Anti-Adipogenic Lanostane-Type Triterpenoids from the Edible and Medicinal Mushroom Ganoderma applanatum

Abstract

Our previous research has shown that lanostane triterpenoids from Ganoderma applanatum exhibit significant anti-adipogenesis effects. In order to obtain more structurally diverse lanostane triterpenoids to establish a structure–activity relationship, we continued the study of lanostane triterpenoids from the fruiting bodies of G. applanatum, and forty highly oxygenated lanostane-type triterpenoinds (1–40), including sixteen new compounds (1–16), were isolated. Their structures were elucidated using NMR spectra, X-ray crystallographic analysis, and Mosher’s method. In addition, some of their parts were evaluated to determine their anti-adipogenesis activities in the 3T3-L1 cell model. The results showed that compounds 16, 22, 28, and 32 exhibited stronger anti-adipogenesis effects than the positive control (LiCl, 20 mM) at the concentration of 20 μM. Compounds 15 and 20 could significantly reduce the lipid accumulation during the differentiation process of 3T3-L1 cells, comparable to the untreated group. Their IC₅₀ values were 6.42 and 5.39 μM, respectively. The combined results of our previous and present studies allow us to establish a structure-activity relationship of lanostane triterpenoids, indicating that the A-seco-23→26 lactone skeleton could play a key role in anti-adipogenesis activity.

1. Introduction

Macro-fungi provide crucial food and medicinal resources [1]. Tricholoma matsutake, [2,3] Lentinula edodes, [4,5], and Collybia albuminosa [6] are delicious mushrooms which contain plentiful amino acids, fatty acids, vitamins, crude fiber, and protein. In addition, Fomitopsis pinicola (SW.) [7] Karst, Inonotus obliquus [8,9], Phellinus igniarius [10,11], Ganoderma lucidum [12,13], and Ganoderma sinense [14,15] have been used as edible and medicinal mushrooms for preventing and treating various diseases. The Ganoderma genus plays an important role in the history of Chinese medicine [16]. Shennong’s Herbal Classics recorded its traditional effects to include improving eyesight, strengthening muscles and bones, reinforcing kidney function, soothing the nerves, and prolonging the lifespan. G. lucidum and G. sinense have been registered in the Chinese Pharmacopoeia (2015 version). Meanwhile, G. lucidum was also included in the catalog of the latest edition of “Homology of medicine and Food” in 2020. Modern pharmacological research has further demonstrated that Ganoderma has a variety of pharmacological activities [17,18,19,20,21]. Thus, Ganoderma has great prospects in preventing and treating diseases.

Ganoderma applanatum, belonging to the genus Ganoderma, has traditionally been used to treat various chronic diseases, such as chronic hepatitis, immunological disorders, neurasthenia, arthritis, and nephritis [22]. Meanwhile, G. applanatum has been made into capsules and injections to cure chronic liver fibrosis and inflammation in a clinical setting [23,24,25,26]. G. applanatum is rich in chemical constituents, including polysaccharides, triterpenoids, meroterpenoids, alkaloids, and steroids. The majority of studies relating to it focus on the application and development of polysaccharides [23,27,28,29,30,31]. However, our previous research proved that highly oxygenated triterpenoids showed significant anti-adipogenesis activities [27,28]. In order to search for more active compounds to clarify the structure–activity relationship to lay the foundations for the discovery of lead compounds, we continued to investigate triterpenoids isolated from G. applanatum and 40 lanostane-type triterpenoids; of these (1–40), 16 were new compounds (1–16, Figure 1). Furthermore, their anti-adipogenesis effects were evaluated in the 3T3-L1 cell model, and their structure–activity relationship was established.

2. Materials and Methods

2.1. General Experimental Procedures

NMR spectra were recorded on a Bruker AV-600 MHz (Bruker, Zurich, Switzerland) using TMS as an internal standard for chemical shifts with reference to the TMS resonance. ESIMS and HRTOF-ESIMS were measured on an API QSTAR Pulsar spectrometer. UV spectra were recorded on a Shimadzu UV-2401PC spectrometer. IR was recorded on the Bruker Tensor-27 instrument using KBr pellets. Optical rotations were recorded on a Horiba SEPA-300 polarimeter. CD spectra were measured on a Chirascan instrument. An Agilent 1100 series instrument equipped with an Agilent ZORBAX SB-C18 column (5 μm, 9.6 mm × 250 mm) was used for high-performance liquid chromatography (HPLC) separation.

TLC was performed on precoated TLC plates (200–250 µm thickness, F254 Si gel 60, Qingdao Marine Chemical, Inc., Qingdao, China), with compounds visualized by spraying the dried plates with 10% aqueous H₂SO₄ followed by heating until they were dry. Silica gel ((200–300) mesh, Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (40–63 μm, Fuji), and Sephadex LH-20 (20–150 μm, Pharmacia) were used for column chromatography. Methanol, chloroform, ethyl acetate, acetone, petroleum ether, n-hexane, and 2-propanol were purchased from Tianjing Chemical Reagents Co. (Tianjing, China). All other materials were of the highest grade available.

2.2. Fungal Materials

Ganoderma applanatum (39 kg) was purchased in December 2019 from a traditional Chinese medicine market in Kunming, Yunnan, China, which was identified by Prof. Yang Zhuliang, Kunming Institute of Botany, Chinese Academy of Science (voucher No. 19122201).

2.3. Extraction and Isolation

G. applanatum (39 kg) was chipped and extracted with 95% EtOH under reflux three times (three hours each time). The combined EtOH extracts were evaporated under reduced pressure. The residue was suspended in H₂O and extracted with EtOAc. The volume of the combined EtOAc extracts was reduced to one-third under a reduced pressure. The residue was fractionated by macroporous resin (D-101; MeOH−H₂O, 50:50, 70:30, and 90:10, v/v): fractions I−III. Fraction III (245 g) was further fractioned by a silica gel column with petroleum ether (PE)/ethyl acetate (EA) as the mobile phase, which gave six subfractions (Fr. III-1→Fr. III-6).

Fr. III-2 (156 g) was treated by a silica gel column and CHCl₃/MeOH (80:1→20:1, v/v) was used as an eluent. Ten fractions (Fr. III-2-1→Fr. III-2-10) were obtained, of which Fr. III-2-4 (20 g) was separated using Sephadex LH-20 (MeOH) to obtain three subfractions (Fr. III-2-4a→Fr. III-2-4c). Compound 29 (235 mg) was purified through recrystallization from Fr. III-2-4b. The remaining solution was isolated using semi-preparative HPLC (CH₃CN/H₂O = 52%, v/v) to gain compound 6 (8 mg, t_R = 28.6 min). Fr. III-2-5 (10 g) was treated by a silica gel column, being eluted with PE/EA (20:1, v/v) to obtain five parts (Fr. III-2-5a→Fr. III-2-5e). Subsequently, 5b and 5d were purified using P-TLC (CHCl₃/MeOH = 40:1, v/v) to obtain compounds 18 (11 mg), 16 (26 mg), and 21 (9.2 mg). Fr. III-2-6 (12 g) was separated by Rp-C18 with the elution of MeOH/H₂O (50%→55%) to obtain five fractions. Compounds 36 (6.2 mg) and 13 (12 mg) were obtained from Fr. III-2-6c and Fr. III-2-6d through P-TLC (CHCl₃/MeOH = 40:1, v/v), respectively.

Fr. III-2-7 (25 g) was fractioned by an Rp-C18 column, being eluted with MeOH/H₂O (50%→65% containing 0.3% CF₃COOH, v/v); nine subfractions (7a→7i) were obtained. Furthermore, 7d, 7g, and 7h were purified by semi-preparative HPLC (CH₃CN/H₂O = 45%→60%, v/v) to obtain compounds 14 (5.3 mg, t_R = 19.1 min), 4 (8.3 mg, t_R = 19.1 min), 8 (3.4 mg, t_R = 14.8 min), 9 (3.2 mg, t_R = 17.4 min), 12 (4.2 mg, t_R = 21.3 min), and 17 (5.1 mg, t_R = 22.2 min). Similarly, Fr. III-2-8 (31 g) was also treated using an Rp-C18 column with MeOH/H₂O (50%→55%) to obtain nine subfractions (8a→8i), from which compounds 37 (3.2 mg, t_R = 26.6 min), 38 (3.6 mg, t_R = 27.6 min), 31 (6.1 mg, t_R = 22.1 min), 22 (3.1 mg, t_R = 21.5 min), 25 (14 mg, t_R = 25.9 min), 19 (12.5 mg, t_R = 27.5 min), and 20 (7.2 mg, t_R = 20.7 min) were purified by semi-preparative HPLC (CH₃CN/H₂O = 43%→60% containing 0.3% CF₃COOH, v/v). The Rp-C18 column and semi-preparative HPLC were used to treat Fr. III-2-9 (20 g), and compounds 35 (2.5 mg, t_R = 20.8 min), 28 (2.9 mg, t_R = 20.3 min), 13 (2.1 mg, t_R = 18.3 min), 7 (2.2 mg, t_R = 19.5 min), and 23 (2.9 mg, t_R = 18.5 min) were isolated from 9d-1 and 9d-2. 9e (15 mg) was purified by P-TLC (CHCl₃/MeOH = 30:1, v/v) to obtain compounds 39 (4.2 mg) and 40 (2.8 mg).

The combination of Fr. III-4 and Fr. III-5 weighing 52 g was fractioned using Rp-C18 column elution with MeOH/H₂O (35%→100%, v/v) to obtain six subfractions (Fr. III-4-1→Fr. III-4-6). Among these, Fr. III-4-2→Fr. III-4-5 were treated using Sephadex LH-20 (MeOH). Subsequently, the triterpenoid parts were purified by P-TLC (CHCl₃/MeOH = 20:1 containing 0.3% CF₃COOH, v/v) and semi-preparative HPLC (CH₃CN/H₂O = 38%→53% containing 0.3% CF₃COOH, v/v) to obtain compounds 27 (4.2 mg), 24 (2.1 mg), 30 (6.2 mg), 5 (3.1 mg), 33 (3 mg), 26 (4.8 mg), 17 (5 mg, t_R = 23.5 min), 3 (7.2 mg, t_R = 22.5 min), 34 (6.1 mg, t_R = 12.1 min), 32 (3.3 mg, t_R = 17.8 min), 1 (3.0 mg, t_R = 17.5 min), 10 (2.9 mg), and 2 (3.7 mg).

Ganoapplic acid A (1): white powder (MeOH); [α]²⁸_D −1.2 (c 0.25, MeOH); UV (MeOH); λ_max (log ε): 230 (3.36), and 196 (3.33); IR (KBr) v_max 3428, 2953, 2943, 1653, 1636, 1423, 1364, 1212, and 1131 cm⁻¹; ¹H NMR and ¹³C NMR data: see

Table 1. ¹H NMR spectra of compounds 1–8 (600 MHz, methanol-d₄, J in Hz, δ in ppm).

Position	1	2	3	4	5	6	7	8
1	1.87 dt (13.7 4.5) 2.21 dd (15.4 2.8)	1.86 m 2.20 m	1.82 m 2.16 m	1.85 m 2.17 m	1.91 m 2.29 m	1.87 m 2.16 m	2.02 m 1.51 m	1.50 m 2.02 m
2	2.40 m 2.95 m	2.19 m 2.36 m	2.17 m 2.34 m	2.25 m 2.40 m	2.55 m 2.74 m	2.51 m 2.71 m	1.78 m	1.77 m
3							3.24 t (8.4)	3.23 t (8.4)
5	1.74 dd (14.8 2.8)	2.97 t (8.3)	2.94 t (8.4)	2.95 dd (9.3 7.5)	2.28 m	2.19 m	1.66 dd (14.4 3.6)	1.66 dd (14.4 3.6)
6	2.30 dd (15.4 2.8) 3.30 m	2.03 m	1.99 m	2.03 m	2.44 dd (16.3 3.3)	2.52 m 2.66 m	2.62 m 2.48 m	2.48 m 2.59 m
7		4.93 s	4.95 s	4.98 s
11	5.98 s	6.04 s	6.02 s	6.04 s	6.28 d (9.9)	6.14 d (10.2)	6.31 d (10.2)	6.31 d (10.2)
12					6.59 d (9.9)	6.59 d (10.2)	6.64 d (10.2)	6.65 d (10.2)
15	4.83 s	3.95 d (6.5)	3.91 d (6.5)	3.94 d (6.4)	4.20 s	4.44 d (7.8)	4.39 d (7.2)	4.39 d (7.2)
16	3.36 s	1.80 m 4.65 m	1.73 m 2.64 m	1.76 m 2.67 m	4.57 d (7.8)	2.20 m 2.42 m	2.16 m 2.51 m	2.16 m 2.51 m
17		3.33 m	3.21 dd (10.7 8.7)	3.23 m	2.73 m	2.73 m	2.87 m	2.83 m
18	1.97 s	1.30 s	1.37 s	1.39 s	0.99 s	1.01 s	1.00 s	0.99 s
19	1.65 s	1.01 s	1.04 s	1.05 s	1.14 s	1.27 s	1.16 s	1.16 s
21	1.34 s	4.37 s	5.33 s 5.39 s	5.36 s 5.43 s	1.21 s	2.23 s	2.19 s	2.19 s
22	2.66 d (14.9) 3.00 d (14.9)	5.78 d (8.6)	4.47 d (5.6)	4.50 d (5.6)	6.36 s	6.22 s	6.40 s	6.32 s
23		5.59 dd (8.6 6.6)	4.72 m	4.73 ddd (8.6 5.6 3.2)
24	2.62 dd (18.3 5.6) 3.07 dd (18.3 7.7)	2.19 m	1.97 m 2.16 m	2.00 m 2.56 m	2.61 m 2.90 m	2.54 m 2.94 m	2.64 m 2.90 m	2.53 m 2.55 m
25	1.74 dd (14.8 2.8)	2.82 q (7.7)	2.84 m	2.86 m	2.74 m	2.97 m	2.85 m	2.84 m
26	1.16 d (6.6)	1.29 d (7.3)	1.22 d (6.5)	1.25 d (6.5)	1.18 d (7.0)	1.19 d (7.2)	1.17 d (7.2)	1.17 d (7.2)
28	1.14 s	4.80 s 4.98 s	4.77 s 4.95 s	4.77 s 4.95 s	1.12 s	1.13 s	1.00 s	1.00 s
29	1.07 s	1.80 s	1.78 s	1.80 s	1.10 s	1.14 s	0.90 s	0.90 s
30	0.74 s	1.06 s	1.01 s	1.04 s	1.10 s	0.94 s	0.95 s	0.95 s
OMe				3.65 s		3.68 s	3.65 s

and

Table 2. ¹³C NMR spectra of compounds 1–8 (150 MHz, methanol-d₄).

Position	1	2	3	4	5	6	7	8
1	37.6 CH2	37.7 CH2	37.7 CH2	37.5 CH2	36.5 CH2	35.6 CH2	36.3 CH2	36.3 CH2
2	35.1 CH2	30.4 CH2	30.5 CH2	30.5 CH2	35.0 CH2	34.0 CH2	28.1 CH2	28.1 CH2
3	215.8 C	177.4 C	177.4 C	175.6 C	217.1 C	214.3 C	78.3 CH	78.3 CH
4	48.7 C	146.5 C	146.5 C	146.3 C	51.9 C	46.9 C	39.7 C	39.8 C
5	48.5 CH	45.0 CH	45.3 CH	45.3 CH	50.7 CH	49.3 CH	50.8 CH	50.8 CH
6	35.8 CH2	28.2 CH2	28.1 CH2	28.1 CH2	37.5 CH2	36.8 CH2	37.0 CH2	37.0 CH2
7	205.9 C	64.7 CH	64.8 CH	65.0 CH	203.0 C	210.4 C	204.3 C	204.3 C
8	85.5 C	67.4 C	67.5 C	67.5 C	135.8 C	134.9 C	135.7 C	135.7 C
9	165.0 C	167.2 C	167.8 C	167.3 C	161.4 C	160.6 C	164.5 C	164.5 C
10	41.0 C	45.0 C	45.2 C	45.3 C	39.2 C	37.9 C	39.9 C	39.7 C
11	127.3 CH	130.7 CH	130.6 CH	130.6 CH	123.2 CH	122.0 CH	123.3 CH	123.3 CH
12	204.0 C	206.5 C	207.3 C	207.0 C	147.4 CH	147.5 CH	148.3 CH	148.3 C
13	62.3 C	60.8 C	60.4 C	60.4 C	52.2 C	50.2 C	51.4 C	51.4 C
14	47.0 C	54.4 C	54.4 C	54.4 C	48.0 C	52.5 C	53.9 C	53.8 C
15	80.0 CH	77.4 CH	77.2 CH	77.2 CH	84.9 CH	75.0 CH	76.5 CH	76.6 CH
16	61.2 CH	41.3 CH2	43.7 CH2	43.5 CH2	83.6 CH	36.2 CH2	37.2 CH2	37.2 CH2
17	71.4 C	43.3 CH	41.6 CH	41.6 CH	58.7 CH	48.5 CH	49.7 CH	49.7 C
18	24.6 CH3	20.2 CH3	20.2 CH3	20.2 CH3	19.2 CH3	17.1 CH3	20.2 CH3	21.2 CH3
19	19.9 CH3	24.2 CH3	23.4 CH3	23.4 CH3	21.1 CH3	19.5 CH3	18.0 CH3	18.0 CH3
20	73.4 C	144.1 C	148.7 C	148.7 C	156.5 C	157.3 C	158.8 C	158.4 C
21	27.8 CH3	61.8 CH2	117.6 CH2	117.5 CH2	19.7 CH3	21.4 CH3	21.4 CH3	21.3 CH3
22	53.9 CH2	130.8 CH	76.5 CH	76.5 CH	126.9 CH	124.8 CH	126.0 CH	126.4 CH
23	209.9 C	76.8 CH	80.9 CH	80.9 CH	200.8 C	198.3 C	200.6 C	201.3 C
24	48.7 CH2	38.3 CH2	32.2 CH2	32.2 CH2	48.7 CH2	47.7 CH2	48.8 CH2	49.0 CH2
25	35.8 CH	35.7 CH	35.3 CH	35.3 CH	36.2 CH	34.8 CH	36.6 CH	36.6 CH
26	17.4 CH3	15.9 CH3	16.3 CH3	16.0 CH3	17.5 CH3	17.1 CH3	17.4 CH3	17.7 CH3
27	179.7 C	182.7 C	183.1 C	183.2 C	179.7 C	176.4 C	178.1 C	180.8 C
28	22.3 CH3	115.9 CH2	115.9 CH2	115.9 CH2	21.0 CH3	25.6 CH3	27.8 CH3	27.8 CH3
29	24.9 CH3	23.5 CH3	23.4 CH3	23.4 CH3	25.9 CH3	20.4 CH3	15.7 CH3	15.7 CH3
30	30.7 CH3	21.3 CH3	21.4 CH3	21.4 CH3	19.7 CH3	20.6 CH3	21.2 CH3	20.2 CH3
OMe				52.3 CH3		51.8 CH3	52.3 CH3

; HRMS (ESI-TOF) m/z: 561.2696 [M + H]⁺ (calcd for C₃₀H₄₀O₁₀, 561.2694).

Ganoapplic acid B (2): white powder (MeOH); [α]²⁸_D +48.0 (c 0.13, MeOH); UV (MeOH); λ_max (log ε): 251 (3.75), and 196 (4.08); IR (KBr) v_max 3430, 2953, 2928, 1653, 1636, 1473, 1344, 1211, and 1147 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 527.2653 [M − H]⁻ (calcd for C₃₀H₄₀O₈, 527.2650).

Ganoapplic acid C (3): white powder (MeOH); [α]²⁸_D +42.0 (c 0.09, MeOH); UV (MeOH); λ_max (log ε): 251 (3.59), and 196 (3.93); IR (KBr) v_max 3423, 2955, 2930, 1673, 1635, 1428, 1380, 1219, and 1132 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 527.2654 [M − H]⁻ (calcd for C₃₀H₄₀O₈, 527.2650).

Methyl ganoapplate C (4): white powder (MeOH); [α]²⁸_D +39.5 (c 0.07, MeOH); UV (MeOH); λ_max (log ε): 251 (3.52), and 196 (3.85); IR (KBr) v_max 3458, 2957, 2928, 1689, 1606, 1473, 1375, 1210, and 1132 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 565.2773 [M + Na]⁺ (calcd for C₃₁H₄₂O₈Na, 565.2772).

Ganoapplic acid D (5): white powder (MeOH); [α]²⁸_D −20.1 (c 0.21, MeOH); UV (MeOH); λ_max (log ε): 242 (3.71), and 195 (3.79); IR (KBr) v_max 3503, 2967, 2935, 1663, 1635, 1452, 1390, 1200, and 1125 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 511.2707 [M − H]⁻ (calcd for C₃₀H₄₀O₇, 511.2701).

Methyl gibbosate M (6): white powder (MeOH); [α]²⁸_D −49.64 (c 0.11, MeOH); UV (MeOH); λ_max (log ε): 307 (3.21), 243 (3.80), and 196 (3.77); IR (KBr) v_max 3438, 2966, 2912, 1688, 1634, 1453, 1364, 1212, and 1132 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 533.2873 [M + Na]⁺ (calcd for C₃₁H₄₂O₆Na, 533.2874).

Methyl ganoapplate E (7): white powder (MeOH); [α]²⁸_D −69.3 (c 0.13, MeOH); UV (MeOH); λ_max (log ε): 316 (3.13), 243 (3.74), and 196 (3.72); IR (KBr) v_max 3413, 2953, 2916, 1657, 1620, 1454, 1374, 1209, and 1142 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 535.3033 [M + Na]⁺ (calcd for C₃₁H₄₄O₆Na, 535.3030).

Ganoapplic acid E (8): white powder (MeOH); [α]²⁸_D −143.5 (c 0.19, MeOH); UV (MeOH); λ_max (log ε): 317 (3.57), 244 (4.15), and 196 (4.05); IR (KBr) v_max 3445, 2980, 2906, 1712, 1690, 1458, 1380, 1214, and 1028 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 1 and Table 2; HRMS (ESI-TOF) m/z: 497.2911 [M − H]⁻ (calcd for C₃₀H₄₁O₆, 497.2909).

Methyl gibbosate L (9): white powder (MeOH); [α]²⁸_D +20.27 (c 0.15, MeOH); UV (MeOH); λ_max (log ε): 291 (3.70), 244 (3.94), and 196 (3.85); IR (KBr) v_max 3444, 2976, 2911, 1673, 1658, 1423, 1374, 1219, and 1140 cm⁻¹; ¹H NMR and ¹³C NMR data: see

Table 3. ¹H NMR spectra of compounds 9–16 (600 MHz, J in Hz, δ in ppm).

Position	9 a	10 b	11 b	12 b	13 b	14 b	15 b	16 a
1	2.27 m 1.86 m	1.83 m 2.39 m	1.83 m 2.38 m	2.80 m 2.95 m	1.82 m 2.26 m	1.79 m 2.23 m	1.81 m 2.23 m	1.80 m 1.80 m
2	2.42 m 2.78 m	2.37 m 2.93 m	2.37 m 2.94 m	2.53 m 2.59 m	2.31 m 2.95 m	2.27 m 2.94 m	2.28 m 2.93 m	2.36 m 2.89 m
5	1.65 m	1.78 dd (12.8 3.1)	1.76 dd (11.4 3.60	2.36 dd (15.0 3.0)	1.29 dd (12.4 5.9)	1.67 dd (12.6 6.0)	1.66 m	1.56 dd (12.66.0)
6	1.11 m 2.28 m	2.33 m 2.49 m	2.34 m 2.53 m	2.54 m 2.68 m	2.25 m	2.25 m	2.30 m	2.16 m
7	6.50 br s	6.62 m	6.63 d (7.2)		3.96 d (5.7)	3.93 d (5.4)	3.90 d (3.6)	4.45 d (5.4)
11	5.65 s	5.74 s	5.74 s		6.04 s	6.01 s	5.98 s	6.03 s
12				3.70 s
15	4.58 t (8.3)	4.47 d (3.0)	4.47 d (3.0)	4.40 d (7.8)	4.08 d (6.0)	4.05 d (6.0)	4.35 d (3.0)
16	1.80 m 2.48 m	5.75 d (3.0)	5.74 overlapped	2.07 m 2.43 m	1.94 m 2.43 m	1.91 m 2.42 m	6.20 d (3.0)	5.66 s
17	3.26 m			3.28 t (9.6)	3.24 dd (10.7 7.3)	3.21 dd (10.8 7.8)
18	0.80 s	1.54 s	1.54 s	−0.096 s	1.46 s	1.43 s	1.90 s	1.76 s
19	1.28 s	1.39 s	1.40 s	1.31 s	1.47 s	1.45 s	1.47 s	1.44 s
20
21	2.22 s	1.42 s	1.40 s	2.24 s	2.28 s	2.25 s	2.30 s	1.49 s
22	6.37 s	2.82 d (14.2) 3.01 d (14.2)	2.79 d (14.4) 2.79 dd (14.4)	6.31 s	6.56 s	6.52 s	6.50 s	2.92 d (15.0) 2.98 d (15.0)
24	2.54 m 2.94 m	2.66 dd (18.4 5.2) 3.02 m	2.79 dd (18.6 5.4) 3.02 dd (18.6 8.4)	2.54 m 2.94 m	2.62 m 2.92 m	2.62 m 2.88 m	2.58 m 2.93 m	3.04 m 2.59 dd (18.0 5.4)
25	2.93 m	2.80 m	2.81 m	1.19 m	2.89 m	2.89 m	2.89 m	2.89 m
26	1.17 d (6.7)	1.15 d (7.2)	113 d (7.2)	1.19 d (7.2)	1.20 d (7.0)	1.16 d (7.2)	1.20 d (7.0)	1.15 d (7.2)
28	1.11 s	1.20 s	1.20 s	1.15 s	1.09 s	1.09 s	1.10 s	1.11 s
29	1.15 s	1.11 s	1.06 s	1.13 s	1.13 s	1.13 s	1.38 s	1.12 s
30	1.08 s	1.06 s	3.63 s	1.24 s	0.96 s	0.96 s	1.00 s	1.28 s
OMe	3.68 s		3.56 s	3.69 s		3.65 s	3.63 s	3.65 s

^a Measured in CDCl₃; ^b measured in methanol-d₄.

and

Table 4. ¹³C NMR spectra of compounds 9–16 (150 MHz).

Position	9 a	10 b	11 b	12 b	13 b	14 b	15 b	16 a
1	35.6 CH2	36.7 CH2	36.8 CH2	35.1 CH2	38.4 CH2	38.4 CH2	38.4 CH2	37.2 CH2
2	34.2 CH2	35.4 CH2	35.4 CH2	34.3 CH2	34.9 CH2	34.9 CH2	34.8 CH2	33.8 CH2
3	214.8 C	217.2 C	217.2 C	214.9 C	216.4 C	216.4 C	216.5 C	213.6 C
4	47.0 C	48.4 C	48.8 C	47.1 C	48.7 C	48.5 C	48.8 C	47.8 C
5	49.5 CH	51.1 CH	51.2 CH	49.8 CH	50.7 CH	50.7 CH	51.2 CH	49.9 CH
6	23.9 CH2	25.2 CH2	25.2 CH2	37.6 CH2	22.6 CH2	22.6 CH2	22.4 CH2	21.6 CH2
7	130.9 CH	135.5 CH	135.5 CH	203.4 C	59.7 CH	59.7 CH	58.8 CH	57.1 CH
8	139.8 C	137.3 C	137.3 C	150.2 C	65.7 C	65.8 C	64.4 C	59.0 C
9	161.0 C	165.5 C	165.6 C	151.9 C	162.2 C	162.3 C	163.0 C	165.0 C
10	38.0 C	39.6 C	39.7 C	39.3 C	39.0 C	39.0 C	38.6 C	38.2 C
11	118.2 CH	118.3 CH	118.3 CH	203.9 C	127.6 CH	127.6 CH	127.2 CH	125.0 CH
12	202.5 C	206.5 C	206.5 C	79.6 CH	204.6 C	204.6 C	201.9 C	200.6 C
13	52.3 C	64.0 C	64.0 C	52.9 C	59.4 C	59.4 C	63.1 C	61.8 C
14	57.8 C	54.3 C	54.3 C	50.0 C	51.3 C	51.3 C	50.0 C	54.4 C
15	72.9 CH	79.3 CH	79.4 CH	77.5 CH	79.0 CH	79.0 CH	79.9 CH	202.7 C
16	36.5 CH2	127.5 CH	127.6 CH	34.2 CH2	38.2 CH2	38.2 CH2	134.9 CH	124.4 CH
17	45.7 CH	158.9 C	159.0 C	46.9 CH	49.6 CH	49.6 CH	148.8 C	181.9 C
18	17.8 CH3	29.0 CH3	29.1 CH3	18.6 CH3	20.3 CH3	20.3 CH3	27.0 CH3	30.9 CH3
19	21.3 CH3	21.5 CH3	21.5 CH3	19.0 CH3	21.0 CH3	21.0 CH3	21.2 CH3	20.8 CH3
20	157.6 C	72.6 C	72.6 C	157.5 C	159.0 C	159.2 C	156.0 C	72.6 C
21	20.4 CH3	29.0 CH3	29.1 CH3	20.3 CH3	21.5 CH3	21.5 CH3	17.5 CH3	29.1 CH3
22	125.7 CH	54.6 CH2	54.7 CH2	125.1 CH	127.5 CH	127.5 CH	127.1 CH	52.7 CH2
23	198.3 C	209.8 C	209.7 C	198.6 C	201.0 C	201.0 C	201.7 C	206.3 C
24	47.7 CH2	48.8 CH2	48.7 CH2	47.9 CH2	48.7 CH2	48.7 CH2	48.9 CH2	47.8 CH2
25	34.7 CH	35.8 CH	35.9 CH	35.0 CH	36.3 CH	36.3 CH	36.3 CH	34.5 CH
26	17.0 CH3	17.4 CH3	17.3 CH3	16.9 CH3	17.4 CH3	17.4 CH3	17.5 CH3	17.0 CH3
27	176.4 C	179.7 C	178.2 C	176.7 C	179.7 C	178.2 C	180.1 C	176.2 C
28	25.2 CH3	22.9 CH3	25.5 CH3	26.9 CH3	24.9 CH3	24.9 CH3	24.9 CH3	24.5 CH3
29	22.3 CH3	25.2 CH3	22.9 CH3	20.4 CH3	22.3 CH3	22.3 CH3	22.5 CH3	22.1 CH3
30	18.1 CH3	29.0 CH3	29.1 CH3	27.4 CH3	22.5 CH3	22.5 CH3	24.9 CH3	25.9 CH3
OMe	51.8 CH3		52.2 CH3	52.0 CH3		52.3 CH3	52.0 CH3	51.9 CH3

^a Measured in CDCl₃; ^b measured in methanol-d₄.

; HRMS (ESI-TOF) m/z: 533.2876 [M + Na]⁺ (calcd for C₃₁H₄₂O₆Na, 533.2874).

Ganoapplic acid F (10): white powder (MeOH); [α]²⁸_D +20.86 (c 0.14, MeOH); UV (MeOH); λ_max (log ε): 299 (3.77), and 196 (3.98); IR (KBr) v_max 3437, 2953, 2924, 1678, 1656, 1433, 1374, 1215, and 1132 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 511.2708 [M − H]⁻ (calcd for C₃₀H₄₀O₇, 511.2701).

Methyl ganoapplate F (11): white powder (MeOH); [α]²⁸_D +5.80 (c 0.10, MeOH); UV (MeOH); λ_max (log ε): 301 (3.46), and 196 (3.87); IR (KBr) v_max 3439, 2953, 2929, 1723, 1638, 1445, 1334, 1219, and 1138 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 549.2827 [M + Na]⁺ (calcd for C₃₁H₄₂O₇Na, 549.2823).

Methyl gannosate I (12): white powder (MeOH); [α]²⁸_D +95.0(c 0.14, MeOH); UV (MeOH); λ_max (log ε): 249 (3.99), and 195 (3.73); IR (KBr) v_max 3452, 2985, 2921, 1673, 1618, 1425, 1376, 1221, and 1132 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 565.2769 [M + Na]⁺ (calcd for C₃₁H₄₂O₈Na, 565.2772).

Ganoapplic acid G (13): white powder (MeOH); [α]²⁸_D −32.86 (c 0.14, MeOH); UV (MeOH); λ_max (log ε): 242 (3.99), and 196 (3.86); IR (KBr) v_max 3440, 2978, 2965, 1683, 1628, 1403, 1364, 1200, and 1151 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 535.0000 [M + Na]⁺ (calcd for C₃₀H₄₀O₇Na, 535.0000).

Methyl ganoapplate G (14): white powder (MeOH); [α]²⁸_D +0.80 (c 0.07, MeOH); UV (MeOH); λ_max (log ε): 243 (3.79), and 196 (3.77); IR (KBr) v_max 3445, 2963, 2931, 1683, 1638, 1453, 1384, 1209, and 1142 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 549.2823 [M + Na]⁺ (calcd for C₃₁H₄₂O₇Na, 549.2823).

Methyl applate C (15): white powder (MeOH); [α]²⁸_D +43.44 (c 0.18, MeOH); UV (MeOH); λ_max (log ε): 248 (3.99), and 196 (3.95); IR (KBr) v_max 3443, 2956, 2915, 1665, 1624, 1433, 1376, 1205, and 1132 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 547.2672 [M + Na]⁺ (calcd for C₃₁H₄₀O₇Na, 547.2666).

Methyl gibbosate A (16): white powder (MeOH); [α]²⁸_D +18.94 (c 0.15, MeOH); UV (MeOH); λ_max (log ε): 237 (3.78), and 196 (3.82); IR (KBr) v_max 3430, 2953, 2929, 1703, 1628, 1433, 1354, 1229, and 1147 cm⁻¹; ¹H NMR and ¹³C NMR data: see Table 3 and Table 4; HRMS (ESI-TOF) m/z: 563.2612 [M + Na]⁺ (calcd for C₃₁H₄₀O₈Na, 563.2615)

X-ray Crystallographic Data for Ganoapplic acid A (1): C₃₀H₄₀O₁₀·CH₄O·H₂O, M = 610.68, a = 13.9761(7) Å, b = 6.9089(3) Å, c = 15.8732(7) Å, α = 90°, β = 90.948(2)°, γ = 90°, V = 1532.50(12) ų, T = 100.(2) K, space group P1211, Z = 2, μ(Cu Kα) = 0.844 mm⁻¹, 26,208 reflections measured, 5973 independent reflections (R_int = 0.0593). The final R₁ values were 0.0504 (I > 2σ(I)). The final wR(F²) values were 0.1381 (I > 2σ(I)). The final R₁ values were 0.0543 (all data). The final wR(F²) values were 0.1436 (all data). The goodness of fit for F² was 1.042. Flack parameter = 0.07(9).

2.4. Mosher’s Method

The specific esterification of compound 4 was performed based on the previous method [32]. The ¹H NMR spectroscopic data of the (R)-MTPA ester derivative (4r) of 4 (600 MHz, pyridine-d₅; data were obtained from the reaction NMR tube directly and assigned on the basis of correlations of the ¹H-¹H COSY spectrum): δ 4.253 (1H, m, H-15), δ 2.158 (1H, m, H-16a), δ 2.756 (1H, m, H-16b), δ 3.566 (1H, m, H-17), δ 5.619 (1H, s, H-21a), δ 5.632 (1H, s, H-21b), δ 5.007 (1H, m, H-22), δ 5.067 (1H, m, H-23), δ 2.759 (1H, m, H-24a), δ 1.905 (1H, m, H-24b), δ 3.080 (1H, m, H-25), δ 1.198 (3H, s, Me-26). Meanwhile, the ¹H NMR spectroscopic data of the (S)-MTPA ester derivative (4s) of 4 were: δ 4.255 (1H, m, H-15), δ 2.156 (1H, m, H-16a), δ 2.756 (1H, m, H-16b), δ 3.568 (1H, m, H-17), δ 5.639 (1H, s, H-21a), δ 5.633 (1H, s, H-21b), δ 5.003 (1H, m, H-22), δ 5.062 (1H, m, H-23), δ 2.756 (1H, m, H-24a), δ 1.903 (1H, m, H-24b), δ 3.078 (1H, m, H-25), δ 1.196 (3H, s, Me-26) (see Figures S29–S31 in Section S10).

2.5. Inhibition of Lipogenesis Assay

2.5.1. Cell Culture and Adipocyte Differentiation

3T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). The culture and differentiation of 3T3-L1 cells were performed based on the description reported previously [28]. Firstly, Dulbecco’s modified Eagle’s medium (DMEM) containing 10% bovine calf serum (CS) was used to cultivated 3T3-L1 cells. The whole system was incubated at 37 ℃ in a humidified atmosphere of 5% CO₂ and 95% air. Secondly, different medium systems were used for the different differentiated phases. Confluent cells were grown in DMEM medium containing 1 μg/mL insulin, 1 μM dexamethasone (DEX), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 1 μM rosiglitazone (Rosi). Then, on the third day, post-differentiation medium—namely, DMEM with 10% fetal bovine serum (FBS) and 1 μg/mL insulin—was used to continually cultivate the cells. From the fourth day, DMEM + 10% FBS was used as a maintenance medium for cell differentiation. In this process, it commonly takes two days for the mature adipocytes to form. During the whole differentiation process, the tested compounds or 0.1% DMSO were added to the differentiated 3T3-L1 cells, for which the 0.1% DMSO group was used as the vehicle.

2.5.2. Cell Viability Assay

The viability of cell treated with compounds 15 and 20 was determined by the MTS method. The detailed experimental procedures were similar to those described in our previous study [29].

2.5.3. Lipid Content Analysis

The intracellular lipid contents of 3T3-L1 adipocytes were determined by Oil Red O staining [33]. Briefly, differentiated 3T3-L1 cells were washed twice with PBS and fixed with 10% formaldehyde for 1 h. After another washing with PBS, the fixed cells were stained with 0.5% Oil Red O in 3:2 of Oil Red O/H₂O for 15 min at room temperature and then washed with 60% isopropanol and distilled water. The lipid content was imaged with an inverted light microscope Nikon TS100 (Tokyo, Japan). Finally, 100% isopropanol was used to elute Oil Red O dye and it was quantified at 492 nm absorbance.

3. Results and Discussion

The molecular formula of ganoapplic acid A (1) was established to be C₃₀H₄₀O₁₀ by HRESIMS ion at m/z 561.2696 [M + H]⁺ (calcd. 561.2694), suggesting 11 degrees of unsaturation. Its ¹³C NMR spectra displayed 30 carbon resonances, of which seven methyls, three ketone carbonyls (δ_C 215.8, δ_C 205.9, and δ_C 209.9), an α,β-unsaturated carbonyl (δ_C 165.0, δ_C 127.3, and δ_C 204.0), one carboxyl (δ_C 179.7), two oxygenated methines (δ_C 80.0 and δ_C 61.2), and three quaternary carbons containing oxygen (δ_C 85.5, δ_C 71.4, and δ_C 73.4) were assigned based on the HSQC and ¹³C-DEPT NMR spectra (see Figures S2 and S4 in Section S1). These data indicated that the structure of compound 1 was similar to that of gibbosic acid G with a 7,12,23-trioxo-8,20-dihydroxy-lanosta-9,11-en-26-oic acid skeleton [34], except for the presence of one oxygenated methine and one oxygenated quaternary carbon and the absence of a double bond at C-16 and C-17 in 1. Furthermore, the HMBC spectrum (Figure 2A) of 1 revealed the correlations of H₃-21 with C-20 (δ_C 73.4), C-22, and the oxygenated quaternary carbon (δ_C 71.4); of H₃-30 with C-15 (δ_C 80.0); and of H-15 (δ_H 4.83, s) with the oxygenated methine (δ_C 61.2) and quaternary carbons (δ_C 71.4). Meanwhile, the proton of the methine containing oxygen (δ_H 3.36, s) showed the HMBC correlations with C-13, C-14, C-15, and C-20, as well as the ¹H-¹H COSY correlation with H-15 (Figure 2A), which certified that C-16 and C-17 was substituted by hydroxyls. According to the molecular formula of 1, an epoxy was present in 1 at C-16 and C-17.

In the ROESY spectrum (Figure 2B) of 1, H-15 showed an apparent cross peak with H₃-30, while H-16 correlated with H₃-18, suggesting that H-15 and H-16 were α- and β-oriented, respectively. The X-ray crystallographic analysis (Figure 3A) of 1 (Cu κα) further confirmed that the absolute configurations of C-15, C-16, C-20, and C-25 were R, S, S, and S (see Table S1 in Section S1). Finally, the structure of 1 was determined to be (20S,25S)-7,12,23-trioxo-8β,20-dihydroxy-16α,17α-epoxy-lanosta-9,11-en-26-oic acid.

Ganoapplic acid B (2) was isolated as a colorless solid. Its molecular formula was determined to be C₃₀H₄₀O₈ by the HRESIMS. The 1D NMR and HSQC data of 2 supported the presence of five methyls, an α,β-unsaturated ketone, two carboxyl or ester carbonyls, one oxygenated methylene (δ_H 4.37, s; δ_C 61.8), three oxygenated methines (δ_H 3.95, d, J = 6.5 Hz, δ_C 77.4; δ_H 4.93, s, δ_C 64.7; δ_H 5.59, dd, J = 8.6 and 6.6 Hz, δ_C 76.8), one quaternary carbon containing oxygen (δ_C 67.4), a terminal double bond (δ_C 146.5; δ_C 115.9), and a double bond (δ_C 144.1; δ_H 5.78, d, J = 8.6 Hz, δ_C 130.8), which indicated that compound 2 was an A-seco-lanostane triterpenoid and had the similar structure to that of gibbosicolid F. [35] However, five methyls and an oxygenated methylene were observed in 2, rather than the six methyls found in gibbosicolid F. The detailed analysis of the 2D NMR spectra (see Figures S10–S13 in Section S2) exhibited the long-range HMBC correlations (Figure 2A) of the oxygenated methylene with C-17, C-20, and C-22; of H₃-18 with C-12, C-13, C-14, and C-17; and of H-22 with C-17, C-20, C-23, and C-24, together with the ¹H-¹H COSY correlations of H-22/H-23/H₂-24/H-25/H₃-26. These pieces of evidence confirmed that the hydroxyl was connected to C-21.

The ROESY cross peaks (Figure 2B) of H-7/H₃-18 and H-15/H₃-30 demonstrated that both H-15 and the epoxy between C-7 and C-8 were α. H₂-21 showed a ROESY correlation with H-23, suggesting that the double bond at C-20 and C-22 was Z. In addition, H₃-26 displayed the ROESY correlation with H-23, hinting that H₃-26 and H-23 were on the same face. Moreover, the dd-peak type of H-23 (J = 8.6 and 6.6 Hz) was consistent with that of gibbosicolid B (δ_H 5.31, dd, J = 13.2 and 8.0 Hz), which further indicated that the absolute configurations of C-23 and C-25 were R and S, respectively. [35] Therefore, the structure of 2 was assigned as (23R,25S)-15β,21-dihydroxyl-7α,8α-epoxy-12-oxo-3,4-seco-lanosta-4(28),9-(11),20E(22)-trien-23,26-olide-3-oic acid and named ganoapplic acid B (2).

The molecular formula of ganoapplic acid C (3) was established to be C₃₀H₄₀O₈ by the HRESIMS. The 1D NMR spectra (see Figures S15 and S16 in Section S6) of 3 showed a high similarity with those of ganoapplic acid B (2), suggesting that compound 3 was an A-seco lanostane triterpenoid. However, the comparison of the 1D NMR spectroscopic data of 2 and 3 revealed that another terminal double bond (δ_C 117.6 and δ_C 148.7) and oxygenated methine (δ_C 76.5) were present in 3, while a double bond (C-20-C-22) and an oxygenated methylene (C-21) were observed in 2. Furthermore, the obvious ¹H-¹H COSY correlations of H₃-26/H-25/H₂-24/H-23/H-22, as well as the HMBC correlations of H₃-26 with C-24, C-25, and C-27 and of H₂-24 with C-22, C-23, and C-26, indicated that the hydroxyl was located at C-22 (Figure 2A). Meanwhile, H-22 (δ_H 4.47, d, J = 5.6 Hz) and H-17 displayed HMBC correlations of the terminal double bond, proving that the terminal double bond was at C-21 and C-20.

The 1D NMR spectra and molecular weight (see Figures S22 and S23 in Section S8, and Figure S28 in Section S9) of methyl ganoapplate C (4) showed that compound 4 was the ester derivative of 3, which was confirmed by the HMBC correlation of OMe with C-27 (δ_C 175.6). The analysis of the ROESY spectra of 3 and 4 exhibited cross peaks of H-7/H₃-18, H-23/H₃-26, and H-22/H-25, indicating that the epoxy between C-7 and C-8 was α; meanwhile, H-23 and H₃-26 were cofacial (Figure 2B). Biogenetically, the absolute configuration of C-25 from G. applanatum was S. Thus, C-23 was determined to be R. The absolute configuration of C-22 was established as R based on the revised Mosher’s method (Figure 3B) [33]. Thus, the structures of 3 and 4 were elucidated as (22R,23R,25S)-15β,22-dihydroxyl-7α,8α-epoxy-12-oxo-3,4-seco-lanosta-4(28),9(11),20(21)-trien-23,26-olide-3-oic acid and methyl (22R,23R,25S)-15β,22-dihydroxyl-7α,8α-epoxy-12-oxo-3,4-seco-lanosta-4(28),9(11),20(21)-trien-23,26-olide-3-oate, respectively.

Ganoapplic acid D (5) was isolated as a white powder and its molecular formula was determined to be C₃₀H₄₀O₇ based on the HRESIMS at m/z 511.2701 [M − H]⁻ (calcd. 511.2707). Its ¹³C-DEPT spectra (see Figures S32 and S33 in Section S11) showed thirty carbon resonances belonging to seven methyls, four methylenes, eight methines (including three sp² and two oxygenated), and eleven quaternary carbons (including three ketone carbonyls, one carboxyl, and three sp²). These data indicated that compound 5 was a lanostane-type triterpenoid and had similar structure to that of gibbosic acid M (22) [35], except for the replacement of the methylene (C-16) in 22 with an oxygenated methine in 5. The HMBC spectrum of 5 revealed the correlations of H₃-30 with C-15 (δ_C 84.9), C-13, and C-14; of H₃-18 with C-12, C-13, C-14, and C-17; and of H-17 with C-16, C-15, C-20, C-21, and C-22, suggesting that C-16 was an oxygenated methine in 5, together with the ¹H-¹H COSY correlations of H-15/H-16/H-17 (Figure 2A). The ROESY correlations of H-15/H₃-30 and of H-16/H₃-18 illustrated that H-15 and H-16 were α- and β-oriented, respectively. The E-configuration of ∆^20,22 was determined by the ROESY correlation of H-22/H-17/H-16. Therefore, the structure of 5 was assigned as 15β,16α-dihydroxy-3,7,23-trioxolanosta-8,11,20E(22)-trien-26-oic acid and named ganoapplic acid D (5).

Methyl gibbosate M (6) had the molecular formula of C₃₁H₄₂O₆ based on the positive HRESIMS at m/z 533.2874 [M + Na]⁺ (calcd. 533.2873). The 1D NMR spectra (see Figures S39 and S40 in Section S13) of 6 were the same as those of gibbosic acid M (22) [35], except that the carboxyl at C-27 in 22 was replaced by the ester carbonyl in 6. The key HMBC correlation of OMe with C-27 confirmed the above deduction. The characteristic d-coupling type of H-15 and the ROESY correlation of H-15/H₃-30 indicated that the 15-OH was β-oriented [35]. Finally, the structure of 6 was established to be methyl 15β-hydroxy-3,7,23-trioxolanosta-8,11,20E(22)-trien-26-oate and named methyl gibbosate M (6).

Methyl ganoapplate E (7) was isolated as a white powder and its molecular formula was determined to be C₃₁H₄₄O₆ by the HRESIMS. The analysis of the 1D NMR spectra (see Figures S46 and S47 in Section S15) of 7 showed that compound 7 had a similar structure to that of 6, with the only difference being in the replacement of the ketone carbonyl at C-3 in 6 with the oxygenated methine in 7, which was confirmed by the HMBC correlations of H₃-28, H₃-29, and H-5 with the oxygenated methine (δ_C 78.3). The ROESY correlations of H-3/H-5 and of H-15/H₃-30 indicated that 3-OH and 15-OH were β.

Ganoapplic acid E (8) was deduced to be the demethylated derivative of 7 on the basis of the HMBC correlation regarding the lack of the OMe at C-27 and the low-field shift of C-27. Thus, the structures of compounds 7 and 8 were elucidated as methyl (25S)-3β,15β-dihydroxy-7,23-dioxolanosta-8,11,20E(22)-trien-26-oate and (25S)-3β,15β-dihydroxy-7,23-dioxolanosta-8,11,20E(22)-trien-26-oic acid, respectively.

Methyl gibbosate O (9) was found to be similar to the known compound gibbosic acid O (24) [35] based on the 1D NMR spectroscopic data, except for distinct differences in the chemical shift of C-27 and the presence of an additional methoxyl. The HMBC spectrum of 9 showed the correlation of OMe with C-27, suggesting that 9 was a methyl ester derivative of gibbosic acid O (24). Therefore, the structure of 9 was determined to be methyl 15α-hydroxy-3,12,23-trioxolanosta-7,9(11),20E(22)-trien-26-oate.

The molecular formula of ganoapplic acid F (10) was deduced to be C₃₀H₄₀O₇ based on the HRESIMS and NMR data. Its 1D NMR spectra (see Figures S67 and S68 in Section S21) showed a similar tetracyclic skeleton to that of gibbosic acid O (24) [35] with a 15-hydroxy-3,12-dioxolanosta-7(8),9(11)-diene skeleton, which was confirmed by the 2D NMR spectra. In addition, an oxygenated quaternary carbon signal (δ_C 72.6) and two sp² carbon signals (δ_C 127.5 and δ_C 158.9) were characteristic for the quaternary carbon containing oxygen at C-20 and the double bond at C-16 and C-17. Furthermore, the HMBC correlations (Figure 2A) of H₃-30 with C-15; of H-15 with C-16 and C-17; of H₃-18 with C-17; of H₃-21 with C-17, C-20, and C-22; and of H-22 with C-20, C-23, and C-24 confirmed the above deduction.

Methyl ganoapplate F (11) was an ester derivative at C-27 of ganoapplic acid H (10), according to the HMBC correlation of OMe with C-27. The ROESY spectra of 10 and 11 showed cross peaks of H-15/H₃-30, indicating the β-orientation of 15-OH. Therefore, the structures of 10 and 11 were determined to be 15β,20-dihydroxy-3,12, 23-trioxo-5α-lanosta-7,9(11),16-trien-26-oic acid and methyl 15β,20-dihydroxy-3,12,23-trioxo-5α-lanosta-7,9(11),16-trien-26-oate, respectively.

Based on the NMR data analysis, methyl gibbosate I (12) was found to be close to that of 26 [35], with a 12,15-dihydroxy-3,7,11,23-tetraoxolanosta-8,20(22)-dien structure. The 2D NMR spectra further confirmed its structure and 12 had an additional methoxyl at C-27, which was proven by the key HMBC correlation of OMe with C-27. Moreover, the ROESY correlations of H-12/H₃-18 and H-15/H₃-30 demonstrated that 12-OH was α while 15-OH was β. Thus, the structure of 12 was deduced to be methyl 12α,15β-dihydroxy-3,7,11,23-tetraoxolanosta-8,20(22)-dien-26-ate.

Ganoapplic acid G (13) was isolated as a white powder and its molecular formula was determined to be C₃₀H₄₀O₇ based on the HRMS (ESI-TOF) m/z 535.0000 [M + Na]⁺ (calcd. 535.0000). The 1D NMR spectra of 13 showed the presence of the ketone carbonyl at C-3, 7,8-epoxyl, α,β-unsaturated ketones (C-9/C-11/C-12 and C-20/C-22/C-23), 15-OH, and 27-oic acid, which was further confirmed by the 2D NMR spectra (Figure 2A). The aforementioned information indicated that compound 13 had the same planar structure as gibbosic acid N. [35] The comparison of the ROESY spectra of 13 and gibbosic acid N revealed that they were 15-isomers due to the existence of the ROESY correlation of H-15/H₃-30 and the d-coupling of H-15 [35]. Therefore, the structure of 13 was established to be 15β-hydroxy-7β,8β-epoxy-3,12,23-trioxolanosta-9(11),20E(22)-dien-26-oic acid. In addition, methyl ganoapplate G (14) was deduced to be the methylation product of 13 on the basis of the HMBC correlation of OMe with C-27.

Methyl applanate C (15) was found to have a similar structure to methyl ganoapplate F (14), except for the presence of a double bond in 15, rather than one methylene and one methine in 14. Furthermore, in the HMBC spectrum of 15, the correlations (Figure 2A) of H₃-30 with C-15, of H-15 with the sp² methine and quaternary carbon, and of H₃-18 and H₃-21 with the sp² quaternary carbon were observed, which proved that the double bond was located at C-16 and C-17. The ROESY correlation of H-16/H₃-21 and H₃-18/H-22 suggested that the geometry of the 16,20(22)-conjugated diene was 17,20-Z-(16Z, 20E). Additionally, the ROESY correlation of H₃-30/H-15 demonstrated that 15-OH was β. Finally, the structure of 15 was determined to be methyl 15β-hydroxy-7β,8β-epoxy-3,12,23-trioxolanosta-9(11),16Z,20E(22)-trien-26-oate and named methyl applanate C (15).

Methyl gibbosate A (16) was considered to be the methylation derivative of gibbosic acid A (29) [34] because of their similar 1D and 2D spectra (see Figures S104–S109 in Section S33) and the HMBC correlation of OMe with C-27. 7β,8β-epoxy was proven by the ROESY correlation of H-17/H₃-30. Thus, the structure of 16 was established to be methyl 20-hydroxy-7β,8β-epoxy-3,12,15,23-tetraoxo-lanosta-9,16-dien-26-oate.

In addition, 24 known compounds were identified by comparing their 1D NMR spectra with those reported in the literature, and they were assigned as gibbosic acid G (17) [34], applanoic acid B (18) [36], gibbosicolid E (19) [35], gibbosicolid F (20) [35], gibbosicolid G (21) [35], gibbosic acid M (22) [35], gibbosic acid L (23) [35], gibbosic acid O (24) [35], applanoic acid D (25) [36], gibbosic acid I (26) [35], ganodapplanoic acid D (27) [27], applanoic acid C (28) [36], ganoapplanic acid F (29) [37], elfvingic acid B (30) [37], applanoxidic acid G methyl ester (31) [37], gibbosic acid C (32) [34], gibbosic acid B (33) [34], elfvingic acid C (34) [38], methyl ganoapplaniate D (35) [37], applanone E (36) [36], ganoapplanoid K (37) [28], ganoapplanoid L (38) [28], ganoapplanilactone B (39) [37], and ganoapplanilactone A (40) [37].

Parts of the isolated compounds were evaluated to determine their anti-adipogenesis activities. At a concentration of 20 μM, compounds 16, 22, 28, and 32 showed comparable inhibition for lipid accumulation compared to the positive control (LiCl, 20 mM). Meanwhile, compounds 15 and 20 displayed stronger inhibitory effects than the positive control, even resembling the untreated group (Figure 4). Furthermore, compounds 15 and 20 did not show any toxicity for the 3T3-L1 cells when the concentration was less than 100 μM. At the concentrations of 1.25, 2.5, 5, 10, 20, and 30 or 40, compounds 15 and 20 showed significantly inhibitory activities in a dose-dependent manner, with IC₅₀ values of 6.42 and 5.39 μM, respectively (Figure 5).

The structures of the isolates were divided into nine types, including type I with a 7,12-dioxo-8-hydroxy-9,11-en fraction, type II with a A-seco-7,8-epoxy-9,11-en-12-oxo-23→27 lactone fraction, type III with a 7,23-dioxo-8(9),11(12),20(22)-trien fraction, type IV with a 12-oxo-7(8),9(11)-dien fraction, type V with a 7,11-dioxo-12-hydroxy-8(9)-en fraction, type VI with a 7,8-epoxy-12,23-dioxo-9(11),16(17),20(22)-trien fraction, type VII with a 20-hydroxy-7,8-epoxy-12,23-dioxo-9(11)-en fraction, type VIII with a 12-oxo-7,8-epoxy-9(11)-en-21,22,23,24,25,26,27-norlanostane, and type IX with a 12,23-epoxy-23→27 lactone fraction. The combined results of the previous and present studies showed that bioactive compounds were mainly present in type II (20 and 21), type III (22), type VI (15 and 28), type VII (16 and 32), and type VIII (37). For type II, when the relative configuration of 15-OH was α, the activity was decreased, similar to compound 19, while any changes in the side chain decreased their activities, such as compounds 2–4. For type III, no matter which reactions happened in type III, compounds 5–8 and 23 did not show inhibitory activity. For type VI, compound 27 was the 3-OH analogue of 15 and 28, leading to a decrease in inhibition. In type VII, the carbonyl at C-3 and the carbonyl or hydroxyl at C-15 could be the crucial active functionalities. C24 lanostane triterpenoids possessing a double bond at C-16 and C-17 displayed anti-adipogenesis activity [27,28]. Compared to the other compounds, compound 20 belonging to type II showed the strongest inhibitory activity, suggesting that A-seco-15β-hydroxy-7,8-epoxy-12-oxolanosta-9,11-en-23→27 lactone could play a significant role in the anti-adipogenesis effect (Figure 6).

4. Conclusions

Overall, inspired by our previous studies, we investigate the lanostane-type triterpenoids of G. applanatum; 40 triterpenoids, including 16 new compounds, were isolated. Their anti-adipogenesis activities were evaluated and the results showed that compounds 15 and 20 can significantly inhibit lipid accumulation, with the IC₅₀ values of 6.42 and 5.39 μM, respectively. Furthermore, we established a structure–activity relationship for the lanostane-type triterpenoids from G. applanatum, suggesting that the structure skeleton (A-seco-15β-hydroxy-7,8-epoxy-12-oxolanosta-9,11-en-23→27 lactone) could be of importance for the anti-adipogenic effect. In the next step, we can use type III as a template for further structural modification in order to find the lead compound.