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The fruiting bodies of Ganoderma lucidum are widely used in China, Japan and Korea as medicine for a variety of diseases, such as chronic bronchitis inflammation, hyperlipidemia, hypertension, neurasthenia, hepatitis, leukopenia and adjuvant treatment of cancers.1, 2 Ganoderma spores is the fungus's reproductive cells ejected from the cap of G. lucidum after the fruiting bodies become mature. In recent years, with the development of spores collection, sporoderm-broken technology and application of modern analytical instruments, there has been some progress on chemical constituents, pharmacological effects and mechanisms of action of Ganoderma spores. Moreover, the increasing publications in domestic and international suggest the important bioactivities of the spores of G. lucidum.3, 4

The chemical constituents and bioactivities of the fruiting bodies of G. lucidum have been fully investigated, and >150 triterpenes have been reported from the fruiting bodies of G. lucidum representing five major structural classes.5, 6, 7 However, the chemical investigation of the spores of G. lucidum can only be traced to 1988.8 Up to now, a series of new lanostane-type triterpenes, such as ganosporelactones A, B,9 lucidumol A, ganoderic acid β,10 ganoderic acid γ, δ, ɛ, ξ, η, θ11 and lucidenic acid SP,12 were isolated from the spores of G. lucidum.

Sporoderm-broken technology recently has been applied to Ganoderma spores to improve the extraction rate and improve the remedy effect in the clinical trials.13 However, to the best of our knowledge, an article on the chemistry of the compounds isolated from the sporoderm-broken spores of G. lucidum has not been prepared. As a part of our continuing studies to find novel bioactive compounds from mushroom, we found a new highly oxygenated lanostane-type triterpene aldehyde, lucialdehyde E (1) from the sporoderm-broken spores of G. lucidum, together with seven known triterpenes, lucialdehyde D (2), ganodermanondiol (3), ganoderic acid C1 (4), ganodermanontriol (5), genoderic acid ξ (6), ganoderic acid A (7) and lucidumol A (8) (Figure 1). The structural elucidations of all the compounds were based on the spectroscopic evidences and comparing with literature data. Lanostane-type triterpene aldehydes are very few in the Ganodermataceae, only lucialdehydes A–C and lucialdehyde D were isolated from the fruiting bodies of G. lucidum and G. pfeifferi, respectively.14, 15 Compound 1 is a lanostane-type triterpene aldehyde isolated from the sporoderm-broken spores of G. lucidum for the first time. The cytotoxicity of compounds 1–8 was tested in vitro against esophageal tumor EC109 cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Compounds 1, 2, 3, 4 and 7 showed significant cytotoxic activity with IC50 values of 18.7, 21.4, 16.2 and 20.1 μg ml−1, respectively. In addition, the other four triterpenes only showed weak cytotoxic activity with IC50 values of 124.8, 162.6, 148.1 and 121.5 μg ml−1, respectively.

Figure 1
figure 1

Structures of triterpenes 1–8.

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Lucialdehyde E (1) was obtained as a white amorphous powder, with a positive optical rotation, The presence of two conjugated carbonyl groups (1668 and 1660 cm−1) was suggested by IR spectrometry. The high-resolution electron impact mass (HR-EIMS) spectrum of 1 give an ion peak at m/z 484.3106 (calculated for 484.3114) and corresponded to a molecular formula of C30H44O5. 1H-NMR spectrum of 1 showed signals for seven methyls, and a singlet at δ 9.41 for an aldehyde proton (Table 1). The 13C-NMR spectrum of 1 demonstrated signals characteristic for seven methyls, four olefinic carbons, two oxymethylene carbons (δ 68.6, 72.1), and an aldehyde carbon (δ 194.4). The 13C-NMR spectrum of 1 was close to the structure of ganoderic acid γ,11 except for chemical shifts of C-26 (δ 194.4 in lucialdehyde E vs δ 170.2 in ganoderic acid γ) and C-23 (δ 26.0 in lucialdehyde E vs δ 65.9 in ganoderic acid γ). HMBC correlations were observed between signals of H-5 and C-7; H-30 and C-8; H-19 and C-9, 11; H-17 and C-15; H-18 and C-11, 12. In addition, HMBC correlations between signals of H-22 and C-24 (an olefinic carbon at δ 156.3), and between signals of H-24 and C-26, as well as those of H-24 and C-27 revealed a conjugated aldehyde at C-24–C-26 in the side chain. As the signals of H-28 and H-29 were shift-correlated with that of C-3, a carbonyl group was concluded to be at C-3 (Figure 2). Two equatorial hydroxyl groups at C-7 (β-orientation) and C-15 (α-orientation) were deduced from the multiplicities of H-7 and H-15, which was supported by NOE correlations observed from H-7 to H-5, H-29 and H-30. NOE correlations were also observed from H-15 to H-18, H-19, and H-19 to H-28. As NOE correlations were observed between H-24 and H-26, the configuration of C-24 was suggested to be E with respect to an aldehyde group, which was also supported by comparing to the NMR spectral data of ganoderal A having a (24E)-26-al moiety.16 These data above suggested a highly oxygenated lanostane-type Ganoderma triterpene, consequently, the structure of 1 was determined as 7β, 15α-dihydroxyl-3, 11-dioxo-5α-lanosta-8, 24-dien-26-al.

Table 1 1H and 13C NMR data of lucialdehyde E in CDCl3
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Figure 2
figure 2

Key HMBC correlations of lucialdehyde E.

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Some progresses of chemical and pharmacological research have been made on the spores of G. lucidum. In some cases, extracts of partly purified preparations and pure compounds from Ganoderma spores have been used for in vitro or in vivo testing.17, 18 However, as the biological activities of Ganderma spores are determined by the active ingredients contained, the levels of active ingredients vary from the origin, cultivation, acquisition time and extraction methods.19

At present, the spores of G. lucidum have been widely used in China as a home remedy for the adjuvant treatment of cancers, and its medical value has been widely recognized, however, its biological activities still need further assessment before they can be accepted not only by the traditional Asian medicine, but also by the west science and medicine.

Experimental procedure

General

[α]D was carried out on JASCO-20 polarimeter. IR spectra were obtained on a Bio-Rad FTS-135 IR spectrometer in KBr pellets (Bio-Rad, Philadelphia, PA, USA). 1H, 13C NMR and 2D NMR spectra were recorded on a Bruker AM-400 and DRX-500 instruments (Bruker, Fällanden, Switzerland). Chemical shifts are given as δ in p.p.m. relative to tetramethylsilane as internal standard and coupling constants in Hz. Mass spectra were measured with a VG Autospec 3000 mass spectrometer (VG, Manchester, UK).

Mushroom material

The sporoderm-broken spores of G. lucidum were purchased from Zhengzhou market of Henan Province, People’s Republic of China, in August 2010. 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.

Chemicals

Column chromatography was carried out on silica gel (200–300 mesh), and TLC was carried out on plates precoated with silical gel F254 (Qingdao Marine Chemical Ltd., Qingdao, PR China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden). Fractions were monitored by TLC and spots were detected by spaying 10% sulfuric acid in ethanol followed by heating.

Isolation procedure

The sporoderm-broken spores of G. lucidum (8.0 kg) was extracted with a mixed solvent of CHCl3–MeOH (50 : 50 v/v, 20 L × 3) at room temperature, and the combined solutions were evaporated in vacuo to give a residue (462.8 g). The residue was suspended in 90% MeOH (1000 ml) and extracted with hexane (600 ml × 2). The residual MeOH solution was concentrated in vacuo to give a residue (414.2 g), which was applied to a column chromatography of silica gel. Elution was started with CHCl3, and then CHCl3–MeOH (9 : 1, v/v) to yield two fractions (A: 122.0 g, B: 85.5 g). Column chromatography of fraction A on silica gel (petroleum ether–EtOAc, 5 : 1 → 1 : 1, v/v) yielded five subfractions (A1-A5; 9.8, 13.5, 25.4, 24.6 and 16.8 g, respectively). The subfraction A1 was submitted to repeated column chromatography on silica gel by eluting with hexane–acetone (5 : 1, v/v) and repeated Sephadex LH-20 (CHCl3–MeOH, 1 : 1, v/v), followed by preparative HPLC (a linear gradient of CH3CN (75% → 95%) in 2% AcOH, flow rate 5 ml min−1, detection at 235 nm)to afford compounds 2 (14.6 mg), 3 (9.8 mg), 1 (15.9 mg) and 5 (10.2 mg). The subfraction A2 was further purified by column chromatography on silica gel (hexane–acetone, 4 : 1, v/v) and repeated Sephadex LH-20 (CHCl3–MeOH, 1 : 1, v/v), followed by preparative HPLC (a linear gradient of CH3CN (72% → 90%) in 2% AcOH, flow rate 6 ml min−1, detection at 230 nm) to afford compounds 8 (11.7 mg), 6 (20.1 mg), 4 (22.7 mg) and 7 (19.6 mg).

Cell growth inhibition assay

The growth inhibitor concentrations of compounds 1–8 on esophageal cancer EC109 cell line were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.20 The EC109 cell was exposed to compounds at 0.01, 0.1, 1, 10 and 100 mg l−1 concentrations and each concentration was tested in triplicate. The OD was measured with a microplate reader at 570 nm.