Isolation of an Inhibitor of Platelet Aggregation from a Fungus, Ganoderma lucidum

.AkiraShimizu, Takashi Yano, Yuji Saito and Yuji Inada*

Laboratory of Biological Chemistry, Tokyo Institute of Technology,

Ookayama, Meguro-ku, Tokyo 152, Japan

(Received October 16, 1984)

The water-soluble fraction of Ganoderma lucidum was found to suppress platelet aggregation. It was further shown by gel chromatography, high performance liquid chromatography and nuclear magnetic resonance spectroscopy that the inhibitory substance present in this fraction was adenosine.

Keywords-fungus; Ganoderma lucidum; platelet aggregation: adenosine

Chinese medicines have long been used clinically. Their chemical bases, however, are not necessarily understood in most cases. Some of them have attracted much attention as effective chemotherapeutic agents against cancer.1-3) A fungus, Ganoderma lucidum, has been clinically tested for the treatment of various diseases,4) but despite many positive findings, it has never been reported to suppress thrombus formation. In this work, we have demonstrated for the first time that the water-soluble fraction of this fungus suppresses platelet aggregation.

The aggregation response induced by 0.21unit/ml thrombin in the presence of the G.

lucidum extract is shown in Fig. 1. A clear incubation time-dependent inhibition is apparent.

A significant inhibitory effect was observed until the concentration of the extract was diluted approximately one-hundred times. These results indicate that G. lucidum contains a strong inhibitor(s) of platelet aggregation.

Our initial attempt to isolate the inhibitory substance(s) was made by, gel filtration on a Sephadex G-25 column (1.3 x 47.5 cm) equilibrated with distilled water. The elution profile is shown in Fig. 2. The ultraviolet (UV) absorption and the inhibition of aggregation of each fraction are shown. Most of the materials with UV absorption were eluted within the void volume, but they had no inhibitory effect on platelet aggregation. A small UV absorption peak was observed at 1.6 times the gel volume. This peak (fraction numbers 65-74) contained the platelet aggregation-inhibitory substance(s) of G. lucidum. From this result, it is clear that the inhibitor(s) in the extract is weakly adsorbed by Sephadex G-25.It was reported that a tightly cross-linked dextran gel such as Sephadex G-25 has affinity for aromatic and pseudo-aromatic substances,5) and Sephadex gel is routinely used for separation of ribo ­and deoxyribonucleosides utilizing this feature.6~ Since the absorption spectrum of the active fraction from the gel chromatography showed a maximum at ca. 260 nm, the inhibitor might be a nucleoside-related compound.

Further purification was performed by Bio-Gel P-2 (polyacrylamide gel) chromatog­raphy, because nucleic acid components and related compounds may be separated •by absorption chromatography on polyacrylamide gel.7) The active fractions from Sephadex 0-25 chromatography were concentrated by freeze-drying and the product was applied to a column of Bio-Gel P-2 (1.7 x 26 cm) equilibrated with distilled water. Each fraction of the eluate was checked for absorption at 260 nm (Fig. 3). The extents of inhibition of platelet aggregation are

also shown in Fig. 3. The chromatography yielded two major peaks and several minor ones. The inhibitory activity was associated with only one peak, from fractions 42 through 47.

We confirmed the purity of this fraction by high performance liquid chromatography (HPLC) as shown in Fig. 4. Only a single peak was detected with several different solvent systems. Furthermore, the retention time of this compound always coincided, with that of’ adenosine. Other nucleosides, nucleotides and bases were separated from adenosine under these conditions. We also measured the nuclear magnetic resonance (NMR) spectrum of this compound. The spectrum coincided completely with that of adenosine (data not shown).

Several investigators have reported that adenosine inhibits platelet and thrombocyte aggregation.8-11) Preincubation of platelets with adenosine is apparently required for the inhibition to be observed.8) Adenosine seems to have a stronger effect on platelet aggregation induced by thrombin than on that induced by adenosine diphosphate (ADP).Adenosine clearly inhibits thrombin-induced platelet aggregation at concentrations of the order of ?M.10) These characteristics are in good agreement with what we have observed with the inhibitor obtained from G. lucidum. Adenosine was considered to act competitively with ADP,12) but it has also been suggested that it inhibits the esterolytic activity of thrombin’31 or that it interferes with the energy flow from adenosine triphosphate 14) through its own phosphorylation.The mechanism of this inhibition has yet to be elucidated in detail.

We have thus demonstrated that adenosine is an inhibitor of platelet aggregation found in G. lucidum. The content of adenosine in this fungus was at least 40 mg/l00 g of dried preparation.

Experimental

G. lucidum was a kind gift from Dr. Ikekawa of the National Cancer Center Hospital, Japan. Bovine thrombin and bovine serum albumin (BSA) (fraction V) were purchased from Mochida Pharmaceutical Co. and Sigma Chemical Co., respectively. All other reagents were of analytical grade.
A water-soluble fraction of G. lucidum was prepared as follows; 5 g of the fungus cut into small pieces was added to 300 ml of distilled water, and the mixture was heated at 70-80ºC for 8h under constant stirring, then filtered through layers of gauze. The filtrate was freeze-dried and redissolved in 30 ml of distilled water.
Washed platelets were prepared from bovine blood by the method previously described,’51 and finally suspended in Tris-acid-citrate-dextrose (Tris-ACD) buffer containing 1.0% BSA to give a density of 8 x I 0’/ml. Aggregation of platelets was measured turbidimetrically with a Shimadzu MPS-5000 recording spectrophotometer after the addition of 50 µl of 200 mM CaC12 and 50 µl of thrombin (2-3 units/ml in Tris-ACD) to 0.5 ml of washed platelets (density, 7—8 x l08/ml) in Tris-ACD.
HPLC was conducted with a Shimadzu LC-3A using a Zorbax-ODS column (0.46 x 25cm). The solvent was 10mM potassium phosphate, pH 5.1 or 8.5, containing 15 or 20% methanol, and the column temperature and flow rate were 50 ºC.and 1.0 ml/mm, respectively. The NMR spectrum was measured with a JEOL GX 400 spectrometer operating at 400 MHz.
Acknowledgement: The authors thank Dr. Hironobu Iinuma of the Institute of Microbial Chemistry (Tokyo) for the measurement of the NMR spectrum and Dr. Tetsuro Ikekawa of the National Cancer Center Hospital (Tokyo) for providing G. lucidum.