Hindawi Publishing Corporation International Journal of Endocrinology Volume 2015, Article ID 132436, 7 pages http://dx.doi.org/10.1155/2015/132436 Research Article The Inhibitory Effect of Alisol A 24-Acetate from Alisma canaliculatum on Osteoclastogenesis Kwang-Jin Kim,1 Alain Simplice Leutou,2 Jeong-Tae Yeon,3 Sik-Won Choi,4 Seong Hwan Kim,4 Sung-Tae Yee,1 Kyung Hee Choi,1 Sang-Jip Nam,2 and Young-Jin Son1 1 Department of Pharmacy, Sunchon National University, Suncheon, Jeonnam 540-742, Republic of Korea 2Department of Chemistry and Nano Science, Global Top 5 Program, Ewha Womans University, Seoul 120-750, Republic of Korea 3Research Institute of Basic Science, Sunchon National University, Suncheon 540-742, Republic of Korea 4Laboratory of Translational Therapeutics, Pharmacology Research Center, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea Correspondence should be addressed to Sang-Jip Nam; [email protected] and Young-Jin Son; [email protected] Received 30 September 2014; Revised 19 January 2015; Accepted 14 April 2015 Academic Editor: Ling-Qing Yuan Copyright © 2015 Kwang-Jin Kim et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Osteoporosis is a disease that decreases bone mass. The number of patients with osteoporosis has been increasing, including an increase in patients with bone fractures, which lead to higher medical costs. Osteoporosis treatment is all-important in preventing bone loss. One strategy for osteoporosis treatment is to inhibit osteoclastogenesis. Osteoclasts are bone-resorbing multinucleated cells, and overactive osteoclasts and/or their increased number are observed in bone disorders including osteoporosis and rheumatoid arthritis. Bioactivity-guided fractionations led to the isolation of alisol A 24-acetate from the dried tuber of Alisma canaliculatum. Alisol A 24-acetate inhibited RANKL-mediated osteoclast differentiation by downregulating NFATc1, which plays an essential role in osteoclast differentiation. Furthermore, it inhibited the expression of DC-STAMP and cathepsin K, which are related to cell-cell fusion of osteoclasts and bone resorption, respectively. Therefore, alisol A 24-acetate could be developed as a new structural scaffold for inhibitors of osteoclast differentiation in order to develop new drugs against osteoporosis. 1. Introduction essential roles in the balance of skeletal homeostasis. Osteo- clast differentiation from bone marrow-derived macrophages Bone is a living, dynamic tissue that is constantly remodeled (BMMs) is needed for receptor activator of nuclear factor-B in the process of bone turnover. Bone health in adults ligand (RANKL), which is known to play an important role depends on the synchronized performance of bone-resorbing in osteoclast development [7].Thenuclearfactorofactivated osteoclasts and bone-forming osteoblasts that function T cells c1 (NFATc1), noted master transcription factor for together on the bone surface [1]. Bone remodeling is impor- osteoclast differentiation, is induced by RANKL [8]. NFATc1 tant in vertebrates to maintain bone volume and calcium promotes the expression of osteoclast differentiation-related homeostasis [2]. An imbalance in the activities of bone- factors including tartrate-resistant acid phosphatase (TRAP), resorbing osteoclast cells and bone-depositing osteoblast cells cathepsin K, and dendritic cell-specific transmembrane pro- upon aging or reaching menopause leads to osteoporosis [3]. tein (DC-STAMP) [9–11]. Osteoporosis, Paget’s disease, and rheumatoid arthritis are Plants are valuable sources of medicinal compounds with the result of overactive osteoclasts, which resorb bone [4]. a broad range of biological activity. Approximately 25 to This disorder has been increasing in frequency along with the 50% of current pharmaceuticals are derived from plants [12]. increase in life expectancy [5]. Traditional oriental herbal medicines have been reevaluated Osteoclasts are tissue-specific macrophage polykaryons by clinicians [13] because these medicines have fewer side created by the differentiation of monocyte/macrophage pre- effects and are more suitable for long-term use compared to cursor cells at or near the bone surface [6]. These cells have chemically synthesized medicines [14]. 2 International Journal of Endocrinology O polarity, from 100% CH2Cl2 to 100% MeOH. The fraction containing triterpenoid mixtures eluting with 2% CH2Cl2 O in MeOH was further purified by RP-HPLC [Phenomenex Luna RP-C18(2), 5 m, 250 × 10 mm, 2.5 mL/min] using an HO HO isocratic solvent system with 85% acetonitrile in H2Oto OH afford alisol A 24-acetate (1,7.0mg, 14 min). 1 H 2.4. Alisol A 24-Acetate (1). H NMR (CDCl3,700MHz):H O 4.65 (1H, s, H-24), 3.89 (2H, overlapped, H-11 and H-23), 2.81 H (2H, dd, J = 13.8, 5.9 Hz H-12), 2.68 (1H, m H-20), 2.35 (2H, ddd, J = 15.5, 9.6, 3.3 Hz, H-2), 2.25 (1H, m, Ha-1), 2.20 (3H, s, Figure 1: Molecular structure of alisol A 24-acetate. -COCH3), 2.15 (1H, m, Hb-1), 2.16 (2H, m, H-16), 2.10 (1H, m, H-5), 2.02 (2H, m, H-7), 1.89 (1H, m, H-15a), 1.74 (1H, d, J = 10.8 Hz, H-9), 1.45 (1H, m, H-6a), 1.39 (1H, m, H-6b), 1.38 (2H, Alisma canaliculatum,amemberoftheplantfamily m, H-22), 1.36 (1H, m, H-15b), 1.30 (3H, s, H-27), 1.16 (3H, s, Alismataceae, is a herb commonly used in traditional Ko- H-26), 1.15 (3H, s, H-30), 1.07 (3H, d, J = 11.0 Hz, H-21), 1.06 rean medicine. Rhizoma Alismatis, a dried tuber of A. (3H, s, H-28), 1.00 (3H, s, H-18), 0.99 (3H, s, H-19), 0.98 (3H, canaliculatum, is the main medicinal part of the plant. 13 s, H-29); C NMR (175 MHz, CDCl3): C 220.5 (qC, C-3), A. canaliculatum has diuretic hepatoprotective, antitumor, 171.5 (-COCH3), 138.3 (qC, C-13), 135.5 (qC, C-17), 78.6 (CH, and antibacterial effects [15]. Previous phytochemical and C-24), 73.9 (qC, C-25), 70.0 (CH, C-11), 69.0 (CH, C-23), 57.0 pharmacological investigations of this plant reported the (qC, C-14), 49.6 (CH, C-9), 48.5 (CH, C-5), 47.0 (qC, C-4), isolation of protostane- and seco-protostane-type triterpenes 40.5 (qC, C-8), 39.7 (CH2, C-22), 36.9 (qC, C-10), 34.5 (CH2, [16] such as alisols A, B, and C, alisol A 24-acetate, alisol B C-12), 34.3 (CH2, C-7), 33.8 (CH2, C-2), 30.9 (CH2, C-1), 30.5 23-acetate, alisol C 23-acetate, and alismalactone 23-acetate, (CH2, C-15), 29.6 (CH3, C-28), 29.1 (CH2, C-16), 27.9 (CH, C- and guaiane-type sesquiterpenes [17]suchasalismolsAand 20), 27.5(CH3, C-26), 26.6 (CH3, C-27), 25.7 (CH3, C-19), 24.1 B, sulfoorientalol A, and orientatols AB, C, E, and F. (CH3, C-30), 23.2 (CH3, C-18), 20.1 (-COCH3), 20.1 (CH3,C- In our ongoing investigation of biologically active com- 29), 20.1 (CH3, C-21), 20.0 (CH2, C-6); LCMS /:515[M- pounds from natural products, the dried rhizomes of A. + + H2O+H] ,497[M-2H2O+H] . canaliculatum were examined, and bioactivity-guided frac- tionations and HPLC yielded a triterpenoid, alisol A 24- 2.5. Osteoclast Differentiation. This study was carried out acetate (Figure 1). in strict accordance with the recommendations outlined in Herein, we report the isolation and the biological activi- the Standard Protocol for Animal Study from the Korea ties of alisol A 24-acetate. Research Institute of Chemical Technology (KRICT; Permit number 2012-7D-02-01). The protocol (ID number 7D-M1) 2. Materials and Methods was approved by the Institutional Animal Care and Use 2.1. Reagents. Recombinant mouse receptor activator of Committee of KRICT (IACUC-KRICT). All efforts were nuclear factor-B ligand (RANKL) and recombinant mouse made to minimize the suffering of animals. Bone marrow macrophage-colony stimulating factor (M-CSF) were pur- cells (BMCs) were collected from femur and tibia of 5-6- chased from R&D Systems (MN). Cell culture medium, fetal week-old male ICR mice by flushing femurs and tibias with bovine serum (FBS), and penicillin/streptomycin were pur- -MEM supplemented with antibiotics. BMCs were cultured chased from Invitrogen Life Technologies (NY). The CCK-8 with M-CSF (10 ng/mL) in -MEM containing 10% fetal assay kit was obtained from Dojindo Molecular Technologies bovine serum (FBS) and antibiotics in a culture dish for 1 day. (ML). All reagents used in the reverse transcription (RT) Nonadherent BMCs were cultured for 3 days in a Petri dish in and real-time PCR master mix were from Enzynomics (KR). M-CSF (30 ng/mL), and the adherent cells were used as bone NFATc1 monoclonal and actin polyclonal antibody were from marrow-derived macrophages (BMMs). For the formation of Santa Cruz Biotechnology (CA, USA). osteoclasts, BMMs were cultured with RANKL (10 ng/mL) andM-CSF(30ng/mL)inthepresenceorabsenceofalisolA 2.2. Plant Material. Alisma canaliculatum was purchased 24-acetate for 4 days. The culture medium was changed once from Dongbu plant market in Suncheon in the South Sea in per three days. Osteoclast formation was assessed by TRAP Korea. (tartrate-resistant acid phosphatase) staining. 2.3. Extraction and Isolation. The dried rhizomes of Alisma 2.6. TRAP Staining Assay. Cells were fixed with 3.7% for- canaliculatum (wet weight, 1.2 kg) were minced and extracted malin for 5 min, permeabilized with 0.1% Triton X-100 for with ethanol at room temperature for five days; the ethanol 10 min, and stained with TRAP solution (Sigma-Aldrich, MO, + ≤ was concentrated under vacuum and then partitioned USA) for 10 min.