Methyl Ganoderic Acid DM: A Selective Potent Osteoclastogenesis Inhibitor

Increased osteoclastic bone resorption plays a central role in the pathogenesis of many bone diseases, and osteoclast inhibitors are the most widely used treatments for these diseases. Ganoderic acid DM, the main component of Ganoderma lucidum, has been known for its medicinal effects such as anti-androgen and anti-proliferative activities. In this study, we investigated the inhibitory effects of ganoderic acid DM and its analog (methyl ganoderic acid DM and 7oxo-methyl ganoderic acid Z) on osteoclastogenesis using RAW264 cell in vitro. Methyl ganoderic acid DM blocked osteoclastogenesis completely at 12.5 μM with low cytotoxicity less than 30%. On the other hands, ganoderic acid DM blocked osteoclastogenesis completely at the higher concentration of 50 μM, but 7-oxo-methyl ganoderic acid Z did not up to 100 μM. These results implicated the carbonyl group at C-3 is essentially for selective osteoclastogenesis inhibitory activity, and methyl esters at C-26 should play an important role in enhancing its osteoclastogenesis inhibitory activity. Keyword: Ganoderma lucidum, methyl ganoderic acid DM, osteoporosis, rheumatoid arthritis. INTRODUCTION The balance between bone resorption (by osteoclasts) and bone formation (by osteoblasts) maintains bone homeostasis in a process called bone remodeling [1]. Bone is obviously resistant to dissolution, at least outside the body. Inside the body's highly active milieu, however, bone is remodeled at such a high speed that approximately 10% of the total bone content is replaced per year in adult humans [2]. Incremental changes in the rate of bone resorption can lead to bone disruption. This striking contrast emphasizes what an extraordinary and specific role osteoclasts play in the active maintenance of the bony skeleton. Large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) that are hematopoietic in origin, osteoclasts are capable of resorbing bone [3, 4]. These multinucleated cells help dynamically remodel bones in coordination with osteoblasts, which deposit bone matrix. The excessive osteoclastic bone resorption relative to osteoblastic bone formation is often associated with osteopenic diseases including osteoporosis and rheumatoid arthritis. Osteoclastogenesis progresses through multiple stages, including differentiation, fusion, and activation (maturation) regulated by various factors, including cytokines, hormones, and other cells in the bone microenvironment. So the inhibition of osteoclast differentiation also has great clinical implications. Osteoporosis is a very common disease accompanied by a high level of bone resorption, especially for postmenopausal women. Estrogen treatment, or hormone replacement therapy, is considered by many physicians to be the best *Address correspondence to this author at the Department of Forest and Forest Products Science, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan; Tel: +81-92-642-3002; Fax: +81-92-642-3002; E-mail: [email protected] method to prevent bone loss [5]. However, many women do not tolerate the numerous side effects, or are concerned about the possible increased risk of uterine and/or breast cancer [6-8]. There thus remains a need for highly efficacious anti-resorptive agents with excellent safety and tolerability profiles. Several recent reports whose goal is to identify patterns for preventing osteoporosis through daily diet examined the effects of food components and their bioactive components on bone metabolism [9-10]. We also focused on identifying the lead compound for developmenting inhibitors of osteoclastogenesis among medicinal food stuffs. The fungus G. lucidum (Reishi, Mannentake, or Lingzhi) has been used for centuries in East Asia to treat various human diseases such as hepatitis, hepatopathy, hypertension, nephritis, bronchitis, and cancers [11, 12]. Its dried powder was especially popular as a cancer chemotherapy agent in the Imperial Court of ancient China [13]. G. lucidum has been reported to produce many bioactive oxygenated triterpenoids. Up to now, over 120 kinds of triterpenoids have been isolated from G. lucidum and the genus Ganoderma. Some of the triterpenoids such as ganoderic and lucidic acids, isolated from Ganoderma, have demonstrated cytotoxicity against mouse sarcoma and mouse lung carcinoma cells in vitro [14]. As part of our continuing search for biologically active anti-osteoporotic compounds, we found that osteoclast differentiation was inhibited by the ethanol extracts of G. lucidum and ganoderic acid DM (1) which was isolated as one of the active compounds by bioassay-guided fractionation [15]. Ganoderic acid DM (1) especially suppresses the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1). This suppression leads to the inhibition of dendritic cell-specific transmembrane protein (DC-STAMP) expression and reduces osteoclast fusion. These results prompted us to investigate the ability of gano38 The Open Bioactive Compounds Journal, 2009, Volume 2 Liu et al. deric acid DM (1) analogs (2, 3) (Fig. 1) to inhibit osteoclast differentiation to determine what structural elements are important for the potent inhibition of osteoclast differentiation. MATERIALS AND METHOD