Category Archives: Flavonoids

p-Hydroxycinnamic Acid Inhibits Osteoclast-Like Mouse Cells

Abstract

Phytocomponent p-hydroxycinnamic acid inhibits osteoclast-like cell formation in mouse bone marrow cultures.

The phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have inhibitory effects on bone-resorbing factor-stimulated bone resorption in rat femoral tissues in vitro. The effects of HCA on osteoclast-like cell formation in mouse bone marrow cultures in vitro were investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1-34)] (PTH), prostaglandin E2 (PGE2), or tumor necrosis factor-alpha (TNF-alpha) in effective concentrations. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10(-7) M), PGE2 (10(-5) M), or TNF-alpha (10 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of HCA (10(-8)-10(-5) M). HCA (10(-6) or 10(-5) M) significantly inhibited osteoclast-like cell formation induced by dibutyryl cyclic adenosine monophosphate (10(-5) M) or phorbol 12-myristate 13-acetate (10(-6) M), an activator of protein kinase C. Also, HCA (10(-8)-10(-5) M) had a significant inhibitory effect on osteoclast-like cell formation induced by the receptor activator of NF-kappaB ligand (RANKL) (10 ng/ml) in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/ml). The inhibitory effect of HCA (10(-6) or 10(-5) M) on RANKL plus M-CSF-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis in the transcriptional process, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (10(-6) M), an inhibitor of transcription. This study demonstrates that HCA has a potent inhibitory effect on osteoclast-like cell formation in mouse bone marrow cultures. The inhibitory action of HCA may partly involve a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Lai YL, Yamaguchi M
Int. J. Mol. Med. Jan 2007
PMID: 17143556

Review: Phytochemicals for Bone Osteoporosis

Abstract

Regulatory mechanism of food factors in bone metabolism and prevention of osteoporosis.

Aging induces a decrease in bone mass, and osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. Bone loss with increasing age may be due to decreased bone formation and increased bone resorption. Pharmacologic and nutritional factors may prevent bone loss with aging, although chemical compounds in food and plants which act on bone metabolism are poorly understood. We have found that isoflavones (including genistein and daidzein), which are contained in soybeans, have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption, thereby increasing bone mass. Menaquinone-7, an analogue of vitamin K(2) which is abundant in fermented soybeans, has been demonstrated to stimulate osteoblastic bone formation and to inhibit osteoclastic bone resorption. Of various carotenoids, beta-cryptoxanthin, which is abundant in Satsuma mandarin (Citrus unchiu MARC), has a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. The supplementation of these factors has a preventive effect on bone loss induced by ovariectomy in rats, which are an animal model of osteoporosis, and their intake has been shown to have a stimulatory effect on bone mass in humans. Factors with an anabolic effect on bone metabolism were found in extracts obtained from wasabi leafstalk (Wasabi japonica MATSUM), the marine alga Sargassum horneri, and bee pollen Cistus ladaniferus. Phytocomponent p-hydroxycinnamic acid was also found to have an anabolic effect on bone metabolism. Food chemical factors thus play a role in bone health and may be important in the prevention of bone loss with increasing age.

Yamaguchi M
Yakugaku Zasshi Nov 2006
PMID: 17077614 | Free Full Text

p-Hydroxycinnamic Acid Anabolic and Antiresorptive in Rats

Abstract

Phytocomponent p-hydroxycinnamic acid stimulates bone formation and inhibits bone resorption in rat femoral tissues in vitro.

The effect of cinnamic acid or its related compounds, which is present in many plants, on bone metabolism has not been clarified yet. The effect of cinnamic acid, p-hydroxycinnamic acid (HCA), ferulic acid, caffeic acid, or 3,4-dimethoxycinnamic acid (DCA) on bone calcium content in vitro was investigated. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48,h in Dulbecco’s modified Eagle’s medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of HCA (10(-5) or 10(-4),M) caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. Such an effect was not observed in the presence of cinnamic acid or other compounds at the concentration of 10(-5) or 10(-4),M. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in the presence of HCA (10(-5) or 10(-4),M). The effect of HCA (10(-4),M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal or metaphyseal tissues was completely prevented in the presence of cycloheximide (10(-6),M), an inhibitor of protein synthesis. Thus HCA had anabolic effects on bone components. The presence of parathyroid hormone (PTH; 10(-7),M), a bone-resorbing factor, caused a significant decrease in calcium content and a corresponding elevation in medium glucose consumption, lactic acid production or tartrate-resistant acid phosphatase (TRACP) activity in the diaphyseal or metaphyseal tissues. These alterations were completely prevented in the presence of HCA (10(-5) or 10(-4),M). This study demonstrates that p-hydroxycinnamic acid (HCA) has stimulatory effects on bone formation and inhibitory effects on bone resorption in tissue culture in vitro.

Lai YL, Yamaguchi M
Mol. Cell. Biochem. Nov 2006
PMID: 17036165

p-Hydroxycinnamic Promotes Osteoblasts by Blocking NF-κB in Mouse Cells

Abstract

The bone anabolic carotenoid p-hydroxycinnamic acid promotes osteoblast mineralization and suppresses osteoclast differentiation by antagonizing NF-κB activation.

Numerous plant derived nutritional factors including p-hydroxycinnamic acid (HCA), a member of the carotenoid family, have long been held to possess bone protective properties. Studies in animals have provided a mechanistic basis for these observations by demonstrating the capacity of HCA to promote bone formation and suppress bone resorption in vivo. However, the molecular mechanism by which HCA achieves these effects remains unclear. We have demonstrated that a centralized mechanism by which several other nutritional factors achieve similar effects is through modulation of the nuclear factor-κB (NF-κB) signal transduction pathway. NF-κB activation is essential for osteoclast formation and resorption but potently antagonizes osteoblast differentiation and mineralization. In this study we demonstrate that HCA does indeed antagonize the activation of NF-κB by the key osteoclastogenic cytokine receptor activator of NF-κB (RANKL) in RAW264.7 osteoclast precursors, suppressing their differentiation into osteoclasts. Furthermore, HCA augmented the in vitro differentiation of MC3T3 preosteoblastic cells into mineralizing osteoblasts and relieved the inhibitory action of tumor necrosis factor-α (TNF-α)-induced NF-κB signaling on transforming growth factor-β (TGF-β)- or bone morphogenetic protein-2 (BMP-2)-induced Smad activation, an important pathway in osteoblast commitment and differentiation. Our data provide a mechanism to explain the dual pro-anabolic and anti-catabolic activities of HCA.

Yamaguchi M, Weitzmann MN
Int. J. Mol. Med. Sep 2012
PMID: 22751682

p-Hydroxycinnamic Acid Prevents Bone Loss in Ovariectomized Rats

Abstract

Oral administration of phytocomponent p-hydroxycinnamic acid prevents bone loss in ovariectomized rats.

The preventive effect of phytocomponent p-hydroxycinnamic acid (HCA) on ovariectomy (OVX)-induced bone loss was investigated. HCA (250 or 500 microg/100 g body weight) was orally administered once daily for 30 days to OVX rats. The analysis using a peripheral quantitative computed tomography (pQCT) showed that OVX caused bone loss in the femoral-metaphyseal tissues. This change was significantly restored after the administration of HCA (250 or 500 microg/100 g body weight) to OVX rats. Mineral content, mineral density, and polar strength strain index in the femoral-metaphyseal tissues were significantly decreased in OVX rats. These decreases were significantly restored after the administration of HCA (500 microg/100 g) to OVX rats. Moreover, OVX caused a significant decrease in calcium content or alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. These decreases were significantly restored after the administration of HCA (250 or 500 microg/100 g) to OVX rats. Deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in OVX rats. These increases were significantly restored after oral administration of HCA (500 microg/100 g). This study demonstrates that HCA has preventive effects on OVX-induced bone loss of rats in vivo.

Yamaguchi M, Lai YL, Uchiyama S, Nakagawa T
Mol. Cell. Biochem. Apr 2008
PMID: 18165927

Green Tea Prevents Bone Loss in Rats

Abstract

Protective effect of green tea polyphenols on bone loss in middle-aged female rats.

Recent studies have suggested that green tea polyphenols (GTP) are promising agents for preventing bone loss in women. Findings that GTP supplementation resulted in increased urinary GTP concentrations and bone mass via an increase of antioxidant capacity and/or a decrease of oxidative stress damage suggest a significant role of GTP in bone health of women.
Recent studies suggested that green tea polyphenols (GTP) are promising agents for preventing bone loss in women. However, the mechanism related to the possible protective role of GTP in bone loss is not well understood.
This study evaluated bioavailability, mechanisms, bone mass, and safety of GTP in preventing bone loss in middle-aged rats without (sham, SH) and with ovariectomy (OVX).

A 16-week study of 2 (SH vs. OVX) x 3 (no GTP, 0.1% GTP, and 0.5% GTP in drinking water) factorial design using 14-month-old female rats (n = 10/group) was performed. An additional 10 rats in baseline group were euthanized at the beginning of study to provide baseline parameters.
There was no difference in femur bone mineral density between baseline and the SH+0.5% GTP group. Ovariectomy resulted in lower values for liver glutathione peroxidase activity, serum estradiol, and bone mineral density. GTP supplementation resulted in increased urinary epigallocatechin and epicatechin concentrations, liver glutathione peroxidase activity and femur bone mineral density, decreased urinary 8-hydroxy-2′-deoxyguanosine and urinary calcium levels, but no effect on serum estradiol and blood chemistry levels.
We conclude that a bone-protective role of GTP may contribute to an increase of antioxidant capacity and/or a decrease of oxidative stress damage.

Shen CL, Wang P, Guerrieri J, Yeh JK…
Osteoporos Int Jul 2008
PMID: 18084689

EGCG Suppresses Osteoclasts and Arthritis in Mice

Abstract

(-)-Epigallocatechin-3-gallate suppresses osteoclast differentiation and ameliorates experimental arthritis in mice.

To verify the effects of (-)-epigallocatechin-3-gallate (EGCG) on osteoclast differentiation and on experimental arthritis in mice.
Human osteoclasts were differentiated from peripheral blood monocytes. The effects of EGCG were examined by tartrate-resistant acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, and quantitative real-time polymerase chain reaction.

Arthritis was induced in mice by injecting a cocktail of monoclonal antibodies against collagen. EGCG (20 microg/gm body weight) was administered intraperitoneally every day from day 0 through the end of the experiments (day 15). The effects of EGCG were determined by assessments of joint swelling, histologic changes, and TRAP staining on day 15.
EGCG reduced the generation of TRAP-positive multinucleated cells, bone resorption activity, and osteoclast-specific gene expression without affecting cell viability. EGCG down-regulated expression of nuclear factor of activated T cells c1 (NF-ATc1), but not of NF-kappaB, c-Fos, and c-Jun, suggesting that down-regulation of NF-ATc1 is one of the molecular bases of EGCG action. Additionally, EGCG treatment ameliorated clinical symptoms and reduced histologic scores in arthritic mice (P < 0.05). The in vivo effect of EGCG on osteoclast differentiation was not clear in this model, probably because EGCG suppressed the inflammation itself.
EGCG suppressed osteoclast differentiation and ameliorated experimental arthritis in mice over the short term. It remains to be established whether EGCG is useful for the prevention and treatment of osteoporosis and rheumatoid arthritis.

Morinobu A, Biao W, Tanaka S, Horiuchi M…
Arthritis Rheum. Jul 2008
PMID: 18576345 | Free Full Text

EGCG Inhibits Osteoclasts in Mice

Abstract

Epigallocatechin-3-gallate inhibits osteoclastogenesis by down-regulating c-Fos expression and suppressing the nuclear factor-kappaB signal.

Epigallocatechin-3-gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast differentiation. However, the precise molecular mechanisms underlying the inhibitory action of EGCG in osteoclastogenesis and the effect of EGCG on inflammation-mediated bone destruction remain unclear. In this study, we found that EGCG inhibited osteoclast formation induced by osteoclastogenic factors in bone marrow cell-osteoblast cocultures but did not affect the ratio of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) to osteoprotegerin induced by osteoclastogenic factors in osteoblasts. We also found that EGCG inhibited osteoclast formation from bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor plus RANKL in a dose-dependent manner without cytotoxicity. Pretreatment with EGCG significantly inhibited RANKL-induced the gene expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. EGCG suppressed RANKL-induced activation of c-Jun N-terminal protein kinase (JNK) pathway, among the three well known mitogen-activated protein kinases and also inhibited RANKL-induced phosphorylation of the NF-kappaB p65 subunit at Ser276 and NF-kappaB transcriptional activity without affecting the degradation of IkappaBalpha and NF-kappaB DNA-binding in BMMs. The inhibitory effect of EGCG on osteoclast formation was somewhat reversed by retroviral c-Fos overexpression, suggesting that c-Fos is a downstream target for antiosteoclastogenic action of EGCG. In addition, EGCG treatment reduced interleukin-1-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo. Taken together, our data suggest that EGCG has an antiosteoclastogenic effect by inhibiting RANKL-induced the activation of JNK/c-Jun and NF-kappaB pathways, thereby suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

Lee JH, Jin H, Shim HE, Kim HN…
Mol. Pharmacol. Jan 2010
PMID: 19828731 | Free Full Text

EGCG Improves Bone in Ovariectomized Rats

Abstract

(-)-Epigallocatechin-3-gallate improves bone microarchitecture in ovariectomized rats.

Previously, we reported that (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, increased the osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the messenger RNA expression of osteogenesis-related genes, alkaline phosphatase activity, and, eventually, mineralization. The present study further investigated the effects of EGCG on bone microstructure change and possible mechanisms in ovariectomy (OVX)-induced osteopenic rats.
Rats subjected to OVX were administered EGCG systemically for 12 weeks. Proximal tibial bone mineral densities before and after treatment were compared between groups. Changes in the microarchitecture of both the proximal tibia and the third lumbar spine were compared between EGCG-treated and nontreated groups using micro-CT (μCT). Bone histology and immunohistochemistry in the proximal tibia were evaluated.
Results showed that EGCG 3.4 mg/kg/day (estimated peak serum concentration, 10 μmol/L) hampered the decrease in bone mineral density (from 7.97% to 3.96%) and improved the parameters of μCT measurements, including bone volume (from 18% to 27%), trabecular thickness (from 0.17 to 0.22 mm), trabecular number (from 1.13 to 1.37 mm(-1)), and trabecular separation (from 0.91 to 0.69 mm), compared with nontreated ovariectomized rats. Similar improvements in bone volume (from 30% to 49%) and trabecular thickness (from 0.14 to 0.26 mm) were also found in the third lumbar spine. Bone volume in the tibial cortex also increased after EGCG treatment (from 9% to 28%). A higher trabecular number and greater trabecular volume were also seen in histology, further confirming the results of μCT. The immunolocalized bone morphogenetic protein 2 brown-stained area increased from 31% in the OVX group to 53% in the OVX + 10 EGCG group (P < 0.01). Serial biochemistry data revealed no significant systemic toxic effect of EGCG.
Intraperitoneal treatment with EGCG 3.4 mg/kg/day for 3 months can mitigate bone loss and improve bone microarchitecture in ovariectomized rats, and increased expression of bone morphogenetic protein 2 may contribute to this effect.

Chen CH, Kang L, Lin RW, Fu YC…
Menopause Jun 2013
PMID: 23511703

Naringin Reverses Bone Loss, Increasing Density, Volume, and Thickness in Ovariectomized Rats

Abstract

Naringin promotes osteoblast differentiation and effectively reverses ovariectomy-associated osteoporosis.

Osteoporosis is a common pathological condition that influences 20 % of women over 50 years of age. This condition decreases bone strength and increases the risk of bone fracture. Naringin is a major flavonoid found in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae). Studies have shown that naringin possesses many pharmacological effects. The current study evaluated the influence of naringin on osteoblastic cell differentiation and proliferation, and assessed its therapeutic effects on a rat osteoporosis model.
The proliferation, differentiation, and function of rat bone marrow stromal cells (BMSCs) were determined following treatment with various concentrations of naringin. Ovariectomy (OVX)-induced osteoporotic rats were orally administered naringin daily at low, medium, and high dosages, while a control group received PBS for 2 months. Femoral X-ray images and microCT scans were used for bone mineral density (BMD) and BV/TV (bone volume/total volume) analyses, and histological assessments of left tibiae were employed to check for changes in trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) in the groups.
Naringin was effective at enhancing the proliferation and osteogenic differentiation of BMSCs, and a concentration of 10 μg/ml prompted the highest levels of osteocalcin expression among the in vitro study groups. There appeared to be a delayed response pattern of BMSCs to the naringin treatment. Naringin also effectively reversed OVX-induced bone loss via increasing BMD, bone volume, and trabecular thickness. The medium dose (300 mg/kg) appeared to be the optimal dosage for delivering satisfactory therapeutic effects.
Naringin promotes the proliferation and differentiation of BMSCs, and increases osteocalcin expression. Naringin also effectively reverses ovariectomy-induced osteoporosis in rats. The study suggests that naringin administration may represent an effective treatment for osteoporosis.

Li N, Jiang Y, Wooley PH, Xu Z…
J Orthop Sci May 2013
PMID: 23553541