Six Undescribed Lanostane‐Type Triterpenoids From Ganoderma lucidum

ABSTRACT

Six previously undescribed lanostane triterpenoids (1–6), together with 21 known analogues (7–27) were isolated from the fruiting bodies of the medicinal fungus Ganoderma lucidum. Compounds 1–3 are lucidenate‐type triterpenoids, whereas compounds 4–6 are ganoderate‐type derivatives. The planar structures and relative configurations of the six new compounds were established by comprehensive analysis of HRESIMS and 1D & 2D NMR spectroscopic data. The absolute configurations of compounds 1–3 were determined by ECD calculations or the modified Mosher's method. On the basis of the single‐crystal X‐ray diffraction data for compound 7, together with biogenetic considerations, the absolute configurations at C‐25 of compounds 4–6 were assigned as 25R. Evaluation of the isolated compounds for anti‐inflammatory activity revealed no significant inhibition of LPS‐induced nitric oxide (NO) production in RAW264.7 macrophages.

Six previously undescribed lanostane triterpenoids (1–6), together with 21 known analogues (7–27) were isolated from the fruiting bodies of the medicinal fungus Ganoderma lucidum. Compounds 1–3 are lucidenate‐type triterpenoids, whereas compounds 4–6 are ganoderate‐type derivatives. The planar structures and relative configurations of the six new compounds were established by comprehensive analysis of HRESIMS and 1D & 2D NMR spectroscopic data.

1. Introduction

The dried fruiting bodies of Ganoderma lucidum (Leyss. ex Fr.) Karst. and Ganoderma sinense Zhao, Xu et Zhang are officially listed as the botanical origin of the traditional Chinese medicine “Lingzhi” in the Chinese Pharmacopoeia. This medicinal fungus has been employed to treat a wide range of diseases, including cancer [1], inflammation [2], neurodegenerative disorders [3], Alzheimer's disease [4], and chemotherapy‐related fatigue [5]. As both an edible mushroom and a traditional herbal medicine, Ganoderma is widely cultivated in East Asian countries such as China, Japan, and Korea [6]. It is commonly consumed as a dietary supplement or incorporated as a key ingredient in various herbal formulations. Triterpenoids and polysaccharides are recognized as the two major classes of bioactive constituents in G. lucidum [7]. Triterpenoids obtained from G. lucidum are structurally characterized by a tetracyclic lanostane skeleton, which undergoes extensive diversification through oxidation, hydroxylation, decarboxylation, and skeletal rearrangement. These compounds are biosynthesized predominantly via the mevalonate pathway and exhibit considerable structural complexity [8].

In the present study, six previously undescribed triterpenoids (1–6, Figure 1), together with 21 structurally related analogues (7–27), were isolated from the fruiting bodies of G. lucidum. The structures of the six new compounds were unequivocally established by comprehensive analysis of HRESIMS, 1D & 2D NMR, and ECD spectroscopic data, in conjunction with the modified Mosher's method and single‐crystal X‐ray diffraction analysis. Compounds 1–3 are lucidenate‐type triterpenoids, whereas compounds 4–6 are ganoderate‐type derivatives. Their isolation, structural elucidation, and characterization were described in detail below.

FIGURE 1.

| Structures of compounds 1–7 and X‐ray crystal structure of compound 7.

2. Results and Discussion

Compound 1 was obtained as a pale yellow gel. Its molecular formula was established to be C₃₁H₄₂O₈ based on the HRESIMS (m/z 543.2950 [M + H]⁺, calcd for 543.2952) (Figure SIII‐1), corresponding to 11 degrees of unsaturation. The UV spectrum displayed a maximum absorption at 253 nm [9], suggesting the presence of an 8(9)‐ene‐7,11‐dione moiety. The ¹H NMR spectrum (Table 1) exhibited for one oxygenated methine signal (δ _H 5.85), six singlet methyl groups (δ _H 0.98, 1.23, 1.24, 1.45, 1.93 and 2.30), one doublet methyl (δ _H 1.13), and one ethoxy group (δ _H 1.35, 4.23). The ¹³C NMR spectrum (Table 1) resolved 31 carbon resonances, which were classified with the combination of the HSQC experiment as one ethoxy group, seven methyls, six methylenes, four methines (including one oxygenated carbon), and twelve quaternary carbons, including an 8(9)‐ene‐7,11‐dione moiety (δ _C 200.6, 195.8, 151.2, and 147.5), two ketone carbonyls (δ _C 217.8 and 208.5), and two ester carbonyls (δ _C 175.3 and 171.6). These data indicated that 1 was a typical lanostane‐type triterpenoid [10]. The lanostane‐type 6/6/6/5 ring system of compound 1 and its substitute pattern were further verified by the HMBC correlations (Figure 2) from the geminal dimethyl groups (Me‐28 and Me‐29) to C‐3, C‐4, and C‐5, from the angular methyl Me‐18 to C‐12, C‐13, C‐14, and C‐17, from Me‐19 to C‐1, C‐9, and C‐10, from Me‐30 to C‐8, C‐13, C‐14, and C‐15, and from H‐12/Me‐32 to C‐31. In addition, the HMBC correlations from Me‐34 to C‐33, from H‐33 to C‐24, together with the ¹H‐¹H COSY cross‐peaks of H‐33/H‐34 (Figure 2), demonstrated that the ethoxy group was located at the C‐24 carboxyl terminus. Removal of the C‐12 acetoxy and C‐24 ethoxy substituents, in combination with the molecular formula, indicated that the core skeleton of 1 corresponded to a C₂₇ lucidenic acid derivative. The HMBC correlations from Me‐21 to C‐17, C‐20, C‐22, and from H₂‐22 to C‐17, C‐20, C‐21, C‐23, and C‐24 further confirmed that the side chain attached to C‐17 was a degraded lucidenate‐type fragment. Comparison of the NMR data with those reported in the literature revealed that compound 1 closely resembled lucidenic acid D₂ [11], with the only difference being the presence of an additional ethoxy group at C‐24 in 1. Consequently, 1 was deduced to be an ethoxy derivative of lucidenic acid D₂, and its planar structure was established as depicted in Figure 1.

TABLE 1.

| ¹H and ¹³C NMR spectroscopic data of 1–3 (δ in ppm).

No.	1 a		2 b		3 a
	δ H (J in Hz)	δ C, type	δ H (J in Hz)	δ C, type	δ H (J in Hz)	δ C, type
1a 1b	2.63, m; 1.89, m	34.7, CH2	2.60, m	36.8, CH2	2.59, dt (13.4, 3.6); 0.97, m	35.5, CH2
2	2.41, m	32.7, CH2	2.72, m	32.1, CH2	1.66, m	27.9, CH2
3		217.9, C	3.25, dd (11.4, 5.0)	77.6, CH	3.16, dd (11.6, 4.7)	78.8, CH
4		48.0, C		37.8, C		39.7, C
5	2.46, m	51.8, CH	1.63, m	51.5, CH	0.95, d (1.8)	50.3, CH
6a; 6b	2.67, m; 2.43, m	38.2, CH2	2.66, m 2.45, m	27.5, CH2	2.22, m; 1.62, m	28.0, CH2
7		200.6, C		199.0, C	4.90, m	67.2, CH
8		147.4, C		146.1, C		158.3, C
9		151.2, C		151.7, C		144.1, C
10		40.6, C		39.3, C		39.6, C
11		195.8, C		194.2, C		194.1, C
12	5.74, s	80.6, CH	5.62, s	79.5, CH	5.66, s	81.5, CH
13		48.9, C		48.1, C		51.2, C
14		59.9, C		58.6, C		61.3, C
15		208.6, C		206.3, C		217.2, C
16a; 16b	2.92, dd (18.5, 9.9) 2.05, dd (18.5, 8.0)	38.3, CH2	2.79, dd (18.4, 9.8); 2.08, dd (18.4, 8.3)	40.6, CH2	2.74, dd (19.4, 8.5) 2.32, m	38.3, CH2
17	2.60, m	46.2, CH	2.57, dd (14.5, 2.6)	45.6, CH	2.47, m	47.3, CH
18	0.86, s	12.5, CH3	0.83, s	12.3, CH3	0.98, s	13.8, CH3
19	1.34, s	19.1, CH3	1.32, s	18.1, CH3	1.24, s	19.0, CH3
20	1.80, m	34.0, CH	1.83, m	30.3, CH	1.79, m	32.8, CH
21	1.02, d (6.7)	20.9, CH3	0.99, d (6.7)	21.0, CH3	0.99, d (6.8)	21.0, CH3
22a; 22b	1.96, m 1.31, m	31.2, CH2	1.85, m 1.29, m	33.1, CH2	1.89, m 1.29, m	30.7, CH2
23a 23b	2.81, m; 1.90, m	35.0, CH2	2.36, m	33.4, CH2	2.44, m 2.36, m	32.9, CH2
24		175.3, C		173.5, C		175.3, C
25
26
27
28	1.12, s	20.6, CH3	0.88, s	15.7, CH3	0.84, s	16.2, CH3
29	1.13, s	27.6, CH3	1.02, s	28.0, CH3	1.02, s	28.6, CH3
30	1.82, s	20.8, CH3	1.72, s	21.5, CH3	1.49, s	24.7, CH3
31		171.6, C		170.3, C		171.9, C
32	2.19, s	20.9, CH3	2.21, s	20.3, CH3	2.18, s	20.9, CH3
33	4.12, q (7.1)	61.5, CH2	4.12, q (7.1)	60.6, CH2	4.12, q (7.1)	61.5, CH2
34	1.24, t (7.1)	14.6, CH3	1.25, t (7.1)	14.4, CH3	1.25, t (7.1)	14.6, CH3

^a Recorded in Methanol‐d4 at 500/125 MHz.

^b Recorded in Chloroform‐d at 500/125 MHz.

FIGURE 2.

| Key 2D NMR correlations of compounds 1–3.

In the ROESY spectrum of 1 (Figure 2), the correlation of Me‐19/Me‐28 indicated that both methyl groups were β‐oriented. The correlations of H‐5/Me‐29, H‐17/Me‐21, H‐17/Me‐30, demonstrated the α‐orientation of H‐5 and H‐17 and established the configuration at C‐20 as R*. The correlation of H‐12/Me‐30 indicated the α‐orientation for H‐12. These relative configurations were consistent with those reported for lucidenic acid D₂. To determine the absolute configuration, ECD calculations were performed for (5R,10S,12S,13R,14R,17R,20R)‐1 (Figure 4). The close agreement between the calculated and experimental ECD curves of 1 allowed the assignment of the absolute configuration as 5R,10S,12S,13R,14R,17R,20R. Accordingly, compound 1 was identified and named as ethyl lucidenate D₂.

FIGURE 4.

| Experimental ECD spectra of 1–6 and calculated ECD spectra of 1 and 3.

Compound 2 was obtained as a pale white gel. Its molecular formula was deduced to be C₃₁H₄₄O₈ based on the HRESIMS analysis (m/z 567.2928 [M + Na]⁺, calcd for 567.2928) (Figure SIII‐9), implying 10 degrees of unsaturation. Comparison of the UV and NMR data with those of 1 revealed that 2 was also a lucidenate‐type triterpenoid. The molecular weight of 2 was two mass units higher than that of 1. Analysis of the ¹H and ¹³C NMR spectroscopic data (Table 1) showed that one nonconjugated carbonyl in 1 was replaced by an oxygenated methine (δ _H 3.25, δ _C 77.6) in 2, suggesting that a carbonyl group in 1 had been reduced to a hydroxy group in 2. The HMBC correlations (Figure 2) from Me‐28, Me‐29, H‐2, and H‐5 to C‐3, together with the ¹H‐¹H COSY correlations of H‐2/H‐3, unequivocally located the hydroxy group at C‐3. In the ROESY spectrum, correlations of H‐3/H‐5, H‐3/Me‐29 established the β‐orientation of 3‐OH (Figure 2). The remaining ROESY correlations were identical to those observed for 1, indicating that the relative configurations at the other stereogenic centers were the same as those of 1; therefore, the relative configuration of 2 was defined as 3S*,5R*,10S*,12S*,13R*,14R*,17R*,20R*. The absolute configuration of 2 was assigned by comparison of its experimental ECD curve with that of 1 (Figure 4), which exhibited very similar Cotton effects. To further verify the absolute configuration of the 3‐OH, the modified Mosher's method was applied. Compound 2 was esterified with (R)‐ and (S)‐MTPA‐Cl to afford the corresponding (S)‐ and (R)‐MTPA esters (2a and 2b). Analysis of the Δδ _{H( S ‐ R )} values (Figure 3) according to the Mosher's method model confirmed the S configuration at C‐3. Collectively, the structure of 2 was established and designated as ethyl lucidenate E₂.

FIGURE 3.

| Δδ _{H( S–R )} values (in ppm) for the (S)‐ and (R)‐MTPA esters of compounds 2 and 3.

Compound 3 was isolated as a pale yellow gel. The molecular formula of C₃₁H₄₆O₈ was established by HRESIMS at m/z 569.3086 [M + Na]⁺ (calcd for C₃₁H₄₆NaO₈ 569.3085) (Figure SIII‐17). The molecular weight of 3 was two mass units higher than that of 2. Comparative analysis of their NMR data (Table 1) revealed that one nonconjugated carbonyl in 2 was replaced by an oxygenated tertiary carbon (δ _H 4.90, δ _C 67.2) in 3, indicating the reduction of a carbonyl to a hydroxy group in 3. The HMBC correlations (Figure 2) from H‐7 to C‐5 and C‐8, along with the ¹H‐¹H COSY spin system of H‐7/H₂‐6, confirmed that the hydroxy group was located at C‐7. The relative configuration at C‐7 was assigned as 7S* based on the ROESY correlation (Figure 2) of H‐5/H‐7. All other ROESY correlations were consistent with those of 2, establishing the relative configuration of 3 as 3S*,5R*,7S*,10S*,12S*,13R*,14R*,17R*,20R*. The absolute configuration of 3 was confirmed by the excellent match between its experimental ECD spectrum and the calculated ECD curve for (3S,5R,7S,10S,12S,13R,14R,17R,20R)‐3 (Figure 4). To further corroborate the absolute configuration of the C‐7 hydroxy group, the modified Mosher's method was employed. Esterification of 3 with (R)‐ and (S)‐MTPA‐Cl furnished the C‐7 mono‐MTPA esters (3a and 3b), as confirmed by HRESIMS and HMBC correlations (Figure SI‐2). Analysis of the Δδ _{H( S ‐ R )} values (Figure 3) unequivocally established the S configuration at C‐7. Thus, the structure of 3 was fully elucidated and named ethyl lucidenate P.

Compound 4 was obtained as a pale yellow gel. Its HRESIMS spectrum exhibited protonated molecular ion peaks at m/z 539.3001 [M + H]⁺ (calcd 539.3003 for C₃₂H₄₃O₇) (Figure SIII‐25), corresponding to the molecular formula of C₃₂H₄₂O₇. The ¹H NMR spectrum displayed signals for one ethoxy group (δ _H 1.25, 4.13), one doublet methyl (δ _H 1.19), and six singlet methyls (δ _H 0.73, 1.12, 1.14, 1.26, 1.68, and 2.14) (Table 2). The ¹³C NMR spectrum, in conjunction with the HSQC experiment, resolved 32 carbon signals, which were assigned to one ethoxy group, seven methyls, six methylenes, four methines, and thirteen quaternary carbons. In the HMBC spectrum (Figure  5), there were correlations from the geminal dimethyls (Me‐28 and Me‐29) to C‐3/C‐4/C‐5, from Me‐18 to C‐12/C‐13/C‐14/C‐17, from Me‐19 to C‐1/C‐9/C‐10, and from Me‐30 to C‐8/C‐13/C‐14/C‐15, indicative of the skeleton and the substitution pattern on the ring system. Removal of the ethoxy group, in combination with the molecular formula, indicated that the core skeleton of 4 was a C₃₀ ganoderic acid derivative. HMBC correlations from Me‐21 to C‐17/C‐20/C‐22, from H‐22 to C‐17/C‐20/C‐21/C‐23, and from H‐25 to C‐23/C‐24/C‐26/C‐27 confirmed that the side chain was a ganoderate‐type fragment. Comparison of the NMR data with those reported in the literature revealed that 4 was highly similar to ganoderenic acid F [12], the only difference was an extra ethoxy group at C‐26 in 4. The HMBC correlations from Me‐34 to C‐33, from H‐33 to C‐26, combined with the ¹H‐¹H COSY correlations of H‐33/H‐34, placed the ethoxy group at C‐26. In the ROESY spectrum of 4, correlations of H‐22/H₂‐16 indicated an E configuration for the Δ ^20, 22 double bond (Figure 5), which is consistent with the relative configuration reported for ganoderenic acid F. The principal chromophore of 4 was identical to that of 1; therefore, the absolute configuration of the tetracyclic core was assigned as 5R,10S,13R,14R by comparison of its experimental ECD curve with that of 1 (Figure 4). The absolute configuration at C‐25 could not be determined by ECD calculations [9]. To address this, a single crystal of the known compound 7, which possesses an identical side chain to that of 4, was obtained and subjected to single‐crystal X‐ray diffraction analysis (Figure 1). The X‐ray crystallographic data unambiguously established the absolute configuration at C‐25 as R, with a flack parameter of 0.10 (CCDC No. 2531233). Moreover, all previously reported ganoderic acid derivatives from G. lucidum possess the 25R configuration [13]. Based on biosynthetic considerations, the C‐25 configuration of 4 was assigned as R. Consequently, compound 4 was identified as ethyl ganoderenate F.

TABLE 2.

| ¹H and ¹³C NMR spectroscopic data of 4–6 (δ in ppm).

No.	4 a		5 a		6 b
	δ H (J in Hz)	δ C, type	δ H (J in Hz)	δ C, type	δ H (J in Hz)	δ C, type
1a 1b	2.93, m 1.75, m	34.7, CH2	2.69, m 1.15, m	33.3, CH2	2.81, m	36.1, CH2
2	2.61, m	33.9, CH2	1.66, m	27.4, CH2	2.56, m	29.6, CH2
3		215.2, C	3.24, dd (11.3, 4.9)	77.5, CH		218.9, C
4		47.1, C		39.2, C		49.6, C
5	2.31, dd (14.9, 2.8)	50.9, CH	1.54, dd (14.4, 2.5)	51.5, CH	1.71, m	50.0, CH
6a 6b	2.69, m 2.51, m	37.3, CH2	2.64, d (14.5) 2.51, m	36.7, CH2	2.18, dd (11.4, 8.2) 1.68, m	29.0, CH2
7		199.0, C		199.1, C	4.91, m	66.8, CH
8		146.8, C		145.9, C		159.2, C
9		150.2, C		151.8, C		142.1, C
10		39.6, C		40.5, C		39.0, C
11		198.2, C		194.1, C		201.5, C
12a 12b	2.93, m 2.63, t (9.7)	47.9, CH2	5.61, m	79.3, CH	4.44, s	79.7, CH
13		45.2, C		48.0, C		52.5, C
14		56.7, C		58.6, C		61.2, C
15		206.2, C		205.9, C		218.2, C
16a 16b	2.68, m 2.47, m	36.4, CH2	2.75, m 1.90, dd (18.2, 8.3)	38.0, CH2	2.69, m 2.27, m	39.0, CH2
17	3.19, t (9.1)	48.6, CH	2.51, m	44.8, CH	2.58, m	47.4, CH
18	0.73, s	17.3, CH3	0.80, s	12.3, CH3	0.88, s	12.8, CH3
19	1.26, s	17.8, CH3	1.31, s	18.0, CH3	1.39, s	18.8, CH3
20		153.4, C	2.27, m	29.5, CH	2.47, m	35.2, CH
21	2.14, s	21.4, CH3	0.96, d (5.7)	21.8, CH3	1.13, s o	21.5, CH3
22	6.05, s	125.1, CH	2.31, m	48.5, CH2	2.62, m	47.9, CH2
23		198.6, C		207.7, C		211.0, C
24	2.51, m	47.9, CH2	2.80, dd (17.7, 8.9)	46.8, CH2	2.86, m	47.3, CH2
25	1.39, d (7.1)	35.2, CH	2.91, ddd (8.8, 7.0, 4.9)	34.9, CH	1.53, m	36.2, CH
26		175.9, C		175.8, C		177.6, C
27	1.19, d (6.6)	18.6, CH3	1.16, d (7.0)	17.2, CH3	1.17, d (6.5)	17.3, CH3
28	1.12, s	20.5, CH3	0.87, s	14.3, CH3	1.12, s	22.1, CH3
29	1.14, s	27.6, CH3	1.01, s	28.0, CH3	1.14, s	26.9, CH3
30	1.68, s	21.5, CH3	1.71, s	21.4, CH3	1.46, s	23.9, CH3
31				170.4, C
32			2.22, s	21.0, CH3
33	4.13, q (7.1)	60.7, CH2	4.10, q (7.1)	60.8, CH2	4.12, q (7.1)	61.7, CH2
34	1.25, t (7.1)	14.3, CH3	1.23, t (7.1)	15.7, CH3	1.25, t (7.1)	14.5, CH3

^a Recorded in Chloroform‐d at 500/125 MHz.

^b Recorded in Methanol‐d4 at 600/150 MHz.

^o Overlapped signals.

FIGURE 5.

| Key 2D NMR correlations of compounds 4–6.

Compound 5 was obtained as a pale yellow gel. Its HRESIMS displayed a sodium adduct ion at m/z 623.3193 [M + Na]⁺ (calcd for C₃₄H₄₈NaO₉, 623.3191) (Figure SIII‐33), consistent with the molecular formula C₃₄H₄₈O₉. Comparison of its UV and NMR data with those of 4 indicated that 5 was also a ganoderate‐type triterpenoid. Analysis of the ¹H and ¹³C NMR spectroscopic data of 5 and 4 revealed that 5 lacked the characteristic double bond signals and one nonconjugated carbonyl present in 4, and instead displayed two oxygenated methines (δ _H 3.24, 5.61; δ _C 77.5, 79.3) together with an acetyl group (δ _H 2.22; δ _C 21.0, 170.4). HMBC correlations (Figure 5) from Me‐28/Me‐29/H₂‐2/H‐5 to C‐3, along with the ¹H‐¹H COSY cross‐peaks between H₂‐2 and H‐3, located a hydroxy group at C‐3. HMBC correlations from H‐12 and Me‐32 to C‐31 placed an acetoxy group at C‐12. HMBC correlations from H‐22 to C‐20, C‐21, and C‐23, together with the ¹H‐¹H COSY couplings of H‐20/H₂‐22, indicated that the Δ ^20, 22 double bond in 4 had been reduced to a single bond in 5. In the ROESY spectrum (Figure 5), correlations of H‐3/H‐5, H‐3/Me‐29 established the β‐orientation of 3‐OH, while the correlation of H‐12/Me‐30 indicated the α‐orientation for H‐12. The absolute configuration of 5 was assigned by comparison of its experimental ECD curve with those of 1, 2, and 4 (Figure 4), leading to the assignment of 3S,5R,10S,12S,13R,14R,17R,20R. The configuration at C‐25 was assigned as 25R based on biogenetic considerations. Thus, the structure of 5 was confirmed and designated as ethyl ganoderate H.

Compound 6 was isolated as a pale white gel. The molecular formula of C₃₂H₄₆O₈ was determined by its HRESIMS data [m/z 581.3082 [M + Na]⁺ (calcd for C₃₂H₄₆NaO₈, 581.3085)] (Figure SIII‐41). The molecular weight of 6 was 42 mass units lower than that of 5. Comparison of their NMR data revealed that 6 lacked the characteristic acetyl signals (δ _C‐31 170.4, δ _H‐32 2.22, δ _C‐32 21.0) observed in 5, suggesting that the C‐12 acetoxy group in 5 was hydrolyzed to a hydroxy group in 6. This was corroborated by the shielded chemical shift of H‐12 (Δδ _H 1.17) and the HMBC correlations (Figure 5) from H‐12 to C‐11 and C‐13. Moreover, the HMBC correlations from H‐7 to C‐5 and C‐8, along with the ¹H‐¹H COSY correlations of H‐6/H‐7, located the hydroxy group at C‐7. HMBC correlations from Me‐28/Me‐29/H‐2/H‐5 to the carbonyl carbon C‐3 indicated that the 3‐OH in 5 had been oxidized to a ketone in 6. In the ROESY spectrum, the correlation of H‐7/H‐5 established the β‐orientation of 7‐OH (Figure 5). The remaining ROESY correlations were consistent with those of 5, indicating identical relative configurations at the other stereogenic centers. Compound 6 exhibited a similar experimental ECD spectrum with that of 3 (Figure 4), allowing the absolute configuration of 6 to be assigned as 5R,7S,10S,12S,13R,14R,17R,20R. The absolute configuration at C‐25 was assigned as 25R based on biogenetic considerations. Accordingly, compound 6 was identified and named ethyl ganoderate D.

Biosynthetically, both lucidenate‐type (1–3) and ganoderate‐type (4–6) triterpenoids are considered to originate from the mevalonate‐derived lanosterol pathway. Compared with the intact side chain of ganoderate‐type triterpenoids, the shortened C‐17 side chain of lucidenate‐type derivatives may result from oxidative cleavage of the terminal isopentenyl unit of a ganoderate‐type precursor [14]. The ethyl group present in compounds 1–6 might be generated from pyruvate produced by glycolysis in G. lucidum [15, 16]. Although ethyl ester‐containing lanostane‐type triterpenoids have previously been reported as natural products [17], the origin of lanostane‐type triterpenoid ethyl esters from G. lucidum should be assessed on the basis of direct experimental evidence, such as LC‐MS analysis or chemical transformation studies. In the present study, LC‐MS analysis supported compounds 2 and 5 as genuine natural products (Figures SI‐4 and SI‐6), whereas compounds 1 and 3 were identified as artifacts formed during ethanol extraction (Figures SI‐3 and SI‐5). Owing to the limited amounts available, the origins of compounds 4 and 6 could not be conclusively determined.

The structures of known compounds (Figure SI‐1) were identified by comparison of their MS and 1D NMR data with those reported in the literature as ethyl ganoderenate D (7) [17], lucidenic acid N (8) [18], methyl lucidenate N (9) [19], lucidenic acid P (10) [20], methyl lucidenate P (11) [20], methyl lucidenate E₂ (12) [12], lucidenic acid C (13) [21], methyl lucidenate A (14) [22], ethyl lucidenate A (15) [10], methyl lucidenate Q (16) [20], methyl lucidenate D (17) [11], ethyl ganoderate F (18) [1], ganodernoid E (19) [23], ganoderic acid A (20) [24], methyl ganoderate A (21) [24], ganoderic acid K (22) [25], methyl ganoderate E (23) [22], ganoderenic acid A (24) [26], methyl ganoderenate A (25) [26], ganoderenic acid K (26) [27], and 12β‐acetoxy‐3β‐hydroxy‐7,11,15,23‐tetraoxo‐lanosta‐8,20E‐diene‐26‐oic acid (27) [28].

All compounds with sufficient quantities were evaluated for their inhibitory effects on NO production in LPS‐induced RAW264.7 macrophage cells. However, none of the tested compounds exhibited significant inhibition at a concentration of 20 µM. The most active compound 6 showed an inhibition rate of 11.15% at 20 µM, whereas the remaining compounds exhibited virtually no inhibitory effect on NO production.

3. Conclusions

In this study, six previously undescribed triterpenoids (1–6), together with 21 known structural analogues (7–27), were isolated from the fruiting bodies of G. lucidum. Compounds 1–3 are lucidenate‐type triterpenoids, whereas compounds 4–6 are ganoderate‐type derivatives. Their structures were unambiguously elucidated by comprehensive analysis of 1D and 2D NMR spectroscopic data, HRESIMS, and ECD calculations. The absolute configurations of the 3‐OH in 2 and the 7‐OH in 3 were further corroborated by the modified Mosher's method. Among the six compounds, compounds 2 and 5 were identified as natural products, while compounds 1 and 3 were determined to be artifacts (Compounds 4 and 6 could not be determined due to their insufficient amounts). Although no significant activity was observed in the preliminary NO inhibition assay, these structurally interesting lanostane‐type triterpenoids may still warrant further pharmacological evaluation, particularly with respect to cytotoxic or senotherapeutic activities [17, 29].

4. Experimental Section

4.1. General Experimental Procedures

HRESIMS spectra were acquired on an Agilent UPLC‐Q‐TOF 6520B mass spectrometer. Analytical HPLC was performed on an Agilent 1260 series instrument equipped with a DAD detector and a Shim‐pack VP‐ODS column (4.6 µm, 250 × 4.6 mm). Preparative HPLC was carried out on a Shimadzu LC‐20AR system equipped with an SPD‐20A dual‐wavelength detector and a Shim‐pack RP‐C18 column (10 µm, 200 × 20 mm). Optical rotations were measured on a JASCO P‐1020 automatic digital polarimeter. UV spectra were recorded on a Shimadzu UV‐2450 UV/Vis spectrophotometer. ECD spectra were recorded on a JASCO J‐810 circular dichroism spectrometer. NMR spectra were acquired on Bruker Avance III‐500 and Avance III‐600 spectrometers, with TMS as an internal standard. Silica gel (100–200 and 200–300 mesh) for column chromatography was purchased from Qingdao Haiyang Chemical Co., Ltd. (China). ODS RP‐C18 material (40–63 µm) was obtained from Fuji Silysia Chemical Co., Ltd. (Japan), and Sephadex LH‐20 gel (40–70 µm) was purchased from Amersham Biosciences (Sweden).

4.2. Fungal Material

Fruiting bodies of Ganoderma lucidum were collected in November 2023 from the cultivation base of Limin Pharmaceutical Co., Ltd. (Suichang County, Lishui City, Zhejiang Province, China). The specimen was identified by Prof. Guoyong Xie, Department of Medicinal Plant Resources, China Pharmaceutical University. A voucher specimen (2023‐LZ) was deposited at the State Key Laboratory of Natural Medicines, China Pharmaceutical University.

4.3. Extraction and Isolation

Air‐dried and powdered fruiting bodies of G. lucidum (20.1 kg) were extracted with anhydrous ethanol under reflux (3 h × 3). The combined extracts were filtered and concentrated under reduced pressure to afford a crude residue (760 g). The residue was suspended in water and partitioned successively with petroleum ether, dichloromethane, and n‐butanol. The dichloromethane‐soluble fraction (211.7 g) was subjected to silica gel column chromatography eluted with a stepwise gradient of CH₂Cl₂─MeOH (100:1 – 1:1, v/v) to afford 11 fractions (Fr.1–11) based on TLC profiles.

Fraction 3 (8.8 g) was purified over a Sephadex LH‐20 column to yield four subfractions (Fr.3A–3D). Fr.3B (4.7 g) was further separated by silica gel column chromatography using a petroleum ether‐EtOAc gradient, giving eight subfractions (Fr.3B1–3B8). Fr.3B8 (2.1  g) was purified on a preparative column (Shim‐pack RP‐C18 column, 10 µm, 200  ×  20 mm) by preparative HPLC (Acetonitrile‐H₂O, 55:45, 10  mL/min, 254 nm) to afford compounds 1 (42.9  mg, t _R = 19.9  min), 2 (35.5  mg, t _R = 12.2  min), 3 (23.9  mg, t _R = 11.1  min), 4 (13.4  mg, t _R = 17.3  min), 6 (6.2  mg, t _R = 15.7  min), 7 (13.6  mg, t _R = 15.9  min), 15 (8.2  mg, t _R = 18.3  min), and 18 (83.2  mg, t _R = 19.3  min).

Fraction 5 (60.5 g) was subjected to RP‐C18 column chromatography eluted with a gradient of Acetonitrile‐H₂O to give nine subfractions (Fr.5A–5I). Fr.5H (2.7 g) was subjected to preparative HPLC (Shim‐pack RP‐C18 column, MeOH─H₂O, 7:3, 10 mL/min, 254 nm) to yield compounds 5 (24.1 mg, t _R = 10.6 min), 17 (142 mg, t _R = 10.3 min), and 23 (5.9 mg, t _R = 9.2 min). Fr.5G (4.2 g) was purified by the same preparative HPLC (MeOH─H₂O, 7:3, 10 mL/min) to afford compound 14 (33.5 mg, t _R = 13.3 min, 254 nm).

Fraction 6 (62.8 g) was separated by ODS column chromatography eluted with a MeOH‐H₂O gradient, yielding five subfractions (Fr.6A–6E). Fr.6C (10.4 g) was further purified by Sephadex LH‐20 column chromatography followed by preparative HPLC (Shim‐pack RP‐C18 column, MeOH─H₂O, 6:4, 10 mL/min, 254 nm) to afford compounds 8 (43.9 mg, t _R = 11.8 min), 11 (3.9 mg, t _R = 21.2 min), and 13 (10.6 mg, t _R = 11.3 min). Preparative HPLC (Shim‐pack RP‐C18 column, Acetonitrile‐H₂O, 35:65, 10 mL/min, 254 nm) of Fr.6C gave compounds 22 (31.4 mg, t _R = 18.4 min), 26 (40.6 mg, t _R = 17.4 min), and 27 (6.9 mg, t _R = 20.3 min). Further preparative HPLC purification (Shim‐pack RP‐C18 column, Acetonitrile‐H₂O, 45:55, 10 mL/min, 254 nm) of Fr.6C yielded compounds 9 (22.1 mg, t _R = 15.2 min), 10 (72.2 mg, t _R = 16.1 min), and 12 (82.5 mg, t _R = 18.7 min).

Fraction 11 (27.2 g) was chromatographed over an ODS column eluted with a Acetonitrile‐H₂O gradient to give six subfractions (Fr.11A–11F). Fr.11D (7.4  g) was successively purified by silica gel column chromatography, Sephadex LH‐20 column chromatography, and preparative HPLC (Shim‐pack RP‐C18 column, Acetonitrile‐H₂O, 45:55, 10  mL/min, 254 nm) to afford compounds 16 (52.5  mg, t _R = 19.1  min), 19 (28.1  mg, t _R = 11.4  min), 20 (653.3  mg, t _R = 8.4  min), 21 (259.6  mg, t _R = 15.5  min), 24 (165.3  mg, t _R = 18.8  min), and 25 (72.8  mg, t _R = 16.6  min).

4.3.1. Ethyl Lucidenate D2 (1)

Pale yellow gel; ${[\alpha ]}_{\mathrm{D}}^{20}$ +54.8 (c 0.03, MeOH); UV (MeOH) λ _max nm (log ε) 203 (3.16), 218 (3.07), 253 (3.37); ECD (MeOH) λ _max (Δε) 226 (+33.25), 252 (–9.83), 276 (+14.71), 306 (–12.23); ¹H NMR (500 MHz, Methanol‐d ₄) and ¹³C NMR (125 MHz, Methanol‐d ₄), see Table 1; HRESIMS m/z 543.2950 [M + H]⁺ (calcd for C₃₁H₄₃O₈, 543.2952).

4.3.2. Ethyl Lucidenate E₂ (2)

Pale white gel; ${[\alpha ]}_{\mathrm{D}}^{20}$ +51.8 (c 0.01, MeOH); UV (MeOH) λ _max nm (log ε) 204 (3.46), 219 (3.42), 256 (3.65); ECD (MeOH) λ _max (Δε) 225 (+48.43), 253 (–19.13), 277 (+32.13), 305 (–27.97); ¹H NMR (500 MHz, Chloroform‐d) and ¹³C NMR (125 MHz, Chloroform‐d), see Table 1; HRESIMS m/z 567.2928 [M + Na]⁺ (calcd for C₃₁H₄₄NaO₈, 567.2928).

4.3.3. Ethyl Lucidenate P (3)

Pale yellow gel; ${[\alpha ]}_{\mathrm{D}}^{20}$ +9.6 (c 0.02, MeOH); UV (MeOH) λ _max nm (log ε) 202 (3.13), 216 (3.03), 255 (3.49); ECD (MeOH) λ _max (Δε) 253 (+114.49), 289 (–30.69), 318 (–5.29); ¹H NMR (500 MHz, Methanol‐d ₄) and ¹³C NMR (125 MHz, Methanol‐d ₄), see Table 1; HRESIMS m/z 569.3086 [M + Na]⁺ (calcd for C₃₁H₄₆NaO₈, 569.3085).

4.3.4. Ethyl Ganoderenate F (4)

Pale yellow gel; ${[\alpha ]}_{\mathrm{D}}^{20}$ +6.8 (c 0.01, MeOH); UV (MeOH) λ _max nm (log ε) 209 (3.23), 243 (3.64); ECD (MeOH) λ _max (Δε) 223 (+14.17), 248 (–5.31), 276 (+9.93), 305 (–9.35); ¹H NMR (500 MHz, Chloroform‐d) and ¹³C NMR (125 MHz, Chloroform‐d), see Table 2; HRESIMS m/z 539.3001 [M + H]⁺ (calcd for C₃₂H₄₃O₇, 539.3003).

4.3.5. Ethyl Ganoderate H (5)

Pale yellow gel; ${[\alpha ]}_{\mathrm{D}}^{20}$ +46.5 (c 0.01, MeOH); UV (MeOH) λ _max nm (log ε) 206 (3.46), 228 (3.31), 254 (3.45); ECD (MeOH) λ _max (Δε) 223 (+67.46), 251 (–28.11), 277 (+40.20), 306 (–36.51); ¹H NMR (500 MHz, Chloroform‐d) and ¹³C NMR (125 MHz, Chloroform‐d), see Table 2; HRESIMS m/z 623.3193 [M + Na]⁺ (calcd for C₃₄H₄₈NaO₉, 623.3191).

4.3.6. Ethyl Ganoderate D (6)

Pale white gel; ${[\alpha ]}_{\mathrm{D}}^{20}$ +90.0 (c 0.02, MeOH); UV (MeOH) λ _max nm (log ε) 202 (3.44), 214 (3.39), 253 (3.64); ECD (MeOH) λ _max (Δε) 256 (+43.27), 305 (–11.87); ¹H NMR (600 MHz, Methanol‐d ₄) and ¹³C NMR (150 MHz, Methanol‐d ₄), see Table 2; HRESIMS m/z 581.3082 [M + Na]⁺ (calcd for C₃₂H₄₆NaO₈, 581.3085).

4.4. ECD Calculations

Detailed procedures for the ECD calculations are provided in the Supporting Information.

4.5. Anti‐inflammatory Assay

Mouse monocytic macrophage leukemia cells (RAW264.7, RRID: CVCL_0493) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells in the logarithmic growth phase were seeded into 96‐well plates at a density of 2.5 × 10⁴ cells per well (100 µL per well) and incubated at 37°C in a 5% CO₂ atmosphere for 18 h. Subsequently, 10 µL of the test compounds were added to the treatment groups, with L‐NMMA (IC₅₀ = 37.2 ± 2.2 µmol·L⁻¹) as the positive control, while the blank and model groups received 10 µL of blank medium. After 1 h of incubation, 10 µL of LPS (final concentration 1 µg·mL⁻¹) was added to stimulate NO production, and the cells were incubated for an additional 18 h. An aliquot of 50 µL of supernatant from each well was collected and processed according to the instructions provided with the nitric oxide assay kit. The absorbance (A) of each well was measured at 540 nm using a microplate reader. The NO inhibition rate was calculated using the following formula: Inhibition rate (%) = [(A _model— A _sample) / (A _model—A _blank)] × 100%.

4.6. Preparation of (S)‐MTPA and (R)‐MTPA Esters of Compounds 2 and 3

Compound 2 (4.9 mg) was dissolved in 1 mL of anhydrous CHCl₃. DMAP (15 mg), triethylamine, and (R)‐MTPA‐Cl (7.0 mg) were added sequentially. The mixture was stirred at room temperature for 12 h, and the reaction was quenched by addition of 1 mL of MeOH. The solvent was removed under reduced pressure, and the residue was dissolved in MeOH and purified by preparative HPLC to afford the (S)‐MTPA ester of 2 (2.1 mg). The (R)‐MTPA ester of 2 (2.9 mg) was prepared following the same procedure using (S)‐MTPA‐Cl. The (S)‐ and (R)‐MTPA esters of compound 3 were prepared analogously.

Conflicts of Interest

The authors declare no conflict of interest.

Supporting information

Supporting file 1: cbdv71265‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available within the article and its supplementary materials.