Highly oxygenated lanostane-type triterpenoids and their bioactivity from the fruiting body of Ganoderma gibbosum

Fitoterapia 119 (2017) 1–7

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Highly oxygenated lanostane-type triterpenoids and their bioactivity from the fruiting body of Ganoderma gibbosum De-Bing Pu a,d,1, Xi Zheng b,1, Jun-Bo Gao a,d, Xing-Jie Zhang c, Yan Qi b, Xiao-Si Li b, Yong-Mei Wang a,d, Xiao-Nian Li a, Xiao-Li Li c,⁎, Chun-Ping Wan b,⁎, Wei-Lie Xiao a,c,⁎⁎ a

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China Central Laboratory, The No. 1 Affiliated Hospital of Yunnan University of Traditional Chinese Medicine, Kunming 650021, PR China Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, and State Key Laboratory for Conservation and Utilization of BioResources in Yunnan, Yunnan University, Kunming 650091, PR China d University of Chinese Academy of Sciences, Beijing 100049, PR China b c

a r t i c l e

i n f o

Article history: Received 24 January 2017 Received in revised form 9 March 2017 Accepted 10 March 2017 Available online 11 March 2017 Keywords: Ganoderma gibbosum Polyporaceae Lanostane triterpenes Gibbosic acid Immunoregulatory effect

a b s t r a c t Eight new highly oxygenated lanostane triterpenes, gibbosic acids A–H (1–8), along with three known ones (9– 11), were isolated from the fruiting body of Ganoderma gibbosum. The structures of new isolates were assigned by NMR and HRESIMS experiments. The absolute configurations of 1 were further confirmed by single crystal X-ray diffraction data and computational ECD methods. Immunoregulatory effect and anti-inflammatory activities of these compounds were screened in murine lymphocyte proliferation assay and in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages, respectively. Compound 2 exhibited immunostimulatory effect both in lymphocyte proliferation assay without any induction and ConA-induced mitogenic activity of T-lymphocyte, and the proportion of lymphocyte proliferation at the concentration of 0.1 μM are 20.01% and 21.40%, respectively. © 2017 Elsevier B.V. All rights reserved.

1. Introduction The species of genus Ganoderma, a group of wood-decay mushrooms with hard fruiting bodies, belongs to the family of Polyporaceae (Ganodermataceae). More than 300 species of this genus have been recorded and most of them are distributed in the tropical regions and grow on the rotten wood [1,2]. Some species of genus Ganoderma were used as folk medicine. For example, Ganoderma lucidum, known as “Lingzhi” in Traditional Chinese Medicine (TCM), has been widely used as a panacea for chronic diseases, such as insomnia, bronchitis, asthma hypertension, hepatopathy, diabetes, and cancer [3–7]. It was also a well-known traditional drug which has long been used for the promotion of longevity and maintenance of vitality. G. applanatum has been widely used as a folk medicine for the treatment and prevention of various diseases [8–10]. Up to now, the phytochemical studies led to the isolation of more than 400 secondary metabolites from the genus Ganoderma, which include lanostane-type triterpenoids, ⁎ Corresponding authors. ⁎⁎ Correspondence to: W.-L. Xiao, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China. E-mail addresses: [email protected] (X.-L. Li), [email protected] (C.-P. Wan), [email protected] (W.-L. Xiao). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2017.03.007 0367-326X/© 2017 Elsevier B.V. All rights reserved.

meroterpenoids, steroids and other types of chemical constituents [11]. Recently, a number of meroterpenoids reported from this genus have aroused attention of chemists [12–16]. Ganoderma gibbosum mainly grows at the bases of dead and rotten deciduous tree stumps in damp places [17]. This mature mushroom is similar to G. lucidum and infantile mushroom similar to G. applanatum from their shape and color. Therefore, local residents inaccurately use the mushrooms as G. applanatum and G. lucidum to cure diseases [18]. However, there are rarely reports about the investigations on its chemistry and bioactivity. In the present study, eight new oxygenated lanostane triterpenes (1–8), including three known ones (9–11), were isolated from G. gibbosum. Herein, the structure elucidation and bioactivity of the isolates are reported. 2. Experimental 2.1. General experimental procedures X-ray data were collected using a Bruker APEX DUO instrument. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained on a Tenor 27 spectrophotometer with KBr pellets. One-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded on Bruker DRX-600 spectrometers with

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D.-B. Pu et al. / Fitoterapia 119 (2017) 1–7

TMS as the internal standard. Chemical shifts (δ) were expressed in parts per million with reference to the solvent signals. HRESIMS was performed on an Agilent G6230 TOF MS. Semi-preparative HPLC was performed on an Agilent 1260 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm) column. Column chromatography (CC) was performed on silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20 (Pharmacia). Fractions were monitored by TLC, and spots were visualized by UV light (254 nm) and sprayed with 8% H2SO4 in ethanol, followed by heating.

purified by semi-preparative HPLC (64% MeOH–H2O) to give 4 (15.1 mg) and 10 (12.5 mg). Compound 9 (1.2 g) was crystallized from fraction B3. The remaining part of fraction B3 was purified by silica gel CC (200–300 mesh), eluted with CHCl3–MeOH (gradient system: 50:1–10:1), and then isolated by semi-preparative HPLC with solvent system (60% MeOH–H2O) to yield 1 (80.4 mg), 2 (15.5 mg), and 3 (24.8 mg). B4 was purified by silica gel CC to yield fractions B4a–B4e. Fraction B4c was then isolated by semi-preparative HPLC (58% MeOH– H2O) to yield 6 (5.5 mg), 7 (10.3 mg), and 8 (9.8 mg). Compounds 5 (8.5 mg) and 11 (15.1 mg) were isolated from B4d by semi-preparative HPLC (55% MeOH–H2O).

2.2. Plant material

2.3.1. Gibbosic acid A (1) White crystal; [α]25D + 2.0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 240 (4.2) nm; IR νmax (KBr) 3442, 2978, 1713, 1378, 1121 cm− 1; 1H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 549 [M + Na]+; HRESIMS m/z 549.2459 [M + Na]+ (calcd. for C30H38NaO8, 549.2464).

The fruiting bodies of Ganoderma gibbosum were collected from Ciba county, Kunming City, P. R. China, in July 2015. A voucher specimen (Xiaowl 20150714) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. The specimen was identified by Dr. Zai-Wei Ge, who is working in the Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The air-dried and powdered fruiting bodies of Ganoderma gibbosum (0.7 kg) were extracted with 70% aqueous acetone (3 L) four times (two days each time) at room temperature and then filtered. The filtrate was evaporated under reduced pressure at 40 °C and then partitioned between ethyl acetate and H2O. The ethyl acetate soluble portion (63 g) was subjected to RP-18 gel CC (5 cm × 45 cm), eluted with a CH3OH– H2O gradient system (30%, 60%, 90%, 100%), that afforded fractions A–E. Fraction B (5 g) was subjected to Sephadex LH-20 CC (MeOH) to detach red pigment, then purified by RP-18 gel CC (2.5 cm × 30 cm, MeOH–H2O gradient, 30%–100%) to give fractions B1–B8. B2 was

2.3.2. Gibbosic acid B (2) White amorphous powder; [α]21D + 29.6 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 241 (4.2) nm; IR νmax (KBr) 3424, 1719, 1377 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 551 [M + Na]+; HRESIMS m/z 551.2620 [M + Na]+ (calcd. for C30H40NaO8, 551.2621). 2.3.3. Gibbosic acid C (3) White microcrystal; [α]20D + 22.5 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 208 (3.8), 250 (3.7) nm; IR νmax (KBr) 3426, 2977, 1708, 1380 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 528 [M − H]−; HRESIMS m/z 527.2652 [M − H]− (calcd. for C30H39O8, 527.2645).

Table 1 1 H NMR data of compounds 1–8 measured at 600 MHz (δ in ppm, J in Hz). Position

1a

1a 1b 2a 2b 3 5 6a 6b 7 11 12 15 16a 16b 17 18 19 21 22a 22b 24a 24b 25 27 28 29 30 8-OH 3-OCHO

2.21, overlap 1.81, overlap 2.84, td (14.8, 5.2) 2.37, br d (14.8)

a b

2a

1.84, br d (13.0) 1.46, overlap 1.70, overlap 1.66, overlap 3.15, dd (11.5, 4.1) 1.58, dd (12.3, 5.7) 1.11, overlap 2.15, overlap 2.21, overlap 2.15, overlap 1.97, t (14.3) 4.44, d (5.3) 4.33, d (6.2) 6.04, s 5.96, s

3a

4a

5a

6a

7b

2.17, overlap 1.81, t (12.8) 2.85, overlap 2.37, d (15.0)

2.21, overlap 1.81, m 2.83, td (15.0, 5.3) 2.37, br d (15.0)

6.36, s

2.04, br d (12.9) 1.78, td (12.9, 3.9) 2.90, td (15.0, 5.5) 2.47, overlap

1.58, dd (12.3, 4.9) 2.15, overlap 2.12, overlap 3.73, d (5.4) 5.98, s

1.55, dd (12.7, 5.5) 2.19, overlap 2.19, overlap 3.78, d (5.8) 5.99, s

1.93, m 2.30, overlap 2.30, overlap 3.85, d (4.2) 6.19, s

1.93, d (13.4) 1.64, t (13.4) 1.86, overlap 1.79, overlap 4.61, dd (11.5, 4.0) 1.31, dd (13.5, 4.4) 2.25, m 2.15, dd (26.2, 12.5) 3.82, d (6.2) 6.04, s

6.09, s

6.13, s

4.23, d (2.7) 5.66, d (2.7)

4.24, d (2.5) 5.64, d (2.5)

5.41, d (2.8) 5.91, d (2.9)

5.44, d (2.9) 5.93, d (3.0)

1.86, s 1.47, s 1.43, s 3.03, d (14.1) 2.80, d (14.1) 3.10, dd (18.4, 8.3) 2.67, dd (18.4, 5.0) 2.93, m 1.21, d (7.1) 1.28, s 1.15, s 1.10, s

1.81, s 1.22, s 1.42, s 3.02, d (14.2) 2.81, d (14.2) 3.08, dd (18.5, 8.3) 2.67, dd (18.5, 4.9) 2.91, m 1.20, d (7.2) 0.95, s 0.99, s 1.01, s

2.26, s 1.69, overlap 1.69, overlap 3.22, d (13.4) 3.14, d (13.4) 3.55, dd (18.2, 7.6) 2.97, dd (18.2, 5.8) 3.30, m 1.38, d (7.2) 1.17, s 1.12, s 1.02, s 7.94, s

2.26, s 1.55, s 1.71, s 3.24, d (13.5) 3.16, d (13.5) 3.56, dd (18.2, 7.6) 2.98, dd (18.2, 5.7) 3.32, overlap 1.38, d (7.2) 1.17, s 1.11, s 1.05, s 7.84, s

3.97, d (6.2) 2.28, m 1.94, dd (14.8, 8.7) 2.45, br t (11.4) 1.76, s 1.68, s 1.48, s 1.71, s 1.45, s 1.13, overlap 1.43, overlap 1.43, s 1.49, s 1.43, s 1.43, overlap 1.35, s 3.00, overlap 2.93, overlap 2.99, overlap 2.89, d (14.9) 2.93, overlap 2.85, overlap 2.81, overlap 2.52, overlap 3.03, overlap 2.97, overlap 2.96, overlap 3.15, dd (18.4, 7.7) 2.61, dd (18.2, 5.1) 2.56, dd (18.6, 4.4) 2.68, dd (18.5, 3.9) 2.56, dd (18.4, 5.5) 2.92, overlap 2.85, overlap 2.83, overlap 2.97, m 1.20, d (7.2) 1.13, overlap 1.18, d (7.1) 1.23, d (7.2) 1,12, overlap 0.97, s 1.10, s 1.11, s 1.12, overlap 0.81, s 1.12, s 1.12, s 1.29, s 1.23, s 1.08, s 0.87, s 5.67, s

In CDCl3. In pyridine-d5.

5.59, s

4.92, s 5.34, s

8.12, s

8b

1.82, br d (12.4) 1.53, overlap 2.02, dd (9.5, 6.7) 2.02, dd (9.5, 6.7) 3.49, t (9.0) 1.88, dd (14.7, 2.6) 1.46, dd (14.8, 2.5) 3.38, t (15.1) 3.30, overlap 2.48, overlap 2.61, dd (15.4, 2.4)

D.-B. Pu et al. / Fitoterapia 119 (2017) 1–7

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Table 2 13 C NMR data of compounds 1–8 measured at 150 MHz (δ in ppm). Position

1a

2a

3a

4a

5a

6a

7b

8b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3-OCHO

37.2, CH2 33.8, CH2 213.8, C 47.8, C 49.9, CH 21.6, CH2 57.1, CH 59, C 165.1, C 38.2, C 125, CH 200.6, C 61.9, C 54.5, C 202.9, C 124.4, CH 182.1, C 31.0, CH3 20.8, CH3 72.6, C 29.1, CH3 52.7, CH2 206.3, C 47.5, CH2 34.4, CH 180.3, C 16.8, CH3 24.6, CH3 22.1, CH3 26.0, CH3

36.5, CH2 26.9, CH2 77.9, CH 39.3, C 48.4, CH 20.9, CH2 57.5, CH 58.9, C 167.1, C 38.5, C 124.6, CH 201, C 61.9, C 54.5, C 203.2, C 124.4, CH 182.2, C 31.1, CH3 21.7, CH3 72.6, C 29.1, CH3 52.7, CH2 206.4, C 47.6, CH2 34.4, CH 180.1, C 16.8, CH3 27.8, CH3 15.2, CH3 26.0, CH3

37.2, CH2 33.9, CH2 214.0, C 47.8, C 49.9, CH 21.9, CH2 58.2, CH 60.3, C 164.2, C 38, C 125.5, CH 203.4, C 62.4, C 53.7, C 73.4, CH 127.4, CH 152.1, C 24.6, CH3 20.9, CH3 71.7, C 29.7, CH3 53.3, CH2 208.4,C 47.6,CH2 34.4,CH 180.6, C 17.2, CH3 24.6, CH3 22.1, CH3 16.8, CH3

37.4, CH2 33.9, CH2 213.6, C 47.7, C 49.6, CH 21.7, CH2 57.8, CH 64.6, C 160.2, C 37.8, C 126.3, CH 204.7, C 57.2, C 50.2, C 76.9, CH 35.4, CH2 48.9, CH 19.0, CH3 20.6, CH3 73.2, C 28.2, CH3 52.2, CH2 210.5, C 47.7, CH2 34.3, CH 179.5, C 16.8, CH3 24.5, CH3 22.0, CH3 22.6, CH3

120.8, CH 142.9, C 198.4, C 43.8, C 46.3, CH 21.1, CH2 57.4, CH 63.3, C 158.5, C 39.8, C 125.8, CH 202.8, C 63.2, C 47.1, C 79.3, CH 125.5, CH 158.8, C 27.5, CH3 23.2, CH3 71.7, C 29.4, CH3 53.9, CH2 208.0, C 47.9, CH2 34.3, CH 178.6, C 16.9, CH3 24.6, CH3 21.9, CH3 25.0, CH3

36.1, CH2 23.5, CH2 79.2, CH 38.1, C 48.2, CH 20.8, CH2 57.8, CH 63, C 163.1, C 38.2, C 126, CH 203.5, C 63.4, C 46.8, C 79.4, CH 125.2, CH 158.9, C 27.6, CH3 21.6, CH3 71.7, C 29.7, CH3 53.8, CH2 208.1, C 47.9, CH2 34.5, CH 179.7, C 16.9, CH3 27.5, CH3 16.3, CH3 25.1, CH3 160.7

36.5, CH2 34.2, CH2 212.7, C 47.6, C 47.7, CH 35.6, CH2 205.5, C 81.3, C 164.0, C 40.0, C 124.7, CH 205.1, C 66.5, C 48.6, C 81.4, CH 126.1, CH 159.5, C 29.5, CH3 19.5, CH3 72.3, C 30.5, CH3 54.8, CH2 208.0, C 48.8, CH2 35.5, CH 178.4, C 17.6, CH3 24.6, CH3 21.6, CH3 25.7, CH3

36.5, CH2 27.7, CH2 76.8, CH 40.0, C 47.0, CH 35.2, CH2 206.4, C 81.2, C 166.0, C 40.4, C 124.0, CH 205.4, C 66.5, C 48.6, C 81.4, CH 126.0, CH 159.5, C 29.6, CH3 20.6, CH3 72.3, C 30.4, CH3 54.7, CH2 208.2, C 48.9, CH2 35.7, CH 179.3, C 17.7, CH3 27.9, CH3 15.7, CH3 25.7, CH3

a b

In CDCl3. In pyridine-d5.

2.3.4. Gibbosic acid D (4) White amorphous powder; [α]21D + 75.3 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 239 (3.9) nm; IR νmax (KBr) 3441, 1708 cm− 1; 1 H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 553 [M + Na]+; HRESIMS m/z 553.2776 [M + Na]+ (calcd. for C30H42NaO8, 553.2777). 2.3.5. Gibbosic acid E (5) Brown amorphous powder; [α]21D − 40.0 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 205 (3.9), 252 (3.9) nm; IR νmax (KBr) 3441, 1708 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 541 [M − H]−; HRESIMS m/z 565.2422 [M + Na]+ (calcd. for C30H38NaO9, 565.2414). 2.3.6. Gibbosic acid F (6) White amorphous powder; [α]20D − 26.0 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 204 (3.9), 250 (4.0) nm; IR νmax (KBr) 3429, 2975, 1717, 1459, 1377, 1178 cm− 1; 1H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 581 [M + Na]+; HRESIMS m/z 581.2734 [M + Na]+ (calcd. for C31H42NaO9, 581.2727). 2.3.7. Gibbosic acid G (7) White amorphous powder; [α]21D − 46.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 203 (4.1), 237 (4.1) nm; IR νmax (KBr) 3433, 2977, 1722, 1677, 1457, 1381, 1170, 660 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2; ESIMS m/z 567 [M + Na]+; HRESIMS m/z 567.2575 [M + Na]+ (calcd. for C30H40NaO9, 553.2570). 2.3.8. Gibbosic acid H (8) White amorphous powder; [α]20D − 28.5 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 202 (4.1), 244 (4.1) nm; IR νmax (KBr) 3424, 1715, 1660, 1402 cm−1; 1H (600 MHz) and 13C NMR (150 MHz) data,

see Tables 1 and 2; ESIMS m/z 545 [M − H]−; HRESIMS m/z 545.2757 [M − H]− (calcd. for C30H41O9, 545.2751).

2.3.9. X-ray crystallographic study of gibbosic acid A C30H38O8·H2O, M = 544.62, orthorhombic, a = 6.8662(4) Å, b = 12.0343(8) Å, c = 33.453(2) Å, α = 90°, β = 91.773(3)°, γ = 90°, V = 2762.9(3) Å3, T = 100(2) K, space group P21, Z = 4, μ(CuKα) = 0.790 mm−1, 18,799 reflections measured, 7685 independent reflections (Rint = 0.0714). The final R1 values were 0.1558 (I N 2σ(I)). The final wR(F2) values were 0.4024 (I N 2σ(I)). The final R1 values were 0.1633 (all data). The final wR(F2) values were 0.4106 (all data). The goodness of fit on F2 was 1.870. Flack parameter = 0.19(17). Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1524931).

2.4. Lymphocyte proliferation assay in vitro The proliferation of splenocytes in response to ConA was determined by CCK-8 as described previously [19]. Briefly, BALB/c splenocytes suspension was cultured with ConA (5 μg/mL) as stimulator in 96-well flat-bottomed plate (Costar). The cultures were incubated for 48 h, 20 L of CCK-8 was then added to each well and before the end of culture, O.D value was read at 570 nm. The MTT method was used to measure the activity of lymphocyte without any stimulation. Splenocytes were cultured in triplicates in the absence or presence of samples in 96-well flat-bottomed plate (Costar) for 48 h. MTT (5 mg/mL) was pulsed 4 h prior to end of the culture, upon removal of MTT/medium, 150 μL of DMSO was added to each well and the plate was agitated at oscillator for 5 min to dissolve the precipitate. The assay plate was read at 570 nm using a microplate reader.

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2.5. Anti-inflammatory activity

are the excitation energies and rotational strengths for transition i, respectively. σ = 0.35 eV and Rvelocity were used in this work.

The anti-inflammatory activity was investigated as those described previously [20]. Briefly, murine RAW 264.7 macrophages (American Type Culture Collection, Manassas) were plated in 96 well plate at a density of 1 × 105 cells/well and stimulated with 1 μg/mL LPS in the present or absence of various concentration of compound for 24 h, the culture supernatant were harvested to measure NO. The production of NO was determined by assaying culture supernatant for NO− 2 , a stable reaction product of NO, 100 μL of supernatant was mixed with an equal volume of Griess reagent at room temperature for 10 min. Absorbance was measured at 540 nm in a microplate reader. Nitrite concentration was calculated from a NaNO2 standard curve. 2.6. ECD calculation The theoretical calculations of compound 1 were carried out using Gaussian 09 [21]. Conformational analysis was initially performed using Maestro 9.0 with the OPLS_2005 force field. The conformers were optimized at B3LYP/6-31+G(d,p) level. Room-temperature equilibrium populations were calculated according to Boltzmann distribution law. The theoretical calculations of ECD were performed using TDDFT [22,23] at B3LYP/6-31 ++G(d,p) level in MeOH. The ECD spectra of compound 1 were obtained by weighing the Boltzmann distribution rate of each geometric conformation. The ECD spectra are simulated by overlapping Gaussian functions for each transition according to

ΔεðEÞ ¼

1 −39

2:297  10

A 2 1  pffiffiffiffiffiffiffiffiffiffi ∑ ΔEi Ri e−½ðE−Ei Þ=ð2σ Þ 2πσ i

where σ represents the width of the band at 1/e height, and ΔEi and Ri

3. Results and discussion The air-dried and powdered fruiting bodies of Ganoderma gibbosum was extracted three times with 70% acetone aqueous at room temperature to give a crude extract, which was suspended in H2O and successively partitioned with EtOAc. Various column chromatographic separations of the EtOAc extract afforded compounds 1–11 (Fig. 1). Compound 1 was isolated as an optically active colorless crystals with [α]25D + 2.0 (c 0.15, MeOH). Its HRESIMS data showed a sodium adduct ion [M + Na]+ at m/z 549.2459 (calcd. for C30H38NaO8, 549.2464). The 13C NMR and DEPT data (Table 2) were consistent with a molecular formula of C30H38O8, representing 12 degrees of unsaturation. The IR absorption bands at 3442 and 1713 cm−1 indicated the presence of the hydroxyl and carbonyl groups, respectively. Analysis of the 1H NMR (Table 1) revealed the presence of seven methyl groups [δH 1.12 (3H, overlap), 1.12 (3H, overlap), 1.20 (3H, d, J = 7.2 Hz), 1.29 (3H, s), 1.45 (3H, s), 1.49 (3H, s), and 1.76 (3H, s)], an oxygenated methine [δH 4.44 (1H, d, J = 5.3 Hz)], two olefinic proton [δH 5.67 (1H, s), 6.04 (1H, s)], and a series of aliphatic methylene or methine multiples. The 13C NMR and DEPT spectra obviously resolved 30 carbon resonances attributable to seven methyls, five methylenes, five methines [two aromatic carbon (δC 124.4, 125.0)], thirteen quaternary carbons [four ketone groups (δC 213.8, 206.3, 202.9, and 200.6), one carboxyl group (δC 180.3), two double-bond carbons (δC 165.1 and 182.1), and one oxygenated carbon (δC 72.6)], which were quite similar to those of the known compound elfvingic acid B (9) [24], except for the presence of a conjugated ketone signal (δC 202.9) in 1 instead of an oxygenated-methine signal for C-15 (δH 3.83; δC 79.3) in 9. Thus, an α, βunsaturated ketone was assigned to C-15/C-16/C-17, which was supported by HMBC correlations from H-16 to C-13/C-14/C-15/C-17, H-18

Fig. 1. Structures of compounds 1–11.

D.-B. Pu et al. / Fitoterapia 119 (2017) 1–7

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Fig. 2. Key HMBC and 1H\ \1H COSY correlations of 1 and 7.

Fig. 3. Key 1H\ \1H ROESY correlations of 1 and 7.

and H-21 to C-17, and H-30 to C-15 (Fig. 2). The locations of other functional groups were also confirmed through the analysis of HMBC and 1 H\\1H COSY spectra. In ROESY experiment (Fig. 3), the correction of H-7 with H-5 and H-30 indicated that H-7 was α-orientation, and the configuration of 7, 8-epoxy group was correspondingly inferred to be β-orientated. In order to confirm the structure, a single crystal X-ray crystallographic analysis (Fig. 4) with anomalous scattering of Cu Kα radiation [Flack parameter = 0.19(17)] was employed. However, as the value of Flack parameter was slightly large, which still cannot assign the absolute configuration of 1. Ultimately, the absolute configuration of 1 was assigned as 5R, 7S, 8S, 10S, 13R, 14R, 20S, 25S by the comparison of its experimental (black line) and calculated (red line) ECD spectra (Fig. 5). The two curves rose up an almost identical trend. Consequently, the structure of 1 was established as 20-hydroxy-7β, 8β-epoxy-3, 12, 15, 23-tetraoxo-lanosta-9, 16-dien-26-oic acid, and named as gibbosic acid A. Compound 2 was obtained as a white amorphous powder. Its molecular formula was established as C30H40O8 by HRESIMS data, being the same as that of elfvingic acid B (9) [24]. But there were obviously

Fig. 4. ORTEP plot of 1 drawn with 30% probability displacement ellipsoids.

differences from their 1H NMR spectrum. The 13C NMR spectra of 2 were very similar to those of 9, except for the presence of the oxygenated methine in 9 at C-15 replaced by a conjugated ketone (δC-15 203.2) in 2, and the ketone group at C-3 in 9 changed to be an oxygenated methine in 2. This was further confirmed by the HMBC correlations of two methyl protons [δH-28 0.97(3H, s), δH-29 0.81(3H, s)] with an oxygenated methines (δC-3 77.9), and of H-30 with C-15, and 1H\\1H COSY correlations of H-1/H-2/H-3. The configuration of OH-3 was determined to be β-orientated by the correlations of H-3/H-5/H-28 in the ROESY spectrum. In addition, the observed ROESY correlations of H-7 with H-6α and of H-7 with H-30 indicated that 7, 8-epoxy group was β-orientated as 1. Thus, the structure of 2 was determined as 3β, 20-dihydroxy-7β, 8β-epoxy-12, 15, 23-trioxo-lanosta-9, 16-dien-26-oic acid and named as gibbosic acid B.

Fig. 5. Experimental (black line) and calculated (red line) ECD spectra of 1. Shift = +5 nm. Experimental CD spectra of 1 were observed in MeOH.

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Compound 3 was obtained as white microcrystal, with a molecular formula of C30H40O8, as established by HRESIMS data. Comparison of the 1H and 13C NMR data of 3 with those of the co-isolated known compound 9 [24] showed that 3 was quite similar to 9. Compounds 3 and 9 possessed the same planar structure by analysis of their 1D and 2D NMR data. However, the 1H and 13C chemical shifts of C-15, and 13C chemical shift of C-17 showed obvious differences between them. The reason can be rationalized by the α-orientated OH-15 in 3, instead of β-orientated OH-15 in 9, which was further supported by the obvious ROESY correlation of H-18/H-15 in 3. Therefore, compound 3 was determined as 15α, 20-dihydroxy-7β, 8β-epoxy-3, 12, 23-trioxo-lanosta-9, 16-dien-26-oic acid and given the trivial name as gibbosic acid C. Compound 4 was isolated as white amorphous powder. It had a molecular formula of C30H42O8 by the HRESIMS experiment, indicating nine degrees of unsaturation. The structure of 4 was deduced to be a lanostane-type triterpene as compared with its NMR data with those of compounds 1–3 and 9. Compared with those of 9, the 1H and 13C NMR data of 4 showed the absence of the signals of an double bond and the appearance of two aliphatic carbons (a methylene at δC 35.4 and a methine at δC 48.9). These two aliphatic carbons were further assigned to C-16 (δC 35.4) and C-17 (δC 48.9), respectively, deduced by the HMBC correlations of H-18/H-21 with C-17 (δC 48.9) and H-16 with C-13, C-14, and C-20. Furthermore, 1H\\1H COSY correlations of H-15/H-16/H-17 also supported this deduction. The configuration of OH-15 was assigned as β-orientation determined by the ROESY correlations of H-15 with H-30 and H-16α. Thus, this compound was established as 15β, 20-dihydroxy-7β, 8β-epoxy-3, 12, 23-trioxolanosta-9-en-26-oic acid, and called as gibbosic acid D. Compound 5 had the molecular formula C30H38O9 as determined by HRESIMS. From the NMR spectra, most of the signals of 5 were similar to those of 9, expect for the appearance of the signals of a double bond [δH 6.36 (H, s), δC 120.8; δC 142.9] in 5 instead of two aliphatic methylenes in 9. In addition, a relative high-field carbonyl signal at δC 198.4 in 5 was observed instead of C-3 at δC 213.6 in 9. Above information suggested the double bound may locate between C-1 and C-2. This deduction was supported by the HMBC correlations from H-19 to C-1 (δC 120.8) and from H-1 [δH 6.36 (H, s)] to C-2, C-3, C-5, and C-19. An enol structure existed between C-1 and C-2 was deduced by the relative low-field chemical shift at δC 142.9 (C-2) and MS data of 5. The configuration of OH-15 was determined as β-orientation by the ROESY correlation of H-30/H-15. Thus, its structure was established as 2, 15β, 20-trihydroxy-7β, 8β-epoxy-3, 12, 23-trioxo-lanosta-1, 9, 16-trien-26-oic acid, and named as gibbosic acid E. The molecular formula of compound 6 was determined by HRESIMS as C31H42O9. The 1H and 13C NMR data showed an additional signal for formic ester [δH 8.12; δC 160.7] when compared to those of elfvingic acid C (10) [24]. This formic ester located at C-3 was determined by the HMBC correlation of the hydrogen of formic ester with C-3, and of H-3 with the carbon of formic ester. The 1H\\1H ROESY correlations of H-3/H-28 and H-3/H-1α in 6 suggested that configuration of the formic group was β-orientation. The β-orientation of OH-15 was deduced by the ROESY correlation of H-30/H-15. Therefore, this structure was established as 3β-formyl-15β, 20-trihydroxy-7β, 8β-epoxy-12, 23dioxo-lanosta-9, 16-dien-26-oic acid, and given the name as gibbosic acid F. A molecular formula of C30H40O9 was assigned to compound 7 on the basis of its HRESIMS data. The NMR spectra indicated that this compound was similar to elfvingic acid G (11) [24]. The differences were two olefinic carbons (δC 126.1 and 159.5) in 7 replaced two aliphatic carbons in 11. The double bond assigned between C-16 and C-17 was supported by the HMBC correlations of H-16 with C-13, C-14, C-15 and C-17. The configuration of OH-8 was assigned as β-orientation by the ROESY correlations of the proton of OH with H-18 and H-19. The β-orientation OH-15 was established through ROESY correlation of H-30/H-15. Thus, compound 7 was determined as 8β, 15β, 20-trihydroxy-3, 8, 12, 23tetraoxo-lanosta-9, 16-dien-26-oic acid, and named as gibbosic acid G.

The 1H and 13C NMR spectroscopic signals of compound 8 were close to those of 7, expect for the presence of an oxygenated methine [δH 3.49 (1H, t, J = 9.0 Hz); δC 76.8] in 8 instead of a keto-carbonyl signal (δC-3 212.7) in 7. The oxygenated methine was assigned to C-3 by the HMBC correlations from H-28 and H-29 to C-3 and the 1H\\1H COSY correlations of H-3/H-2/H-1. The configuration of OH-3 was determined as β-orientation by the ROESY correlation of H-3 with H-28. Therefore, compound 8 was established as 3β, 8β, 15β, 20-tetrahydroxy-8, 12, 23-trioxo-lanosta-9, 16-dien-26-oic acid and named as gibbosic acid H. The known compounds were identified as elfvingic acid B (9) [24], elfvingic acid C (10) [24], elfvingic acid G (11) [24], by comparison of their physical and spectroscopic data with those of published values. Abundant evidence indicated that the chemical constituents isolated from genus Ganoderma exerted significantly immunoregulatory effect, anti-inflammatory and anti-cancer activities [25–30]. In the present study, immunoregulatory effect and anti-inflammatory of these compounds were screened in murine lymphocyte proliferation assay and in lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages, respectively. Compound 2 exhibited immunostimulatory effect both in lymphocyte proliferation assay without any induction and ConA-induced mitogenic activity of T-lymphocyte, and the proportion of lymphocyte proliferation at the concentration of 0.1 μM are 20.01% and 21.40%, respectively. Compound 2 display similar immunostimulatory activities in vitro compared to reference drug lentinan (19.06% and 15.28%, respectively). However, these compounds did not obvious influenced the production of NO in LPS-induced RAW-264.7 macrophages at the concentration of 40 μM using dexamethasone as positive control (the proportion of inhibition at the concentration of 40 μM is 20.50%). Acknowledgments This project was supported financially by the NSFC for Outstanding Young Scholar (81422046), Natural Science Foundation of China (81460624), the Application Basic Research Program of Yunnan Province (2015FB199), and sponsored by SRF for ROCS, SEM to Wei-Lie Xiao (Y317821261). The calculation sections were supported by the HPC Center of KIB of CAS. Appendix A. Supplementary data Supplementary data (1D and 2D NMR spectra, MS, HRMS, UV and IR of compounds 1–8, and crystal structure and data of 1) in this article can be found online at http://dx.doi.org/10.1016/j.fitote.2017.03.007. References [1] C. Richter, K. Wittstein, P.M. Kirk, et al., An assessment of the taxonomy and chemotaxonomy of Ganoderma, Fungal Divers. 71 (2015) 1–15. [2] G.S. Seo, P.M. Kirk, Ganodermataceae: nomenclature and classification, in: J. Flood, P.D. Bridge, P. Holderness (Eds.), Ganoderma Disease of Perennial Crops, CABI Publishing, Wallingford, UK 2000, pp. 3–22. [3] S. Fatmawati, K. Shimizu, R. Kondo, Ganoderic acid Df, a new triterpenoid with aldose reductase inhibitory activity from the fruiting body of Ganoderma lucidum, Fitoterapia 81 (2010) 1033–1036. [4] Y. Mizushina, L. Hanashima, T. Yamaguchi, et al., A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases, Biochem. Biophys. Res. Commun. 249 (1998) 17–22. [5] T. Nishitoba, H. Sato, K. Oda, et al., Novel triterpenoids and a steroid from the fungus Ganoderma lucidum, Agr. Biol. Chem. Tokyo 52 (1988) 211–216. [6] S.P. Wasser, A.L. Weis, Therapeutic effects of substances occurring in higher basidiomycetes mushrooms: a modern perspective, Crit. Rev. Immunol. 19 (1999) 65–96. [7] S.P. Wasser, Reishi or Ling Zhi (Ganoderma lucidum), Encyclopedia of Dietary Supplements, Marcel Dekker, New York, USA 2005, pp. 603–622. [8] M. Adams, M. Christen, I. Plitzko, et al., Antiplasmodial lanostanes from the Ganoderma lucidum mushroom, J. Nat. Prod. 73 (2010) 897–900. [9] C.W. Lieu, S.S. Lee, S.Y. Wang, The effect of Ganoderma lucidum on induction of differentiation in leukemic U937-cells, Anticancer Res. 12 (1992) 1211–1216. [10] X.M. Wang, M. Yang, S.H. Guan, et al., Quantitative determination of six major triterpenoids in Ganoderma lucidum and related species by high performance liquid chromatography, J. Pharm. Biomed. Anal. 41 (2006) 838–844. [11] S. Baby, A.J. Johnson, B. Govindan, Secondary metabolites from Ganoderma, Phytochemistry 114 (2015) 66–101.

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