New Azulene-Type Sesquiterpenoids from the Fruiting Bodies of Lactarius deliciosus

1 Introduction

The genus Lactarius belongs to the family Russulaceae (class Basidiomycetes) and is widely distributed allover the world. Many Lactarius species are edible; chemically, these mushrooms are appreciated for their metabolites, including sesquiterpenes, steroids, nitrogen-containing compounds and other secondary metabolites with e.g. antitumor or antiviral activities [1, 2]. Lactarius deliciosus, L. aureus, L. hatsudake, and others are well known for their colorful pigments, some of which are formed as response to injury of the fruiting bodies [3-5]. Previous chemical and biological investigation of this genus showed that these pigments belong to the group of azulene-type sesquiterpenoids, some of them possessing potent biological activities [6-10]. This class of metabolites is also considered as chemotaxonomic marker of the genus Lactarius [11].

As part of our search for fungal metabolites, we are reporting herein the structures and complete ¹H and ¹³C NMR assignments of two new pigments 1 and 2 from the edible mushroom L. deliciosus collected in the forests near Göttingen (Germany). In addition to these two new compounds, four other pigments (3-6) and three fatty acids were also isolated. Although ¹H NMR data of the unstable dihydroazulene alcohol 3 have been reported [3, 12], to the best of our knowledge, no ¹³C NMR data were published for this compound. We also evaluated the antimicrobial activities of the pigments 1 and 4-6.

2 Results and Discussion

After extraction of the freshly collected mushrooms with methanol and repetitive chromatography on Sephadex LH-20, compound 1 was obtained as a purple amorphous solid, with violet color in solution. Electrospray high resolution mass spectrometry (ESI HRMS) displayed an [M + H]⁺ ion peak at m/z 227.1075, suggesting C₁₅H₁₄O₂ as molecular formula. The ¹H and ¹³C NMR data of 1 (Table 1) were very similar to those reported for lactaroviolin (4) [13], a further Lactarius constituent also isolated in the present investigation. The ¹H-¹H COSY spectrum of 1 exhibited three spin systems of H-2/H-3, H-5/H-6/H-8/H-14 and H-12/H₂-13, indicating that 1 and 4 might have the same azulene substitution pattern. Interestingly, the ¹H NMR spectrum (Table 1) of 1 displayed a proton at δ_H 10.0 (d, J = 2.1 Hz), which was attached to a carbon at δ_C 136.8 (HSQC), excluding thereby an aldehyde. This finding corroborated with the ¹³C NMR spectrum (Table 1) that did not show resonances at lower field of an aldehyde group, but exhibited the characteristic signal of a conjugated carboxyl group at δ_C 170.0. Further comparison of ¹H and ¹³C NMR data of 1 with those of lactaroviolin (4) and 7-acetyl-4-methylazulene-1carboxylic acid [13] were in good agreement with the presence of the carboxy group at C-1. This was supported in the HMBC experiment by long-range couplings between the aromatic proton at δ_H 8.43 (H-2) and the carboxy group (Fig. 2). The HMBC spectrum also exhibited cross-peaks of the olefinic methylene protons at δ_H 5.46 and 5.33 (H₂-12) with the carbon atoms at δ_C 23.3 (C-13), 146.9 (C-11) and 141.2 (C-7), and of the methyl protons at δ_H 2.96 (H-14) with the carbons at δ_C 143.3 (C-10), 148.0 (C-4) and 129.9 (C-5). From the above data, the structure of 1 was established as 7-isopropenyl-4-methyl-azulene-1-carboxylic acid (Fig. 1). Related azulene-1-carboxylic acids have been characterized previously from L. deliciosus [13] and L. hadsudake [8].

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Further compounds were isolated as inseparable orange pigments, which decomposed rapidly. ESI MS exhibited pseudo-molecular ion peaks at m/z 237 [M + Na]⁺, 253 [M + K]⁺, and 467 [2M + K]⁺, consistent with the molecular formula C₁₅H₁₈O. The ¹H and ¹³C NMR spectra indicated, however, that it was a mixture of two isomeric compounds 2 and 3 in a ratio of 1:3. The ¹H and ¹³C NMR spectra of compounds 2 and 3 showed significant differences to those of 1 and 4, notably the signals of two aliphatic methylene groups in 2 and one methylene and one methine group in 3. Carbonyl signals were absent, and the methyl signal of C-14 at the azulene system was shifted upfield. The ¹H NMR spectra of both compounds indicated two additional oxymethylene protons, which were connected with sp³ carbons at δ_C 58.1 and 56.6, respectively, indicating the presence of the dihydroazulene alcohols 2 and 3; the latter is known to be very unstable [3, 6, 12].

We were unable to separate both compounds, however, succeeded to assign all NMR signals in the mixture and elucidated the structures of 2 and 3 unambiguously; the ¹H-¹H COSY spectrum was clearly resolved and indicated three spin systems for each component (see Fig. 2). The different signal intensities in the 1:3 mixture of 2 and 3 allowed us to assign the HSQC and HMBC signals without doubt. For the less intensive peaks (compound 2), the methylene protons at δ_H 4.33 (H₂-15) correlated with the carbons at δ_C 147.4 (C-1) and 125.6 (C-2); the expected correlation with C-9 was observed for compound 3, but not for 2 (Fig. 2). These two carbons in turn correlated with the methylene protons at δ_H 3.20 (H₂-3). HMBC correlation was also observed between C-1 and the olefinic proton at δ_H 6.49 (H-8), which showed further correlations with the carbons at δ_C 28.5 (C-6), 141.7 (C-11), and 145.7 (C-10). A long-range correlation was seen from the methyl at δ_H 1.93 (H-14) to C-10, and to the carbons at δ_C 132.7 (C-4) and 115.8 (C-5). Thereby, the minor component was finally characterized as 15-hydroxy-3, 6-dihydrolactarazulene (2).

In a similar way, the main component was identified as 15-hydroxy-6, 7-dihydrolactarazulene (3). Its structure was further supported by comparison of the ¹H NMR data with those published by Sterner et al. [3]. To the best of our knowledge, the ¹³C NMR data (Table 1) for this compound are reported here for the first time.

Further components of the extract were identified on basis of their NMR data as lactaroviolin (4) [13, 14], 7-acetyl-4-methylazulene-1-carbaldehyde (5) [13], quinizarin (6) [15], stearic acid, and a mixture of oleic and linoleic acid. Among them, quinizarin (6) is reported here from higher fungi for the first time.

Compounds 1 and 4-6 were screened for their antimicrobial activities against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Mucor miehei. Only 7-acetyl-4-methylazulene-1-carbaldehyde (5) displayed a moderate antibacterial activity against S. aureus in the agar diffusion test, with an inhibition diameter of 15 mm at a concentration of 50 μg/disc.

3 Experimental Section

3.1 General Experimental Procedures

The NMR spectra were recorded on a Varian Inova-500 spectrometer at 599.737 MHz (¹H) or 150.818 MHz (¹³C), respectively. The chemical shifts are given in δ values with TMS as internal reference, and coupling constants are given in [Hz]. The ESI and ESI HR mass spectra were recorded on a Bruker micrOTOF mass spectrometer. Open column chromatography was done on silica gel 60 (0.063-0.20 mm), and PTLC was performed on silica gel P/UV₂₅₄ (both obtained from Macherey-Nagel, Düren, Germany). Size exclusion chromatography was performed on Sephadex LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd., purchased from Sigma-Aldrich Chemie, Steinheim, Germany). Pre-coated silica gel plates (Polygram SIL G/UV₂₅₄, Macherey-Nagel & Co.) were used for TLC. Spots were visualized at 254 or 365 nm, and sprayed with an anisaldehyde/sulfuric acid reagent followed by heating.

3.2 Extraction and Isolation

The fruiting bodies of L. deliciosus were collected in forests near Göttingen (Germany) in September 2015. The fresh material (7.8 kg) was grinded and macerated with 2 × 4 L of MeOH at ~20 ℃. The solution was evaporated under reduced pressure to afford a dark brown crude extract (110 g) that was suspended in water and further extracted with ethyl acetate (EtOAc). The evaporation residue (38 g) of the EtOAc fraction was chromatographed on Sephadex LH-20 (column 7 × 60 cm) with MeOH to give four main fractions A-D. According to their ¹H NMR profiles, the colorless fractions A (8.5 g) and B (6.3 g) contained fatty acids and glycerol derivatives, while the orange and violet fractions C (11.7 g) and D (10.5 g), respectively, contained azulene-type sesquiterpenoids. Fraction C was again chromatographed on Sephadex LH-20 (column 4 × 90 cm) and further on silica gel (column, CH₂Cl₂) to afford a mixture of the unstable compounds 2 and 3 (35 mg). Fraction D crystallized from MeOH to yield stearic acid (200 mg). The mother liquor from D was purified again on Sephadex LH-20 (column, CH₂Cl₂/MeOH 1:1) to afford compound 1 (5 mg), quinizarine (6; 2 mg, R_f = 0.90 CH₂Cl₂/MeOH 95:5) and two subfractions D-1 (violet) and D-2 (red). By PTLC (CH₂Cl₂/MeOH 99:1), D-1 gave lactaroviolin (4; 70 mg, R_f = 0.78 CH₂Cl₂/MeOH 95:5). Purification of D-2 on a silica gel (column, CH₂Cl₂/MeOH 98:2) yielded 7-acetyl-4-methylazulene-1-carbaldehyde (5; 7 mg, R_f = 0.72 CH₂Cl₂/MeOH 95:5) and a mixture of stearic, oleic and linoleic acid (150 mg).

3.3 7-Isopropenyl-4-methyl-azulene-1-carboxylic acid (1)

Purple amorphous solid, R_f = 0.41 (CH₂Cl₂/MeOH 95:5); UV (MeOH) λ_max (log ε) 238 (4.10), 300 (4.10), 380 (3.59); IR (film) ν_max 3300-2500 (br, OH), 2920, 1652 (C=O), 1496, 1415, 1252, 890 cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data, see Table 1; (+)-ESI MS m/z 227 ([M + H]⁺), 249 ([M + Na]⁺), 475 ([2M + Na]⁺); (+)-ESI HRMS; m/z 227.1075 (calcd for C₁₅H₁₅O₂ [M + H]⁺, 227.1067).

3.4 15-Hydroxy-3, 6-dihydrolactarazulene (2) and 15-hydroxy-6, 7-dihydrolactarazulene (3)

Orange gum, R_f = 0.30 (CH₂Cl₂/MeOH 95:5); ¹H NMR (DMSO-d₆, 300 MHz) and ¹³C NMR (DMSO-d₆, 125 MHz) data, see Table 1; (+)-ESI MS: m/z = 237 ([M + Na]⁺), 253 ([M + K]⁺), 475 ([2M + K]⁺); (+)-ESI HRMS; m/z 237.1250 (calcd for C₁₅H₁₈ONa [M + Na]⁺, 237.1255).

3.5 Antimicrobial Test

The antimicrobial test was performed according to a previously described procedure [16].

Notes

Acknowledgements

We thank Dr. H. Frauendorf and Dr. M. John for MS and NMR measurements, respectively. JQ thanks the Natural Science Foundation for a Chinese Government Scholarship Fund for Study Abroad (31470414, 20140101126JC).

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.