Isohericenone, a new cytotoxic isoindolinone alkaloid from Hericium erinaceum

Abstract

In our continuing search for structurally interesting and cytotoxic metabolites from Korean wild mushrooms,1–5 we have collected scores of Korean mushroom species annually and evaluated their MeOH extracts for their antitumor activity in our screening test. Among the collected wild mushrooms, the extract of Hericium erinaceum showed significant cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT15 cell lines using a sulforhodamine B (SRB) bioassay. This mushroom H. erinaceum (Yamabushitake in Japanese) belonging to the family Hericiaceae is widely known as edible mushroom that grows on dead trunks of hard woods in Korea, Japan, China and Europe. This mushroom has been used as a medicine for the treatment of dyspepsia, gastric ulcer and enervation in traditional Chinese medicine for a long time.6 This medicinal mushroom is a rich source of unique metabolites. Chemical components of this mushroom have been reported to have hericenones A and B as cytotoxic constituents,7 hericenones C, D and E as stimulators of nerve growth factor synthesis,8 hericenone J as an endoplasmic reticulum stress-suppressive substance,9 hericerin as a pollen growth inhibitor10 and antitumor-active polysaccharides11 as the bioactive components. A bioassay-guided fractionation and chemical investigation of its MeOH extract resulted in the isolation of a new isoindolinone alkaloid named isohericenone (1), together with nine known compounds, namely isohericerin (2),6 erinacerin B (3),12 hericenone A (4),7 hericenone J (5),9 3,4-dihydro-5-methoxy-2methyl-2-(40-methyl-20-oxo-30-pentenyl)-9(7H)-oxo-2H-furo[3,4-h] benzopyran (6),13 erinacerin A (7),12 hericenone F (8),14 hericenone D (9)8 and hericenone E (10)8 (Figure 1). Here, we describe the isolation and structural elucidation of (1) as well as the cytotoxic activities of compounds 1–10. The half dried fruiting bodies of H. erinaceum (5.0 kg) were extracted with 80% aqueous MeOH twice at room temperature and then filtered. The filtrate was evaporated under vacuum to afford a MeOH extract (500 g), which was partitioned with hexane, CH2Cl2, EtOAc and n-BuOH subsequently with H2O, yielding hexane (63.3 g), CH2Cl2 (4.5 g), EtOAc (2.0 g) and n-BuOH fractions (17.5 g). Each fraction was evaluated for its cytotoxicity against A549, SK-OV-3, SKMEL-2 and HCT-15 cell lines using a SRB bioassay. We selected the hexane-soluble and CH2Cl2-soluble fractions for the current phytochemical investigation, because the CH2Cl2-soluble fraction was the most active and hexane-soluble fraction also had significant cytotoxicity against the tested tumor cell lines. The active hexane-soluble fraction (60 g) was separated on a silica gel column with a gradient solvent system of hexane-EtOAc (50 : 1–1 : 1) to give five fractions (H1–H5). Fraction H1 (11 g) was separated on a RP-C18 silica gel column using a gradient solvent of increasing MeOH in H2O from 10 to 100% to give three subfractions (H11–H13). Fraction H13 (5 g) was separated twice on a RP-C18 silica gel column with a gradient solvent system of MeOH-H2O (2 : 3–7 : 3) and then purified by RPC18 preparative HPLC (Econosil RP-18 10m column (Alltech, Nicholasville, KY, USA), 250 10 mm2) using a solvent of MeOHH2O (1 : 1) to yield compound (5) (9 mg). Fraction H2 (12 g) was subjected to repeated RP-C18 silica gel column separation using a gradient solvent system of MeOH-H2O (1 : 1–7 : 3) and then purified by RP-C18 preparative HPLC (60% MeOH) to afford compound (6) (4 mg). Fraction H5 (10 g) was separated on a RP-C18 silica gel column using a gradient solvent of increasing MeOH in H2O from 10 to 100% to give three subfractions (H51-H53). Fraction H51 (1 g) was isolated using repeated RP-C18 silica gel column separation with a gradient solvent system of MeOH-H2O (2 : 3–7 : 3) and then purified by RP-C18 preparative HPLC (60% MeOH) to give compound (9) (38 mg). Fraction H53 (1 g) was subjected to repeated RP-C18 silica gel column separation using a gradient solvent system of MeOH-H2O (1 : 1–7 : 3) and then purified by RP-C18 preparative HPLC (50% MeOH) to afford compound (8) (36 mg). The most active CH2Cl2soluble fraction (4 g) was separated on a silica gel column with a gradient solvent system of CH2Cl2-MeOH (50 : 1–1 : 1) to yield five fractions (C1–C5). Fraction C2 (1 g) was separated on a RP-C18 silica gel column using a gradient solvent of increasing MeOH in H2O from