Three New Triterpenoids from European Mushroom Tricholoma terreum.

Three rare triterpenoids, saponaceolides Q-S (1-3), have been isolated from fruiting bodies of the mushroom Tricholoma terreum. Their structures were characterized based on extensive spectroscopic data. Compound 1 showed certain cytotoxicities against four human tumor cell lines.

Introduction

Our previous work has identified Tricholoma terreum as a hitherto unknown poisonous European mushroom [1]. From which fifteen new triterpenoids terreolides A–F and saponaceolides H–P have been isolated. Terreolides A–F possessed novel frameworks, while saponaceolides B and M were the main toxins in the mushroom. The structural diversity, as well as important bioactivity discovery, prompted us to make a further study on this mushroom. According to an investigation on chloroform extract of T. Terreum collected in Arcachon in southwestern France, three new triterpenoids, saponaceolides Q–S (1–3), have been obtained (Fig. 1). Their structures were established by extensive spectroscopic methods. Compounds 1–3 were evaluated for their cytotoxicities against five human cancer cell lines.

Fig. 1

Structures of compounds 1–3

Results and Discussion

Compound 1 was isolated as a colorless oil. The HRESIMS data (m/z 525.3193 [M + Na]+) indicated the molecular formula C30H46O6, requiring eight degrees of unsaturation. The IR absorption bands at 3436 and 1723 cm−1 suggested the presence of hydroxy and carbonyl groups, respectively. In the 1H NMR spectrum (Table 1), five singlets from δH 0.54 to 1.29 were readily identified signals for five methyls, while a singlet at δH 7.11 (1H, br s, H-11) revealed the existence of a double bond that might have an α,β-unsaturated keto moiety. The 13C NMR and DEPT spectra displayed thirty carbon resonances that was classified into five CH3, thirteen CH2, four CH, and eight C, as shown in Table 2. These data displayed high similarities to those of saponaceolide O, a triterpenoid isolated from the same mushroom by Yin et al. [1]. The key difference in 1 was that C-7 was identified as a methylene (δH 1.82 and 1.67, m; δC 23.1) rather than a carbonyl carbon in saponaceolide O, which was supported by 1H–1H COSY fragments of H-7 with δH 1.68 (1H, m, H-6) and 2.48 (1H, m, H-8a), as well as HMBC correlations from H-7 to δC 53.5 (d, C-6) and 24.7 (t, C-8) (Fig. 2). Analyses of 2D NMR data suggested that the other parts of 1 were the same to those of saponaceolide O (Fig. 2) [1]. Compound 1 was, therefore, elucidated as shown in Fig. 1 and named saponaceolide Q.

Table 1

1H NMR data for compounds 1–3 (δ in ppm, J in Hz)

Entry	1	2	3
2	1.09 overlapped	1.06, overlapped	1.15 overlapped
3	1.78 m; 1.09 m	1.76 m; 1.07 m	1.79 m; 1.13 m
4	2.32 m; 1.92 m	2.29 m; 1.88 m	2.34 m; 1.98 m
6	1.68 m	1.60 m	2.13 m
7	1.82 m; 1.67 m	4.13 dd (7.6, 5.2)	4.10 d (10.4)
8	2.48 m; 2.14 m	2.01 m	1.86 m; 1.63 m
10	7.11 br s	7.35 br s	7.33 br s
11	4.78 br s	4.84 d (6.0)	4.84 d (17.0)
12	0.99 s	0.94, s	0.99, s
13	0.54 s	0.53, s	0.52, s
14	4.88 br s; 4.59 br s	4.87, br s; 4.64, br s	4.94, br s; 4.88, br s
3′	2.00 m; 1.87 m	2.00 m; 1.87 m	2.00 m; 1.88 m
4′	2.16 m; 1.68 m	2.16 m; 1.67	2.17 m; 1.67
7′	1.97 m; 1.51 m	1.96 m; 1.50 m	1.97 m; 1.51 m
8′	1.67 m	1.65 m	1.67 m
9′	1.48 m	1.47 m	1.49 m
10′	1.26 m; 1.03 m	1.23 m; 1.01 m	1.26 m; 1.04 m
11′	1.55 m; 0.81 m	1.53 m; 0.79 m	1.56 m; 0.82 m
12′	1.29 s	1.29 s	1.29 s
13′	1.21 s	1.21 s	1.22 s
14′	1.09 s	1.09 s	1.10 s
15′	3.69 dd (11.0, 10.9)3.60 dd (11.0, 4.3)	3.68 dd (11.0, 10.8)3.58 dd (11.0, 4.2)	3.69 dd (11.3, 10.8)3.60 dd (11.3, 4.0)
MeO-		3.30 s	3.30 s

Data (δ) were measured in CDCl3. The assignments were based on DEPT, 1H–1H COSY, HSQC, and HMBC experiments

Table 2

13C NMR data for compounds 1–3 (δ in ppm)

No.	1	2	3
1	39.8 C	39.7 C	39.5 C
2	48.0 CH	47.9 CH	48.1 CH
3	30.3 CH2	29.1 CH2	30.5 CH2
4	37.3 CH2	37.2 CH2	37.5 CH2
5	148.0 C	148.8 C	148.1 C
6	53.5 CH	49.2 CH	49.3 CH
7	23.1 CH2	75.2 CH	75.4 CH
8	24.7 CH2	30.5 CH2	32.0 CH2
9	134.8 C	135.0 C	136.5 C
10	143.8 CH	146.8 CH	144.9 CH
11	70.1 CH2	70.2 CH2	70.6 CH2
12	26.5 CH3	26.4 CH3	26.6 CH3
13	14.9 CH3	14.9 CH3	15.3 CH3
14	106.7 CH2	106.5 CH2	107.9 CH2
15	174.4 C	172.8 C	173.2 C
1′	77.5 C	77.5 C	77.7 C
2′	96.6 C	96.6 C	96.8 C
3′	27.9 CH2	27.9 CH2	28.1CH2
4′	28.5 CH2	28.5 CH2	28.4 CH2
5′	72.8 C	72.8 C	73.0 C
6′	101.4 C	101.2 C	101.5 C
7′	29.2 CH2	29.2 CH2	29.5 CH2
8′	24.8 CH2	24.8 CH2	25.1 CH2
9′	35.7 CH	35.6 CH	35.9 CH
10′	31.7 CH2	31.6 CH2	31.9 CH2
11′	27.6 CH2	27.6 CH2	28.1 CH2
12′	25.9 CH3	25.9 CH3	26.1 CH3
13′	22.4 CH3	22.4 CH3	22.6 CH3
14′	20.9 CH3	20.9 CH3	21.1 CH3
15′	65.8 CH2	65.9 CH2	66.2 CH2
MeO-		57.2 CH3	58.4 CH3

Data (δ) were measured in CDCl3. The assignments were based on DEPT, 1H–1H COSY, HSQC, and HMBC experiments

Fig. 2

Key 2D NMR correlations of saponaceolide R (2)

Compound 2 was isolated as a colorless oil. The HRESIMS ion peak at m/z 555.3290 ([M + Na]+) (calcd. 555.3292) indicated the molecular formula C31H48O7, requiring eight degrees of unsaturation. The 1H and 13C NMR spectra displayed similar patterns to those of 1 except for the signals at δH 4.13 (1H, dd, J = 7.6, 5.2 Hz, H-7) and 3.30 (3H, s, MeO-) and δC 75.2 (d, C-7) and 57.2 (q, MeO-), suggesting the existence of a methoxy substituent. The HMBC correlation from δH 3.30 (3H, s, MeO-) to C-7 indicated that a methoxy group placed at C-7, which was also supported by 1H–1H COSY correlations of H-7 with δH 1.60 (1H, m, H-6) and 2.01 (2H, m, H-8). Detailed analyses of 2D NMR data suggested that other parts of 2 were the same to those of 1. The coupling constant of H-7 (J = 7.6, 5.2 Hz) suggested an S configuration of C-7 referring to the data of the literature [1-4]. Therefore, compound 2 was determined to be saponaceolide R.

The 1D NMR (Tables 1 and 2) and HRESIMS data of 3 were almost the same to those of 2, which informed that 3 might have the same framework to that of 2. Detailed analyses of 2D NMR data suggested that 3 did possess the same planar structure to that of 2. However, tiny changes of NMR data of CH-6, CH-7, and CH2-8 suggested that the stereoconfiguration of C-7 was different from that of 2. The coupling constant of H-7 (d, J = 10.4 Hz) was also significantly different to those reported previously (less than 8 Hz) [1-4], which allowed an R configuration of C-7 in 3, that was also in agreement with those described in the literature [3]. Therefore, compound 3 was established and named saponaceolide S.

Many triterpenoids in this type, such as saponaceolides B, E, and F, have been reported to possess cytotoxicities to several cancer cell lines [2-4]. Compounds 1–3 were, therefore, evaluated for their cytotoxicities to five human cancer cell lines. As a result, compound 1 showed moderate activities as shown in Table 3.

Table 3

Cytotoxicities of compounds 1–3 (IC50, μM)

Entry	HL-60	SMMC-7721	A-549	MCF-7	SW480
1	12.2	19.3	>40	12.2	1.4
2	>40	>40	>40	>40	>40
3	>40	>40	>40	>40	>40
Cisplatin	2.4	11.2	17.6	18.7	14.9

Experimental

General Experimental Procedures

Optical rotations were measured on a Jasco-P-1020 polarimeter. IR spectra were obtained using a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with instrument of a Bruker DRX-600 with tetramethylsilane (TMS) used as an internal standard at room temperature. HRESIMS were recorded on an API QSTAR pulsar spectrometer. Silica gel (200–300 mesh), Sephadex LH-20 and RP-18 gel (20–45 µm) were used for column chromatography (CC). Fractions were monitored by thin layer chromatography and spots were visualized by heating silica gel plates immersed in H2SO4 in EtOH, in combination with the Agilent 1200 series HPLC system (Eclipse XDB-C18 column, 5 μm, 4.6 × 150 mm).

Mushroom Material

Wild mushrooms, T. terreum, were collected from Arcachon in southwestern France in December 2012 and identified by Prof. Zhu-Liang Yang of Kunming Institute of Botany, Chinese Academy of Sciences. A specimen (No. KIB20121205.2) was deposited at the Kunming Institute of Botany, Chinese Academy of Sciences. For details of this mushroom please see that reported previously [1].

Extraction and Isolation

The fresh fruiting bodies of T. Terreum (3 kg) were extracted with chloroform (24 h × 3), and then partitioned with water (1:1). Finally, a chloroform extract (12 g) was obtained, which was submitted to silica gel CC using petroleum-acetone (from 1:0 to 0:1) to give six fractions (A–F). Fraction B (1.8 g) was separated by reverse-phased CC eluted with gradient mixture of MeOH and H2O (30:70–100:0, v/v) to afford five sub-fractions (B1–B5). Fraction B2 (32 mg) was purified by Sephadex LH-20 (MeOH) to give compound 1 (2.2 mg), while fraction B4 (57 mg) was also purified by Sephadex LH-20 (MeOH) to give compounds 2 (2.3 mg) and 3 (1.3 mg).

Saponaceolide Q (1)

Colorless oil,  + 7.2 (c 0.12 MeOH); IR (KBr) νmax 3436, 2937, 1723, 1448, 1367, 1201, 1068, 991 cm−1; for 1H (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2, respectively; HRESIMS: m/z 525.3193 (calcd for C30H46O6Na, [M + Na]+, 525.3187).

Saponaceolide R (2)

Colorless oil,  + 19.7 (c 0.10 MeOH); IR (KBr) νmax 3443, 2926, 1726, 1457, 1381, 1065, 998 cm−1; for 1H (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2, respectively; HRESIMS: m/z 555.3290 (calcd for C31H48O7Na, [M + Na]+, 555.3292).

Saponaceolide S (3)

Colorless oil,  + 13.7 (c 0.11 MeOH); IR (KBr) νmax 3441, 2926, 1724, 1452, 1382, 1120, 997 cm−1; for 1H (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 2, respectively; HRESIMS: m/z 555.3290 (calcd for C31H48O7Na, [M + Na]+, 555.3292).

Cytotoxicity Assay

Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7 and colon cancer SW480 cell lines were used in the cytoxic assay. All cell lines were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10 % fetal bovine serum (Hyclone, USA) in 5 % CO2 at 37 °C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates [5]. Cisplatin was used as a positive control.

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