Mushrooms are a prolific source of bioactive metabolites [1]. Gymnopilus aeruginosus, called magic blue gym in English and “midori-sugitake”, meaning green cedar mushroom in Japanese, belongs to the family Cortinariaceae and is widespread in northwest pacific coast including Japan, often found on decaying conifers. Although a bitter taste is known for this mushroom, only four metabolites, psilocybin [2], two gymnopilins A11 and A12, and gymnoprenol A11 [3] are known from this species.

As part of our program to study underexplored organisms as a source of new natural products, cytotoxic and antimicrobial constituents were pursued to identify polyprenols (data not shown). Phaeolschidin F (1, Fig. 1), a new symmetrical bis(styrylpyrone), was isolated as a side product.

Fig. 1
figure 1

Structures of phaeolschidin F (1) and related metabolites

Frozen fruiting bodies (233.7 g) were extracted with 70% EtOH, and the combined extract was fractionated by three successive solvent-solvent partitions using water and n-BuOH, 90% aqueous MeOH and n-hexane, and 60% aqueous MeOH and CH2Cl2 systems, giving water-, 60% aqueous MeOH-, CH2Cl2-, and n-hexane-soluble fractions. The third fraction was sequentially chromatographed on octadecyldimethylsilyl-bonded silica gel and Sephadex LH-20. Fractions exhibiting m/z 547.2 in a positive ion QTOFMS analysis were combined and purified on a pentabromobenzyl-bonded silica gel HPLC column eluted with aqueous acidic n-PrOH, which yielded 1 (3.7 mg, 1.6 × 10–3% wet weight).

The molecular formula of 1 was determined to be C30H26O10 based on a pseudomolecular ion m/z 545.1453 ([M – H], calcd for C30H25O10, 545.1453, Δ0.0 mmu) in a negative ion QTOF HRMS measurement. Eighteen degrees of unsaturation, calculated from the molecular formula, was in good agreement with UV absorption maxima at 490 and 374 nm, indicating an extended conjugated system in 1. Moreover, IR absorption bands were observed at 3172, 1661, 1549, and 1283 cm−1, implying the presence of exchangeable protons, carbonyl, and phenyl functionalities. 1H and 13C NMR spectra in CD3OD (Table 1) exhibited only 10 and 17 resonances, respectively, which were considerably less in number than those expected from the molecular formula. Because resonances at the deshielded regions, for protons at 6.15 ppm and above and carbons at 103.1 ppm and above, had double the intensity of those in the shielded regions, two identical substructures were assumed to be symmetrically arranged in 1. Of six deshielded 1H resonances, a pair of doublet signals (δH 7.27 and 6.55) was mutually coupled with J = 15.9 Hz, suggesting a 1,2-disubstitued E-olefin bond (H7-H8, Fig. 2). In addition, three resonances in doublet, doublet of doublet, and doublet splitting patterns (δH 7.01, 6.92, and 6.76) implied a 1,3,4-trisubstituted benzene ring (H10-H13-H4) as judged from two coupling constants J = 8.2 Hz and 1.6–1.8 Hz.

Table 1 1H and 13C NMR data for phaeolschidin F (1) in CD3OD and DMSO-d6
Fig. 2
figure 2

Selected COSY (bold lines) and HMBC (arrows) correlations for 1

COSY, TOCSY, and HSQC correlations supported these two units and further identified a n-butylidene chain (=CH15-CH216-CH217-CH318, Fig. 2). One of the three substituents on the aromatic ring proved to be the disubstituted E-olefin by HMBC correlations from the olefinic proton H8 to a non-protonated sp2 carbons (δC 128.9: C9) and two aromatic methine carbons C10 and C14, thus forming a styryl unit. The remaining two substituents were found to be both phenolic hydroxy groups, which were supported by HMBC correlations in DMSO-d6 from two exchangeable protons to two oxygenated aromatic carbons (δC 147.2: C11 and 145.6: C12) and to two aromatic methine carbons C10 and C13 (Fig. 2). The protons at the open termini of the styryl (H7) and n-butylidene units (H15) showed HMBC correlations to the same carboxy carbon (δC 169.2: C2). Additionally, H7 was correlated with an oxygenated aromatic carbon at δC 159.6 (C6) and an aromatic methine carbon (δC 103.1: C5), while H15 coupled with another oxygenated aromatic carbon at δC 170.7 (C4) and an aromatic tertiary carbon (δC 105.7: C3), suggesting a γ-oxy-α-pyrone substructure between the olefin and butylidene units. This was corroborated by HMBC correlations within the pyrone ring, from H5 to C3, C4, and C6. A broad exchangeable proton (δH 11.65), attributed to a hydroxy group on the pyrone, was observed in DMSO-d6 (Table 1), thus completing the structure of 1.

Styrylpyrones, represented by kavalactones [4] or mushroom luciferin [5] (Fig. 1), are unique to plants [6] and mushrooms [7]. As often compared to lignin, these polyphenols are diversified by oligomerization of the monomeric members or by fusion with phenylpropanoids or isoprenoids, and exhibit a variety of bioactivities commonly seen in flavonoids. Within this metabolite class, symmetrical bis(styrylpyrone) derivatives are distinguished from others by incorporation of an aliphatic chain, the origin of which is biosynthetically intriguing. Seven metabolites precede 1 (Fig. 1), squarrosidine from Pholiota squarrosa (Agaricales) [7], pinillidine from Phellinus pini (Hymenochaetales) [7], Inonotus xeranticus [8], and Phellinus sp [9], phaeolschidins A–D from Phaeolus schweinitzii (Polyporales) [10], and rheadinin from Inonotus rheades (Hymenochaetales) [11].

As expected from the previous study on phaeolscidins A–D [1], 1 exhibited antioxidant activity, inhibiting Fenton reaction-initiated chemiluminescence of luminol [12] by 50% at 2 μM (Fig. S20). This was more potent than a flavonoid, quercetin, requiring 8 μM to show the same extent of extinction. Trolox equivalent antioxidant capacity of 1 was evaluated to be 1.65 (Fig. S21). However, 1 at 10 μM induced nicking of a plasmid DNA in the presence of CuCl2, a Fenton catalyst, which demonstrated pro-oxidant capacity (promoting oxidative damage) of 1 [13] (Fig. S22). Quercetin at the same concentration was more detrimental, inducing a linearized plasmid. Collectively, 1 is concluded to be a redox catalyst having more anti-oxidant and less pro-oxidant activities than quercetin [14]. Detailed studies on its anti-oxidant/pro-oxidant properties are now underway.