.15 m | 45.8 | 2.61 m | 45.5 | 2.62 m |
| 18′ | 16.1 | 0.86 s | 11.1 | 0.66 s | 11.0 | 0.66 s | 16.1 | 0.86 s | 11.1 | 0.67 s | 11.1 | 0.67 s |
| 19′ | 18.4 | 1.40 s | 18.5 | 1.50 s | 18.5 | 1.49 s | 18.4 | 1.39 s | 18.5 | 1.50 s | 18.5 | 1.50 s |
| 20′ | 35.7 | 1.52 m | 32.9 | 1.75 m | 32.9 | 1.75 m | 35.8 | 1.51 m | 32.9 | 1.75 m | 32.8 | 1.76 m |
| 21′ | 18.5 | 1.01 d (6.4) | 20.5 | 1.15 d (6.4) | 20.5 | 1.15 d (6.4) | 18.4 | 1.00 d (6.4) | 20.5 | 1.16 d (6.4) | 20.5 | 1.14 d (6.4) |
| 22′ | 34.4 | 1.51 m | 33.9 | 1.55 m | 33.8 | 1.62 m | 34.4 | 1.52 m | 33.9 | 1.56 m | 33.9 | 1.62 m |
| 1.22 m | 1.22 m | 1.55 m | 1.21 m | 1.20 m | 1.57 m | |||||||
| 23′ | 25.7 | 2.27 m | 26.7 | 2.29 m | 26.7 | 2.30 m | 25.5 | 2.29 m | 26.6 | 2.27 m | 26.8 | 2.29 m |
| 2.13 m | 2.16 m | 2.18 m | 2.14 m | 2.16 m | 2.17 m | |||||||
| 24′ | 143.9 | 6.84 t (7.3) | 144.5 | 6.84 t (7.3) | 144.5 | 6.85 t (7.3) | 143.9 | 6.84 t (7.3) | 144.5 | 6.84 t (7.3) | 144.6 | 6.85 t (7.3) |
| 25′ | 127.5 | 127.5 | 127.3 | 127.4 | 127.3 | 127.3 | ||||||
| 26′ | 172.3 | 171.9 | 172.1 | 172.2 | 171.8 | 172.0 | ||||||
| 27′ | 12.1 | 1.84 s | 12.3 | 1.84 s | 12.3 | 1.83 s | 12.1 | 1.84 s | 12.3 | 1.84 s | 12.3 | 1.84 s |
| 28′ | 22.1 | 1.23 s | 22.2 | 1.22 s | 22.1 | 1.23 s | 22.1 | 1.23 s | 22.2 | 1.23 s | 22.1 | 1.23 s |
| 29′ | 65.6 | 4.59 d (11.7) | 65.7 | 4.58 d (11.7) | 65.7 | 4.58 d (11.7) | 65.7 | 4.60 d (11.7) | 65.7 | 4.57 d (11.7) | 65.7 | 4.59 d (11.7) |
| 4.11 d (11.7) | 4.10 d (11.7) | 4.10 d (11.7) | 4.11 d (11.7) | 4.11 d (11.7) | 4.09 d (11.7) | |||||||
| 30′ | 21.1 | 1.61 s | 20.3 | 1.73 s | 20.3 | 1.73 s | 21.1 | 1.61 s | 20.3 | 1.73 s | 20.3 | 1.72 s |
| 1″ | 171.7 | 171.8 | 171.7 | 171.6 | 171.4 | 171.6 | ||||||
| 2″ | 44.4 | 2.85 d (15.6) | 44.4 | 2.85 d (15.6) | 44.6 | 2.85 d (15.6) | 44.6 | 2.75 d (15.6) | 45.1 | 2.79 d (15.6) | 44.7 | 2.75 d (15.6) |
| 2.55 d (15.6) | 2.56 d (15.6) | 2.56 d (15.6) | 2.56 d (15.6) | 2.61 d (15.6) | 2.56 d (15.6) | |||||||
| 3″ | 69.9 | 69.9 | 69.9 | 69.9 | 69.9 | 69.9 | ||||||
| 4″ | 45.6 | 2.69 d (14.3) | 45.6 | 2.68 d (14.3) | 45.6 | 2.68 d (14.3) | 45.4 | 2.67 d (14.3) | 45.5 | 2.69 d (14.3) | 45.4 | 2.67 d (14.3) |
| 2.64 d (14.3) | 2.65 d (14.3) | 2.64 d (14.3) | 2.64 d (14.3) | 2.66 d (14.3) | 2.65 d (14.3) | |||||||
| 5″ | 172.2 | 172.4 | 171.5 | 171.5 | 172.9 | 171.4 | ||||||
| 6″ | 27.7 | 1.37 s | 27.8 | 1.38 s | 27.8 | 1.38 s | 27.7 | 1.37 s | 27.8 | 1.38 s | 27.8 | 1.37 s |
1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data were measured in CDCl3.
Ganoleucocontin N (14): amorphous powder; UV (MeOH) λmax (log ε) 245 (4.05) nm; [α]25 D + 27.3 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 222 (+2.2), 278 (+1.7), 305 (−2.6) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 1127.6667 (calcd. for C66H95O15, 1127.6665); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin O (15): amorphous powder; UV (MeOH) λmax (log ε) 238 (4.01) nm; [α]25 D + 17.2 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 224 (+4.6), 277 (+3.5), 311 (−1.8) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 1127.6666 (calcd. for C66H95O15, 1127.6665); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin P (16): amorphous powder; UV (MeOH) λmax (log ε) 251 (4.10) nm; [α]25 D + 50.1 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 221 (+1.3), 276 (+0.8), 307 (−1.7) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 1111.6720 (calcd. for C66H95O14, 1111.6716); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin Q (17): amorphous powder; UV (MeOH) λmax (log ε) 245 (4.02) nm; [α]25 D + 28.7 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 215 (+2.1), 278 (+1.9), 305 (−1.7) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 1127.6671 (calcd. for C66H95O15, 1127.6665); its 1H and 13C NMR (CDCl3) data, see Table .
Ganoleucocontin R (18): amorphous powder; UV (MeOH) λmax (log ε) 241 (4.10) nm; [α]25 D + 60.2 (c 0.1 MeOH); CD (MeOH): λmax (Δε) 223 (+2.3), 274 (+1.5), 302 (−1.6) nm; HRESIMS (positive-ion mode) m/z [M + H]+ 1127.6668 (calcd. for C66H95O15, 1127.6665); its 1H and 13C NMR (CDCl3) data, see Table .
Molecular Networking Analysis
The MS/MS metabolome-based data were recorded using a Q-TOF instrument (Agilent Technologies), coupled with an Agilent Eclipse XDB C8 column (150 × 4.6 mm, 5 μm). Gradient elution consisted of H2O (A) and MeCN (B) in a linear gradient manner (20–30% B (0.0–10.0 min); 30–37% B (10.1–20.0 min); 37–55% B (20.1–30.0 min); 55–100% B (30.1–60.0 min)). The file data were transformed by MSConver software, then uploaded to http://gnps.ucsd.edu for the MN analysis. Spectral networks were visualized through Cytoscape 3.8.2 with the force-directed layout. The molecular network files are available via https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=45cd17c395d14ec595ab674bbba7735e.
Alkaline Hydrolysis of Compounds 11 and 13–16
Compounds 11 and 13–16 (each 3.0 mg) were hydrolyzed in 2% MeONa/MeOH (4 mL), as described in our early work. The obtained product was further purified by preparative TLC (CH2Cl2-MeOH, 20:1) to give compounds 39 (1.3 mg) and 38 (1.0 mg), respectively.
Mass Spectrometry Imaging and Data Analysis
The frozen fresh G. leucocontexum fruiting body embedded in 10% gelatin (wt/vol) solutions was cut into sections of 30 μm thickness with a freezing microtome (Leica CM 1950, Nussloch, Germany) at −20 °C. The slices were placed on coated glass slides. 2,5-Dihydroxybenzoic acid (DHB) was used for the MALDI experiments in a positive mode at a thickness of 1.5 μm. Mass spectrometry imaging (MSI) of the fresh tissue was done on the iMScope instrument (Shimadzu, Kyoto, Japan) with an AP-MALDI source, as well as an ion trap-TOF mass spectrometer. Reconstruction of mass images was carried out using IMAGEREVEAL MS (Shimadzu, Kyoto, Japan).
Cytotoxicity on Huh7
The Huh7 cell line was generously provided by professor George F Gao (Institute of Microbiology, Chinese Academy of Sciences) and cultured in DMEM medium (Gibco, C11995500BT) supplemented with 10%v/v fetal bovine serum (Gibco, A3160801), mixed antibiotics (Gibco, 15140–122), with a humidified atmosphere at 37 °C. Huh7 cell was seeded and treated with compounds at a concentration of 25 and 50 μM in 96-well plates for 96 h. Subsequently, cell viability was assessed according to the instructions of the Cell Counting Kit-8 (Biosharp, BS350B). The cell sizes of Huh7 cells were measured and analyzed by using ImageJ software. Cisplatin (CDDP, TCI, D3371) and dimethyl sulfoxide (DMSO, MP Biomedicals, 196055) were used as controls, respectively.
Flow Cytometry Analysis
Huh7 cell was treated with 10 and 15 for 48 h, and subsequently detected using the FITC-Annexin V apoptosis detection kit with propidium iodide (PI) (BioLegend, 640914) staining. Cell cycle was detected by PI (BioLegend, 421301) staining and flow cytometry analysis using BD LSRFortessa SORP (BD Biosciences, USA).
Caspase–3/7 Activity Assay
The Huh7 cells were plated and treated with compounds 10 and 15 in the 96-well plates for 24 h. Cells were collected and examined according to the instructions of the Caspase-Glo 3/7 Assay (Promega, G8091). Luminescence readings were recorded by a GloMax 20/20 Luminometer (Promega, USA).
Western Blot
Huh7 cells were treated with 10 and 15 for 48 h. The total protein was extracted from cells by using SDS lysis buffer (Beyotime, P0013G), and isolated by SDS-PAGE, then incubated with RARP Antibody (CST, 9542S) and β-Actin Mouse Monoclonal Antibody (Tianjin Sungene Biotech Co., Ltd., KM9001L).
Real-Time PCR
The cell total RNA was extracted with the TRIzol reagent (Ambion, 15596018) as described in the manufacturer’s instructions. The RNA was reversely transcribed, and a real-time PCR method was conducted on the SYBR real-time PCR master mix (NEB M3003L). Relative expression of target genes was quantified by using the 2–ΔΔCt method.
Results and Discussion
Isolation and Structural Identification
Given that triterpenoids are the main biologically active compounds of G. leucocontexum, ganoleucoins A (27), K (28), and N (40) obtained from our previous work were submitted to GNPS as reference probes to locate the triterpenoid clusters within the MN (Figure ). A total of 25 clusters and 1681 connected nodes were found in the MN. The probes indicated the presence of triterpenoid analogs within clusters 1–3. Notably, an intriguing cluster (cluster 4), characterized by a high molecular mass range within m/z 1050–1250, suggested the probable presence of dimeric triterpenoids. Through the meticulous separation process under the guidance of LC-MS/MS, a total of 32 known compounds and 18 hitherto undescribed lanostane triterpenoids were successfully identified. These newly discovered triterpenoids were designated as ganoleucocontin A–R (1–18) (Figures and , Table S1).
Ganoleucocontin A (1) was characterized with a molecular formula of C30H42O7, indicating 10 degrees of unsaturation. The 1H NMR data of 1 showed signals corresponding to an olefinic proton at δH 6.84 (t, J = 7.3 Hz), one methine bearing an oxygen atom at δH 4.83 (dd, J = 9.6, 7.5 Hz), one oxygenated methylene at δH 4.08 (d, J = 11.3 Hz) and 3.44 (d, J = 11.3 Hz), a doublet methyl at δH 1.02 (d, J = 6.5 Hz) and five singlet methyls at δH 1.00, 1.21, 1.33, 1.34, and 1.85. The 13C NMR data, in conjunction with HSQC spectra (Figure S11), revealed 30 carbon resonances including three carbonyl carbons at δC 197.8, 218.1, and 220.3, one carboxyl carbon at δC 172.1, two pairs of olefinic carbons at δC 158.0, 140.8, 144.2, and 127.5, six methyls, eight methylenes (one oxygenated at δC 65.7), and four methines (one oxygenated at δC 66.3). The comprehensive NMR data supported a highly oxygenated lanostane-type triterpene backbone in compound 1 (Table ). The 1H–1H COSY correlations (Figure ) of H-20–H2-22–H2-23–H-24, H2-16–H-17–H-20–H3-21, H-5–H2-6–H-7, and H2-1–H2-2, along with the key HMBC correlations as indicated in Figure confirmed the complete planar structure of 1 as 7,29-dihydroxy-3,11,15-trioxolanosta-8,24-dien-26-oic acid.
3.
Key 1H–1H COSY, HMBC correlations of compounds 1, 7, and 10–13.
The relative configuration of 1 was assigned by NOE correlations of H3-30 with H-7 and H-17, H-5 with H3-28 and H3-30, and H3-19 with H3-18 and H2-29, indicated α-configurations for CH3-28, H-5, H-7, CH3-30, and H-17, and β-configurations for CH3-18, CH3-19, and CH2OH-29. The NOE correlation between H3-27 and H-23 confirmed the E-configuration at the C-24/C-25 double bond (Figure ). Furthermore, by comparing the experimental ECD spectrum with the calculated ECD spectra of two possible isomers of 1 (4S,5R,7S,10S,13R,14R,17R, 20R-1a and 4R,5S,7R,10R,13S,14S,17S,20S-1b), 1 was assigned the complete absolute configuration of 4S, 5R, 7S, 10S, 13R, 14R, 17R, and 20R (Figure S3).
4.
Key NOE correlations of compounds 1, 7, 10, 11, and 13.
According to the HRESIMS data, ganoleucocontin B (2) had a molecular formula of C30H42O8. A comparison of spectroscopic data between 2 and 1 revealed the presence of an extra oxygenated methine at δH/δC 3.82 (s)/78.9 in 2 (Table ). The HMBC correlation from H3-18 to C-12, as well as H-12 to C-14, C-13, C-9, and C-11, established the substitution of a hydroxy group at C-12 (Figure S1). The NOE correlations of H-12 with H3-19 and H3-18 assigned the β-orientation for H-12 (Figure S2). The similar Cotton effects in the CD spectra of compounds 1 and 2, along with their shared biosynthetic origin, indicated the absolute configurations in 2 to be 4S, 5R, 7S, 10S, 12R, 13R, 14R, 17R, and 20R. Thus, the structure of 2 was confirmed as 7,12,29-trihydroxy-3,11,15-trioxolanosta-8,24E-dien-26-oic acid, named ganoleucocontin B. The structural divergence between ganoleucocontin C (3) and 2 resides in the functional group at the C-12 position. Compound 3 features a carbonyl group at this C-12 position, while 2 has a hydroxyl group at the same position. The structure of 3 was identified through MS and NMR spectral analysis (Table , Figures S1 and S2), especially the key HMBC correlation between H3-18 and C-12 (δC 197.6).
Analysis of the HRESIMS and NMR data revealed that ganoleucocontin D (4) and ganoleucocontin E (5) have chemical structures similar to that of leucocontextin G (19). Compound 4 is different from 19 by an additional 12β-hydroxy group (Table ), as evidenced by the key HMBC correlations (Figure S1) along with NOE correlations of H-12 with H-17 and H3-30 (Figure S2). Compound 5 differs from 19 in that it has a hydroxyl moiety at C-15 instead of a carbonyl group, which was ascertained by the key HMBC correlations (Figure S1) of H-15 (δH 4.28) with C-13, C-14, C-17, and C-30, and H3-30 with C-15 (δC 72.3). The NOE correlation of H-15 with H3-18 indicated the β-configuration for H-15 (Figure S2). Ganoleucocontin F (6) exhibited an identical planar structure to compound 19 as supported by mass and NMR data (Table ). The NOE correlations of H3-19 with H3-28, and H2-29 with H-5 indicated the α-configuration for CH2OH-29 and the β-configuration for CH3-28, different from the corresponding relative configurations in 19 (Figure S2).
Ganoleucocontin G (7) was isolated as a yellow powder. It possessed the molecular formula C38H50O13 by HRESIMS data. Examination of the spectroscopic data (Table ) of 7 indicated a lanostane-type triterpene moiety identical to leucocontextin M (Figures and ). The remaining NMR signals, including one methyl group at δC/H 27.4/1.37 (s), two carbonyl carbons at δC 171.6, 175.0, one quaternary carbon bearing an oxygen atom at δC 69.9, and two methylene groups, were assigned as a 3-hydroxy-3-methylglutaryl (HMG) group. This assignment was also corroborated by HMBC correlations from H2-2′, H2-4′, and H3-6′, as indicated in Figure . The attachment of the HMG moiety to C-29 of the triterpenoid skeleton was verified by HMBC correlations from H2-29 (δH = 4.51, 4.15) to C-1′ (δC = 171.6) (Figure ). The HMG group is rarely reported in fungi, with known occurrences in terpenoids from Naematoloma fasciculare, Clavariadelphus truncates, and Ganoderma species, , as well as flavonoids from Oxytropis falcata. The absolute configuration at C-3′ within the HMG moiety was ascertained to be S by comparing the NMR data of compound 7, ganoleucoins K (28) and L (29), while also considering their shared biosynthetic origins.
Ganoleucocontin H (8) was assigned to be an analogue of 7 through MS and NMR analyses, belonging to the triterpene-HMG hybrids (Table ). The HMBC correlations from H-3 (δH 3.72) to C-1, C-5, C-28, and C-29, from H3-28 and H2-29 to C-3 (δC 69.9), from H2-12 (δH 2.89 and 2.72) to C-13, C-18, and C-11, and from H3-18 and H-17 to C-12 (δC 49.5) evidenced the presence of OH-3 and CH2–12 in 8 (Figure S1), different to the substitution groups at C-12 and C-3 in 7. The relative configuration at H-3 was determined to be the β-orientation by the NOE correlations of H-3 with H2-29 and H3-19 (Figure S2). Ganoleucocontin I (9) was identified as the acetylated derivative at 3-OH of 8, as supported by the presence of an acetoxyl group (δH/δC 2.06/21.7 for the methyl group and δC 170.7 for carbonyl carbon) in NMR data (Table ) and HMBC correlation (Figure S1) from H-3 to δC 170.7.
Ganoleucocontin J (10) possessed the molecular formula of C30H46O5. The NMR spectroscopic data of 10 were similar to that of ganodermanontriol (38), except for an extra carbonyl moiety at δC 198.9 (Table ). The HMBC correlations from H3-30 to C-8, C-13, and C-15, from H-5 to C-6, C-7, C-9, and C-19, from H2-12 to C-10, C-11, and C-14 indicated the presence of an α, β-unsaturated ketone moiety (Figure ) and a carbonyl moiety (δC 198.9) at C-6. The four isomers of the triol moiety in the side chain of ganodermanontriol (38) were reported in a previous study. The δC values (in CDCl3) of C-24, C-25 and C-26 were 79.3, 74.0, and 67.6 (isomer24S25R ), 77.2, 74.1, and 69.3 (isomer24R25S ), 78.5, 73.9, 67.6 (isomer24S25S ), 76.2, 74.1, and 69.4(isomer24R25R ), and 79.7, 74.7, and 67.2 (10), respectively. Subsequently, by comparison of the chemical shift values of the triol moiety in compound 10 with the literature values, the absolute configurations at C-24 and C-25 in compound 10 were confirmed to be S and R, respectively.
A molecular formula of C51H76O7 was indicated for ganoleucocontin K (11) based on its HRESIMS data. The 1H NMR spectrum of 11 exhibited ten methyl groups, one oxygenated methylene at δH 3.49 (d, J = 12.1 Hz) and 3.32 (d, J = 12.1 Hz), two oxygenated methines at δH 3.26 (dd, J = 11.4, 4.4 Hz) and 4.98 (1H, dd, J = 10.7, 1.8 Hz), and eight olefinic protons. The 13C NMR spectrum of 11 exhibited fifty-one carbon signals (Table ). Analysis of its spectroscopic data identified 11 as a triterpene-farnesyl hydroquinone hybrid similar to ganoleuconin N (40), featuring a 3β–OH in 11 instead of a 3-carbonyl in 40. Ganoleucocontin L (12) shares a similar planar structure to 11, with the difference being the chemical shift values of C-24 and C-25. To confirm the configuration in the side chain, compounds 11 and 12 were hydrolyzed to produce the corresponding triterpenes. The absolute configurations at C-24 and C-25 in the triol side chain were confirmed as 24S, 25R for 11, and 24R, 25S for 12 by comparing the chemical shift values with the literature data.
Ganoleucocontin M (13) possessed the molecular formula C66H94O14 as deduced from its HRESIMS data. The 1H NMR spectrum of 13 (Table ) showed 14 methyl groups including two doublet methyl group, two oxygenated methylene at δH 4.59 (d, J = 11.7 Hz)/4.11 (d, J = 11.7 Hz), δH 3.57 (d, J = 11.9 Hz)/3.32 (d, J = 11.9 Hz), one oxygenated methine at δH 4.82 (dd, J = 10.3, 2.1 Hz), and three olefinic protons at δH 5.49 (d, J = 6.6 Hz), 5.38 (d, J = 6.1 Hz), and 6.84 (t, J = 7.3 Hz). The 13C NMR spectrum of 13 demonstrated the presence of sixty-six carbons. Comparing the above characteristic NMR data with those of leucocontextin G (19), ganoleucoin L (29), and ganodermanontriol (38) suggested that compound 13 incorporated structural fragments corresponding to 29 and 38, which coincides with the molecular formula. Inspection of 2D NMR data fully established the structure of 13 in which two triterpenoid parts from 19 and 38 were linked with one HMG group by ester bonds from C-24 to C-5″ and from C-29′ to C-1′′, as indicated by the key HMBC correlations of H-24 (δH 4.82) with C-5″ (δC 172.2), and H2-29′ (δH 4.59 and 4.11) with C-1″ (δC 171.7) (Figure ). Ganoleucocontins N–P (14–16) and ganoleucocontins Q–R (17–18) were determined to be analogues of 13 through detailed analysis of their NMR spectral data and comparison with 13 (Table ). The absolute configurations at C-24 and C-25 were affirmed as 24S, 25R for 13–16 and 24R, 25S for 17–18 based on the alkaline hydrolysis and NMR comparison as described for 10 and 11. Compounds 13–18 represent a new type of triterpenoid dimmers.
Compounds Inhibit Tumor Cell Growth by Altering Cell Morphology, Inducing Cell Apoptosis, and Disturbing Cell Cycle
Ganoderma has been reported to possess the mitigation of alcoholic liver disease, NAFLD, fibrosis, and liver cancer effects. Furthermore, triterpenoids from Ganoderma demonstrated cytotoxic activity against various cell lines, such as K562, PC-3, MCF-7, Vero, BEL-7402, and SGC-7901. ,, In this study, we explored whether these isolated compounds could prohibit tumor cell growth. We initially treated hepatocellular carcinoma Huh7 cells with 50 compounds for 96 h and subjected them to the CCK-8 assay. The results showed that the cell viability of Huh7 cells was reduced to less than 50% by treating with compounds 10, 13, 15, 16, 18, 30, 40, 44, and 49 at a dose of 50 μM (Figure S4). The IC50 values of new compounds 10, 13, 15, 16, and 18 ranged from 24.39 to 47.29 μM (Table S2).
Considering the remaining amount of these five new bioactive compounds, 10 and 15 were selected for investigation of their anticancer properties. Huh7 cells were treated with 10 and 15 for 12, 24, and 48 h, and cell morphologies were imaged. As shown in Figure S5, the cell size decreased following treatment with 10 and 15 compared to DMSO-treated cells (Figure A), indicating the occurrence of early apoptosis. Furthermore, the apoptotic effects of 10 and 15 on Huh7 cells were investigated. Caspase 3/7 activities in cells treated with 10 and 15 were significantly upregulated, which subsequently triggered the cleavage of PARP-1 (Figure B,C). Moreover, flow cytometry analysis using FITC-Annexin V/PI staining demonstrated increased ratios of apoptotic cells following treatment with 10 and 15 (Figures D and S76). The expression levels of pro-apoptotic genes Bad, Puma, and Noxa were significantly upregulated in Huh7 cells treated with 10 and 15, whereas the expression of the anti-apoptotic gene Bcl-xl was downregulated (Figure E). These results indicated that treatments with 10 and 15 significantly promote tumor cell apoptosis, thereby inhibiting tumor cell growth.
5.
Compounds inhibit tumor cell growth by altering cell morphology, inducing cell apoptosis, and disturbing cell cycle. (A) Cell size of Huh7 cells treated with 50 μM 10 and 15 for 48 h was measured by using ImageJ software, and each point represented the average size of 5 cells per field of view. (B) Relative caspase 3/7 activity of Huh7 cells treated with 50 μM 10 and 15 for 24 h was detected by luciferase assay. (C) PARP-1 cleavage of Huh7 cells treated with 50 μM 10 and 15 for 48 h was detected by Western blot. (D) Ratio of apoptotic cells treated with 50 μM 10 and 15 for 48 h was detected by flow cytometry. (E) The expression levels of Bad, Puma, Noxa and Bcl-xl genes in Huh7 cells treated with 50 μM 10 and 15 for 48 h was detected by real-time PCR. (F) Cell cycle analysis of Huh7 cells after treatment with 50 μM 10 and 15 for 48 h was detected by flow cytometry. Data represent mean ± SEM *p < 0.05; **p < 0.01; ***p < 0.001 and not significant (ns) from one-way ANOVA. Abbreviations: Control, no treatment; CDDP, 10 μg/mL cisplatin treatment; DMSO, 0.5% DMSO treatment.
Additionally, we assessed the cell cycle of Huh7 cells treated with 10 and 15 using PI staining and flow cytometry analysis. The results showed that the S phase and G2/M phase of Huh7 cells treated with 10 were significantly reduced, indicating that 10 treatment led cells to arrest on the G0/G1 phase (Figures F and S7). After treatment with 15, the G0/G1 phase and S phase of Huh7 cells were significantly reduced, demonstrating that 15 treatment triggered cells to arrest on the G2/M phase (Figures F and S7). In summary, our findings demonstrate that 10 and 15 could inhibit tumor cell growth by altering cell morphology, inducing cell apoptosis, and inhibiting cell proliferation.
Spatial Distribution of Metabolites in the Fruiting Body of G. leucocontextum Using MSI
Mass spectrometry imaging (MSI) has emerged as a powerful tool for visualizing the spatial distribution of metabolites in the tissues of herbal medicines, owing to its extensive coverage, high sensitivity, and no need for labeling. In a recent research, MSI was employed to clarify the spatial dynamics of ganoderic acids at different maturity stages of G. lingzhi, revealing the distribution of ganoderic acids in the bottom of the cap’s mediostratum layer, as well as the stipe’s shell and context layers.
Herein, we established a suitable MALDI-MSI method to unveil the spatial distribution of functional metabolites in G. leucocontexum, aiming to provide a basis for the rational utilization of its medicinal parts. 80 compounds (Table S3) from this study and our previous research on Ganoderma were selected as a reference database for visualization. These compounds were detected by MALDI-MS/MS within the mass ranges of m/z 300–600 and m/z 600–900. A total of 20 metabolites were visualized in both the cap and the stipe of G. leucocontexum (Figure A,B). Most of the representative bioactive components, such as ganodermatriol (33), ganoderiol F (34), ganoderic acid S (35), ganoderol A (36), ganoderal A (37), and ganodermanontriol (38), were predominantly distributed at the edge of the upper shell layer in both the cap and stipe. Leucocontextins G (19) and D (20), ganoleucoins E (24), D (25), F (26), A (27), K (28), and L (29), and ganomycins B (47), C (48), and I (49) were detected in all tissue sites but primarily enriched at the edge as well. The concentrations of ganoderiol A (39) and ganoleuconin N (40) were extremely low in the cap and stipe. Subsequently, we analyzed the extracts of the peel and inner layers of the cap and stipe using HPLC analysis. The results also indicate that triterpenoids are mainly distributed on the peel layers of both the cap and stipe (Figure C,D). From a chemical ecological point of view, the distribution of triterpenes on the peels of the fruiting body may help protect this mushroom from abiotic and biotic stresses.
6.
Spatial distribution of metabolites in fruiting body of G. leucocontextum using mass spectrometry imaging (MSI). (A) Slice positions in the cap and stipe of G. leucocontextum. Sampling sites including a and b in the slice of cap and c and d in the slice of stipe. (B) Distribution of the bioactive components in diverse tissue parts of G. leucocontextum fruiting body visualized via MSI. Compounds were detected by MALDI-MS/MS in positive ionization mode within the mass ranges of m/z 300–600 (samples a and c) and m/z 600–900 (samples b and d) in the cap and stipe. Color scales encode arbitrary ion relative strength. (C) HPLC analysis of peels and inner layer extracts of cap and stipe at 254 nm. (D) The chromatographic peak area of compounds 24, 28, 34, 38, and 49 in peels and inner layer extracts of cap and stipe at 254 nm, data represent mean ± SD *p < 0.05; **p < 0.01; ***p < 0.001 from two-tail t test.
Conclusions
In this study, guided by MS/MS molecular networking, a total of 50 compounds, including 18 new lanostane triterpenoid metabolites, ganoleucocontins A–R (1–18), were obtained from the EtOAc extract of the fruiting body of G. leucocontextum. The elucidation of the new compounds based on comprehensive spectral analysis reveals the structural characteristics of G. leucocontextum-derived triterpenoids, featuring the occurrence of hydroxylation at Me-29 (1–9), substitution of the HMG moiety via an ester bond at C-24, C-26, and C-29 (7–9, 13–16, 17 and 18), and dimerization of two triterpenes through HMG (13–16, 17 and 18). Compounds 10, 13, 15, 16, and 18 exhibited inhibitory effects on Huh7 cells with IC50 values in the range 24.39–47.29 μM. Furthermore, compounds 10 and 15 were demonstrated to induce cell apoptosis and cell cycle arrest. In addition, a MALDI-MSI method was established to visualize the spatial distribution of functional metabolites in G. leucocontexum. These findings provide deeper insights into the potential applications of this important Ganoderma mushroom as a functional food.
Supplementary Material
Acknowledgments
This work was supported by the Science and Technology Service Network Initiative (KFJ-STS-QYZD-2021-22-004) and the National Natural Science Foundation of China (Grant No. 82204257). Dr. Jinwei Ren (State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences) is appreciated for their help in measuring the NMR data.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c01716.
Information of known compounds (19–50), key 2D correlations for compounds 2–9, 12, and 14–18, NMR spectra of 1–18, the calculated ECD results for 1, and results of bioactivity evaluation (PDF)
W.D., X.J., and J.D. contributed equally to this work. All authors have given approval to the final version of the manuscript.
The authors declare no competing financial interest.
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