Anti-Proliferative and Anti-Inflammatory Lanostane Triterpenoids from

The mushroom Macrolepiota procera, also called “parasol mushroom” due to its large fruiting body resembling a parasol, is widespread in temperate ...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. 2018, 66, 3146−3154

Anti-Proliferative and Anti-Inflammatory Lanostane Triterpenoids from the Polish Edible Mushroom Macrolepiota procera He-Ping Chen,† Zhen-Zhu Zhao,†,‡ Zheng-Hui Li,† Ying Huang,† Shuai-Bing Zhang,† Yang Tang,†,‡ Jian-Neng Yao,†,‡ Lin Chen,† Masahiko Isaka,§ Tao Feng,*,† and Ji-Kai Liu*,† †

College of Pharmaceutical Sciences, South-CentralUniversity for Nationalities, Wuhan 430074, China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China § National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand ‡

S Supporting Information *

ABSTRACT: This study features the isolation and identification of 12 lanostane-type triterpenoids, namely lepiotaprocerins A− L, 1−12, from the fruiting bodies of the Poland-collected edible mushroom Macrolepiota procera. The structures and the absolute configurations of the new compounds were ambiguously established by extensive spectroscopic analyses, ECD calculation, and single-crystal X-ray diffraction analyses. Structurally, lepiotaprocerins A−F, 1−6, are distinguished by the presence of a rare “1-en1,11-epoxy” moiety which has not been previously described in the lanostane class. Biologically, lepiotaprocerins A−F, 1−6, displayed more significant inhibitions of nitric oxide (NO) production than the positive control L-NG-monomethyl arginine (LNMMA) (IC50 47.1 μM), and lepiotaprocerins G−L, 7−12, showed various cytotoxicity potencies against a panel of human cancer cell lines. Compound 9 also displayed antitubercular activity against Mycobacterium tuberculosis H37Ra with a minimal inhibitory concentration (MIC) 50 μg/mL. KEYWORDS: Mushroom, Macrolepiota procera, lanostane triterpenoid, anti-inflammatory activity, cytotoxicity

■

INTRODUCTION

lepiotaprocerins A−L, 1−12, from the fruiting bodies of M. procera (Figure 1).

According to the World Health Organization, cancer is the second leading cause of death worldwide, and was responsible for 8.8 million deaths in 2015.1 Many cancers arise from infection, chronic irritation, and inflammation. Recent researches expanded the concept that inflammation is a critical component of tumor progression.2,3 Therefore, anti-inflammatory therapy is efficacious toward early neoplastic progression and malignant conversion. A large number of scientific publications have shown that natural products from medicinal/edible mushrooms play a dominant role in the discovery of leads for the development of drugs for prevention and treatment of this disease.4−6 Moreover, a meta-analysis of results of observational studies suggested that consumption of more mushrooms may be associated with decreased risk of breast cancer.7 The mushroom Macrolepiota procera, also called “parasol mushroom” due to its large fruiting body resembling a parasol, is widespread in temperate regions. In Europe, M. procera is a highly sought-after and popular item due to its large size fruiting bodies, frequent seasonal accessibility, and versatility in the kitchen. However, no reports have addressed the secondary metabolites of this kind of famous edible mushroom so far. As our continuous research aiming at discovery drug leads from edible mushroom, a chemical investigation on the constituents of the Poland-origin parasol mushroom M. procera was carried out. Herein, we report the isolation, structure elucidation, and biological evaluation of 12 lanostane triterpenoids, namely © 2018 American Chemical Society

■

MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured by a JASCO P-1020 digital polarimeter (Horiba, Kyoto, Japan). A UV-2401PC UV−visible recording spectrophotometer (Shimadzu, Kyoto, Japan) was used to record the ultraviolet (UV) spectra. A Chirascan circular dichroism spectrometer (Applied Photophysics Limited, Leatherhead, Surrey, UK) was used to record the CD spectra. 1D and 2D NMR spectra were obtained on Bruker Avance III 600 MHz or Ascend 800 MHz spectrometers (Bruker Corporation, Karlsruhe, Germany). An Agilent 6200 Q-TOF MS system (Agilent Technologies, Santa Clara, CA) was used to acquire the HRESIMS data. A Waters AutoSpec Premier P776 MS system (Waters Corporation, Milford, MA) was used to acquire the HREIMS datum. An APEX II DUO spectrophotometer (Bruker AXS GmbH, Karlsruhe, Germany) was applied for performing the single-crystal Xray diffraction experiment. Column chromatography (CC) was run on Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) and silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). A Büchi Sepacore System (pump manager C-615, pump modules C-605, and fraction collector C-660) (Büchi Labortechnik AG, Flawil, Switzerland) was used to perform medium-pressure liquid chromatography (MPLC). It was equipped with a column (400 mm × 7.4 mm i.d., 40− 75 μm, flow rate 40 mL/min) filled with Chromatorex C-18 (Fuji Received: Revised: Accepted: Published: 3146

January 17, 2018 March 3, 2018 March 4, 2018 March 6, 2018 DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of lepiotaprocerins A−L (1−12).

Table 1. 1H NMR Data of Compounds 1−6 (CDCl3) 2 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 CH3COCH3Oa

1a

2b

3a

5.19, s 1.36, dd (13.2, 2.0) 1.65, m 1.78, m 2.18, m 2.18, m 5.14, br. dd (9.6, 8.0) 2.50, dd (12.5, 8.0) 1.83, dd (12.5, 9.6) 2.22, dd (13.0, 8.0) 1.49, dd (13.0, 5.5) 5.36, ddd (8.0, 8.0, 5.5) 1.96, dd (10.7, 8.0) 1.14, s, 3H 1.25, s, 3H 1.92, m 1.06, d (6.4), 3H 1.40, m 1.08, m 2.49, m; 2.60, m 6.02, t (7.0) 1.91, s, 3H 1.15, s, 3H 1.10, s, 3H 1.02, s, 3H 2.04, s, 3H

5.18, s 1.36, dd (13.2, 2.0) 1.79, m 1.65, m 2.18, m 2.19, m 5.14, br. dd (9.6, 8.5) 2.51, dd (12.5, 8.0) 1.83, dd (12.5, 9.6) 2.22, dd (13.0, 9.0) 1.50, dd (13.0, 5.5) 5.35, ddd (9.0, 8.0, 5.5) 1.96, dd (10.7, 8.0) 1.14, s, 3H 1.25, s, 3H 1.92, m 1.06, d (6.5), 3H 1.38, m 1.06, m 2.44, m; 2.55, m 5.87, t (8.0) 1.88, s, 3H 1.16, s, 3H 1.10, s, 3H 1.02, s, 3H 2.05, s, 3H 3.73, s, 3H

5.19, s 1.38, dd (13.0, 2.3) 1.79, m 1.67, m 2.22, m 2.22, m 5.14, br. t (7.5) 2.29, dd (12.0, 7.8) 1.90, overlapped 1.99, dd (12.5, 8.0) 1.66, dd (12.5, 7.5) 4.54, ddd (8.0, 7.5, 6.5) 1.82, m 1.10, s, 3H 1.26, s, 3H 1.81, overlapped 1.12, d (6.0), 3H 1.87, overlapped 1.87, overlapped 6.86, 1.91, 1.16, 1.10, 1.02,

s s, s, s, s,

4b

3H 3H 3H 3H

5.19, 1.38, 1.68, 1.78, 2.21, 2.23, 5.15, 2.28, 1.85, 1.96, 1.65, 4.10, 1.62, 1.15, 1.26, 1.90, 1.06, 1.55, 1.68, 4.86, 6.16, 1.95, 1.16, 1.11, 1.00,

s dd (13.0, 2.0) m m m m dd (8.0, 8.0) dd (13.0, 8.0) dd (13.0, 8.0) overlapped overlapped dd (13.0, 8.0) dd (16.0, 8.0) s, 3H s, 3H m d (6.5), 3H m m m m s, 3H s, 3H s, 3H s, 3H

5b

6b

5.19, s 1.36, br. d (13.0, 2.0) 1.79, m 1.65, m 2.18, m 2.19, m 5.14, dd (9.5, 8.0) 1.86, dd (12.0, 9.5) 2.50, dd (12.0, 8.0) 1.48, dd (13.5, 6.5) 2.25, dd (13.5, 8.0) 5.33, ddd (8.0, 8.0, 6.5) 2.08, dd (11.0, 8.0) 1.15, s, 3H 1.25, s, 3H 2.12, m 1.17, d (6.5), 3H 1.61, m 1.50, m 4.95, br. t (6.5) 7.06, br. s 1.92, s, 3H 1.16, s, 3H 1.10, s, 3H 1.04, s, 3H 2.05, s, 3H

5.19, s 1.36, br. d (13.0, 2.0) 1.79, m 1.66, m 2.17, m 2.19, m 5.16, dd (9.5, 8.0) 1.86, dd (12.0, 9.5) 2.53, dd (12.0, 8.0) 1.52, dd (13.5, 6.5) 2.20, dd (13.5, 8.0) 5.32, ddd (8.0, 8.0, 6.5) 1.92, dd (11.0, 8.0) 1.18, s, 3H 1.25, s 3H 2.39, m 1.16, d (6.5), 3H 1.34, m 1.31, m 4.99, br. t (6.5) 6.96, br. s 1.90, s, 3H 1.16, s, 3H 1.10, s, 3H 1.02, s, 3H 2.09, s, 3H

Recorded at 600 MHz. bRecorded at 800 MHz. Prof. Yu-Cheng Dai, who is a mushroom specialist of Beijing Forestry University. A specimen (No. 20141005PL) was kept by Herbarium of Ethnic Medicinal Plants of South-Central University for Nationalities (SCUEC). The dried collection was permitted by the local authorities. Extraction and Isolation. The dried fruiting bodies of M. procera (2.0 kg) were powdered and then extracted with 10 L of 90% ethanol three times (24 h each). The extract was evaporated and redissolved in

Silysia Chemical Ltd., Kasugai, Japan) RP-C18 silica gel. An Agilent 1260 liquid chromatography system (Agilent) equipped with an ODS column (Zorbax SB-C18, 150 mm × 9.4 mm i.d., 5 μm, flow rate 10 mL/min) was used for preparative high-performance liquid chromatography (prep-HPLC). Fungal Material. The mushroom M. procera was collected at a meadow near Wroclaw, Poland in October 2014, and identified by 3147

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry Table 2. 1H NMR Data of Compounds 7−12 (CDCl3) 7a 1 2 5 6 7 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 CH3COCH3O15-OH a

8.04, d (10.5) 5.91, d (10.5) 1.80, overlapped 1.80, overlapped 1.65, m 2.40, m 2.40, m 2.76, d (16.0) 2.49, d (16.0) 2.07, dd (13.0, 8.0) 1.86, overlapped 4.55, ddd (8.0, 8.0, 6.5) 1.88, dd (10.5, 5.5) 1.00, s, 3H 1.35, s, 3H 1.76, m 1.05, d (6.5), 3H 1.88, m 1.88, m 6.86, 1.92, 1.15, 1.16, 1.18,

br. s s, 3H s, 3H s, 3H s, 3H

8b 8.11, 5.89, 1.79, 1.67, 1.78, 2.38, 2.48, 2.57, 2.67, 2.09, 1.87, 4.56, 1.76, 1.04, 1.34, 1.90, 0.98, 1.17, 1.58, 2.31, 6.11, 1.89, 1.15, 1.16, 1.11, 3.73,

d (10.5) d (10.5) overlapped m m dd (11.0, 6.0) overlapped d (17.0) d (17.0) dd (13.0, 8.0) dd (13.0, 5.5) m overlapped s, 3H s, 3H overlapped d (6.5), 3H overlapped overlapped m; 2.48, m t (7.5) s, 3H s, 3H s, 3H s, 3H s, 3H

9a

10b

11b

12b

8.08, d (10.3) 5.89, d (10.3) 1.78, overlapped 1.62, m 1.78, overlapped 2.40, dd (20.0, 6.0) 2.35, dd (10.7, 7.0) 2.72, d (17.0) 2.63, d (17.0) 2.28, dd (13.0, 8.0) 1.72, dd (13.0, 5.0) 5.34, ddd, (8.0, 8.0, 5.0) 2.01, dd (10.6, 8.0) 1.02, s, 3H 1.32, s, 3H 1.88, m 0.96, d (6.6), 3H 1.41, m 1.07, m 2.58, m; 2.50, m 6.02, t (7.4) 1.91, s, 3H 1.14, s, 3H 1.15, s, 3H 1.15, s, 3H 2.05, s, 3H

8.09, d (10.5) 5.90, d (10.5) 1.79, overlapped 1.63, overlapped 1.79, overlapped 2.38, overlapped 2.34, overlapped 2.63, d (17.0) 2.72, d (17.0) 2.28, dd (13.0, 8.0) 1.72, dd (13.0, 5.0) 5.34, ddd (8.0, 8.0, 5.0) 2.01, dd (11.0, 8.0) 1.03, s, 3H 1.34, s, 3H 1.88, overlapped 0.97, d (6.5), 3H 1.39, m 1.06, m 2.53, m; 2.43, m 5.87, t (7.5) 1.89, s, 3H 1.15, s, 3H 1.16, s, 3H 1.16, s, 3H 2.07, s, 3H 3.73, s, 3H

8.07, d (10.5) 5.91, d (10.5) 1.80, overlapped 1.80, overlapped 1.62, overlapped 2.37, dd (11.0, 6.8) 2.40, ddd (20.0, 7.0, 1.6) 2.75, d (17.0) 2.62, d (17.0) 2.31, dd (13.0, 7.8) 1.72, dd (13.0, 5.0) 5.31, ddd (7.8, 7.8, 5.0) 2.15, dd (11.0, 7.8) 1.04, s, 3H 1.34, s, 3H 2.08, m 1.08, d (6.5), 3H 1.62, overlapped 1.50, m 4.95, br. d (9.5) 7.06, s 1.92, s, 3H 1.15, s, 3H 1.16, s, 3H 1.18, s, 3H 2.06, s, 3H

8.09, d (10.5) 5.90, d (10.5) 1.80, overlapped 1.81, overlapped 1.65, overlapped 2.41, dd (20.0, 5.0) 2.36, overlapped 2.74, d (17.0) 2.65, d (17.0) 2.25, dd (13.0, 8.0) 1.75, dd (13.0, 5.0) 5.32, ddd (8.0, 8.0, 5.0) 1.97, dd (11.0, 8.0) 1.08, s, 3H 1.34, s, 3H 2.35, overlapped 1.07, d (6.5), 3H 1.35, overlapped 1.32, overlapped 4.98, br. d (9.5) 6.96, s 1.91, s, 3H 1.15, s, 3H 1.16, s, 3H 1.16, s, 3H 2.11, s, 3H

2.95, d (4.0)

Recorded at 600 MHz. bRecorded at 800 MHz. Lepiotaprocerin B, 2. White powder; C33H46O6; (+)-HRESIMS m/ z 539.3375 [M + H]+, calcd for C33H47O6, 539.3367; [α]24 D + 81.7; UV (MeOH) λmax (log ε) 203 (3.87), 261 (3.99) nm; 1H NMR data (Table 1); 13C NMR (Table 3). Lepiotaprocerin C, 3. White powder; C30H38O5; (+)-HRESIMS m/ z 479.2794 [M + H]+, calcd for C30H39O5, 479.2792; [α]24 D + 58.9; UV (MeOH) λmax (log ε) 198 (3.03), 214 (2.96), 260 (3.12) nm; CD (MeOH) λmax (Δε) 208 (− 1.79), 223 (+ 5.67), 261 (+ 35.37), 306 (− 10.64) nm; 1H NMR data (Table 1); 13C NMR (Table 3). Lepiotaprocerin D, 4. White powder; C30H40O5; (+)-HRESIMS m/ z 481.2950 [M + H]+, calcd for C30H41O5, 481.2949; [α]25 D + 78.6; UV (MeOH) λmax (log ε) 220 (3.11), 260 (3.20) nm; 1H NMR data (Table 1); 13C NMR (Table 3). Lepiotaprocerin E, 5. White powder; C32H42O6; (+)-HRESIMS m/ z 523.3054 [M + H]+, calcd for C32H43O6, 523.3054; [α]25 D + 282.3; UV (MeOH) λmax (log ε) 193.4 (4.03), 206 (4.32), 261 (4.34) nm; CD (MeOH) λmax (Δε) 196 (+ 7.56), 215 (+ 7.26), 261 (+40.98), 308 (−10.98) nm; 1H NMR data (Table 1); 13C NMR (Table 3). Lepiotaprocerin F, 6. White powder; C32H42O6; (+)-HRESIMS m/ z 523.3055 [M + H]+, calcd for C32H43O6, 523.3054; [α]25 D + 106.3; UV (MeOH) λmax (log ε) 206 (4.01), 261 (4.03) nm; CD (MeOH) λmax (Δε) 208 (− 15.96), 215 (− 12.06), 261 (+ 50.53), 307 (− 14.11) nm; 1H NMR data (Table 1); 13C NMR (Table 3). Lepiotaprocerin G, 7. Colorless crystals (MeOH); C30H38O5; (+)-HRESIMS m/z 479.2792 [M + H]+, calcd for C30H39O5, 479.2792; [α]21 D + 179.8; UV (MeOH) λmax (log ε) 204 (3.96), 254 (3.76) nm; 1H NMR data (Table 2); 13C NMR (Table 3). Lepiotaprocerin H, 8. White powder; C31H44O5; (+)-HRESIMS m/ z 497.3261 [M + H]+, calcd for C31H45O5, 497.3262; [α]25 D + 132.1; UV (MeOH) λmax (log ε) 223 (4.11), 251 (3.85) nm; 1H NMR data (Table 2); 13C NMR (Table 3). Lepiotaprocerin I, 9. White powder; C32H44O6; (−)-HRESIMS m/ z 523.3064 [M − H]−, calcd for C32H43O6, 523.3065; [α]25 D + 199.7;

water followed by partition between H2O/EtOAc three times (3 h each). The EtOAc layers were combined and concentrated in vacuum to afford a crude extract (85 g). This residue was separated by MPLC with MeOH/H2O (from 20:80 → 100:0, v/v, totally 4 L) to give 11 main fractions (A−K). Fraction F was separated by Sephadex LH-20 CC (acetone) to give four subfractions F1−F4. Subfraction F1 was subjected to normal-phase silica gel CC (petroleum ether/acetone: 5:1 → 2:1, v/v, totally 1 L) to afford three subfractions F1a−F1c. Subfraction F1a was purified by prep-HPLC (MeCN/H2O, 55:45 → 75:25, v/v, 10 mL/min) to obtain compounds 8 (tR = 16.5 min, 1.2 mg), 9 (tR = 18.4 min, 3.5 mg), 10 (tR = 20.1 min, 1.3 mg). Subfraction F2 was purified by normal-phase silica gel CC (petroleum ether/acetone, 5:1 → 1:1, v/v, totally 1 L) to give compound 1 (1.8 mg) and F2b. Subfraction F2b was further separated by prep-HPLC (MeCN/H2O, 50:50 → 75:25, v/v, 10 mL/min) to yield compounds 11 (tR = 17.4 min, 1.5 mg) and 12 (tR = 18.4 min, 1.4 mg). Subfraction F3 was subjected to normal-phase silica gel CC (petroleum ether/ acetone, 5:1 → 1:1, v/v, totally 1.2 L) to give three subfractions F3a− F3c. Subfraction F3a was further separated by Sephadex LH-20 CC (acetone) to give three subfractions F3aa−F3ac. Subfraction F3aa was purified by HPLC (MeCN/H2O, 45:55 → 80:20, v/v, 10 mL/min) to give compound 2 (tR = 14.6 min, 0.8 mg). Subfraction F3ab was purified by HPLC (MeCN/H2O, 45:55 → 75:25, v/v, 10 mL/min) to give compounds 3 (tR = 18.2 min, 2.2 mg) and 7 (tR = 14.8 min, 2.2 mg). Subfraction F3ac was purified by HPLC (MeCN/H2O, 35:65 → 65:35, v/v, 10 mL/min) to give compounds 5 (tR = 21.4 min, 0.6 mg) and 6 (tR = 22.0 min, 0.6 mg). Subfraction F4 was purified repeatedly by HPLC to yield compound 4 (MeCN/H2O, 45:55 → 75:25, v/v, 10 mL/min, tR = 15.6 min, 1.0 mg). Lepiotaprocerin A, 1. Colorless prisms (MeOH); C32H44O6; (+)-HRESIMS m/z 525.3211 [M + H]+, calcd for C32H45O6, 525.3211; [α]24 D + 114.2; UV (MeOH) λmax (log ε) 222 (3.01), 261 (3.30) nm; 1H NMR data (Table 1); 13C NMR (Table 3). 3148

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

a

3149

187.6, C 97.1, CH 206.8, C 44.1, C 48.8, CH 18.3, CH2 25.8, CH2 137.6, C 132.6, C 41.7, C 81.7, CH 33.6, CH2 48.4, C 49.2, C 39.3, CH2 74.9, CH 52.6, CH 16.7, CH3 19.4, CH3 30.5, CH 18.7, CH3 35.8, CH2 27.3, CH2 146.1, CH 126.2, C 171.8, C 20.8, CH3 21.0, CH3 29.3, CH3 26.4, CH3 170.8, C 21.4, CH3

187.5, C 97.0, CH 206.7, C 44.1, C 48.8, CH 18.3, CH2 25.8, CH2 137.6, C 132.5, C 41.6, C 81.6, CH 33.5, CH2 48.3, C 49.2, C 39.3, CH2 74.8, CH 52.6, CH 16.6, CH3 19.4, CH3 30.5, CH 18.6, CH3 35.8, CH2 27.0, CH2 143.4, CH 126.9, C 168.3, C 20.8, CH3 21.0, CH3 29.3, CH3 26.4, CH3 170.7, C 21.4, CH3 51.3, CH3

2b

3a 187.3, C 97.0, CH 206.5, C 44.0, C 48.7, CH 18.1, CH2 25.8, CH2 137.9, C 132.4, C 41.5, C 81.2, CH 34.3, CH2 47.3, C 48.6, C 37.3, CH2 75.0, CH 52.7, CH 19.0, CH3 19.4, CH3 25.3, CH 21.1, CH3 37.7, CH2 106.9, C 145.8, CH 131.1, C 172.1, C 10.4, CH3 20.9, CH3 29.2, CH3 26.1, CH3

Recorded at 150 MHz. bRecorded at 200 MHz.

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 CH3COCH3CO-OMe

1a

Table 3. 13C NMR Data of Compounds 1−6 (CDCl3) 187.7, C 97.0, CH 206.7, C 44.1, C 48.8, CH 18.3, CH2 25.9, CH2 138.2, C 132.4, C 41.6, C 81.6, CH 34.2, CH2 47.8, C 48.4, C 37.9, CH2 76.2, CH 52.1, CH 18.8, CH3 19.4, CH3 24.3, CH 21.8, CH3 37.4, CH2 71.3, CH 145.5, CH 128.2, C 169.6, C 20.6, CH3 21.0, CH3 29.3, CH3 26.4, CH3

4b 187.4, C 97.2, CH 206.7, C 44.1, C 48.8, CH 18.3, CH2 25.8, CH2 137.5, C 132.7, C 41.6, C 81.4, CH 33.6, CH2 48.4, C 49.2, C 39.4, CH2 74.8, CH 52.6, CH 16.6, CH3 19.4, CH3 28.3, CH 19.9, CH3 39.9, CH2 80.1, CH 148.8, CH 130.0, C 174.1, C 10.9, CH3 21.0, CH3 29.3, CH3 26.5, CH3 170.4, C 21.5, CH3

5b 187.5, C 97.1, CH 206.7, C 44.1, C 48.9, CH 18.3, CH2 25.8, CH2 137.5, C 132.7, C 41.7, C 81.5, CH 33.7, CH2 48.5, C 49.2, C 39.3, CH2 73.8, CH 52.9, CH 16.7, CH3 19.4, CH3 28.0, CH 18.4, CH3 40.7, CH2 78.3, CH 149.4, CH 129.9, C 174.2, C 10.8, CH3 21.0, CH3 29.3, CH3 26.4, CH3 171.4, C 21.4, CH3

6b 158.6, CH 126.1, CH 204.6, C 44.5, C 48.8, CH 17.9, CH2 29.3, CH2 165.8, C 134.7, C 40.0, C 197.7, C 51.1, CH2 46.4, C 49.6, C 39.4, CH2 74.3, CH 53.0, CH 19.3, CH3 24.4, CH3 25.5, CH 20.4, CH3 37.4, CH2 106.8, C 145.7, CH 131.2, C 172.0, C 10.4, CH3 21.3, CH3 28.2, CH3 26.7, CH3

7

51.6, CH3

159.1, CH 125.9, CH 204.8, C 44.5, C 48.8, CH 17.9, CH2 29.2, CH2 166.6, C 134.8, C 40.0, C 198.5, C 51.4, CH2 46.9, C 49.7, C 42.5, CH2 71.4, CH 54.7, CH 17.3, CH3 24.5, CH3 30.8, CH 18.4, CH3 36.3, CH2 28.0, CH2 143.9, CH 127.4, C 168.4, C 20.5, CH3 21.3, CH3 28.2, CH3 26.9, CH3

8 158.8, CH 126.0, CH 204.7, C 44.5, C 48.7, CH 17.9, CH2 29.1, CH2 165.7, C 135.0, C 39.9, C 198.1, C 51.1, CH2 47.2, C 50.1, C 41.4, CH2 73.9, CH 52.9, CH 17.2, CH3 24.5, CH3 30.1, CH 17.9, CH3 35.3. CH2 27.0, CH2 146.0, CH 126.2, C 172.5, C 20.6, CH3 21.3, CH3 28.2, CH3 27.0, CH3 170.7, C 21.2, CH3

9 158.8, CH 126.0, CH 204.7, C 44.5, C 48.7, CH 17.9, CH2 29.1, CH2 165.7, C 135.0, C 39.9, C 198.0, C 51.1, CH2 47.2, C 50.1, C 41.5, CH2 73.9, CH 52.9, CH 17.2, CH3 24.5, CH3 30.1, CH 17.8, CH3 35.3, CH2 26.8, CH2 143.2, CH 126.9, C 168.2, C 20.7, CH3 21.3, CH3 28.2, CH3 27.0, CH3 170.7, C 21.2, CH3 51.3, CH3

10 158.6, CH 126.1, CH 204.6, C 44.5, C 48.6, CH 17.9, CH2 29.1, CH2 165.5, C 135.1, C 39.9, C 197.6, C 51.1, CH2 47.2, C 50.0, C 41.5, CH2 73.8, CH 52.7, CH 17.1, CH3 24.5, CH3 27.8, CH 19.1, CH3 39.3, CH2 79.6, CH 148.6, CH 129.9, C 173.8, C 10.7, CH3 21.3, CH3 28.2, CH3 27.0, CH3 170.4, C 21.3, CH3

11

158.6, CH 126.1, CH 204.6, C 44.5, C 48.7, CH 17.8, CH2 29.1, CH2 165.5, C 135.1, C 40.0, C 197.7, C 51.1, CH2 47.2, C 50.0, C 41.3, CH2 72.8, CH 53.2, CH 17.3, CH3 24.5, CH3 27.7, CH 17.7, CH3 40.3, CH2 78.2, CH 149.2, CH 129.8, C 174.0, C 10.7, CH3 21.3, CH3 28.2, CH3 27.0, CH3 171.2, C 21.2, CH3

12

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry UV (MeOH) λmax (log ε) 221 (4.10), 248 (3.92) nm; 1H NMR data (Table 2); 13C NMR (Table 3). Lepiotaprocerin J, 10. White powder; C33H46O6; (+)-HREIMS m/ z 538.3033 [M]+, calcd for C33H46O6, 538.3294; [α]25 D + 85.7; UV (MeOH) λmax (log ε) 219 (3.85), 251 (3.49) nm; 1H NMR data (Table 2); 13C NMR (Table 3). Lepiotaprocerin K, 11. White powder; C32H42O6; (+)-HRESIMS m/z 523.3055 [M + H]+, calcd for C32H43O6, 523.3054; [α]25 D + 179.9; UV (MeOH) λmax (log ε) 213 (4.16), 246 (3.88) nm; CD (MeOH) λmax (Δε) 201 (− 3.50), 215 (+ 4.28), 258 (+ 26.58), 340 (− 4.75) nm; 1H NMR data (Table 2); 13C NMR (Table 3). Lepiotaprocerin L, 12. White powder; C32H42O6; (+)-HRESIMS m/z 523.3060 [M + H]+, calcd for C32H43O6, 523.3054; [α]25 D + 166.0; UV (MeOH) λmax (log ε) 214 (4.23), 246 (3.97) nm; CD (MeOH) λmax (Δε) 207 (− 26.91), 215 (− 22.93), 257 (+ 38.98), 338 (− 6.93) nm; 1H NMR data (Table 2); 13C NMR (Table 3). Single-Crystal X-ray Diffraction Data for 1. Crystal data for Cu_1_0m: C32H44O6, M = 524.67, a = 5.9650(5) Å, b = 16.1958(11) Å, c = 14.5295(11) Å, α = 90°, β = 91.695(5)°, γ = 90°, V = 1403.05(18) Å3, T = 100(2) K, space group P21, Z = 2, μ(CuKα) = 0.674 mm−1, 13050 reflections measured, 4533 independent reflections (Rint = 0.0798). The final R1 values were 0.0595 (I > 2σ(I)). The final wR(F2) values were 0.1450 (I > 2σ(I)). The final R1 values were 0.0981 (all data). The final wR(F2) values were 0.1601 (all data). The goodness of fit on F2 was 1.065. Flack parameter = −0.2(2). The crystallographic data were deposited to the Cambridge Crystallographic Data Centre (CCDC) with the number CCDC 1559796. Copies of the data are available for free from Cambridge Crystallographic Data Centre.8 Single Crystal X-ray Diffraction Data for 7. Crystal data for Cu_7_0m: C30H38O5, M = 478.60, orthorhombic, a = 6.2081(3) Å, b = 19.8715(11) Å, c = 20.2149(11) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2493.8(2) Å3, T = 100(2) K, space group P212121, Z = 4, μ(CuKα) = 0.680 mm −1, 13924 reflections measured, 4386 independent reflections (Rint = 0.0502). The final R1 values were 0.0493 (I > 2σ(I)). The final wR(F2) values were 0.1274 (I > 2σ(I)). The final R1 values were 0.0547 (all data). The final wR(F2) values were 0.1319 (all data). The goodness of fit on F2 was 1.061. Flack parameter = 0.4(3). The Hooft parameter is 0.27(13) for 1786 Bijvoet pairs. The crystallographic data were submitted to the Cambridge Crystallographic Data Centre (CCDC) with the number CCDC 1559976. These data can be accessed free of charge from Cambridge Crystallographic Data Centre.9 Nitric Oxide Production in RAW 264.7 Macrophages. The RPMI 1640 medium (Hyclone, Logan, UT) containing 10% FBS was used to culture the murine monocytic RAW 264.7 macrophages. The compounds were dissolved in DMSO and further diluted in medium to produce different concentrations. The culture medium and cell mixture were dispensed into 96-well plates (2 × 105 cells/well) and maintained at 37 °C under 5% CO2 in a humidified atmosphere. After preincubation for 24 h, serial dilutions of the test compounds were added into the cells, up to the maximum concentration 25 μM, then added with LPS to a concentration 1 μg/mL and continued to incubate for 18 h. NO production in each well was assessed after addition of 100 μL of Griess reagent (reagent A and reagent B, Sigma, St. Louis, MO) to 100 μL of each supernatant from the LPS-treated or LPS- and compound-treated cells in triplicates and incubation for 5 min, NO production of each cell was assessed. The sample absorbance was measured at 570 nm by a 2104 Envision Multilabel Plate Reader. L-NG-monomethyl arginine (L-NMMA) were used as positive controls. Cytotoxicities Against Five Human Cancer Cell Lines. The following five human cancer cell lines were used: the HL-60 (ATCC CCL-240) human myeloid leukemia; SMMC-7721 human hepatocellular carcinoma; A-549 (ATCC CCL-185) lung cancer; MCF-7 (ATCC HTB-22) breast cancer; SW-480 (ATCC CCL-228) human colon cancer. The cell line SMMC-7721 was bought from China Infrastructure of Cell Line Resources (Beijing, China), and others were bought from American Type Culture Collection (ATCC, Manassas, VA). All cells were cultured in RPMI-1640 medium containing 10%

fetal bovine serum (FBS) (Hyclone) and maintained at 37 °C under 5% CO2 in a humidified atmosphere. Colorimetric measurements of the amount of insoluble formazan produced in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) was used to assess cell viability. In brief, each well of a 96-well cell culture plate was seeded with 100 μL of adherent cells and kept for 12 h for adherence, and then test compounds were added. However, suspended cells were seeded before addition of test compounds with both the same density of 1 × 105 cells/mL every 100 μL of culture medium. After different concentrations of test compounds were added, each cancer cell line was incubated for 48 h in triplicates. Cisplatin was used as positive control. After the incubation, MTT (100 μg) was added to each well and incubation was continued for 4 h at 37 °C. After removal of the 100-μL culture medium, the cells were lysed with 20% SDS-50% DMF (100 μL). The remained lysates were subjected to measurement of the optical density at 595 nm with a 96-well microtiter plate reader. The IC50 value for each compound was calculated by a published method.10 Antimycobacterial Assay. Antimycobacterial activity against Mycobacterium tuberculosis H37Ra was determined using the green fluorescent protein microplate assay.11

■

RESULTS AND DISCUSSION Structural Elucidations of Lepiotaprocerins A−L, 1− 12. Lepiotaprocerin A, 1, was isolated as colorless needles. It

Figure 2. Key 2D NMR correlations of compounds 1, 3−6.

was determined to have the molecular formula C32H44O6 based on the protonated molecule ([M + H]+) on HRESIMS analysis, corresponding to 11 degrees of unsaturation. The 1D NMR data (Tables 1 and 2) displayed resonances assignable to eight methyls (one doublet), six methylenes, seven methines, and 11 quaternary carbons. All these data are reminiscent of those for (24Z)-3,11-dioxo-lanosta-8,24-dien-26-oic acid, a lanostane triterpenoid from the mushroom Jahnoporus hirtus,12 indicating the same lanostane skeleton of compound 1. A thorough analysis using a combination of 1H−1H COSY and HMBC spectra allowed the completion of the planar structure of 1. The 1 H−1H COSY correlations between H-15 (δH 2.22, 1.49)/H-16 (δH 5.36), and H-16/H-17 (δH 1.96), and the HMBC correlations from H-16 and a methyl (δH 2.04) to a carbonyl at δC 170.8 revealed the presence of an acetoxy group attached to C-16. In addition, the HMBC correlations from H-11 (δH 3150

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry

Figure 6. ORTEP drawing of compound 7. Figure 3. Oak Ridge Thermal Ellipsoid Plot Program (ORTEP) drawing of compound 1.

Figure 7. Experimental CD and calculated ECD spectra for compound 7.

Figure 4. Experimental CD and calculated ECD spectra for compound 3, and calculated ECD for 3a (enantiomer of 3).

Figure 8. CD spectra of compounds 11 and 12.

Table 4. Inhibitory Activity of NO Production of Compounds 1−6

Figure 5. CD spectra of compounds 5 and 6.

5.14) to C-8 (δC 137.6)/C-9 (δC 132.6), from Me-19 (δH 1.25) to a low field quaternary carbon at δC 187.6 (C-1), from H-2 (δH 5.19) to C-4 (δC 44.1) and C-10 (δC 41.7), and C-1 were observed (Figure 2). All these data indicated that the existence of a β-oxygenated-α,β-unsaturated functionality located at C-1 and C-2, leading to the assignment of 10 degrees of unsaturation. Although the HMBC correlation from H-11 to C-1 was absent, the remaining one degree of unsaturation was ascribed to a ring constructed by an epoxy group between C-1 and C-11, which was confirmed by the downfield shifted of C-1, C-11 (δC 81.7), and the HRESIMS result. Thus, the planar

sample

IC50 (μM)

L-NMMA 1 2 3 4 5 6

47.1 33.8 24.3 34.9 17.9 29.9 26.7

structure of compound 1 was established as shown in Figure 1. Structurally speaking, the highly constrained 1-en-1,11-epoxy group is unprecedented in the lanostane class. The relative 3151

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry Table 5. Cytotoxicity of 7−12 Against Five Human Cancer Cell Line HL-60

A-549

SMMC-7721

7 8 9 10 11 12 Cisplatin

MCF-7

SW480

IC50 (μM ± SD)

sample 3.52 2.88 14.59 12.70 14.09 3.09 2.95

± ± ± ± ± ± ±

0.79 0.08 0.39 0.06 0.86 0.03 0.13

6.99 4.96 18.86 17.68 21.03 5.70 15.97

± ± ± ± ± ± ±

0.29 0.10 0.36 0.52 0.74 0.18 0.69

5.00 3.17 10.77 12.61 15.28 3.14 10.17

± ± ± ± ± ± ±

0.11 0.04 0.85 0.16 0.61 0.06 0.41

12.95 8.27 25.89 15.50 23.23 12.48 23.64

± ± ± ± ± ± ±

0.73 0.12 0.87 0.80 0.88 0.50 1.6

5.21 3.57 16.77 17.38 19.73 4.38 9.26

± ± ± ± ± ± ±

0.39 0.22 0.84 0.71 0.60 0.39 0.98

correlation between H-20 (δH 1.81) and H-24 (δH 6.86). The absolute configuration of compound 3 was determined by comparison of experimental and theoretical electronic circular dichroism (ECD) spectra.14−17 A conformation search by MMFF94s force field of 3 only gave two conformers. Both the conformers were optimized at B3LYP/6-31G** level of theory. The theoretical calculations of ECD were performed using time-dependent density functional theory (TDDFT) at B3LYP/6-31G** level in MeOH with the IEFPCM model. As shown in Figure 4, the calculated ECD for 23R matched with the experimental curve and thus determined the absolute configurations. On the basis of the foregoing evidence, lepiotaprocerin C was established as (23R,24Z)-1,11α-16β,23diepoxy-3-oxo-lanosta-1,8,24-trien-26,23-olide, 3. The white powder, lepiotaprocerin D, 4, was determined to have the molecular formula C30H40O5 based upon the protonated molecule ([M + H]+) on HRESIMS analysis, indicating 11 degrees of hydrogen deficiency. Comparison of its 1D NMR data with those of 3 indicated that 4 is an analogue of 3 (Tables 1 and 2). In the 13C NMR and DEPT spectra of 4, the absence of the ketal carbon (δC 106.9 in 3) and the presence of an oxygenated methine at δC 71.3 suggested that the difference between them was that the α,β-unsaturated-γlactone group in 3 was opened to give 4 which presented an α,β-unsaturated carboxylic acid. These changes were further confirmed by key HMBC correlation from H-16 (δH 4.10) to C-23 (δC 71.3), and one degree of unsaturation less than compound 3. The configuration of C-23 was determined as S by the ROESY correlation between H-16 and H-23 (δH 4.86) (Figure 2). Thus, lepiotaprocerin D was identified as (23S,24Z)-1,11α-16β,23-diepoxy-3-oxo-lanosta-1,8,24-trien-26oic acid, 4. Lepiotaprocerins E, 5, and F, 6, were two compounds with close retention time on preparative HPLC, nearly the same NMR spectroscopic patterns, and the same molecular formula C32H42O6 established by HRESIMS, indicating that they were isomeric with each other. Both the 1D NMR spectra exhibited resemblance with those of compound 1 (Tables 1 and 2). The HMBC correlations from H-16 (δH 5.33) and a methyl singlet at δH 2.05 to a carbonyl at δC 170.4, and from H-23 (δH 4.95) to C-26 (δC 174.1) observed for 5 suggested the absence of the 16,23-epoxy bond in the structure of 5, but an acetoxy group attached to C-16 (Figure 2). The ROESY correlation between H-16 and H-17 (δH 2.08) enabled the determination of 16acetoxy group as β-oriented. The remarkable discrepancies between the 1H and 13C NMR chemical shifts of the position 23 revealed that compounds 5 and 6 differed from each other by virtue of the absolute configurations of C-23. In many cases, S or R configuration of C-23 were responsible for the respective positive or negative signs of Cotton effects at approximately 215 nm, involving π→π* transitions due to the 5-substituted

configuration of 1 was determined by a ROESY experiment. In the ROESY spectrum, the cross peaks between Me-18/H-11/ Me-19, Me-30/H-16/H-17, and H-24/Me-27 suggested an α orientation of 1,11-epoxy group, β orientation of the 16-acetoxy group, and Z configuration of C-24−C-25 double bond (Figure 2). A single crystal X-ray diffraction analysis of 1 further confirmed the above assignments, and provided solid evidence for the existence of the highly rigid 1,11-epoxy group (Figure 3). Therefore, the structure of lepiotaprocerin A, 1, was determined as (24Z)-16β-acetoxy-1,11α-epoxy-3-oxo-lanosta1,8,24-trien-26-oic acid. Lepiotaprocerin B, 2, was acquired as white powder. The molecular formula C33H46O6 was established based on the protonated molecule ([M + H]+) on HRESIMS analysis. Both the 1H and 13C NMR spectra of 2 showed high similarities with those of 1 (Tables 1 and 2). The only difference was the presence of a methoxy group, which was substantiated by the 1 H and 13C resonances at δH 3.73, δC 51.3, respectively. Elucidating the HMBC spectrum of 2 revealed that the 26carboxylic group in 1 was transformed into the corresponding methyl ester in 2, which was supported by the HMBC correlation from δH 3.73 to C-26 (δH 168.3). Therefore, lepiotaprocerin B was established as methyl (24Z)-16β-acetoxy1,11α-epoxy-3-oxo-lanosta-1,8,24-trien-26-oate, 2. It may be an artifact produced during the isolation processes. Lepiotaprocerin C, 3, was purified as white powder. Its molecular formula was established as C30H38O5 by HRESIMS on the basis of sodium adduct ion peak, indicating 12 degrees of hydrogen deficiency. The 13C and DEPT data (Tables 1 and 2) exhibited resonances assignable to seven methyls, five methylenes, seven methines, and 11 quaternary carbons. The overall 1D NMR spectra displayed significant similarities to those of 1, except for the absence of an acetyl group and a methylene, while with the presence of dioxygen-bearing nonprotonated carbon at δC 106.9. In the HMBC spectrum, H-16, H-22, and H-24 were correlated to the aforementioned carbon (δC 106.9) (Figure 2), suggesting that the methylene C23 in 1 was oxygenated into a ketal carbon in 3 and formed an epoxy bond between C-16 and C-23. Additionally, all above assignments accounted for 11 degrees of unsaturation; the residuary one degree of unsaturation was assigned as a γ-lactone group connected by C-23 and C-26 carbonyl, which showed typical 1H and 13C resonance patterns the same as those of spirochensilide A (3: δC 145.8 (C-24), 131.1 (C-25), 172.1 (C26), 10.4 (C-27); spirochensilide A: δC 147.2 (C-24), 132.0 (C25), 171.9 (C-26), 10.5 (C-27)).13 Thus, the planar structure of 3 was established as depicted in Figure 1. The absolute configurations of the chiral centers in the lanostane nucleus of 3 were established to be the same as those of compound 1 by ROESY spectrum. The relative configuration of C-23 was assigned as R* based upon the key ROESY 3152

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry 2(5H)-furanone moiety.18−20 In accordance with these empirical rules, the C-23 configurations of compounds 5 and 6 were determined as S and R, respectively (Figure 5). The structures of compounds 5 and 6 were designated as (23S,24Z)- or (23R,24Z)-16β-acetoxy-1,11α-epoxy-3-oxo-lanosta-1,8,24-trien-26,23-olide, respectively. The colorless crystals compound 7 gave a protonated molecular ([M + H]+) on HRESIMS analysis, indicating a molecular formula of C30H38O5 (calcd for C30H39O5, 479.2792) with 12 degrees of unsaturation. The NMR data of compound 7 were highly similar to those of compound 3 (Tables 2 and 3), where the discrepancies involved the absence of the sp2 quaternary carbon at δC 187.3 (C-1) and the oxygenated carbon at δC 81.2 (C-11), and the presence of an sp2 methine at δC 158.6 (C-1) and a carbonyl at δC 197.7 (C-11). These changes were proved to be the removal of 1,11-ether bond and assignment of a carbonyl at C-11 of 3 to give the resultant 7, which could be corroborated by the 1H−1H COSY correlation between H-1 (δH 8.04, d, J = 10.5 Hz) and H-2 (δH 5.91, d, J = 10.5 Hz), and HMBC correlations from H-12 (δH 2.76, d, J = 16.0 Hz; δH 2.49, d, J = 16.0 Hz) to C-11. Other assignments were same to those of compound 3. The relative configuration of 7 was established by X-ray single crystal diffraction due to the poor Flack parameter 0.4(3), which led to the assignment of C-23 as R* configuration which was the same as that of 3 (Figure 6). Although no natural lanostane enantiomers exist to the best of our knowledge, a definite conclusion of the absolute configuration of C-23 as R could not be drawn. Finally, the absolute configuration of 7 was unequivocally determined as 23R by ab initio calculation of its circular dichroism spectrum. As shown in Figure 7, the calculated ECD for 23R configuration showed similarity to those of the experimental CD spectra. Compounds 8, 9, and 10 were three lanostane congeners with similar NMR spectroscopic characteristics. Their molecular formulas were established by HRESIMS/HREIMS analyses as C31H44O5, C32H44O6, and C33H46O6, respectively. Their spectroscopic data shared similarities with those of 7 (Tables 2 and 3), revealing the presence of three α,β-unsaturated carbonyl groups and oxygenated pattern for C-16 in the structures of 8−10. However, the absence of the spiroketal carbon at δC 106.8 (C-23) and the presence of methylenes (C23, δC 28.0 for 8, 27.0 for 9, and 26.8 for 10) suggested that the C-17 side chain remained uncyclized. The 1H−1H COSY correlation between the hydroxy proton 16-OH (δH 2.95, d, J = 4.0 Hz) and H-16 (δH 4.56) and the HMBC correlations from methoxy at δH 3.73 to the carbonyl at δC 168.4 (C-24), and from H3-27 to C-24, C-25, C-26, suggested that compound 8 possessed a hydroxy group at C-16 and an α,β-unsaturated carboxylic group with methyl esterification. As for compounds 9 and 10, C-16 were substituted by an acetoxy group deduced by the HMBC correlation from H-16 (δH 5.34, ddd, J = 8.0, 8.0, 5.0 Hz) and a methyl at δH 2.05/2.07 to the carbonyl at δC 170.7. Furthermore, the HMBC correlation from a methoxy at δH 3.73 to the carbonyl (δC 168.2, C-26) demonstrated that the C-26 carboxylic group of 10 was methyl esterified compared to that of 9. The ROESY spectra of 8−10 which demonstrated cross peaks between H-16/H-17 implied that the hydroxy/ acetoxy of C-16 were β-oriented. Therefore, compounds 8−10 were established as shown in Figure 1, and trivially named as lepiotaprocerins H−J, respectively. Lepiotaprocerins K, 11, and L, 12, were isolated as white powders. HRESIMS results showed that these two compounds had the identical molecular formula of C32H42O6. Comparison

of their 1D NMR spectroscopic data which bore high resemblance to those of compounds 5 and 6 revealed that these two compounds had the same planar structure while differentiated at the configuration of C-23 (Tables 2 and 3). The HMBC correlations from H-12 (δH 2.75, 2.62 for 11, δH 2.74, 2.65 for 12) to C-11 (δC 197.6/197.7), C-8, and C-9, as well as the 1H−1H COSY correlations between H-1 (δH 8.07/ 8.09) and H-2 (δH 5.90/5.91), enabled the assignments of two α,β-unsaturated carbonyl groups at rings A and C. The configuration of C-23 was determined via the rules used in the structure elucidation of compounds 5 and 6. As shown in Figure 8, the respective positive (Δε + 4.28) and negative (Δε − 22.93) Cotton effects around 215 nm for compounds 11 and 12 indicated the S and R configuration of C-23, respectively. Thus, lepiotaprocerins K, 11, and L, 12, were determined as (23S)-16β-acetoxy-3,11-dioxo-lanosta-1,8,24-trien-26,23-olide and (23R)-16β-acetoxy-3,11-dioxo-lanosta-1,8,24-trien-26,23olide, respectively. Biological Activities of Lepiotaprocerins A−L, 1−12. Anti-Inflammatory Activity. All the compounds were evaluated for their inhibitory activity against NO production. However, compounds 7−12 were toxic to the subject murine monocytic RAW 264.7 macrophages at the concentration of 25 μM. As shown in Table 4, compounds 1−6 showed notable inhibitory activity on NO production in RAW 264.7 macrophages in vitro, which was more significant than the positive control L-NMMA. The most pronounced was compound 4, possessing an IC50 value of 17.9 μM. Comparison of compounds 3 and 4 suggested that the structures with 26,23-lactone functionality were less potent than those with the free carboxylic acid group. Comparison of 5 and 6 revealed that the 23R or 23S configuration of γ-lactone group showed no differentiations for the inhibitory activities of NO production. Antiproliferative Activity Against Five Human Cancer Cell Lines. All the compounds were screened for their cytotoxicity against five human cancer cell lines (HL-60, A-549, SMMC7721, MCF-7, SW480). Compounds 1−6 were inactive in the cytotoxicity assay (IC50 > 40 μM). Compounds 7−12 displayed inhibitory activity against the five human cancer cell lines. Among them, compounds 7, 8, and 12 exhibited significant cytotoxicity while compounds 9−11 display moderated cytotoxicity (Table 5). Anti-Tubercular Activity. The scarcity of the compounds at our disposal precluded the screen for other promising activity except for 9. Compound 9 was evaluated for its anti-tubercular activity against the strain Mycobacterium tuberculosis H37Ra. The results indicated that this compound showed weak antitubercular activity with an MIC value of 50 μg/mL. MIC values of the positive controls were isoniazid 0.0469 μg/mL and ethambutol 0.938 μg/mL. In summary, chemical investigation on the Polish edible mushroom Macrolepiota procera acquired six minor lanostane triterpenoids harboring a rare and rigid 1-en-1,11-epoxy moiety. The absolute configurations of all isolates were unambiguously determined via single crystal X-ray diffraction analysis, Cotton effects, and ECD calculation. Among the structures, lepiotaprocerin C, 3, is a lanostane triterpenoid with inflexible polycyclic structure. All these lanostanoids displayed remarkable anti-inflammatory activities in RAW 264.7 macrophages or anti-proliferative activity against five human cancer cell lines in vitro. This work represents the first report of triterpenoids or even secondary metabolites from the well-known edible mushroom M. procera, and also discloses its medicinal value 3153

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154

Article

Journal of Agricultural and Food Chemistry

(5) Guillamón, E.; García-Lafuente, A.; Lozano, M.; DArrigo, M.; Rostagno, M. A.; Villares, A.; Martínez, J. A. Edible mushrooms: role in the prevention of cardiovascular diseases. Fitoterapia 2010, 81, 715− 723. (6) Chen, H. P.; Liu, J. K. Secondary metabolites from higher fungi. Prog. Chem. Org. Nat. Prod. 2017, 106, 1−201. (7) Li, J.; Zou, L.; Chen, W.; Zhu, B.; Shen, N.; Ke, J.; Lou, J.; Song, R.; Zhong, R.; Miao, X. Dietary mushroom intake may reduce the risk of breast cancer: evidence from a meta-analysis of observational studies. PLoS One 2014, 9, e93437. (8) CCDC Number 1559796. https://www.ccdc.cam.ac.uk/ structures/Search?Ccdcid=1559796 (Accessed 28 February 2018). (9) CCDC Number 1559976. https://www.ccdc.cam.ac.uk/ structures/Search?Ccdcid=1559976 (Accessed 28 February 2018). (10) Reed, L. J.; Muench, H. A simple method of estimating fifty percent endpoint. Am. J. Epidemiol. 1938, 27, 493−497. (11) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob. Agents Chemother. 2003, 47, 3682−3687. (12) Liu, X. T.; Winkler, A. L.; Schwan, W. R.; Volk, T. J.; Rott, M. A.; Monte, A. Antibacterial compounds from mushrooms I: a lanostane-type triterpene and prenylphenol derivatives from Jahnoporus hirtus and Albatrellus flettii and their activities against Bacillus cereus and Enterococcus faecalis. Planta Med. 2010, 76, 182−185. (13) Zhao, Q. Q.; Song, Q. Y.; Jiang, K.; Li, D. G.; Wei, W. J.; Li, Y.; Gao, K. Spirochensilides A and B, two new rearranged triterpenoids from Abies chensiensis. Org. Lett. 2015, 17, 2760−2763. (14) Goto, H.; Ö sawa, E. Corner flapping: a simple and fast algorithm for exhaustive generation of ring conformations. J. Am. Chem. Soc. 1989, 111, 8950−8951. (15) Goto, H.; Ö sawa, E. An efficient algorithm for searching lowenergy conformers of cyclic and acyclic molecules. J. Chem. Soc., Perkin Trans. 2 1993, 2, 187−198. (16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2013. (17) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis: Quantifying the comparison of calculated and experimental electronic circular dichroism spectra. Chirality 2013, 25, 243−249. (18) Hu, Z. X.; Hu, K.; Shi, Y. M.; Wang, W. G.; Du, X.; Li, Y.; Zhang, Y. H.; Pu, J. X.; Sun, H. D. Rearranged 6/6/5/6-fused triterpenoid acids from the stems of Kadsura coccinea. J. Nat. Prod. 2016, 79, 2590−2598. (19) Tanaka, R.; Wada, S. I.; Aoki, H.; Matsunaga, S.; Yamori, T. Spiromarienonols A and B: Two new 7(8→9)abeo-lanostane-type triterpene lactones from the stem bark of Abies mariesii. Helv. Chim. Acta 2004, 87, 240−249. (20) Wang, G. W.; Lv, C.; Yuan, X.; Ye, J.; Jin, H. Z.; Shan, L.; Xu, X. K.; Shen, Y. H.; Zhang, W. D. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry 2015, 116, 221−229.

from the chemical aspects. From these points of view, efforts to promote the artificial cultivation of this kind of mushroom put into practice could bring economic value to the food industry.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00287. Spectroscopic data including 1D and 2D NMR, HRMS, CD, X-ray crystallographic data, and calculation details of compounds 1−12 (PDF) C32 H44 O6 (CIF) C30 H38 O5 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zheng-Hui Li: 0000-0003-1284-0288 Masahiko Isaka: 0000-0002-9229-3394 Tao Feng: 0000-0002-1977-9857 Funding

This work was financially supported by National Natural Science Foundation of China (31560010, 81561148013), the Key Projects of Technological Innovation of Hubei Province (2016ACA138), and the Fundamental Research Funds for the Central Universities, South-Central University for Nationalities (CZW17094, CZY16010, CSP17061, CZQ17008). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Analytical and Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities for MS and NMR spectra tests. The computational work was supported by HPC Center, Kunming Institute of Botany, CAS, China.

■

ABBREVIATIONS USED HRESIMS, high-resolution electrospray ionization mass spectroscopy; MPLC, medium-pressure liquid chromatography; DAD, diode array detector; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation; 1H−1H COSY, 1H−1H correlation spectroscopy; ROESY, rotating frame nuclear Overhauser effect spectroscopy; ORTEP, Oak Ridge Thermal Ellipsoid Plot Program; RMPI, Roswell Park Memorial Institute medium; LPS, lipopolysaccharide; MIC, minimal inhibitory concentration; SCUEC, South-Central University for Nationalities

■

REFERENCES

(1) World Health Organization. Cancer, 2017; http://www.who.int/ mediacentre/factsheets/fs297/en/ (Accessed January 15, 2018). (2) Mantovani, A.; Allavena, P.; Sica, A.; Balkwill, F. Cancer-related inflammation. Nature 2008, 454, 436−444. (3) Coussens, L. M.; Werb, Z. Inflammation and cancer. Nature 2002, 420, 860−867. (4) Friedman, M. Chemistry, nutrition, and health-promoting properties of Hericium erinaceus (lion’s mane) mushroom fruiting bodies and mycelia and their bioactive compounds. J. Agric. Food Chem. 2015, 63, 7108−7123. 3154

DOI: 10.1021/acs.jafc.8b00287 J. Agric. Food Chem. 2018, 66, 3146−3154