Screening of Korean medicinal plants for possible osteoclastogenesis effects in vitro
© Springer-Verlag 2007
Published: 16 November 2007
Bone undergoes continuous remodeling through bone formation and resorption, and maintaining the balance for skeletal rigidity. Bone resorption and loss are generally attributed to osteoclasts. Differentiation of osteoclasts is regulated by receptor activator of nuclear factor NF-kB ligand (RANKL), a member of tumor necrosis factor family. When the balance is disturbed, pathological bone abnormality ensues. Through the screening of traditional Korean medicinal plants, the effective molecules for inhibition and stimulation of RANKL-induced osteoclast differentiation in mouse bone marrow macrophages were identified. Among 222 methanol extracts, of medicinal plants, 10 samples exhibited ability to induce osteoclast differentiation. These include Dryobalanops aromatica, Euphoria longana, Lithospermum erythrorhizon, Prunus mume, Prunus nakaii, and Polygonatum odoratum. In contrast, Ailanthus altissima, Curcuma longa, Solanum nigrum, Taraxacum platycarpa, Trichosanthes kirilowii, and Daphne genkwa showed inhibitory effects in RANKL-induced osteoclast differentiation.
Bone destruction is observed in advanced cases of rheumatoid arthritis and the neoplastic diseases, osteoporosis, and periodontitis. The balance between bone formation and resorption is tightly regulated by osteoblast and osteoclast, respectively, to maintain the homeostasis of our skeleton. Osteoclasts are sole bone-resorptive multinucleated cells (MNCs) that derived from hematopoietic cells. Excessive osteoclastogenesis or activation of mature osteoclasts causes the bone destruction which is implicated in rheumatoid arthritis, osteoporosis, multiple myeloma and bone metastasis.
Bone mineral density (BMD) and bone metabolism are affected by genetic, endocrine, mechanical and nutritional factors, with interactions among the different factors . Bone mass in adult humans decreases with age, leading to an increased risk of fractures. Osteoporosis occurs frequently in postmenopausal women due to decrease in estrogen levels. Despite its positive effects on the bone physiology, estrogen replacement therapy is no longer recommended as the first choice therapy for the prevention and treatment of the postmenopausal osteoporosis because it increases the risks of cardiovascular, thromboembolic, and breast cancer . As an alternative way to the hormone therapy, the use of phytoestrogens has attracted attention . Nutritional factors are of particular importance to bone health because they are modifiable .
Treating perimenopausal and postmenopausal women with 40 g/day soy protein isolate providing 80–90 mg/day of isoflavones attenuated the loss of bone mineral density in the spine but not in other sites [5–8]. Onion and mixtures of vegetables, salads and herbs inhibit the bone resorption when metabolic acid is completely buffered with potassium citrate [9, 10]. Sage, rosemary and thyme, which are the herbs rich in essential oil, also strongly inhibit bone resorption. There is a long history in the use of essential oils as medical applications for the relief of head and chest colds as well as pain . Natural products of plant origin are still a major part of traditional medicinal systems in Korea. Korean herbal formulations, such as Yukmi-jihang-tang and Dae-bo-won-chun, were reported for their preventive effect on the progress of bone loss in the rats [11, 12].
To examine the inhibitory effects of Korean traditional medicinal plant extracts on the bone resorption in the mouse macrophage cells, we have screened the inhibitory activities. Here, we report that the methanol extracts of Ailanthus ltissima, Curcuma longa, Solanum nigrum, Taraxacum platycarpa, Trichosanthes kirilowii and Daphne genkwa inhibit osteoclastogenesis.
Materials and methods
The methanol extract was of medicinal plants provided by Dr. Young Seop Kim (Korea Research Institute of Chemical Technology, South Korea). Minimal essential medium alpha modification (α-MEM), fetal bovine serum (FBS), and antibiotics were purchased from Well Gene (Dae gu, South Korea). Macrophage colony stimulation factor(M-CSF) were purchased from R&D Systems. TRANCE was provided by Dr. Lee (University of Ewha Women’s University, Korea). TRAP staining kit (Leukocyte acid phosphatase kit) was obtained from Sigma (St. Louis, Miss., USA).
Culture of mouse bone marrow mononuclear cells
The bone marrow cells were isolated from the long bones of 4-week-old C57BL/6 male mice, and cultured with α-MEM/10% FBS/1% antibiotics with M-CSF (25 ng/ml) in a humidified incubator (5% CO2 in air) at 37°C. After 24 h of cultures, the non-adherent cells were collected and centrifuged to obtain the bone marrow macrophage (BMM) cells which were the depleted stromal cells. For the osteoclast differentiation experiments, the BMM cells were cultured in 96-well plates (3 × 104/well) with M-CSF (50 ng/ml), TRANCE (400 ng/ml) and stimuli for 6–9 days.
Tartrate-resistant acid phosphatase (TRAP) assay
To determine the characteristics of osteoclast differentiation, cells were fixed with 3.7% formaldehyde for 10 min and then washed with distilled water. Then the cells were stained for TRAP with 0.1 M acetate solution containing 6.76 mM sodium tartrate, 0.12 mg/ml naphthol AS-MX phosphate, and 0.07 mg/ml of fast Garnet GBC solution as described in the manufacturer’s instructions (Leukocyte acid phosphatase kit) for 30 min at room temperature.
Results and discussion
Korean medicinal plants for possible osteoclastogenesis in this experiment
Brassica chinensis var.oleifera
Phyllostachys nigra var.henonis
Melia azedarach var.japonica
Sambucus williamsii var.coreana
Trigonella foenum- graecum
Rehmania glutinosa var.purpurea
The positive effects of the crude compounds in osteoclastogenesis
Sambucus williamsii var.coreana
The negative effects of the crude compounds in osteoclastogenesis
The positive effects of nutritional supplement with herbal formulation extracts on bone mineral density and height in prepubescent children were reported .
The methanol extract and its major bioactive compound, gallic acid of Orostachys japonicus, greatly enhanced the activities of hepatic alcohol dehydrogenase, the microsomal ethanol-oxidizing enzyme, and aldehyde dehydrogenase in a dose dependent manner . In additions, the inhibitory effects on the formation of carcinogenic N-nitrosodimethylamine were observed .
The main root of Aconitum carmichaelli has been used in Chinese herbal medications mainly for the treatment of musculoskeletal disorders, and the herbal formulations containing it have been used for the treatment of rheumatism and heart failure as well as improvement of the immune system and retarding aging [16, 17]. The plant contained the highly toxic C19 diterpenoid alkaloids of aconitine, mesaconitine and hypaconitine. A. altissima (synonym A. glandulosa) has been used to treat cold, gastric diseases, and cancer. From the bioassay-oriented study, it is reported that it has cytotoxicity and antiproliferative activities. It contains quassinoids, indole and β-carboline alkaloids. These compounds are reported for antitubercular, antimalarial, and inhibitory effects against Epstein-Barr virus . Furthermore, other Ailanthus species have anti-cancer agents . Curcuma longa has been in use for centuries as a dye and also as a component of curry powder . Daphne genkwa has an antitumor activity. The anti-tumor constituent, daphnodorin complex, was reported to have inhibitory effects on tumor growth and metastasis by protecting host immunocyte viability and proliferation potential, thus selectively inhibiting tumor cell proliferation . Daphnane diterpene esters from flower buds induced apoptosis in human pro-myelocytic leukemia HL-60 cells. This esters were found to have suppressed the growth of Lewis lung carcinomas (LLC) inoculated into mice . An anticoagulant purified from Taraxacum platycarpum has been used as an inflammatory agent to treat colitis and ulcer. In addition, this anticoagulant protein, when treated to the murine macrophage cell line RAW 264.7, induced expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase, and production of anti-tumor necrosis factor-α . The rhizome extract of Rheum undulatum was reported to have vasorelaxant, anti-allergic and anti-platelet aggregation activities [23, 24]. The methanolic extract of the cortex of Eugenia caryophyllata exerted the COX-2 inhibitor activity in RAW264.7 cells .
Eugenol is a major component of essential oil isolated from the E. caryophyllata, which was reported as an anti-cancer agent . The root tuber protein of Richosanthes kirilowii suppressed the HSV-1 infection by targeting the mitogen-activated protein kinase (MAPK) family pathway . Water extract of the root of Lindera strychnifolia slowed down the progression of diabetic nephropathy in db/db mice . Citrus fruits were found to be a potentially important source of anti-inflammatory flavonoids in the human diet . The peel of citrus fruits is a rich source of flavones. Nitric oxide (NO) has been implicated in a variety of pathophysiological conditions, including inflammation, carcinogenesis, and atherosclerosis . The ethyl acetate soluble fraction of Cnidium officinale MAKINO inhibited neuronal cell death by reducing excessive NO production in LPS-treated rat hippocampal slice cultures and microglia cells . The extract of Phaeseolus angularis (Adzuki bean) exhibited estrogen-like activities  but in different ways from Phaseolus lunatus L.
Some medicinal plants showed stimulatory effects on osteoclast differentiation. The aqueous extract from the medicinal plant Dryobalanops aromatica specifically inhibited catecholamine secretion that is important in stressful states and emotional behavior . The kaempferol derived from Polygonatum odoratum has been used for the treatment of chronic airway diseases . Euphoria longana and Prunus mume fruit was reported to have an anti-cancer effects [34, 35]. The naphthoquinone pigment, shikonin, isolated from Lithospermum erythrorhizon, has several therapeutic potential including anti-inflammatory and anti-tumor effects .
From these results, we found several compounds with stimulatory or inhibitory effects on osteoclastogenesis (Tables 2, 3). There were several medicinal plants that showed strong effects on osteoclast differentiation (Table 2), even though we could not find a relationship through these results in osteoclastogenesis. The next step will be a study with single compounds purified from 30 verified plants.
This work was supported by the Project of Bio-Food Research from the Korea Science and Engineering Foundation (KOSEF) under the Ministry of Science and Technology in Korea, and also supported by grant No. R01-2006-000-10515-0 from the Basic Research Program of the Korea Science & Engineering Foundation.
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