Structure, cytotoxic activity and mechanism of protoilludane sesquiterpene aryl esters from the mycelium of Armillaria mellea

Journal of Ethnopharmacology 184 (2016) 119–127

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Structure, cytotoxic activity and mechanism of protoilludane sesquiterpene aryl esters from the mycelium of Armillaria mellea Zhijin Li a,1, Yunchao Wang b,1, Bin Jiang a, Wenliang Li a,d,n, Lihua Zheng a, Xiaoguang Yang a, Yongli Bao a, Luguo Sun a, Yanxin Huang a, Yuxin Li c,nn a

National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China Shijiazhuang No.4 Pharmaceutical Co., ltd, Shijiazhuang 050000, China c Jilin Province Key Laboratory on Chemistry and Biology of Natural Drugs in Changbai Mountain, School of life Sciences, Northeast Normal University, Changchun 130024, China d School of Pharmaceutical Science, Jilin Medical University, Jilin 132013, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 12 September 2015 Received in revised form 28 January 2016 Accepted 28 February 2016 Available online 4 March 2016

Ethnopharmacological relevance: Armillaria mellea (Vahl. ex. Fr.) Karst is an important traditional Chinese medicine used in dispelling wind and removing obstruction in the meridians, and strengthening tendons and bones. Armillaria mellea has been recorded in the book Caobenshiyi which was written by ancestor for the function of suppressing hyderactive liver for calming endogenous wind medicine. The aim of this study is to investigate the cytotoxic activity for liver cell lines (normal and cancerous) of protoilludane sesquiterpene aryl esters from the mycelium of A. mellea. Materials and methods: A systemic fractionation of the mycelium extracts of A. mellea and relative activity mechanisms were studied. Results: Two new protoilludane sesquiterpene aryl esters named 5′-methoxy-armillasin (1) and 5-hydroxyl-armillarivin (2) were isolated. In addition, eight known protoilludane sesquiterpene aryl esters armillaridin (3), armillartin (4), armillarin (5), melleolide B (6), armillarilin (7), armillasin (8), armillarigin (9) and melleolide (10) were also isolated from the mycelium of A. mellea. The relative configurations of the two new compounds were confirmed by NOESY spectra. Among ten protoilludane sesquiterpene aryl esters, compounds 2, 3, 4, 7, 8, 9 and 10 were active constituents with highly cytotoxic activity against HepG2 cells (4.95–37.65 μg/mL). We reported here for the time, that compound 10 (melleolide) showed anti-tumor ability on hepatoma cell. The relative mechanism was assessed on HepG2 cells. Conclusions: Among all the ten protoilludane sesquiterpene aryl esters, melleolide (10) showed the best cytotoxic activity for HepG2 cells (4.95 μg/mL) and lower activity for L02 cells (16.05 μg/mL). Mechanism study showed that melleolide decreased the viability of the cancer cells with varying levels of cleavedcaspase 3, caspase 8, caspase 9, Bax and Ki67 expression. On the other hand, melleolide induced HepG2 cell cycle arrest at the G2/M phase. & 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Armillaria mellea Protoilludane Sesquiterpene aryl esters Cytotoxic activity Melleolide Chemical compounds studied in this article: Armillaridin (PubChem CID: 126031) Armillartin (PubChem CID: 131866) Armillarin (PubChem CID: 134206) Armillarilin (PubChem CID: 195529) Armillasin (PubChem CID: 134206) Armillarigin (PubChem CID: 51351607) Melleolide (PubChem CID: 435402)

1. Introduction Armillaria mellea (Vahl.ex. Fr.) Karst is a basidiomycete fungus in the genus Armillaria (Sun et al., 2009). It is also called honey mushroom (Wang et al., 2013a, 2013b, 330–336) by locals and has been recorded in some medicinal books, such as Ganoderma lucidum
Armillaria melle
Pleurotus nebrodensis (Yan and Zeng, 2013) and Bencaogangmushiyi (Zhao, 1963). A. mellea is a widely n Corresponding author at: National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China. nn Corresponding author. E-mail addresses: [email protected] (W. Li), [email protected] (Y. Li). 1 First authorship determined by coin toss.

http://dx.doi.org/10.1016/j.jep.2016.02.044 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.

used traditional medicine with the function of suppressing hyderactive liver for calming endogenous wind, dispelling wind and removing obstruction in the meridians, and strengthening tendons and bones (Yan and Zeng, 2013). It has been used to treat dizziness, vertigo, neurasthenia, palsy, headache, hypertension, and insomnia (Chen et al., 2014). A. mellea is one of primary constitutes in traditional Chinese compound medicine, such as Compound A. mellea Polysaccharide and Polypeptide Tablets, which is a clinical medicine in China (Zhao, 2009). In the modern research, the function of extracts from A. mellea was anti-tumor, anti-inflammation, anti-radiation and immunomodulation (Yu et al., 2001; Lin et al., 1988). The extracts from A. mellea are complex, including compounds such as diterpenes, triterpenes, steroids (Guo et al., 2007), sesquiterpenoid

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aromatic esters (Alberto et al., 1986; Donnelly et al., 1986), pigments (Guo et al., 2007), alkaloids (Wang et al., 2013a, 2013b), protoilludane sesquiterpene aryl esters (Momose et al., 2000), protoilludane norsesquiterpenoid esters (Yang et al., 1991a, 1991b, 478–480), phenolic compounds, sugars, fatty acid, tocopherols, ascorbic acid (Vaz et al., 2011) and so on. These compounds exhibit a diverse array of activities, including antibacterial (Alves et al., 2012), hypnotic, anticonvulsant, anti-tumor, anti-aging (Song and Yan, 2010), antioxidant potential and DNA protecting (Gao and Wang, 2012). Many recent studies have focused on the chemical constituents and pharmacological characterization of A. mellea. In our previous work, we obtained five new compounds, one diketopiperazine (Wang et al., 2013a, 2013b, 203–208) and four alkaloids (Wang et al., 2013a, 2013b, 330–336), from the liquid fermentation broth of A. mellea. However, the comprehensive and systematical pharmacological studies on this medicinal plant, especially on the effects related to its traditional uses were rare. In this work, systematical separation, structure elucidation and cytotoxic activities were performed. Ten protoilludane sesquiterpene aryl esters, including two new esters, were isolated. Furthermore, with the aim of clarifying the possible mechanism, western blot, DAPI, immunofluorescence detection and flow cytometry were tested for representative compound.

by shaking in 500 mL of media (glucose (12 g), soy bean (12 g), cane sugar (24 g), silkworm chrysalis (6 g), MgSO4 (0.9 g) and KH2PO4 (1.8 g/L) using five 250 mL flasks at 25 °C and was kept in the dark and shaking at 180 rpm for 5 days. Then, in the same conditions (25 °C, dark and 180 rpm), the liquid seed culture was further cultured in a 10 L liquid fermentation tank using the same media for 6 days. At the end of the process, the culture was filtered to obtain A. mellea mycelium. 2.3. Antibodies and reagents Antibodies against Bax (mouse monoclonal, sc-20067), Ki67 (polyclonal, sc-7846) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against GAPDH (mouse monoclonal, KC-5G4) were purchased from KangCheng Bioengineering Company. Antibodies against caspase 3 (polyclonal, 9662), caspase 8 (mouse monoclonal, 9746) and caspase 9 (polyclonal, 9504) were purchased from Cell Signaling Technology (Beverly, MA). 2.4. Extraction and isolation

1 H NMR and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively, with TMS as internal standard at 25 °C on a Bruker AV-400 spectrometer. 2D NMR spectra were obtained on the same spectrometer. LC and ESI-TOF-MS mass spectra were obtained on an Agilent 1200 HPLC/Micro TOF II mass spectrometer in positive ionization mode with dithranol as a matrix. Other equipments and methods were collected in Table 1.

The extraction and isolation process of Armillaria mellea were collected in Table 2. 5′-methoxy-armillasin (1): colorless powder, m.p. 229–230 °C; [α]D 20 ¼ þ189.29 (0.28 mg/mL, CHCl3); UV/Vis (CHCl3): λmax ¼259 nm; IR(neat)υmax: 3684.38, 3018.72, 2955.76, 2993.54, 2400.25, 2851.33, 1673.41, 1462.59, 1377.49, 1215.47, 1024.10, 761.30, 668.87 cm  1; ESI-TOF-MS: m/z 386.2166 [M] þ (calcd for C23H30O5, 386.21); 1H NMR and 13C NMR data are listed in Table 4. 5-hydroxyl-armillarivin (2): colorless powder, m.p. 214–216 °C; [α]D (0.96 mg/mL, CHCl3); UV/Vis (CHCl3): λmax 20 ¼ þ20.83 ¼257 nm; IR(neat)υmax: 3415.95, 3121.78, 2827.86, 2613.80, 1717.84, 1667.42, 1567.35, 1446.52, 1285.55, 1188.17, 980.01, 743.36, 610.76 cm  1; ESI-TOF-MS: m/z 400.1963 [M] þ (calcd for C23H28O6, 400.19); 1H NMR and 13C NMR data are listed in Table 4.

2.2. Fungal material

2.5. Cell lines and cell culture

A. mellea strain was purchased from the Institute of Microbiology Chinese Academy of Science (50,063). The voucher specimen (No. 2,008,016) was deposited in the National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun, China. The strain was first cultured

Human hepatocellular carcinoma cell line (HepG2) and human normal liver cell line (L02) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). They were maintained in DMEM media with 10% fetal bovine serum (TBD Science, Tianjin, China), 100 U/mL penicillin and 100 mg/mL

2. Materials and methods 2.1. General experimental procedures

Table 1 The method, equipment and company of general experimental procedures. Method

Equipment

Company

NMR LC-ESI-TOF-MS IR Spectrum UV Preparation HPLC

Bruker AV-400 spectrometers Agilent 1200 HPLC/MicrOTOFII Nicolet 6700 Fourier transform infrared spectrophotometer (FT-IR) Lambda 3B UV/vis-spectrophotometer Waters PrepHPLC system with a Waters 2487 UV detector and Delta-Pak™ C18 25 mm  100 mm column Silica gel GF254 plates (20  20 mm2, 1 mm thick)

Bruker corporation, Switerland Agilent Technologies Thermo Scientific Fisher Perkin-Elmer Waters Corporation, Milford, MA, USA

Preparation TLC

UV of TLC 2F  1B UV analysis Column chromatography Silica gel, 200–300 mesh Sephadex LH-20 gel, 40–70 mm MTT test Immunofluorescence Flow cytometry Western blot DAPI Data analysis

Micro ELISA reader BD Pathway 855 Bio Cellular Imaging BD FACS Calibur MicroChemi OLYMPUS 60 ImageJ/ Microsoft Excel/ SPSS

Qingdao Marine Chemical Factory, Qingdao, China Shanghai BiLang company Qingdao Marine Chemical Factory, Qingdao, China Amersham Pharmacia Biotech AB, Uppsala, Sweden Bio-Rad, Hercules, CA BD company BD company DNA Biology system company OLYMPUS National Institutes of Health, Microsoft, SPSS

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Table 2 The process of extraction and purification. Start material

Separation method

Resulting fraction/Compound

Air-dried–Armillaria mellea (A. mellea)–(3.5 kg) Crude residue of CHCl3 extraction (280.5 g) AM4 (36.5 g) AM4-4(22.6 g) AM4-4-2 (9.5 g) AM4–2–1(181.0 mg) AM4-4-2-1-1 (170.5 mg) AM4-4-2-1-1-3(162.1 mg) AM4-4-2-1(181.0 mg) AM4-4-2-1-2 (50.3 mg) AM4-4-2-1(181.0 mg) AM4-4-2-1-3 (84.6 mg) AM4-4-2-1-3_3(53.9 mg) Crude residue of CHCl3 extraction (280.5 mg) AM5 (81.3 g) AM5-4(12.0 g) AM5-4-4 (2.5 g) AM5-4-4-3(149.6 mg) AM5-4(12.0 g) AM5-4-2 (3.8 mg) AM5-4-2-3(94.0 mg) AM5 (81.3 g) AM5-5(15.9 g) AM5-5-2 (11.1 g) AM5-5-2-2(3.2 g) AM5-5-2–2-1 (295.80 mg) AM5-5-2–2-1 (295.80 mg) AM5-5-2-2-1-3(50.78 mg) AM5-5-2-2(3.2 g) AM5-5-2-2-2 (900.30 mg) AM5-5-2-2-2-1, AM5-5-2-2-3 AM5-5-2-2-2 (900.30 mg) AM5-5-2-2-2–4(706.0 mg) AM5-5-2–2-4-2 (160.4 mg) AM5-5-2-2-2-4-2-1(102.3 mg) AM5-5-2-2-3 (1.4 g) AM5-5-2-2-3-1(558.9 mg) AM5-5-2-2-3-1-3 (67.7 mg)

Three times CHCl3 extraction, RT Silica gel, 10  10 cm2, 200–300 mesh, CHCl3–MeOH ¼ 96:4

Crude residue of CHCl3 extraction (280.5 g) AM4 (36.5 g)

Silica gel, 5  40 cm2, 200–300 mesh, PE-CHCl3 ¼ 20:80 Silica gel, 4.6  40 cm2, 200–300 mesh, PE-EtOAc¼ 90:10 Silica gel, 4.6  40 cm2, 200–300 mesh, PE-EtOAc¼10:1 PTLC, 20  20 cm2, PE-EtOAc¼ 5:1 PTLC, 20  20 cm2, PE-acetone¼4:1 PTLC, 20  20 cm2, PE-EtOAc¼ 5:1PHPLC, 90% MeOH-H2O, 230 nm TR ¼6 min, 1.0 mL/min PTLC, 20  20 cm2, PE-EtOAc¼ 5:1 PTLC, 20  20 cm2, CHCl3-EtOAc¼1:1 PTLC, 20  20 cm2, PE-EtOAc¼ 5:1 PTLC, 20  20 cm2, PE-EtOAc¼ 5:1 PTLC, 20  20 cm2, CHCl3-EtOAc¼1:1 Silica gel, 10  100 cm2, 200–300mesh, CHCl3–MeOH ¼94:6

AM4 4(22.6 g) AM4-4-2 (9.5 g) AM4-4-2-1(181.0 mg) AM4-4-2-1-1 (170.5 mg) AM4-4-2-1-1-3(162.1 mg) Compound 3 (2.3 mg) AM4-4-2-1-2 (50.3 mg) Compound 1 (6.9 mg) AM4-4-2-1-3 (84.6 mg) AM4-4-2-1-3-3(53.9 mg) Compound 5 (14.5 mg) AM5 (81.3 g)

Silica gel, 10  100 cm2, 200–300mesh, CHCl3–MeOH ¼94:6 LH-20, 5  40 cm2, CHCl3–MeOH ¼ 1:1 PTLC, 20  20 cm, CHCl3-EtOAc¼ 1:1 PTLC, 20  20 cm2, CHCl3–EtOAc¼ 1:1 LH-20, 5  40 cm2, CHCl3–MeOH ¼ 1:1 PTLC, 20  20 cm2, PE-EtOAc¼ 1:1 PHPLC, 80% MeOH–H2O, 230 nm TR ¼7.6 min, 1.0 mL/min Silica gel, 10  100 cm2, 200–300 mesh, CHCl3–MeOH ¼90:10 Silica gel, 5  40 cm2, 200–300mesh, CHCl3–MeOH ¼10:1 LH-20, 5  40 cm2, CHCl3-MeOH ¼1:1 Silica gel, 4  40 cm2, 200–300 mesh, CHCl3 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PTLC, 20  20 cm2, PE-EtOAc¼ 5:1 Silica gel, 4  40 cm2, 200–300 mesh, CHCl3 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PHPLC, 80% MeOH–H2O, 230 nm TR ¼10 min, 1.0 mL/min PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PHPLC, 70% MeOH–H2O, 230 nm TR ¼ 12 min, 1.0 mL/min PTLC, 20  20 cm2, CHCl3-EtOAc¼4:1 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1 PTLC, 20  20 cm2, PE-EtOAc¼ 2:1

AM5-4(12.0 g) AM5-4-4 (2.5 g) AM5-4-4-3(149.6 mg) Compound 4 (25.5 mg) AM5-4-2 (3.8 mg) AM5-4-2–3(94.0 mg) Compound 6 (8.1 mg) AM5-5(15.9 g) AM5-5-2 (11.1 g) AM5-5-2-2(3.2 g) AM5-5-2-2-1 (295.80 mg) Compound 9 (24.3 mg) AM5-5-2–2-1–3(50.78 mg) Compound 2 (4.50 mg) AM5-5-2-2-2 (900.30 mg) AM5-5-2-2-2–1, AM5-5-2-2-2–3 Compound 8 (6.2 mg) AM5–5-2-2-2–4(706.0 mg) AM5–5-2–2-2–4-2 (160.4 mg) AM5-5-2–2-2–4-2–1(102.3 mg) Compound 10 (12.39 mg) AM5–5-2–2-3–1(558.9 mg) AM5–5-2–2-3–1-3 (67.7 mg) Compound 7 (8.3 mg)

TR: time retention. CHCl3: Trichloromethane; PE: Petroleum ether; MeOH: Methanol; EtOAc: ethyl acetate.

Fig. 1. The key HMBC correlations of Compounds 1 (A) and 2 (B).

streptomycin (Ameresco, US) in 5% CO2 at 37 °C. 2.6. MTT assay Compounds 1–10 were further screened using the MTT assay. Melleolide samples were dissolved in DMSO to make 10 mg/mL stock solutions. Cells were plated in 96-well plates (0.8  104 cells/ well) in 100 mL of growth media and permitted to grow for 24 h. The cells were then treated with the isolated compounds (0, 0.1, 1, 10, 100 mg/mL) in the presence of 3% serum (in order to avoid possible interference arising from the complicated composition of the serum). The incubation time for all drugs was 48 h. After

incubation, 20 μL of MTT solution in PBS at a concentration of 5 mg/mL was added and the plates were incubated for another 4 h at 37 °C, followed by removal of the culture media containing MTT and addition of 100 μL of DMSO to each well to dissolve the formazan crystals formed. Finally, the plates were shaken for 10 minutes, and the absorbance of formazan product was measured at 570 nm by a micro ELISA reader (Bio-Rad, Hercules, CA). Cell viability was expressed as a percentage of the control and shown as the mean 7standard deviation (S.D.), and IC50 values were determined by calculation.

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Table 3 Chemical structures of compounds 1–10.

Table 5 Cytotoxic Activity of Compounds for HepG2 and L02 Cell Lines. Compound IC50 values (μg/mL)

1 2 3 4 5 6 7 8 9 10

R1

R2

R3

R4

R5

R6

H CHO CHO CHO H CH2OH CHO H CHO CHO

OH H OH OH Octadec-9-enoic ester OH OH OH OH OH

H H H OH H H OH H H H

OCH3 OH OCH3 OH OH OCH3 OCH3 OH OCH3 OH

H H Cl H H H H H H H

H OH H H H H H H H H

Table 4 1 H and 13C NMR data for compounds 1 and 2 in CDCl3. Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 5′–OCH3 1

1

2

δH ( J in Hz)

δc

5.45 d (10.2) 5.67 d (10.2)

135.2 124.7 73.5 79.2 32.3

5.12 t 1.98 m 1.72 m

37.6 21.8 44.0 41.9

1.27 s 2.17 m 1.45 m 1.36 m

38.0 47.4

1.82 m 1.45 m 2.67 m 0.99 s 1.02 s

38.7 31.8 31.6 172.2 104.7 165.8 98.8 164.2 111.3 143.5 24.5 55.3

11.66 s 6.28 d (2.4) 3.80 s 6.33 d (2.4)

H NMR recorded at 400 MHz,

13

δH ( J in Hz)

δc

9.34 s

193.9 135.7 159.0 45.3 102.7 44.4

6.81 s 3.23 s 2.26 m 2.26 m 1.38 s 2.26 m 1.45 m 0.94 m 2.00 m 1.69 m 3.00 t 0.96 s 1.03 s

6.38 s 6.40 s

C NMR recorded at 100 MHz.

30.7 26.2 45.5 42.3 37.5 47.1 40.5 31.9 31.7 161.7 106.7 160.0 101.7 158.1 114.5 146.0 21.9

1 2 3 4 5 6 7 8 9 10

HepG2

L02

– 18.03 7 6.03 13.377 2.69 12.26 7 3.02 – – 13.25 7 0.95 15.63 7 3.35 37.65 7 3.46 4.95 7 1.79

– 22.707 1.42 12.157 0.95 31.95 7 1.05 – – 18.00 73.80 14.38 7 3.60 69.10 75.86 16.05 7 2.89

Ratio of IC50 values (L02 to HepG2)

1.26 0.91 2.61 1.36 0.92 1.84 3.24

“-”: no significant effect.

2.7. Mechanistic studies of melleolide cytotoxicity 2.7.1. Western blot analysis The HepG2 cells were plated to give a concentration of 6  104 cells in 2 mL of DMEM in a 6-well plate. HepG2 cells were treated with melleolide (0, 0.5, 1, 2, 4 mg/mL) in the presence of 3% serum for 12 h. After 12 h, cells were collected and rinsed twice with phosphate-buffered saline (PBS). Cell extracts were prepared with lysis buffer (1% Nonidet P-40; 50 mmol/L Tris–HCl, pH 7.5; 1 mmol/L phenylmethylsulfonyl fluoride; 150 mmol/L NaCl; 1 mmol/L NaF; 1 mg/mL aprotinin; 4 mg/mL leupeptin) for 30 min, with occasional rocking, followed by centrifugation at 12,000 rpm for 15 min at 4 °C. Identical amounts (100 mg of protein) of cell lysate were solved by 10% sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE), and the resolved proteins were electrophoretically transferred to a polyvinylidene fluoride membrane and blocked with 5% fat-free dry milk in TBST (20 mmol/L Tris–HCl, pH 7.6; 0.02% Tween-20; 150 mmol/L NaCl) for 1 h at room temperature. The membrane was immunoblotted with antibodies that are about apoptosis in 5% defatting milk in TBST. First, the antibodies were incubated overnight at 4 °C. The membranes were washed three times with TBST, incubated with horseradish peroxidase-conjugated secondary antibodies for 40 min at room temperature, and washed extensively before detection. The membranes were subsequently developed using enzymatic chemiluminescence (ECL) reagent (Beyotime, Shanghai, China) and exposed to film according to the manufacturer's protocol (Zhang et al., 2015; Li et al., 2013). 2.7.2. Nuclear staining assay HepG2 cells were treated with melleolide (0, 0.5, 1, 2, 4 mg/mL) for 12 h, 24 h, and 48 h, harvested, washed three times with PBS and then fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature. Then, cells were washed three times with PBS and stained with DAPI for 10 min in the dark at room temperature. After another three washes with PBS, the cells were analyzed under an Olympus BX50 fluorescencemicroscope

Fig. 2. The relative configuration of Compounds 1 (A) and 2 (B).

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Fig. 3. (a) Western blot analyses of cleaved-caspase 3, caspase 8, caspase 9 and bax expression levels in cell lysates from melleolide (0, 0.5, 1, 2, 4 mg/mL) treated HepG2 cells. Cells were treated for 12 h. (b) Western blotting data were quantified using Image J software. The results are presented as the mean 7 SD of three independent experiments. *po 0.05,**p o 0.01 determined by Student’s t-test, reprisents significant differences of treated vs. the control group.

Fig. 4. The effect of melleolide on the morphology of the nuclear chromatin in HepG2 cells. HepG2 cells were treated with 0, 1 and 4 mg/mL melleolide in serum-free medium for 12 h, 24 h and 48 h, respectively, and fixed and stained with DAPI. The morphological changes in nuclear chromatin were then viewed under a fluorescence microscope. The arrow points to the apoptotic HepG2 cells. The results were from one experiment representative of three experiments. (magnification, 40  ).

(Olympus Tokyo, Japan) (Yao et al., 2012). 2.7.3. Immunofluorescence detection HepG2 cells seeded on the 96-well plates. After incubation in media for 24 h, HepG2 cells were treated with 0, 0.5, 1, 2, and 4 mg/mL melleolide in the presence of 3% serum for 48 h. The treated HepG2 cells were harvested and washed three times with PBS. The cells were then fixed in 4% paraformaldehyde (PFA) in PBS for 30 min and washed three times with PBS. After washing three times with PBS, the cells were treated with Triton X-100 in PBS for 10 min. Non-specific sites were then blocked with PBS containing 5% bovine serum albumin (BSA) for 30 min at room temperature. Thereafter, the cells were flooded with a solution

containing a specific antibody (anti–Ki67) and incubated at 4 °C overnight. After washing with PBS, the cells were further incubated with FITC-conjugated goat anti-rabbit IgM (Beyotime) for 30 min at room temperature, followed by washing with PBS. The images were acquired using BD Pathway 855 Bio Cellular Imaging (Yang et al., 2012). 2.7.4. Flow cytometry analysis of cell cycle Flow cytometry was used to evaluate the number of cells in specific phases of the cell cycle. The cell cycle distribution was determined by staining DNA with propidium iodide (PI). HepG2 cells were plated at a density of 1  106 cells/well. After treatment, the cells were collected, washed twice with ice-cold PBS buffer

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Fig. 5. Flow cytometry analysis of melleolide-treated HepG2 cells on cell cycle distribution. Cells were treated with 0 (A), 2 (B), 4 (C) mg/mL. After 48 h, cells were collected and stained with PI. (D) Percentages of cells in different phases of the cell cycle were measured by flow cytometry. The results are presented as the mean7 S.D. of three independent experiments.*p o 0.05,**po 0.01 versus the control group.

(pH 7.4), fixed with 70% alcohol at 4 °C overnight and then stained with PI (20  ) in the presence of RNase A (50  ) for at least 30 min. The percentages of cells in different phases of the cell cycle were measured with a flow cytometer (Beckman Coulter Epics) and the percentage of cells in G0/G1, S and G2/M phases were analyzed with the MultiCycle software (Yang et al., 2012). 2.7.5. Flow cytometry analysis of apoptosis Flow cytometry analysis of HepG2 cell apoptosis induced by melleolide was conducted using Annexin V/PI staining. The extent of apoptosis was measured with the annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, Jiangsu, China) as described by the manufacturer's instruction (Zhang et al., 2013). The samples were then analyzed with MultiCycle software. 2.8. Statistical analysis From more than three independent experiments, results were obtained. Statistical analysis of data was shown by the Student’s ttest.*Po0.05 and **P o 0.01 are two significance levels were set. Error bars express the standard deviation (S.D.).

3. Results and discussion Two new protoilludane sesquiterpene aryl esters (compounds 1 and 2) and eight analogues (Table 1) were separated from a CHCl3 extract of the mycelium of A. mellea. The known compounds were identified as armillaridin (3) (2.3 mg) (Yang et al., 2013; Yang, 1984), armillartin (4) (25.5 mg) (Yang et al., 1991a, 1991b, 117– 122), armillarin (5) (14.5 mg) (Yang et al., 1992), melleolide B (6) (8.1 mg) (Yang et al., 1992), armillarilin (7) (8.3 mg) (Yang, 1984; Yang et al., 1992), armillasin (8) (6.2 mg) (Yang et al., 1992), armillarigin (9) (24.3 mg) (Yang et al., 1992) and melleolide (10) (12.39 mg) (Yang et al., 1992; Midland et al., 1982). By comparison of their NMR spectroscopic data (Table S1–5, Figs. S13–S28) with those reported in the literature, these known compounds were identified without any difficulty. Compound 1 (6.9 mg) was obtained as a colorless powder. The ESI-TOF-MS spectrum showed a molecular ion peak at m/z 386.2166 [M] þ (calcd 386.21), consistent with a molecular formula of C23H30O5. The 1H NMR and 13C NMR spectroscopic data showed prominent signals of protoilludane sesquiterpene aryl ester (Table 4, Figs. S1–S6). Compound 1 closely resembled those of armillasin (8) (Yang, 1984), except for a remarkable singlet signal at δ

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Fig. 6. (a) Immunofluorescense detection of ki67 in melleolide-induced apoptosis. HepG2 cells were incubated with 0, 0.5, 1, 2, 4 mg/mL melleolide for 48 h. Cells were stained by an antibody recognizing activated Ki67 (green fluorescence), and the nuclei were visualized with the nuclear dye DAPI (blue fluorescence). The results were from one experiment representative of three experiments. (magnification, 20  ). (b) Immunofluorescense detection data were quantified using Image J software. The results are presented as the mean7 SD of three independent experiments.*p o 0.05,**p o0.01 determined by Student’s t-test, reprisents significant differences of treated vs. the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

H 3.80 ppm, which is the characteristic signal from a methoxy group. This newly appearing methoxy group was assigned to the C-5′ position because of the HMBC correlation between  OCH3 and C-5′ (Fig. 1A and Fig. S4). As discussed above, compound 1 was named as 5′-methoxy-armillasin (Table 3). Compound 2 (4.5 mg) was obtained as a colorless powder. The ESI-TOF-MS spectrum showed a molecular ion peak at m/z 400.1963 [M] þ (calcd 400.19), consistent with a molecular formula of C23H28O6. The 1H NMR and 13C NMR spectra (Table 4, Figs. S7– S12) displayed signals similar to those of 4, except that the hydroxyl groups of C-4 and C-13 in 4 was replaced by H [δ H 3.23, s; δ C 45.3; δ H 3.00, t; δ C 40.5], an assertion which was supported by HMBC correlations between the H-4/C-2, H-4/C-5 and H-13/C12, H-13/C-3positions (Fig. 1B and Fig. S10). There were no signals of δH 4–5 in 1H NMR and δC 70–80 in 13C NMR, and there was a new signal of δC 102.7 in 13C NMR. The δC 102.7 belong to C-5 as confirmed by HMBC spectra (Fig. 1B and Fig. S10). Therefore, the R6 group was deemed as a hydroxyl. Compound 2 was named 5-hydroxyl- armillarivin (Table 3). The structure of these two novel compounds 1 and 2 are similar with previous protoilludane sesquiterpene aryl esters. As shown in Table 3, the aldehyde group on R1, the hydroxyls on R2 and R4, and the methoxy group on R4 can be found frequently in many sesquiterpene aryl esters. The relative configurations of the two new compounds were confirmed by NOESY spectra. The NOESY spectrum of compound 1 (Fig. S5) showed correlation between CH38 (1.27) and H9 (2.17), and between CH38 (1.27) and H5 (5.12), as well as between H9 (2.17) and H13 (2.67). From the above, the relative configuration of compound 1 is shown in Fig. 2A. In the same way, the NOESY correlation (Fig. S11) between CH38 (1.38) and H9 (2.26), between H9 (2.26) and H13 (3.00), and between CH38 (1.38) and H4 (3.23) discovered the relative configuration of compound 2 as shown in Fig. 2B. Armillaria mellea is an important traditional Chinese medicine with the function of suppressing hyderactive liver for calming

endogenous wind medicine. We hypothesized that the protoilludane sesquiterpene aryl esters separated from Armillaria mellea have the cytotoxic activity to liver cells. To test the hypothesis, MTT assay were carried out on human hepatocellular carcinoma cell line (HepG2) and human normal liver cell line (L02). The cytotoxic activity of these protoilludane sesquiterpene aryl esters were shown in Table 5. Among these protoilludane sesquiterpene aryl esters, compounds 3, 4, 7, 8 and 10 were active constituents with relative less IC50 values. Compound 10, melleolide, was more effective against HepG2 than others with the IC50 value of 4.95 mg/mL. On the other hand, melleolide has the biggest ratio of IC50 values (L02 to HepG2). It indicates that melleolide showed good cytotoxic activity to cancerous liver cell line HepG2 and no cytotoxicity to normal liver cell line L02. As far as we know, there are no other activities reported for melleolide, except inhibitory activity against K-562 (Bohnert et al., 2011) and Gram-positive bacteria in vitro (Kin et al., 2008). We reported here for the time, that melleolide showed anti-tumor ability on hepatoma cell. With the cytotoxic activity data in hand, it seems a logical step forward to study the mechanism. Melleolide was selected as a key compound for further study. It has been recognized that the Bcl-2 family plays crucial roles in regulating apoptosis by functioning as promoters (e.g., bax) of cell death (Chao and Korsmeyer, 1998). To assess the molecular mechanism of apoptosis induced by melleolide, we examined the expression of a pro-apoptotic protein bax. Fig. 3a shows a dosedependent increase of bax protein induced by melleolide. Because activation of caspases is affected by bax, we next investigated the involvement of caspase 3, caspase 8 and caspase 9 in melleolideinduced apoptosis. The results in Fig. 3 indicated that melleolide may cause apoptosis in HepG2 cells in part by increasing these caspase proteins activity. In addition, the different concentration of melleolide showed the different degree of up-modulation of bax protein and/or caspase proteins. It indicates that the melleolideinduced apoptosis on HepG2 cells are dose-dependent. The induction of apoptosis by melleolide in HepG2 cells was

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Fig. 7. Flow cytometry analysis of HepG2 cell apoptosis induced by melleolide using Annexin V/PI staining after 48 h. Cells were treated with 0 (A), 2 (B), 4 (C) mg/mL. (D) Percentages of cells in different phases of the cell cycle were measured by flow cytometry. The results are presented as the mean 7 S.D. of three independent experiments.*p o 0.05,**po 0.01 versus the control group.

further confirmed in fluorescence photomicrographs of HepG2 cells stained with DAPI after treatment with 1 or 4 mg/mL melleolide for 12, 24 and 48 h (Fig. 4 and S11). The morphological features of apoptosis, condensation of chromatin, and fragmentation of the nucleus were examined. As shown in Fig. 4, Control cells (0 mg/mL) presented round and homogeneous nuclei, whereas melleolide-treated cells showed condensed and fragmented nuclei (arrows). To examine whether the anti-proliferative effect of melleolide is associated with cell cycle arrest, we tested the effect of melleolide on cell cycle distribution by flow cytometry (Fig. 5A–C). In the control group (0 mg/mL), cell populations in the G0/G1, S and G2/M phases were 57.22%, 33.83% and 8.94%, respectively. After 48 h of incubation with 4 mg/mL of melleolide, the percentage of G0/G1 phase cells was almost unchanged, while the cell population in the G0/G1, S and G2/M phases changed to 62.31%, 15.42%, and 22.27%, respectively (Fig. 5D). The above data showed that the proportion of G2/M phase cells was significantly increasing. Such changes in the cell cycle distribution showed that melleolide induced cell cycle arrest at the G2/M phase.

Ki67 is a nuclear protein and is widely used as a cell proliferation marker (Li et al., 2014). We performed Ki67 staining on HepG2 cells for 48 h with five different concentrations of melleolide (0, 0.5, 1, 2, 4 mg/mL) by immunofluorescence. As shown in Fig. 6, treatment of melleolide induced a dramatic reducing of Ki67 activity in HepG2 cells at 48 h. The level of green fluorescence was inversely proportional to drug concentration (Fig. 6B). These results suggest that melleolide could kill HepG2 cells by antiproliferation. The annexin V-FITC apoptosis detection kit was then employed to examine the influence of melleolide on HepG2 cell apoptosis by flow cytometry. As shown in Fig. 7A–C, HepG2 cells bound to annexin V-FITC highly increased in a concentration-dependent manner after treatment with melleolide (Q2: 8.95% to 28.29%, Fig. 7D). To summarize, dots were dispersed and shifted to the Q2 side in a dose-dependent manner when HepG2 cells were treated with melleolide, indicating that the cells moved to the late apoptotic stage. These experimental results also demonstrate that melleolide induced apoptosis of HepG2 cells. From the above evidence, we can conclude that melleolide played a role in

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inducing apoptosis in HepG2 cells.

4. Conclusions Two new and eight known protoilludane sesquiterpene aryl esters were isolated from the mycelium of A. mellea. The relative configuration of compound 1 and 2 were identified by NOESY. Compounds 2, 3, 4, 7, 8, 9 and 10 showed better cytotoxic activity than other compunds. Melleolide (10) had the lowest IC50 values (4.95 71.79 μg/mL) on HepG2 cells, and a relative higher IC50 values on L02 cells. No anti-tumor activity on human hepatocellular carcinoma cells was reported for melleolide before this work. We examined the molecular mechanisms involved in melleolide (10) induced apoptosis in human hepatocellular carcinoma HepG2 cells. Melleolide induced apoptosis in HepG2 cells via activation of cleaved-caspase 3, caspase 8, caspae 9, and bax. Moreover, the different concentration of melleolide (10) showed the different degree of apoptosis on HepG2 cells by Western Blot, DAPI, flow cytometry analysis and immunofluorescence detection. So it is concluded the degree of apoptosis of melleolide was dose-dependent. The investigation in the cell cycle distribution showed that melleolide induced cell cycle arrest at the G2/M phase. These findings establish a link between Armillaria mellea and cancer therapy.

Author contribution Zhijin Li contributed to the writing of the paper and revising it for important intellectual content; Yunchao Wang contributed to acquiring the results in Table 1, Fig. 1 and Fig. 2; Bin Jiang contributed to acquiring the results in Table 2; Wenliang Li made substantial contribution to the conception and design of the study in the natural pharmaceutical chemistry; Lihua Zheng and Xiaoguang Yang contributed to acquiring the results in Fig. 3 and Fig. 4; Yongli Bao made an important contributions to the the analysis and interpretation of the results in Fig. 4; Luguo Sun contributed to acquiring the results in Fig. 5; Yanxin Huang contributed to acquiring the results in the data of UV, IR and Optical rotation; Yuxin Li made contribution to the conception and design of the study for bioactivities.

Conflict of interest The authors declare no conflict of interest in the present study.

Acknowledgments This work is supported by the National Natural Science Foundation of China (Nos. 51403031, 31170324 and 31070318), by the China Postdoctoral Science Foundation (No. 2014M550163, 2015T80281) and by the Research Funds from Science & Technology Department of Jilin Province (Nos. 20140520049JH, and 20130201008ZY).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2016.02.044.

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