New autophagy-modulating lanostane-type triterpenoids from a hallucinogenic poisonous mushroom Gymnopilus orientispectabilis

doi:10.21203/rs.3.rs-3118031/v1

Download PDF

Article

New autophagy-modulating lanostane-type triterpenoids from a hallucinogenic poisonous mushroom Gymnopilus orientispectabilis

https://doi.org/10.21203/rs.3.rs-3118031/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Gymnopilus orientispectabilis, also known as "big laughter mushroom,” is a hallucinogenic poisonous mushroom that causes excessive laughter upon ingestion. From the fruiting bodies of G. orientispectabilis, eight lanostane-type triterpenoids (1-8), including seven novel compounds: gymnojunols A-G (2-8), were isolated. The chemical structures of these new compounds (2-8) were determined by analyzing their 1D and 2D NMR spectra and HR-EISMS, and their absolute configurations were unambiguously assigned by quantum chemical ECD calculations and a computational method coupled with a statistical procedure (DP4+). Upon evaluating autophagic activity, compounds 2, 6, and 7 increased LC3B-II levels in HeLa cells to a similar extent as bafilomycin, an autophagy inhibitor. In contrast, compound 8 decreased the levels of both LC3B-I and LC3B-II, and a similar effect was observed following treatment with rapamycin, an autophagy inducer. Our findings provide experimental evidence for new potential autophagy modulators in the hallucinogenic poisonous mushroom G. orientispectabilis.

Physical sciences/Chemistry/Organic chemistry/Structure elucidation

Biological sciences/Chemical biology/Natural products

Biological sciences/Drug discovery/Medicinal chemistry/Drug discovery and development

Gymnopilus junonius (Cortinariaceae), formerly known as G. spectabilis (Fr.) Singer, is one of the well-known hallucinogenic poisonous mushrooms^1,2. It is also known as the big laughter mushroom, as its ingestion causes odd behavior, which results in hysterical laughing episode³. Recently, the scientific name of G. junonius that originated in Asia, was corrected to G. orientispectabilis⁴. As G. junonius is relatively not studied in terms of its chemical constituents, few chemical analyses have been performed on this mushroom. G. junonius has been reported to possess gymnopilins as the major constituent responsible for mushroom's bitter taste and neurotoxic properties¹. Along with gymnopilins, psilocybin is another well-reported constituent that is represented as a hallucinogen of G. junonius². Researchers also reported steroids and acetylenic compounds in the mushroom^1-3. Our group recently identified gymnopilin derivatives from G. spectabilis with cytotoxic activities against several cancer cell lines⁵, and the styrylpyrone-fused ergosterol derivative (ergopyrone), a novel steroid and phenyl heptadienoic acid dimer, from G. orientispectabilis along with its biosynthetic analogs⁶. Furthermore, the identification of monoacylglycerol, a phenolic compound, and sterols, which show anti-proliferative activities and nitric oxide inhibitory effects, was also reported in this mushroom⁷.

As a part of the continuing natural product discovery for bioactive molecules from diverse natural resources^8-12, we further investigated the hallucinogenic poisonous mushroom G. orientispectabilis to identify potential unreported bioactive compounds. Phytochemical analysis of the MeOH extract of G. orientispectabilis fruiting bodies aided by liquid chromatography-mass spectrometry (LC-MS) led to the isolation of eight lanostane-type triterpenoids (1-8), including seven new compounds, gymnojunols A-G (2-8). The structures of the newly isolated compounds were determined using NMR spectroscopic data, HR-EISMS analysis, computational methods for ECD calculations, and DP4+ analysis. The current study presents the structural elucidation and biosynthetic proposal of compounds 1-8 and their biological activity in terms of autophagy modulation.

Isolation of compounds 1–8 and their structural elucidation

The dried fruiting bodies of G. orientispectabilis were extracted with 80% MeOH at room temperature, and the resulting MeOH extract was successively partitioned with four representative organic solvents to obtain four main fractions. Each fraction was analyzed by LC-MS using a house-built UV library database, which revealed that the CH₂Cl₂-soluble fraction was rich in triterpenoid analogs with a high molecular formula between m/z 750 and m/z 850. Column chromatography procedures, including semi-preparative HPLC purification, were efficiently applied to the CH₂Cl₂ fraction, leading to the isolation of eight lanostane-type triterpenoids (1–8) (Fig. 1). Compound 1 was structurally determined to be fasciculic acid A (1)¹³ by comparison of its spectroscopic data with those previously reported and by HR-ESI-MS analysis; other compounds (2–8) were identified as new lanostane-type triterpenoids, which were named gymnojunols A-G.

Gymnojunol A (2) was obtained as an amorphous powder. The molecular formula was confirmed to be C₄₆H₇₁NO₉ based on the molecular ion peak [M + H]⁺m/z 782.5207 (calcd. for C₄₆H₇₂NO₉, 782.5207) using positive-ion high-resolution electrospray ionization mass spectrometry. The IR spectrum exhibited absorption bands corresponding to hydroxyl (3668 cm^-1) and aromatic (1662 cm^-1) functionalities. The ¹H NMR data (Table S1) of 2 showed the presence of proton signals for two oxygenated methines at δ_H 3.13 (1H, d, J = 10.5 Hz), 3.35 (1H, t, J = 6.0 Hz), a de-shielded oxygenated methine at δ_H 4.99 (1H, ddd, J = 11.5, 10.5, 4.0 Hz), seven tertiary methyl groups at δ_H 0.70 (3H, s), 0.89 (6H, s), 1.04 (3H, s), 1.10 (3H, s), 1.17 (3H, s) and 1.22 (3H, s), and a secondary methyl group at δ_H 0.91(3H, d, J = 6.5 Hz), suggesting a lanostane-type triterpenoid skeleton. The ¹³C NMR (Table S1) data of 2, obtained with the assistance of HSQC spectra, confirmed the presence of oxygenated methines at δ_C 73.8, 78.5, and 80.1, and eight methyl groups at δ_C 15.5, 16.3, 18.3, 19.8, 23.1, 24.1, 26.3, and 28.2. Based on this information, compound 2 was predicted to possess a lanostane-type triterpenoid backbone. In addition, signals assignable to 3-hydroxy-3-methylglutaryl (HMG) moiety [δ_H 1.33 (3H, s), 2.53 (2H, s), 2.39 (1H, d, J = 14.5 Hz), 2.58 (1H, d, J = 14.5 Hz), and δ_C 27.8, 45.5, 45.8, 70.5, 171.4, 171.7]¹⁴ were observed, and signals that corresponded to the phenylalanine methyl ester attached to the HMG moiety [δ_H 3.08 (1H, dd, J = 14.0, 7.0 Hz), 3.18 (1H, dd, J = 14.0, 5.5 Hz), 3.74 (3H, s), 4.90 (1H, m), 7.01 (1H, d, J = 8.0 Hz), 7.14 (2H, d, J = 7.5 Hz), 7.24 (1H, t, J = 7.5 Hz), 7.29 (2H, t, J = 7.5 Hz), and δ_C 37.5, 51.4, 52.9, 126.9, 128.5, 129.0, 135.9, 172.3]¹⁵ were also observed. Collectively, compound 2 appeared to be highly similar to 1, fasciculic acid A¹³, with the only difference being the attachment of phenylalanine methyl ester to the HMG moiety. Further analysis of the HMBC and ¹H-¹H COSY spectra (Fig. 2) revealed the complete planar structure of 2, as shown in Fig. 1.

A comparison of the optical rotation value (\({\left[\alpha \right]}_{\text{D}}^{25}\) +10.8) of 2 with that of fasciculic acid A¹³ suggested that compound 2 had an absolute configuration identical to that of fasciculic acid A. Furthermore, the biosynthetic perspective also indicated that the stereochemistry of 2 was the same as that of fasciculic acid A. The NOEs between H-2/H₃-19, H-3/H-5, H-5/H₃-30, and H₃-18/H-20, as well as ¹³C chemical shifts at C-24 (δ_C 78.9 in fasciculic acid A¹³ and δ_C 78.5 in 2) further supported the above evidence (Fig. 3). The experimental ECD spectra of 1 and 2 were compared to confirm the absolute configuration of the triterpenoid backbone of 2 (Fig. 4A). In addition, to assign the absolute configuration at C-2'' of the phenylalanine methyl ester, a computational method coupled with a statistical procedure (DP4+) was performed at the mPW1PW91/6-311* level for the two diastereomers of the phenylalanine methyl ester moiety, and the structural equivalence of 2 to 2''S-2 with 95.96% probability was confirmed (Fig. S8). Thus, the complete assignment of the absolute configuration of 2, namely gymnojunol A, was elucidated, as shown in Fig. 1, and its hypothetical biosynthetic pathway was predicted (Fig. 5).

Gymnojunol B (3), obtained as an amorphous powder, possessed a molecular formula of C₄₅H₆₉NO₁₀ deduced from the molecular ion peak [M + H]⁺m/z 784.5006 (calcd. for C₄₅H₇₀NO₁₀, 784.5000) in positive-ion HR-ESIMS. The IR spectrum exhibited absorptions corresponding to the hydroxyl (3677 cm^-1) and aromatic (1660 cm^-1) groups. The ¹H and ¹³C NMR (Table S1) of 3, obtained by the aid of HSQC and HMBC spectra, were almost identical to those of 2, with the absence of a methoxy group in phenylalanine residue and the presence of a hydroxyl group at C-12. Compound 3 was also highly similar to pardinol A, with the only difference being the absence of a methoxy group at the phenylalanine residue¹⁶. The complete planar structure of 3 is shown in Fig. 1. The NOESY analysis revealed that compound 3 had the same NOEs as compound 2, with an additional NOE between H-12 and H₃-18 (Fig. 3). Because the presence or absence of methoxy and hydroxyl functionalities did not notably impact the Cotton effect, the experimental ECD spectrum of 3 was compared with that of 2; both exhibited a strong positive Cotton effect at 212 nm (Fig. 4A). Collectively, the absolute configuration of 3, namely gymnojunol B, was assigned, as illustrated in Fig. 1, and its biosynthetic pathway is illustrated in Fig. 5.

Gymnojunol C (4) was obtained as an amorphous powder. The molecular formula was confirmed to be C₄₇H₇₁NO₁₁, based on the molecular ion peak [M + H]⁺m/z 826.5099 (calcd. for C₄₇H₇₂NO₁₁, 826.5105) in positive-ion HR-ESIMS. The IR spectrum showed absorption bands for the hydroxyl (3666 cm^-1) and aromatic (1670 cm^-1) groups. The ¹H NMR data (Table S1) of compound 4 was similar to those of compound 3, with two de-shielded oxygenated methines [δ_H 4.79 (1H, d, J = 10.5 Hz) and 5.11 (1H, ddd, J = 11.5, 10.5, 4.5 Hz)], an oxygenated methine [δ_H 4.01 (1H, d, J = 8.0 Hz)] and a singlet methyl attributable to an acetoxyl group [δ_H 1.97 (3H, s)]. Compared to compound 3, one of the hydroxyl groups appeared to be acetylated in 4. Based on the HMBC of H₂-1/C-2, H₂-1/C-3, H-2/C-3, H-2/C-4, H-3/C-2, H-3/C-28, and H-3/C-29, along with the cross peaks between H₂-1/H-2/H-3 in ¹H-¹H COSY spectrum (Fig. 2), the phenylalanine-attached HMG moiety appeared to be attached at C-3, and the acetoxyl group was confirmed to be attached at C-2. Hydroxylation at C-12 was confirmed by further analysis of the HMBC and ¹H-¹H COSY spectra (Fig. 2). The complete planar structure of compound 4 was elucidated in Fig. 1. The NOESY spectrum revealed identical correlations between the protons as in 2 and 3, suggesting the same absolute configuration of 4 as that of compounds 2 and 3 (Fig. 3). Thus, the experimental ECD spectrum of 4 was compared with those of 2 and 3, and the spectrum of 4 exhibited a strong positive Cotton effect at 214 nm (Fig. 4A). Collectively, the absolute configuration of compound 4, gymnojunol C, was assigned, as illustrated in Fig. 1, and its hypothetical biosynthetic pathway was predicted (Fig. 5).

Gymnojunol D (5) was obtained as an amorphous powder. Its molecular formula was determined as C₃₁H₅₂O₆ from the molecular ion peak [M + Na]⁺m/z 543.3661 (calcd. for C₃₁H₅₂NaO₆, 543.3662) using positive-ion HR-ESIMS. The IR spectrum exhibited absorptions corresponding to hydroxyl (3668 cm^-1) and alkene (1662 cm^-1) functionalities. The ¹H NMR (Table S2) spectrum of 5 exhibited the presence of proton signals attributable to four oxygenated methines at δ_H 3.03 (1H, d, J = 9.5 Hz), 3.43 (1H, dd, J = 11.5, 2.0 Hz), 3.68 (1H, m), 3.71 (1H, m), a de-shielded oxygenated methine at δ_H 4.92 (1H, d, J = 2.5 Hz), seven tertiary methyl groups at δ_H 0.62 (3H, s), 0.85 (3H, s), 1.04 (3H, s), 1.05 (3H, s), 1.07 (3H, s), 1.16 (3H, s), and 1.22 (3H, s), and a methoxyl group at δ_H 3.38 (3H, s). The ¹³C NMR (Table S2) data of 5, obtained with the assistance of HSQC and HMBC spectra, confirmed the existence of oxygenated methines at δ_C 69.3, 72.4, 74.4, and 83.5; a downfield-shifted oxygenated methine at δ_C 99.5; tertiary methyl groups at δ_C 16.4, 16.8, 20.0, 23.9, 24.1, 26.1, and 28.3, and a methoxy group at δ_C 53.7. The ¹H and ¹³C NMR data of 5 closely resembled those of crustulinol¹⁷, a lanostane-type triterpenoid. The only difference was the replacement of the hydroxyl group at C-21 with a methoxy group in 5, which caused a downfield shift of H-21. This was supported by the HMBC correlations of 21-OCH₃ with C-21 (δ_C 99.5), and H-21/21-OCH₃, C-22 and C-24, as well as by cross-peaks of COSY between H₂-15/H₂-16/H-17/H-20/H-21 and H-20/H₂-22/H₂-23/H-24 (Fig. 2). Further analysis of the HMBC and ¹H-¹H COSY spectra allowed the determination of the complete planar structure of compound 5, as shown in Fig. 1. The relative configuration of 5 was assigned using the NOESY correlations of H-12/H-21, H₃-18/H-20, and H-20/H-24 (Fig. 3). Based on the reported structure of crustulinol¹⁷, the absolute configurations of C-12 and C-21 are S and R forms, respectively; however, NOESY analysis of 5 revealed that these carbons have different stereochemistry¹⁷. Thus, the relative configurations of 5 were unambiguously determined to be 2R*,3R*,5R*,10S*,12R*,13R*,14S*,17R*,20R*,21S*, and 24S*. Quantum chemical calculations of the ECD simulations were performed to verify the absolute configuration of 5. The ECD data of the two possible enantiomers, 5a (2R,3R,5R,10S,12R,13R,14S,17R,20R,21S, and 24S) and 5b (2S,3S,5S,10R,12S,13S,14R,17S,20S,21R, and 24R), were calculated, and the experimental ECD spectrum of 5 was in good agreement with that of 5a, as shown in Fig. 4B. Thus, the chemical structure of 5, namely gymnojunol D, was elucidated, as shown in Fig. 1, and its hypothetical biosynthetic pathway was revealed (Fig. 5).

Gymnojunol E (6) was obtained as an amorphous powder. The molecular formula was established as C₄₇H₇₁NO₁₁ based on the molecular ion peak [M + H]⁺m/z 826.5115 (calcd. for C₄₇H₇₂NO₁₁, 826.5105) in positive-ion HR-ESIMS. The IR spectrum exhibited absorption bands for the hydroxyl (3700 cm^-1) and aromatic (1660 cm^-1) groups. The ¹H and ¹³C NMR (Table S2) of 6, obtained by the aid of HSQC and HMBC spectra, presented high similarity with those of 5, with additional signals assignable to the phenylalanine methyl ester attached to the HMG moiety^14,15. With the absence of a proton signal for H-2 in 6 [δ_H 3.71 (m)], which appeared to be downfield shifted to δ_H 4.97 (1H, ddd, J = 11.5, 10.5, 4.5 Hz), the HMG moiety was predicted to be attached at C-2. This was confirmed by careful analysis of HMBC and ¹H-¹H COSY spectra (Fig. 2); H-2/C-1', H-2'/C-1', H₃-6'/C-2' and C-4', H-4'/C-5', H-2''/C-1'', 1''-OCH₃/C-1'', H-3''/C-1'' and C-4''. Further analysis of the ¹H-¹H COSY spectrum facilitated determining the complete planar structure of 6, as indicated in Fig. 1. The relative configuration of the triterpenoidal moiety of 6 was predicted to be the same as 5 because the similar NOEs were observed between the protons (Fig. 3). In accordance with the absolute configuration of the phenylalanine methyl ester-attached HMG moiety that was previously determined as 3'S, 2''S, and based on the prediction that the phenylalanine methyl ester-attached HMG moiety does not notably affect the Cotton effect, the experimental ECD spectrum of compound 6 was compared with that of 5. As shown in Fig. 4C, the ECD spectrum of 6 exhibits a strong positive Cotton effect at 219 nm. Thus, the absolute configuration of 6, namely gymnojunol E, was determined, as shown in Fig. 1 and its biosynthetic pathway is illustrated in Fig. 5.

Gymnojunol F (7) was then obtained as an amorphous powder. The molecular formula was established as C₄₇H₇₁NO₁₁, based on the molecular ion peak [M + H]⁺m/z 826.5128 (calcd. for C₄₇H₇₂NO₁₁, 826.5105) in positive-ion HR-ESIMS. The IR spectrum exhibited absorptions corresponding to hydroxyl (3700 cm^-1) and aromatic (1677 cm^-1) functionalities. The ¹H and ¹³C NMR (Table S2) of 7, obtained with the assistance of HSQC and HMBC spectra, were almost identical to those of 6, except for the chemical shifts of the oxygenated methines at C-2 and C-3. The proton signal of H-2 was upfield shifted from δ_H 4.97 (1H, ddd, J = 11.5, 10.5, 4.5 Hz) to δ_H 3.85 (1H, td, J = 11.5, 4.0 Hz), and that of H-3 was rather downfield shifted from δ_H 3.12 (1H, d, J = 10.5 Hz) to δ_H 4.60 (1H, d, J = 10.0 Hz) in 7, suggesting the esterification of C-3 instead of C-2. This was confirmed by the HMBC correlations of H₂-1/C-2, H₂-1/C-3, H-2/C-1, H-2/C-3, H-2/C-4, H-3/C-2, H-3/C-4, and H-3/C-5, as well as the cross-peaks between H₂-1/H-2/H-3 in ¹H-¹H COSY spectrum (Fig. 2). NOESY analysis suggested that compound 7 possessed the same relative configuration as 5 and 6 (Fig. 3); thus, the experimental ECD spectra of the three compounds were compared. As shown in Fig. 4C, the ECD spectrum of 7 shows the same pattern as those of 5 and 6, exhibiting a strong positive Cotton effect at 213 nm. Thus, the chemical structure of 7, namely gymnojunol F, was elucidated, as shown in Fig. 1, and its hypothetical biosynthetic pathway is shown in Fig. 5.

Gymnojunol G (8) was also obtained as an amorphous powder. The molecular formula was confirmed to be C₄₈H₇₁NO₁₂ based on the molecular ion peak [M + H]⁺m/z 854.5052 (calcd. for C₄₈H₇₂NO₁₂ and 854.5055) using positive-ion HR-ESI-MS. The IR spectrum exhibited absorptions corresponding to the hydroxyl (3675 cm^-1) and aromatic (1673 cm^-1) groups. The ¹H and ¹³C NMR data (Table S2) of 8, obtained from the HSQC and HMBC spectra, were highly similar to those of 7. The only differences were the absence of a methoxy group at C-21 and presence of an additional signal for acetyl functionality. Using HMBC correlations between H-2 (δ_H 5.11)/2-OC (δ_C 171.2) and 2-OCOCH₃ (δ_H 1.96)/2-OC, the esterification at C-2 was confirmed, along with its de-shielded proton value, δ_H 5.11 instead of δ_H 3.85 (1H, td, J = 11.5, 4.0 Hz) in 7. The signal for the methoxy group at C-21 was absent, where H-21 was downfield shifted to δ_H 5.51 (1H, br s), while the carbon signal showed upfield shifted value, δ_C 93.1, compared to those of compounds 5–7 that have the methoxy functionality at C-21 (δ_C 99.5, 99.5 and 99.7). This was further supported by the HMBC and ¹H-¹H COSY spectra, in which HMBC correlations of H₂-1/C-2, H₂-1/C-3, H-2/C-3, and H-3/C-2 and cross-peaks between H₂-1/H-2/H-3 were observed (Fig. 2). Using NOESY analysis (Fig. 3), compound 8 was assumed to have the same stereochemistry as the previously discussed compounds; thus, its experimental ECD spectrum was compared with those of 5–7. As shown in Fig. 4C, the ECD spectrum of 8 also showed a strong positive Cotton effect at 217 nm. The absolute configuration of 8, namely gymnojunol G, was confirmed, as depicted in Fig. 1, and its hypothetical biosynthetic pathway was predicted (Fig. 5).

Plausible biosynthetic pathways for compounds 1–8

Plausible biosynthetic pathways for fasciculic acid A (1) and gymnojunols A-G (2–8) have been proposed, starting with fasciculol A as a precursor (Fig. 5). With the addition of a 3-hydroxy-3-methylglutaryl (HMG) moiety at C-2, compound 1 could be formed. The attachment of phenylalanine to the HMG moiety can then occur, yielding an intermediate that could produce compounds 2 and 3 either by oxidation or methylation. The phenylalanine-attached HMG moiety could also be linked at C-3 and further methylation could result in a phenylalanine methyl ester, which could form 4 via oxidation and acetylation. Fasciculol A can also undergo a series of oxidation reactions, followed by cyclization, forming an intermediate that could furnish 5 by methylation and 8 by attaching a phenylalanine methyl ester to the HMG moiety and an acetyl functionality. The addition of this HMG moiety at the C-2 and C-3 positions of compound 5 produced compounds 6 and 7, respectively.

Bioactivity evaluation for compounds 1–8

Autophagy is a cellular mechanism by which long-lived proteins or damaged organelles are eliminated or recycled as energy sources. If autophagy does not proceed normally in cells, it can be the root of various diseases, such as neurodegeneration, aging, and cancer. Because autophagy is involved in both the promotion and inhibition of cancer, compounds that modulate autophagy can be strategically used in treating and preventing cancer^18,19. Immunoblotting analyses of LC3B, an autophagic marker protein, were performed in HeLa cells treated with compounds 1–8 (Fig. 6) to evaluate the autophagic activity. Treatment with compounds 2, 6, and 7 for 24 h increased LC3B-II levels, similar to that induced by bafilomycin, an autophagy inhibitor. Treatment with compound 8 inhibited the levels of both LC3B-I and LC3B-II, comparable to that inhibited by rapamycin, an autophagy inducer. These results suggested that compounds 2 and 6–8 are autophagy modulators. As another method to measure autophagy, compounds 1–8 were also tested for their autophagic activities against HeLa cells stably expressing EGFP-LC3B (Fig. 7). EGFP-LC3B puncta were clearly detected in cells treated with 2 and 6–8, indicating their ability to modulate autophagy. Hydroxychloroquine (HCQ, autophagy inhibitor) was used as a positive control. In addition, compounds 1–8 were evaluated for in vitro cytotoxicity in human cancer cell lines, including HeLa (human cervical cancer), MDA-MB-231 (human triple-negative breast cancer), and HL-60 (human acute promyelocytic leukemia). Compounds 6–8 showed weak cytotoxicity against the tested cancer cell lines, whereas the other compounds exhibited no cytotoxicity even up to 50 µM (Table S3).

These results suggest that lanostane-type triterpenoids with a phenylalanine methyl ester attached to the HMG moiety could play a significant role in their anticancer effects, either by exerting cytotoxicity against human cancer cell lines or by serving as autophagy modulators. Collectively, the active lanostane-type triterpenoids from the poisonous mushroom G. orientispectabilis may be potential autophagy modulators.

General experimental procedures. Optical rotation was measured using a Jasco P-1020 polarimeter (Jasco, Easton, MD, USA). Infrared (IR) spectra were recorded using a Bruker IFS-66/S FT-IR spectrometer (Bruker, Karlsruhe, Germany). Ultraviolet (UV) spectra were obtained through an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA), and the experimental ECD spectra in MeOH were recorded in a quartz cuvette with an optical path length of 1 mm on a Jasco J-1500 spectropolarimeter (Tokyo, Japan). High-resolution (HR)-electrospray ionization (ESI) mass spectra were recorded on an Agilent 1290 Infinity II series instrument with a 6545 LC/Q-TOF mass spectrometer (Agilent Technologies). NMR spectra were recorded using a Bruker AVANCE III spectrometer (Bruker). Preparative high-performance liquid chromatography (HPLC) was conducted using a Waters 1525 binary HPLC pump with Waters 996 photodiode array detector (Waters) and an Agilent Eclipse C₁₈ column (250 × 21.2 mm, 5 µm; flow rate:5 mL/min) (Agilent Technologies), and semi-preparative HPLC used a Shimadzu Prominence HPLC System with SPD-20A/20AV Series Prominence HPLC UV-Vis Detectors (Shimadzu, Tokyo, Japan). Liquid chromatography-mass spectrometry (LC/MS) analysis was performed on an Agilent 1200 Series HPLC system equipped with a diode array detector and a 6130 Series ESI mass spectrometer using an analytical Kinetex^® 5 µm C₁₈ 100 Å column (5 µm, 2.1 × 100 mm, Phenomenex, Torrance, CA, USA). Column chromatography was performed using silica gel 60 (Merck, Darmstadt, Germany; 230–400 mesh) and RP-C₁₈ silica gel (Merck, 230–400 mesh). Sephadex LH-20 (Pharmacia, Uppsala, Sweden) was used as the packing material for the molecular sieve column chromatography. Merck precoated silica gel F₂₅₄ plates and reverse-phase (RP)-18 F_254s plates (Merck) were used for thin-layer chromatography (TLC). The TLC spots were detected under UV light or heating after spraying with anisaldehyde-sulfuric acid.

Fungal material. Fresh fruiting bodies of G. orientispectabilis were collected from the Hongneung Experimental Forest in Seoul, Korea, in September 2018. A voucher specimen (NIFoS 180914-01) of the mushroom was authenticated by one of the authors (R. Ryoo) and was deposited at the Forest Bioresources Department, National Institute of Forest Science, Korea.

Extraction and isolation. Air-dried and chopped fruiting bodies of G. orientispectabilis (1.2 kg) were subjected to extraction three times with 80% aqueous MeOH (each 3 L × 24 h) at room temperature. The resulting extracts were filtered, and the filtrate was evaporated under reduced pressure using a rotary evaporator to obtain a crude MeOH extract (143.3 g). The MeOH extract was suspended in a mixture of distilled water (700 mL) and MeOH (30 mL) and successively solvent-partitioned with hexane, dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), and n-butanol to yield soluble layers of hexane, CH₂Cl₂, EtOAc, and n-butanol at 11.0, 6.6, 14.0, and 19.0 g, respectively. Based on the LC/MS analysis of each fraction obtained, the CH₂Cl₂-soluble fraction was determined to be a promising fraction for investigating new compounds because many peaks detected in the LC/MS analysis of the CH₂Cl₂-soluble fraction showed the characteristic features of triterpenoids with a higher molecular formula between m/z 750 and m/z 850. The CH₂Cl₂ fraction was subjected to silica gel column chromatography (CC) (CH₂Cl₂/MeOH, 60:1 1:1) to yield seven fractions (Fr. A1–A7). Fr. A5 (168.5 mg) was further fractionated using preparative HPLC (MeOH/H₂O, 70:30–100:0), which yielded five subfractions (Fr. A51–A55). Fr. A55 (53.5 mg) was purified using semi-preparative HPLC (MeOH/H₂O, 80:20 to 90:10), and compound 8 (t_R 46.0 min, 3.0 mg) was obtained. Fr. A7 (1.7 g) was subjected to RP-C₁₈ silica gel CC (MeOH/H₂O, 80:20–100:0), which yielded five fractions (Frs. A71–A75). Fr. A72 (233.2 mg) was further fractionated using preparative HPLC (MeOH/H₂O, 85:15–100:0) to obtain four subfractions (Fr. A721–A724). Fr. A723 (35.9 mg) was purified using semi-preparative HPLC (MeOH/H₂O, 76:34) and compounds 1 (t_R 39.5 min, 1.0 mg), 3 (t_R 55.6 min, 1.5 mg), and 4 (t_R 61.5 min, 0.8 mg) were obtained. Fr. A73 (502.3 mg) was subjected to a Sephadex LH-20 column (CH₂Cl₂/MeOH, 2:8) and further fractionated using preparative HPLC (MeOH/H₂O, from 85:15 to 100:0), yielding four subfractions (Fr. A731 – A734). Fr. A733 (12.8 mg) was purified using semi-preparative HPLC (MeOH/H₂O, 85:15) to afford compounds 2 (t_R 26.8 min, 0.5 mg), 5 (t_R 23.0 min, 1.3 mg), 6 (t_R 49.3 min, 1.3 mg), and 7 (t_R 52.0 min, 0.8 mg).

Gymnojunol A (2): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) +10.8 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 205 (3.2) nm; ECD (MeOH) λ_max (Δε) 213 (+ 58.0) nm; IR (KBr) ν_max 3668, 2971, 2868, 1662, 1336 cm⁻¹; ¹H (850 MHz, CD₃OD) and ¹³C (212.5 MHz, CDCl₃) NMR data, see Table S1; HR-ESI-MS (positive-ion mode) m/z 782.5207 [M + H]⁺ (calcd for C₄₆H₇₂NO₉, 782.5207).

Gymnojunol B (3): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) +5.7 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 205 (3.4) nm; ECD (MeOH) λ_max (Δε) 212 (+ 63.1) nm; IR (KBr) ν_max 3677, 2975, 2866, 1660, 1334 cm⁻¹; ¹H (850 MHz, CD₃OD) and ¹³C (212.5 MHz, CD₃OD) NMR data, see Table S1; HR-ESI-MS (positive-ion mode) m/z 784.5006 [M + H]⁺ (calcd for C₄₅H₇₀NO₁₀, 784.5000).

Gymnojunol C (4): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) +17.4 (c 0.08, MeOH); UV (MeOH) λ_max (log ε) 205 (3.2) nm; ECD (MeOH) λ_max (Δε) 214 (+ 51.2) nm; IR (KBr) ν_max 3666, 2969, 2865, 1670, 1339 cm⁻¹; ¹H (850 MHz, CD₃OD) and ¹³C (212.5 MHz, CD₃OD) NMR data, see Table S1; HR-ESI-MS (positive-ion mode) m/z 826.5099 [M + H]⁺ (calcd for C₄₇H₇₂NO₁₁, 826.5105).

Gymnojunol D (5): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) +4.0 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 205 (3.0) nm; ECD (MeOH) λ_max (Δε) 204 (+ 32.9) nm; IR (KBr) ν_max 3668, 2978, 2866, 1662, 1332 cm⁻¹; ¹H (700 MHz, CDCl₃) and ¹³C (175 MHz, CDCl₃) NMR data, see Table S2; HR-ESI-MS (positive-ion mode) m/z 543.3661 [M + Na]⁺ (calcd for C₃₁H₅₂NaO₆, 543.3662).

Gymnojunol E (6): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) -6.1 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 205 (3.8) nm; ECD (MeOH) λ_max (Δε) 219 (+ 78.6) nm; IR (KBr) ν_max 3700, 2975, 1660, 1328 cm^− 1; ¹H (850 MHz, CDCl₃) and ¹³C (212.5 MHz, CDCl₃) NMR data, see Table S2; HR-ESI-MS (positive-ion mode) m/z 826.5115 [M + H]⁺ (calcd for C₄₇H₇₂NO₁₁, 826.5105).

Gymnojunol F (7): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) -16.9 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 205 (3.4) nm; ECD (MeOH) λ_max (Δε) 212 (+ 36.4) nm; IR (KBr) ν_max 3700, 2975, 2866, 1677, 1334 cm^− 1; ¹H (850 MHz, CD₃OD) and ¹³C (212.5 MHz, CDCl₃) NMR data, see Table S2; HR-ESI-MS (positive-ion mode) m/z 826.5128 [M + H]⁺ (calcd for C₄₇H₇₂NO₁₁, 826.5105).

Gymnojunol G (8): Amorphous powder; \({\left[\alpha \right]}_{\text{D}}^{25}\) +7.7 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 210 (3.8) nm; ECD (MeOH) λ_max (Δε) 218 (+ 62.8) nm; IR (KBr) ν_max 3675, 2969, 1673, 1341 cm⁻¹; ¹H (850 MHz, CD₃OD) and ¹³C (212.5 MHz, CDCl₃) NMR data, see Table S2; HR-ESI-MS (positive-ion mode) m/z 854.5052 [M + H]⁺ (calcd for C₄₈H₇₂NO₁₂, 854.5055).

ECD Calculation. Computational density-functional-theory (DFT) calculations were performed to determine the conformational differences between 5a and 5b. The first structural energy minimization of each conformer was performed using Avogadro 1.2.0 with a UFF force field. The ground-state geometries were established by Tmolex 4.3.1 with DFT settings (B3-LYP functional, M3 grid size), geometry optimization options (energy:10^− 6E_h, gradient norm: |dE/dxyz| = 10^− 3E_h/b), and basis set def-SV(P) for all atoms. The calculated ECD spectra of the optimized structures of the sets of conformers were acquired at B3LYP/DFT functional settings using the def-TZVPP basis set for all atoms. The obtained CD spectra were simulated by overlying each transition (Eq. 1), where σ is the width of the band at 1/e height, and ΔE_i and R_i are the excitation energies and rotatory strengths for transition i, respectively. In the present study, the value of σ was 0.10 eV ²⁰.

Δϵ(E) = \(\frac{1}{2.297 \times {10}^{-39}}\frac{1}{\sqrt{2\pi \sigma }}\sum _{a}^{i}\varDelta {E}_{i}{R}_{i}{e}^{\left[{-\left(E-\varDelta {E}_{i}\right)}^{2}/{\left(2\sigma \right)}^{2}\right]}\) (Eq. 1)

Computational NMR chemical shift calculations for DP4 + analysis. Conformational searches were performed by Tmolex 4.3.1 with the DFT settings (B3-LYP functional/M3 grid size), geometry optimization settings (energy 10^− 6 hartree, gradient norm |dE/dxyz| = 10^− 3 hartree/bohr), and the basis set def-SV(P) for all atoms. The NMR shielding constant calculations were performed on the optimized ground-state geometries at the DFT B3-LYP/def-SV(P) level of theory. The NMR chemical shifts of the isomers were obtained by Boltzmann averaging the ¹H NMR and ¹³C NMR chemical shifts of the stable conformers at 298.15 K. Chemical shift values were calculated using the equation below, where \({\delta }_{calc}^{x}\) is the calculated NMR chemical shift for nucleus x, and \({{\sigma }}^{o}\) is the shielding tensor for the proton and carbon nuclei in tetramethylsilane calculated at the DFT B3-LYP/def-SV(P) basis set²¹.

\({\delta }_{calc}^{x} = \frac{{{\sigma }}^{o}- {\sigma }^{x}}{1- {{\sigma }}^{o}/{10}^{6}}\)

(Eq. 2)

The calculated NMR properties of the optimized structures were averaged based on their respective Boltzmann populations and DP4 + probability analysis was performed using the Excel sheet (DP4+) provided by Grimblat et al.²².

Cell lines and cell culture. HeLa (human cervical cancer) and MDA-MB-231 (human triple-negative breast cancer) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, LM 001–05) supplemented with 10% fetal bovine serum (FBS; Welgene, S001-07), 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco, 15140-122), maintained at 37℃ under 5% CO₂ conditions in a humidified atmosphere. HL-60 (human acute promyelocytic leukemia) cells were cultured in Iscove's modified Dulbecco's medium (IMDM; Gibco, 12440-053) supplemented with 20% FBS, penicillin, and streptomycin. HeLa cells stably expressing EGFP-LC3B were cultured in DMEM containing 400 µg/mL G-418 (Roche 4727878001).

Cell viability assay. An EZ-Cytox colorimetric assay kit (Daeil Lab Service, 0793) was used for the cell viability assay. Cells were seeded overnight in 96-well flat-bottomed cell culture plates (HeLa and MDA-MB-231, 7 × 10³ cells/well; HL-60, 1.5 × 10⁴ cells/well) and treated with compounds at the indicated concentrations. After 24 h incubation, each well was supplemented with 10 µL of EZ-Cytox solution and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader (SpectraMax 190; Molecular Devices).

Antibodies and reagents. Primary antibodies against LC3B (L7543) and β-actin (sc-47778) were purchased from Sigma-Aldrich and Santa Cruz Biotechnology, respectively. Horseradish peroxidase (HRP)-conjugated species-specific secondary antibodies (7074 anti-rabbit IgG and 7076 anti-mouse IgG) were purchased from Cell Signaling Technology. Bafilomycin A1 (B1793) and rapamycin (3504A) were purchased from Sigma-Aldrich and Santa Cruz Biotechnology, respectively.

Western blot analysis. HeLa cells were directly lysed with 2× Laemmli sample buffer (ELPISBIO, EBA-1051) for western blotting. Cell lysates were loaded onto SDS-PAGE gels for protein separation and then transferred onto nitrocellulose membranes (0.2 µm; Bio-Rad, 162 − 0112). After blocking with 5% skim milk in TBS-T buffer, the membranes were probed with the indicated primary antibodies at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibodies for 1 h. Protein bands were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, #34080).

Fluorescence microscopy. HeLa cells stably expressing EGFP-LC3B were seeded onto coverslips in 24-well cell culture plates for 16 h. After incubation with the indicated compounds for 24 h, cells were fixed with 4% paraformaldehyde and stained with DAPI. Fluorescent images were captured using a fluorescence microscope (Zeiss Axio Observer Z1).

Acknowledgments

This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT; grant numbers 2019R1A5A2027340 and 2021R1A2C2007937). This work was also supported by grants from the KRIBB Research Initiative Program (KGM5292322 and KGM1222312) funded by the Ministry of Science ICT (MSIT) of the Republic of Korea.

Author contributions

S.K.K., K.H.K.: designed research, S.L., M.J., R.R.: performed the experiments. S.L., M.J.: analyzed data. S.L., M.J.: generated the figures. S.L., M.J., K.H.K.: wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42004-023-00871-z.

Findlay, J. A. & He, Z.-Q. Minor constituents of Gymnopilus spectabilis. J. Nat. Prod. 54, 184–189 (1991).
Kayano, T. et al. Gymnopilins, a product of a hallucinogenic mushroom, inhibit the nicotinic acetylcholine receptor. Toxicon 81, 23–31 (2014).
Tanaka, M., Hashimoto, K., Okuno, T. & Shirahama, H. Neurotoxic oligoisoprenoids of the hallucinogenic mushroom, Gymnopilus spectabilis. Phytochemistry 34, 661–664 (1993).
Thorn, R. G. et al. New species in the Gymnopilus junonius group (Basidiomycota: Agaricales). Botany 98, 293–315 (2020).
Kim, K. H., Choi, S. U. & Lee, K. R. Gymnopilin K: a new cytotoxic gymnopilin from Gymnopilus spectabilis. J. Antibiot. 65, 135–137 (2012).
Lee, S. et al. Ergopyrone, a styrylpyrone-fused steroid with a hexacyclic 6/5/6/6/6/5 skeleton from a mushroom Gymnopilus orientispectabilis. Org. Lett. 23, 3315–3319 (2021).
Suh, W. S. et al. A new monoacylglycerol from the fruiting bodies of Gymnopilus spectabilis. J. Chem. Res. 40, 156–159 (2016).
Lee, B. S. et al. Comparative evaluation of bioactive phytochemicals in Spinacia oleracea cultivated under greenhouse and open field conditions. Arch. Pharm. Res., 1–11 (2022).
Cho, H. et al. Structure determination of heishuixiecaoline A from Valeriana fauriei and its content from different cultivated regions by HPLC/PDA Analysis. Nat. Prod. Sci. 28, 181–186 (2022).
Yu, J. S. et al. New phenalenone derivatives from the Hawaiian volcanic soil-associated fungus Penicillium herquei FT729 and their inhibitory effects on indoleamine 2, 3-dioxygenase 1 (IDO1). Arch. Pharm. Res. 45, 105–113 (2022).
Lee, S. R. et al. Identification of anti-adipogenic withanolides from the roots of Indian ginseng (Withania somnifera). J. Ginseng Res. 46, 357–366 (2022).
Lee, K. H. et al. Ginkwanghols A and B, osteogenic coumaric acid-aliphatic alcohol hybrids from the leaves of Ginkgo biloba. Arch. Pharm. Res. 44, 514–524 (2021).
Nozoe, S., Takahashi, A. & Ohta, T. Chirality of the 3-hydroxy-3-methylglutaric acid moiety of fasciculic acid A, a calmodulin antagonist isolated from Naematoloma fasciculare. Chem. Pharm. Bull. 41, 1738–1742 (1993).
Kamo, T., Hirai, N., Matsumoto, C., Ohigashi, H. & Hirota, M. Revised chirality of the acyl group of 8′-O-(3-hydroxy-3-methylglutaryl)-8′-hydroxyabscisic acid. Phytochemistry 65, 2517–2520 (2004).
Yoshikawa, K., Kuroboshi, M., Ahagon, S. & Arihara, S. Three Novel Crustulinol Esters, Saponaceols A—C, from Tricholoma saponaceum. Chem. Pharm. Bull. 52, 886–888 (2004).
Zhang, S.-B. et al. Lanostane triterpenoids from Tricholoma pardinum with NO production inhibitory and cytotoxic activities. Phytochemistry 152, 105–112 (2018).
Garlaschelli, L., Vidari, G., Virtuani, M., Vita-Finzi, P. & Mellerio, G. The Structures of New Lanostane Triterpenes from the Fruiting Boidies of Hebeloma senescens. J. Nat. Prod. 58, 992–1002 (1995).
Galluzzi, L., Bravo-San Pedro, J. M., Levine, B., Green, D. R. & Kroemer, G. Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles. Nat. Rev. Drug Discov. 16, 487–511 (2017).
Limpert, A. S. et al. Autophagy in cancer: regulation by small molecules. Trends Pharmacol. Sci. 39, 1021–1032 (2018).
Diedrich, C. & Grimme, S. Systematic investigation of modern quantum chemical methods to predict electronic circular dichroism spectra. J. Phys. Chem. A 107, 2524–2539 (2003).
Smith, S. G. & Goodman, J. M. Assigning stereochemistry to single diastereoisomers by GIAO NMR calculation: The DP4 probability. J. Am. Chem. Soc. 132, 12946–12959 (2010).
Grimblat, N., Zanardi, M. M. & Sarotti, A. M. Beyond DP4: An improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMR shifts. J. Org. Chem. 80, 12526–12534 (2015).

There is NO Competing Interest.

supplementaryinformationCC20230628.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

New autophagy-modulating lanostane-type triterpenoids from a hallucinogenic poisonous mushroom Gymnopilus orientispectabilis

Status:

Version 1

Abstract

Figures

Introduction

Results and discussion

Isolation of compounds 1–8 and their structural elucidation

Plausible biosynthetic pathways for compounds 1–8

Bioactivity evaluation for compounds 1–8

Conclusion

Material and methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1