Triterpenoids and Steroids from Ganoderma mastoporum and Their Inhibitory Effects on Superoxide Anion Generation and Elastase Release

The methanol extracts of the fruiting bodies of Ganoderma mastoporum collected in Vietnam was purified to afford eight compounds, including three triterpenoids and five steroids. The purified compounds were examined for their inhibitory effects against superoxide anion generation and elastase release. Among the tested compounds, ergosta-4,6,8(14),22-tetraen-3-one (3) exhibited the most significant inhibition towards superoxide anion generation and elastase release with IC50 values of 2.30 ± 0.38 and 1.94 ± 0.50 µg/mL, respectively.


Introduction
The genus Ganoderma (Polyporaceae) are extensively used as a Chinese remedy for the treatment of tumors [1,2], hepatopathy [3], enhancement of splenic natural killer cell activity, and serum interferon production in mice [4]. Species such as G. lucidum attract much attention because it is a well-known source of various therapeutic agents [5,6]. Previous phytochemical investigations of Ganoderma genus afforded a series of triterpenoids and steroids [7][8][9][10][11][12][13]. These principles displayed significant cytotoxicity and anti-inflammatory bioactivity [14,15]. G. mastoporum is a fungus widely distributed in Southern China, Northern Vietnam, Malaysia, Philippines, and reported to contain sesquiterpenoids [16]. Therefore, additional studies of the phytochemical constituents of G. mastoporum and their bioactivities are merited. In our continuous program aimed at the discovery of anti-inflammatory lead drugs from the natural sources, the chemical composition of the fruiting bodies of G. mastoporum collected in Vietnam was investigated to search for the bioactive constituents. In the present study, we wished to report the identification of eight compounds, as well as their inhibitory effects on superoxide anion generation and elastase release.

The Inhibitory Effects of Isolated Compounds on Superoxide Anion Generation and Elastase Release
The purified compounds were subjected to the examination for inhibition of superoxide anion generation and elastase release by human neutrophils in response to N-formyl-L-methionyl-leucylphenylalanine/cytochalasin B (FMLP/CB) [22], and the data are displayed in Table 1. Only compounds 2 and 8 did not display significant inhibition of superoxide anion generation and elastase release. Other tested compounds 1 and 3-7 demonstrated inhibitory effects towards superoxide anion generation and elastase release in a concentration-dependent manner (data not shown). The IC 50 values were in the range between 2.30 ± 0.38 and 6.36 ± 0.36 μg/mL against superoxide anion generation, and between 1.94 ± 0.50 and 5.01 ± 0.82 µg/mL against elastase release, respectively, compared with the reference compound LY294002 (IC 50 of 0.40 ± 0.02 and 1.53 ± 0.25 µg/mL towards superoxide anion generation and elastase release). Among the tested compounds, ergosta-4,6,8(14),22-tetraen-3-one (3) exhibited the most significant inhibition towards superoxide anion generation and elastase release with IC 50 of 2.30 ± 0.38 and 1.94 ± 0.50 µg/mL. From the above results, the following structure-bioactivity relationships were deduced: triterpenoids or steroids possessing 3-hydroxy rather than 3-ketone functionalities, such as 2, 4, 6, and 8, displayed weaker superoxide anion generation inhibitory effects. Comparison of the bioactivity of 2 and 7 also exhibited this tendency. The 24,25-dihydroxy substitutions in 5 and 6, and the 5-hydroxy group in 8 decreased the inhibition of superoxide anion generation. There is similar tendency in the inhibition of elastase release. However, lucidumol B (6) displayed more significant inhibition of elastase release than 5 and 7.  results are presented as mean ± S.E.M. (n = 3-4); * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the control value. c A phosphatidylinositol-3-kinase inhibitor was used as a positive control for superoxide anion generation and elastase release.
The formation of superoxide anion in neutrophils can be inhibited by modulating cellular signaling pathways, but also by direct radical scavenging. Therefore the purified constituents 1-8 were also subjected to assays of DPPH free radical scavenging activities and ferrous ion chelating capability [23]. None of the examined compounds exhibited significant scavenging of free radicals and capability of chelating ferrous ions at the tested concentration (data not shown). It indicated that the inhibition of superoxide anion generation and elastase release by compounds 1 and 3-7 is mediated by modulating cellular signaling pathways [24]. In the previous literature, Δ 1 -lupenone (1) and ergosterol peroxide (4) were reported to exhibit the inhibitory effects on LPS-induced iNOS-dependent NO production in RAW 264.7 cells [25][26][27]. Moreover, ergosta-7,22-dien-3β-ol (2), ergosterol peroxide (4), and ergosta-7,22-dien-3-one (7) also inhibited superoxide anion generation release by rat neutrophils [15]. In the present study, the inhibitory effects of these isolated compounds on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB were reported for the first time.

General Procedure
Melting points were determined using Yanagimoto MP-S3 apparatus (Kyoto, Japan). Optical rotations were measured using a JASCO DIP-370 polarimeter (Oklahoma City, OK, USA). The UV spectra were obtained on a Hitachi UV-3210 spectrophotometer (Tokyo, Japan), and IR spectra were recorded on a Shimadzu FTIR-8501 spectrophotometer (Kyoto, Japan). 1 H-and 13 C-NMR, COSY, NOESY, HMQC, and HMBC spectra were obtained on the Bruker Avance III-400 NMR spectrometer (Fremont, CA, USA), with tetramethylsilane (TMS) as internal standard and the chemical shifts were reported in δ values (ppm). The electrospray ionization (ESI) mass spectrum was determined using an Agilent 1200 LC-MSD Trap spectrometer (Santa Clara, CA, USA). Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, E. Merck, Darmstadt, Germany). Thin layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck) and the compounds were visualized by spraying with 10% (v/v) H 2 SO 4 followed by heating at 110 °C for 10 min.

Fungus Materials
The basidiomycete (Ganoderma mastoporum (Mont) Pat.) was collected at the Puhuong National Park of Nghean Province, Vietnam, in November 2010 and identified by Dr. Ngo Anh, Department of Biology, Hue University. A voucher specimen (Vinh-TSWu 20100805) was deposited at the herbarium of the Department of Chemistry, Vinh University.

Extraction and Isolation
The fruiting bodies of Ganoderma mastoporum (4.3 Kg) were air-dried, powdered, and extracted with methanol (10 L × 3) at ambient temperature, and the combined extracts were concentrated under reduced pressure to give a deep brown syrup (125 g). The crude extract was suspended in water and partitioned with ethyl acetate to afford ethyl acetate (30 g) and water soluble (95 g) fractions, respectively.

Preparation of Human Neutrophils
Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Blood was drawn from healthy human donors (20-30 years old) by venipuncture into heparin-coated vacutainer tubes, using a protocol approved by the institutional review board at Chang Gung Memorial Hospital [22].

Measurement of Superoxide Anion Generation
The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c [22]. Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm).

Measurement of Elastase Release
Degranulation of azurophilic granules was determined by elastase release as described previously [22]. The results were expressed as the percent of elastase release in the FMLP/CB-activated, drug-free control system.

Assay of Scavenging Activity against Diphenyl Picrylhydrazyl (DPPH) Radical
The DPPH free radical scavenging assay was executed according to the reported method [23]. The DPPH radical scavenging activities of the sample solutions were expressed as inhibition percentage and calculated by using the following equation: Inhibition percentage (%) = [1 − (absorbance of samples at 517 nm)/ (absorbance of control at 517 nm)] × 100 (%).

Chelation of Ferrous Ions
The capability of chelating ferrous ions was determined by the reported method with some modifications [23]. The relative capability of chelating ferrous ions was derived from the formula: [1 − (sample absorbance at 562 nm)/(control absorbance at 562 nm)] × 100 (%) 3.4.6. Statistical Analysis Results were expressed as mean ± S.E.M. Computation of 50% inhibitory concentration (IC 50 ) was computer-assisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using Student's t test. Values of p less than 0.05 were considered to be statistically significant.

Conclusion
In summary, three triterpenoids and five steroids from the fruiting bodies of G. mastoporum collected in Vietnam were characterized and their inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/cytochalasin B was examined. The results provide evidence for the use of the fruiting bodies of G. mastoporum as herbal medicines in the treatment of inflammatory diseases, and these purified principles may be potentially useful in developing new anti-inflammatory therapeutic agents.