Hericium erinaceum is a mushroom belonging to the family Hericiaceae and has been used as medicine or food in China and Japan without harmful effects. H. erinaceum grows on old or dead broadleaf trees and has been used as medicine for treatment of gastricism in traditional Chinese medicine for more than 1000 years. Recently, the chemical constituents of H. erinaceum have been investigated for its interesting and significant bioactivities.1, 2 Hericenones A and B were isolated from the fruiting body of H. erinaceum as cytotoxic principles against Hela cells.3 Hericenones C, D and E exhibited stimulating activity to the synthesis of nerve growth factor in vitro.4, 5, 6, 7 In the presence of hericenones C, D, E and H at 33 μg ml−1, mouse astroglial cells secreted 23.5±1.0, 10.8±0.8, 13.9±2.1 and 45.1±1.1 pg per ml nerve growth factor into the culture medium, respectively. The degree of activity for hericenone D was almost at the same level as that of a potent stimulator, epinephrine. It is of interest that the difference in activity among those compounds was dependent on the nature of the fatty acid. These results suggest the usefulness of H. erinaceum for the treatment and prevention of dementia.

In the course of our continuing search for new active compounds from mushroom, we found two new aromatic compounds, namely hericenone I and hericene D. In this report, we described the isolation, structural elucidation and cytotoxic activity of hericenone I and hericene D (Figure 1).

Figure 1
figure 1

Structures of compounds 1–5.

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The CHCl3-soluble fraction of MeOH extract from the fruiting bodies of H. erinaceum was subjected to repeated chromatography to yield hericenone I and hericene D. Hericenone I was obtained as a white oil. The HRESI-MS of hericenone I gave an ion [M+Na]+ peak at m/z 619.6686 (calc. for C37H56O6Na 619.6702) and corresponded to a molecular formula of C37H56O6. The IR spectrum indicated the presence of a hydroxyl group at 3463 cm−1 and an α, β-unsaturated ketone (1669 cm−1). The 13C and DEPT-NMR spectrum of hericenone I was quite similar to that of hericenone D, except for the presence of two more olefinic CH groups (129.7, CH; 130.0, CH).4 The 1H NMR spectrum of hericenone I showed the signals of three methines at δ 5.37 (1H, t, J=7.2 Hz), 6.15 (1H, s) and δ 6.54 (1H, s); four methyls at δ 0.86 (3H, t, J=7.0 Hz), δ 1.78 (3H, s), δ 1.83 (3H, s) and δ 2.16 (3H, s); and one methoxyl at δ 3.91 (3H, s), together with one CHO group at δ 10.20 (1H, s) (Table 1). Methanolysis of hericenone I gave the methyl ester of oleic acid, which was identified by GC-MS. Considering the molecular formula of hericenone I, it was supposed to be an oleic ester of hericenone. The construction of molecular framework was deduced from the 1H–1H COSY, heteronuclear multiple quantum coherence and heteronuclear multiple bond coherence spectra (Figure 2).

Table 1 1H and 13C NMR data of herienone I in CDCl3
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Figure 2
figure 2

Key HMBC correlations of hericenone I.

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Hericene D was also obtained as a white oil. The HRESI-MS of hericene D gave an ion [M+Na]+ peak at m/z 603.4026 (calc. for C37H56O5Na 603.4031) and corresponded to a molecular formula of C37H56O5, requiring 10 degrees of unsaturation. The IR spectrum indicated the presence of a hydroxyl group at 3446 cm−1. The 13C and DEPT-NMR spectrum of hericene D was similar to that of hericene A, except for the presence of four more olefinic CH groups (δ 127.9, CH; 128.1, CH; 130.0, CH; 130.2, CH).6 The 1H NMR spectrum of hericene D showed the signals of three methines at δ 5.05 (1H, t, J=7.0 Hz), 5.17 (1H, t, J=8.0) and δ 6.53 (1H, s); four methyls at δ 0.87 (3H, t, J=7.0 Hz), δ 1.60 (3H, s), δ 1.63 (3H, s) and δ 1.77 (3H, s); and one methoxyl at δ 3.91 (3H, s), together with one CHO group at δ 10.10 (1H, s) (Table 2). Methanolysis of hericene D yielded the methyl ester of linoleic acid, which was also identified by GC-MS. Considering the molecular formula, hericene D was deduced to be a linoleic ester of hericene.

Table 2 1H and 13C NMR data of hericene D in CDCl3
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Compounds 1–5 were tested in vitro for cytotoxic activity against tumor cells by MTT assay. All the compounds showed cytotoxic activity against EC109 cell line, and the cell growth inhibitor percentages of compounds 1–5 on EC109 cell line are shown in Table 3.

Table 3 Cell growth inhibitor percentages of compounds 1–5 on EC109 cell line
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It may be pointed out that hericenone I and hericene D have a structure related to mycophenolic acid, an antibiotic produced by Penicillium spp.6 Hericenone and hericene D were both tested against the bacteria, Staphylococcus aureus, Bacillus thuringiensis, Escherichia coli, Bacillus megaterium and Bacillus subtilis at 1 mg ml−1, respectively. However, hericenone I and hericene D showed only weak antibacterial activity in vitro against the five bacteria above. The damage of the γ-lactone ring in the structures of hericenone I and hericene D may be the main reason for the deficiency of antibacterial activity.

Experimental procedure

General

IR spectra were measured on a Bio-Rad FTS-135 spectrometer with KBr pellets (Bio-Rad, Philadelphia, PA, USA). NMR spectra were recorded on a Brucker AV-400 spectrometer (Bruker Biospin, Fällanden, Switzerland) in CDCl3 solvent, with tetramethylsilane as the internal standard. MS were recorded on VG Auto-spec-3000 (VG, Manchester, UK) and API QSTAR Plusar spectrometer (Applid Biosystems, Foster City, CA, USA).

Silica gel (200–300 mesh, Qingdao Marine Chemical, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography. Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in ethanol.

Mushroom material

The fresh fruiting bodies of H. erinaceum were collected on Funiu Mountain of He’nan Province, People’s Republic of China, in July 2008. The fungal identification was made by Professor Jin-Wen Shen, Henan Agricultural University. A dried specimen was deposited in the Herbarium of Henan Agricultural University.

Extraction and isolation

The air-dried and powdered fruiting bodies of H. erinaceum (dry weight 2.0 kg) were extracted with MeOH at room temperature to afford a deep brown gum (225 g). Then the MeOH extract was diluted with H2O and partitioned with CHCl3. The CHCl3 extract (62 g) was subjected to column chromatography eluting with petroleum ether/EtOAc. The fraction (0.9 g) from petroleum ether/EtOAc (20 : 1, v/v) was submitted for further purification by repeated Sephadex LH-20 (CHCl3/MeOH=1 : 1, v/v) to give hericenone F (85 mg). The fraction (1.2 g) from petroleum ether/EtOAc (19 : 1, v/v) was submitted for further purification by repeated Sephadex LH-20 (CHCl3/MeOH=1 : 1, v/v) to give a mixture of hericenones C and I. Preparative HPLC with ODS column (250 × 30 mm, 5 ml min−1, MeOH/H2O=96 : 4, detection at 298 nm) to give hericenones C (56 mg) and I (18 mg).

The fraction (1.6 g) from petroleum ether/EtOAc (10 : 1, v/v) was submitted for further purification by repeated Sephadex LH-20 (CHCl3/MeOH=1 : 1, v/v) to give a mixture of hericenes A and D. Preparative HPLC with ODS column (250 × 30 mm, 5 ml min−1, MeOH/H2O=95 : 5, detection at 295 nm) to give hericenes A (150 mg) and D (35 mg).

Hericenone I

White oil: UV λmax (CHCl3) 301 nm. IR (KBr) 3463, 2918, 1742, 1669 and 1567 cm−1. 1H and 13C NMR spectra: see Table 1. HRESI-MS m/z 619.6686 (M+Na, calc. for C37H56O6Na 619.6702).

Hericene D

White oil: UV λmax (CHCl3) 296 nm. IR(KBr) 3446, 2923, 1754 and 1571 cm−1. 1H and 13C NMR spectra: see Table 2. HRESI-MS m/z 603.4026 (M+Na, calc. for C37H56O5Na 603.4031).

Methanolysis of Hericene D and Hericenone I

Hericenone I (5 mg) and Hericene D (5 mg) were dissolved with 0.1 M methanolic KOH (5 ml) and stirred overnight at 4 °C. Water was added to the reaction mixture, and the solution was neutralized with 0.1 M HCl and then extracted with hexane. The hexane layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give the methyl ester of oleic acid and the methyl ester of linoleic acid, which were identified by GC-MS.

Cell growth inhibition assay

The growth inhibitor concentrations of compounds 1–5 on esophageal cancer EC109 cell line were determined by MTT assay.8 The EC109 cell was exposed to compounds at 1 × 10−3, 3 × 10−4 and 1 × 10−4 mol l−1 and each concentration was tested in triplicate. The OD was measured with a microplate reader at 570 nm.