Ganoderma lucidum is a medicinal fungus belonging to the Polyporaceae family which has long been known in Japan as Reishi and has been used extensively in traditional Chinese medicine. We report the isolation and identification of the 26-oxygenosterols ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y and their biological effects on cholesterol synthesis in a human hepatic cell line in vitro. We also investigated the site of inhibition in the cholesterol synthesis pathway. We found that these oxygenated sterols from G. lucidum inhibited cholesterol biosynthesis via conversion of acetate or mevalonate as a precursor of cholesterol. By incorporation of 24,25-dihydro-[24,25-3H2]lanosterol and [3-3H]lathosterol in the presence of ganoderol A, we determined that the point of inhibition of cholesterol synthesis is between lanosterol and lathosterol. These results demonstrate that the lanosterol 14α-demethylase, which converts 24,25-dihydrolanosterol to cholesterol, can be inhibited by the 26-oxygenosterols from G. lucidum. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol.
Higher basidiomycete mushrooms have been used in folk medicine throughout the world since ancient times (25). A large number of mushroom-derived compounds, both cellular components and secondary metabolites, have been shown to affect the immune system and could be used to treat a variety of disease states. Compounds that appear to enhance or potentate host resistance are being sought for the treatment of cancer, immunodeficiency diseases, or generalized immunosuppression after drug treatment (7).
Over the past 20 years, there has been intense interest in the development of an agent from fungi for the treatment of arteriosclerosis and heart disease. Pleurotus ostreatus is one example of a basidiomycete mushroom which can produce lovastatin (5). This compound is the first statin to become available for the treatment of hypercholesterolemia. It inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (mevalonate:NADP+ oxidoreductase; EC 188.8.131.52), which catalyzes the reduction of HMG-CoA to mevalonate during synthesis of cholesterol.
Mevalonate is a common precursor of all isoprenoids, including sterols, ubiquinones, dolichol, and isopentenyladenine, and blocking mevalonate formation may induce undesired side effects in addition to inhibition of sterol synthesis (16). Therefore, it became necessary to find a new hypolipidemic agent to avoid the adverse effects of mevalonate depletion by lovastatin. Consequently, the inhibition of cholesterol biosynthesis beyond the mevalonate production step has recently attracted much attention. Squalene epoxidase, oxidosqualene cyclase, and lanosterol demethylase have been considered as targets to suppress cholesterol biosynthesis.
Ganoderma lucidum, a medicinal fungus belonging to the Polyporaceae family, is used extensively in traditional Chinese medicine. Among the cultivated mushrooms, Ganoderma is unique because it is consumed for its pharmaceutical value rather than as food (8, 26). In 1997, the worldwide production of Ganoderma was approximately 4,300 tons, of which China contributed 3,000 tons (3). Traditionally, Ganoderma is highly regarded as a herbal treatment and is claimed to alleviate or cure virtually all diseases. Current research is focused on purification and characterization of the bioactive components and determination of their clinical value, especially their putative antitumor and anti-ageing properties (2, 4, 12, 14, 21). Many biologically active oxygenated lanostane-type triterpenoids have already been isolated from G. lucidum, and some of them have been shown to inhibit histamine release from rats mast cells (9, 10) and angiotensin-converting enzyme (15). However, no hypocholesterolemic activities have been associated with these molecules so far. Indeed, Komoda and coworkers (10) isolated ganoderic acid B and ganoderic acid C, but no inhibitory activity was detected in vitro. In addition, they derivatized ganoderic acid B by a series of chemical conversions, and the potent inhibitory effect of these derivatives of ganoderic acid was observed with 7-oxo-dehydrolanosterol.
In this study, we tested the effects of G. lucidum on cholesterol metabolism in the hepatic human T9A4 cell line in vitro after incorporation of several key intermediate cholesterol biosynthetic pathways. Here we describe the isolation, identification, and determination of the biological properties in cholesterol synthesis of ganoderol A, ganoderol B, ganoderal A, and ganoderic acid Y. We also investigated the localization of the inhibition site in the cholesterol synthesis pathway.
MATERIALS AND METHODS
General experimental procedures.
Fruiting bodies of G. lucidum were cultivated by Champitec (Fermenta SA, Payerne, Switzerland). Two hundred grams of dried mushrooms was milled and macerated with 80% methanol (MeOH) (2 liters) at room temperature for 2 days. The mixture was then filtered, and the alcohol was evaporated. The extract residue (12.1 g) was recovered with water, the resulting solution was acidified, and the pH was adjusted to 3 with a 2 N chlorydric acid solution.
The aqueous extract was extracted several times with ethyl acetate (EtAOc) (300 ml). The organic phase was dried with anhydrous Na2SO4 and evaporated in a vacuum (30°C) to remove the solvent.
The organic phase residue was dissolved either in an adequate solvent for extraction and purification of active molecules or in a few milliliters of methanol for analysis by chromatography (high-performance liquid chromatography [HPLC] and thin-layer chromatography), spectroscopy (liquid chromatography [LC], mass spectrometry [MS], and LC-MS-MS), and in vitro tests.
Column chromatography was performed with a Kieselgel 60 column (0.063 to 0.200 mm; Merck, Darmstadt, Germany) and a Nucleosil C18 column (250 by 4 mm; Macherey Nagel) with a Lichrospher 100 RP-18 postcolumn (Merck). The mobile phase consisted of a mixture of 0.05% H3PO4 in water and acetonitrile (9:1, vol/vol). The flow rate was 1 ml/min. The detector used was a Hewlett-Packard G1315 A, series 1100 (Hewlett-Packard, Geneva, Switzerland), and the λmax was 254 nm.
The purity of active molecules isolated in this way was controlled by thin-layer chromatography on silica gel with dichloromethane-ethyl acetate (7:3, vol/vol) or dichloromethane-acetone (4:1). The molecules were detected at 254 nm.
Lovastatin (activity enzyme, HMG-CoA reductase [mevalonate:NADP+ oxidoreductase, CoA-acylating; EC 184.108.40.206]) was purchased from Merck.
Radiolabeled 24,25-dihydro-[24,25-3H2]lanosterol and [3-3H]lathosterol were purchased from Amersham Pharmacia Biotech (Cardiff, United Kingdom). Ketoconazole and miconazole were obtained from Sigma Chemicals (Buchs, Switzerland).
Mass spectrometry analyses. (i) HPLC-MS.
Analyses were carried out using either a Micromass Quattro-LC mass spectrometer connected to a Water 2690 HPLC or a Finnigan TSQ-700 triple quadrupole mass spectrometer connected to a Waters HPLC system consisting of a 757 autosampler, a 600-MS pump with a system controller, and a type 486-MS UV detector. The HPLC column used was a Nucleosil 100-5 C18 column (250 by 4 mm). Solvent A was water containing 0.1% trifluoroacetic acid, and solvent B was acetonitrile containing 0.1% trifluoroacetic acid. Using a flow rate of 1 ml/min, separation was started either in the isocratic mode (10% solvent A, 90% solvent B) or with a linear gradient from 90% solvent A and 10% solvent B (5 min) to 10% solvent A and 90% solvent B in 25 min and continued with an isocratic run for 5 min. A flow rate of 1 ml/min was used with a 1/10 postcolumn splitter admitting 0.1 ml/min into the mass spectrometers. Both mass spectrometers worked with an electrospray interface set at a voltage of 4 kV. Mass spectra were acquired from 100 to 800 Da in the positive mode.
Gas chromatography (GC)-MS analyses were performed with an HP 5890 GC combined with a Finnigan MAT 8430 mass spectrometer. The fused silica capillary column employed was a J&W Scientific DB-5 column (30 m by 0.32 mm; film thickness, 0.25 μm). The carrier gas was helium (150 kPa). The temperature program was as follows: 60°C for 1 min, increase at a rate of 30°C/min to 270°C, and increase at a rate of 10°C/min to 320°C (5 min). The splitless injector was heated at 250°C, and the transfer line temperature was 280°C. Mass spectra were obtained in the electronic ionization mode at 70 eV from 20 to 800 Da. The samples were injected before and after trimethylsilyl derivatization performed with a mixture of pyridine and bis(trimethylsilyl) trifluoroacetamide (BSTFA) (1:3, vol/vol) heated for 1 h at 100°C.
The lovastatin content was determined by HPLC. A Nucleosil 100-5 C18 column (250 by 4 mm; Macherey & Nagel) was used with a postcolumn Lichrospher 100 RP-18 postcolumn (Merck). Solvent A was 0.05% H3PO4 in water, and solvent B was acetonitrile. Separation was started with a linear gradient beginning with 95% solvent A and 5% solvent B, followed by 50% solvent A and 50% solvent B at 45 min, 30% solvent A and 70% solvent B at 46 min, 10% solvent A and solvent 90% at 48 min, and 100% solvent B at 50 min and was continued with an isocratic run for 4 min. The initial conditions were maintained for 6 min to reequilibrate the column. The flow rate was 1 ml/min. The detector used was a Hewlett-Packard G1315 A, series 1100, and the λmax was 254 nm.
The spectra were determined with a Bruker DPX-360 nuclear magnetic resonsnce (NMR) spectrometer at room temperature (ca. 20°C). The proton frequency was 360.13 MHz, and the 13C frequency was 90.56 MHz. The following techniques were used: for 1H, normal one-dimensional spectroscopy, two-dimensional homonuclear correlation spectroscopy, and two-dimensional nuclear Overhauser spectroscopy; and for 13C, normal one-dimensional spectroscopy with and without proton decoupling, distortionless enhancement by polarization transfer, and direct and long-range two-dimensional heteronuclear correlation spectroscopy with detection of the 13C frequency. The molecules were dissolved in 99.8% CDCl3.
Human hepatic T9A4 cells were grown in serum-free LCM medium (Biofluids, Rockville, MD) under 3.5% CO2 at 37°C. The cells were seeded into 24-well plates and incubated at confluence with 1 mM [14C]acetate (1 mCi/mmol; Amersham), [14C]mevanolate, 24,25-dihydro-[24,25-3H2]lanosterol, or [3-3H]lathosterol for 20 h in the absence (control) or in the presence of the mushroom extract fractions. Lipid extraction was performed twice by incubation with hexane-isopropanol (3:2) for 30 min at room temperature. The combined extracts were dried under N2, redissolved in hexane, and, in parallel, (i) subjected to high-performance thin-layer chromatography in a hexane-diethyl ether-acetic-acid (75:25:1) solvent mixture and (ii) prepared for direct GC-MS analysis.
For high-performance thin-layer chromatography, the neosynthesis of cholesterol was determined by measuring the incorporation of a labeled precursor into the cholesterol with an instant imager (Camberra Packard, Zurich, Switzerland), and the results were expressed as a percentage of the control.
For GC-MS analysis, the lipid extract was dried under N2 and then resuspended in hexane-propanol (2:1) after addition of 50 nmol of epicoprostanol (5β-cholestan-3α-ol; Sigma) as an internal standard to the sample tube. The sample was purified further by separation on a Bond Elut aminopropyl (NH2) purification spin column (Varian, Zug, Switzerland) used according to the manufacturer’s instructions. The neutral lipid fraction was collected, dried under N2, and then resuspended in 100 μl chloroform-methanol (2:1) prior to injection into the GC-MS.
RESULTS AND DISCUSSION
Extraction, purification, and identification of active molecules.
Cellular extracts were separated into an organic (ethyl acetate) phase and an aqueous phase. Samples from both phases were tested for inhibition of cholesterol synthesis with the human hepatic T9A4 cell model in the presence of [14C]acetate as a precursor. Only the organic phase (referred to below as the “crude extract”) exhibited significant inhibition of cholesterol synthesis. For the crude extract the 50% inhibitory dose (ID50) (the dose needed to inhibit cholesterol synthesis by 50%) was 1 μg/ml, and the ID50 of the positive control lovastatin was 0.5 μg/ml (Table (Table1).1). No statins were detectable in the crude extract by HPLC-MS. From this we concluded that G. lucidum is able to produce molecules other than lovastatin that have the capacity to inhibit cholesterol synthesis in the in vitro model.
|Fraction or compound||ID50 (μg ml−1)|
|G. lucidum aqueous phase||>330|
|G. lucidum organic phase||1.0|
|Lovastatin (positive control)||0.5|
The crude extract (6.7 g) was subsequently partitioned between petroleum ether and MeOH-H2O (90:10, vol/vol). The 90% MeOH extract (6.50 g) was further fractionated by vacuum liquid chromatography using silica gel (Kieselgel 60). The material was eluted stepwise with a chloroform-methanol gradient, and fractions were collected at the following volume ratios: 100:0 (4.70 g), 99:1 (0.22 g), 95:5 (0.54 g), 90:10 (0.60 g), and 0:100 (0.34 g). These preparations were tested for in vitro activity. Inhibitory activity was observed with the 100:0, 95:5, and 90:10 CHCl3-MeOH fractions (Table (Table2).2). The 100% CHCl3 fraction was subsequently rechromatographed on a silica gel column with an EtOAc-hexane gradient, and four fractions were collected at the following volume ratios: 5:95 (1.14 g), 20:80 (1.16 g), 50:50 (1.2 g), and 100:0 (0.5 g). These fractions were also tested for in vitro activity. The most active fractions were the 20:80, 50:50, and 100:0 fractions.
|Fraction||ID50 (μg ml−1)|
The molecules present in these fractions were purified by HPLC. The 20:80 EtOAc-hexane fraction contained active compound 1 with a retention time of 16.51 min. After concentration, it yielded 190 mg of a pure white powder. In the 50:50 EtOAc-hexane fraction, three active molecules were separated: compound 2 (71 mg), compound 3 (50 mg), and compound 4 (60 mg), with retention times of 5.96, 8.82, and 21.82 min, respectively. All of the active molecules isolated had the same UV absorption maxima at 242 and 244 nm, suggesting the presence of a common 7,9(11)-diene tetracyclic triterpene skeleton. Identification was performed by mass spectrometry and NMR. All data obtained for the four compounds described below were in agreement with the data of Arisawa et al. (1) and Morigiwa et al. (15).
The identity of compound 1 purified from the 20:80 EtOAc-hexane fraction was determined by mass spectroscopy. The mass spectrum obtained by electrospray ionization had a [M + H]+ ion peak at m/z 439 and a [M + H − H2O]+ fragment ion peak at m/z 421. The mass spectrum obtained by electron impact analysis had a molecular ion at m/z 438 with a [M − H2O] fragment ion peak at m/z 420. Furthermore, the trimethylsilyl derivative of this compound, measured after electron impact ionization, had a molecular ion at m/z 510 which indicated the presence of one hydroxyl group. The major ions in this spectrum were at m/z 495 ([M-CH3]+), 420 ([M-silanol]+), 405 ([M-silanol-CH3]+) and 309 (base peak).
The 1H-NMR spectrum (CDCl3) included three protons between 5.40 and 5.55 ppm, indicating that there was an olefinic proton. Also, the 1H spectra revealed one secondary group, six tert-methyl groups, and an allylic hydroxymethylene group. The 13C-NMR spectrum included 30 resolved peaks (Table (Table3),3), supporting the molecular formula. The carbons were classified into seven methyl carbons, one hydroxymethylene carbon, eight methylene carbons, three sp2 methine carbons, three methine carbons, three sp2 quarternary carbons, four quarternary carbons, and one carbonyl carbon by analysis of the distortionless enhancement by polarization transfer spectra (Table (Table3).3). The connectivity of proton and carbon atoms was confirmed by the 1H-detected multiple quantum coherence spectrum. The 1H-1H two-dimensional homonuclear correlation spectroscopy spectrum and 13C-1H long-range couplings of 2J and 3J detection of the 1H-detected multiple-bond heteronuclear multiple quantum coherence spectrum confirmed the hypothesized structure of ganoderol A (1, 15). Compound 1 was identified as ganoderol A (Fig. (Fig.11).
|Carbon no. (ppm)||13C chemical shift (ppm)a||1H chemical shift|
The molecular masses of compounds 2, 3, and 4 were determined by LC-MS and GC-MS to be 440, 436, 454 Da, respectively. Compound 2 was identified from the 50:50 fraction as ganoderol B (Fig. (Fig.1).1). Its molecular mass was determined to be 440 Da, and it had an [M − H2O]+ fragment ion at m/z 422 after electrospray ionization. Trimethylsilylation and GC-MS analysis resulted in a mass spectrum having a molecular ion at m/z 584 and fragment ions at m/z 569 ([M-CH3]+), 494 ([M-silanol]+), 479 ([M-silanol-CH3]+), and 383 (base peak).
The 13C-NMR spectrum indicated the presence of the carbon of a CH2OH residue at 62 ppm.
Similarly, compound 3 was identified as ganoderal A (Fig. (Fig.1).1). Its mass spectrum obtained after electrospray ionization had an [M + H]+ ion peak at m/z 437. After trimethylsilylation and GC-MS analysis, this compound had a molecular ion at m/z 508 and fragment ions at m/z 493 ([M-CH3]+), 418 ([M-silanol]+), 403 ([M-silanol-CH3]+), and 309 (base peak). These data indicate the presence of an enolic form, as a carbonyl in the β-position of the olefinic group is present in the ganoderal A structure.
The 1H-NMR spectrum (CDCl3) revealed six tert-methyl and allylic hydroxymethylene groups, and the 13C-NMR spectrum indicated the presence of the carbon of a CHO conjugated residue at 197 ppm.
Compound 4 was identified as ganoderic acid Y (Fig. (Fig.1).1). Its molecular mass was determined to be 454 Da (598 Da after trimethylsilylation). The mass spectrum of the trimethylsilyl derivative revealed fragment ions at m/z 583 ([M-CH3]+), 508 ([M-silanol]+), 493 ([M-silanol-CH3]+), 418 ([M-2 silanol]+), 403 ([M-2 silanol-CH3]+) and 309 (base peak). In addition, the 13C-NMR spectrum of ganoderic acid Y confirmed the presence of a carbonyl in the β-position of the olefinic group at 178 ppm.
Assessment of the point of inhibition in cholesterol biosynthesis.
In the presence of [14C]acetate as a precursor, the concentrations needed to inhibit 50% of cholesterol synthesis (ID50) were similar for ganoderol A (ID50, 2.2 μM) and ganoderic acid Y (ID50, 1.4 μM), while ganoderal A and ganoderol B appeared to be less active (ID50, approximately 20 μM) (Table (Table44).
|Oxygenosterol||ID50 (μM) with:|
|Ganoderic acid Y||1.4||9|
As these active molecules showed various rates of inhibition of cholesterol biosynthesis from [14C]acetate, it was interesting to determine the effects of these compounds on HMG-CoA reductase activity. When incorporated in the presence of [14C]mevalonate as a precursor, these molecules were active and resulted in various inhibition rates (Table (Table4).4). The ID50 of ganoderol A was 4.7 μM, and the ID50 of ganoderic acid Y was 9 μM. In the presence of [14C]acetate, ganoderal A and ganoderol B were less active than other molecules, and their ID50s were 19 and 24 μM, respectively. These results show that one point of inhibition was localized downstream of HMG-CoA reductase. We observed that in the presence of mevalonate ganoderol A was more active than ganoderic acid Y and that the converse was true in the presence of [14C]acetate. The reason for this observation is unknown.
Pathway intermediates downstream of mevalonate, such as lanosterol and lathosterol, were tested to determine more accurately the potential point(s) of inhibition in the cholesterol synthesis pathway. To do this, ganoderol A was tested for inhibition activity at different concentrations using human hepatic T9A4 cells in the presence of a labeled precursor, either 24,25-dihydro-[24,25-3H2]lanosterol or [3-3H]lathosterol (20, 22). Measuring the absorption of the corresponding radioactively labeled derivatives in cells allowed determination of the cellular uptake of these two precursors. Also, ganoderol A at concentrations of 2.3 and 7 μM resulted in reduction of cholesterol synthesis by 15 and 40%, respectively. When [3-3H]lathosterol was used as a precursor, no inhibition was observed. The branch point of inhibition of cholesterol synthesis in the presence of this active compound could therefore be localized between lanosterol and lathosterol (Fig. (Fig.22).
The two demethylation reactions occurring between lanosterol and lathosterol are catalyzed by a cytochrome P-450-dependent enzyme. The first step, 14α-demethylation of lanosterol, results in the removal of a single methyl group, and during the second step (4α-demethylation of demethyl-lanosterol) two methyl groups are removed (Fig. (Fig.2).2). Evidence for the inhibition of P-450 in intact cells by incorporation of ganoderol A was obtained from the acetate incorporation studies. Exponentially growing cells were treated with ganoderol A and with lovastatin as a positive control (Table (Table5).5). The amounts of the main metabolites in the cholesterol pathway were determined by GC-MS. The amounts of squalene, lanosterol, and cholesterol were decreased by addition of lovastatin. In fact, competitive inhibition of HMG-CoA reductase by statins reduced the amounts of all the intermediary metabolites downstream of mevalonate. In contrast, cells treated with ganoderol A showed a twofold increase in the lanosterol content, while the squalene and cholesterol levels were decreased by about 25% compared to control cells (Table (Table5).5). The accumulation of lanosterol in ganoderol A-treated cells could be explained by inhibition of the lanosterol 14α-demethylase activity (Fig. (Fig.2).2). When we tested ganoderic acid Y in the presence of 24,25-dihydro-[24,25-3H2]lanosterol or [3-3H]lathosterol as a precursor, the inhibition was less than the inhibition obtained with ganoderol A (data not shown).
|Prepn||Neutral lipid fraction (ng)|
|Lovastatin (1.3 μM)||169||68,875||6|
|Ganoderol A (3 μM)||374||74,810||36|
Ketoconazole and miconazole are known to be cytochrome P-450 inhibitors and to interact with fungal lanosterol demethylase (17, 24). It has been determined that these compounds cause fungal cell death by blocking the biosynthesis of ergosterol at the lanosterol 14α-demethylation step. In addition, ketoconazole has been shown to reduce total serum cholesterol in humans, and there is a corresponding increase in serum lanosterol concentrations (6, 11, 13). We confirmed this observation by incorporating ketoconazole at a concentration of 2 μM in the presence of [14C]acetate as a precursor. Cholesterol synthesis was reduced by 15%. The same observation was made with miconazole.
In this study we identified several active 26-oxygenosterols from G. lucidum. We believe that the activities of these molecules are similar to the activities of 15-, 24-, 25-, and 32-oxygenosterols.
The metabolic properties of a series of 15- and 32-oxygenosterols have been described previously, and these compounds were found to be very potent inhibitors of sterol synthesis in animal cell culture (18) and inducers of low-density lipoprotein receptor activity (22). In addition, they appear to have significant hypocholesterolemic activity upon oral administration to intact animals (19).
The inhibitory activities of these compounds were concentration dependent and competitive with respect to substrate (23). Some authors have suggested that oxygenated sterols might be endogenous regulators of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, and, consequently, the biosynthesis of cholesterol. We show in this paper that G. lucidum produces molecules that are active against cholesterol synthesis and that these molecules are potent inhibitors of the lanosterol 14α-demethylase. These 26-oxygenosterols could lead to novel therapeutic agents that lower blood cholesterol levels.