Tag Archives: in vitro

Naringin Works Like Statins to Increase Bone Formation In Vitro

Abstract

Effect of naringin on bone cells.

Statin, a HMG-CoA reductase inhibitor, was shown to increase BMP-2 gene expression for bone formation, by blocking the mevalonate pathway in cholesterol production. We investigated the effect of naringin, a flavonoid available commonly in citrus fruits, which was also a HMG-CoA reductase inhibitor, in UMR 106 osteoblastic cell line in vitro. The control group consisted of cells cultured without any intervention for different time intervals (24 h, 48 h, and 72 h), whereas the experimental (naringin) group consisted of cells cultured with naringin of different concentrations (0.001 micromol/L, 0.01 micromol/L, and 0.1 micromol/L) for the same time intervals of the control. Colorimetric Tetrazolium (MTT) assay, total protein content assay, and alkaline phosphatase activity were used to measure the cellular activities. Results for the naringin group showed an increase in MTT assay compared with the control and the effect was dose dependent. At high concentration (0.1 micromol), the increases ranged from 60% to 80%. In the total protein content assay, naringin also showed an increase compared with control and the effect was also dose dependent. At high concentration (0.1 micromol), the increases ranged from 9% to 20%. In the alkaline phosphatase activity assay, naringin at high concentration (0.1 micromol) significantly increased the activity up to 20%. In conclusion, naringin significantly increased bone cell activities in vitro. This is the first study specifically attempted to investigate the effect of naringin on bone cell activities. Besides statin, this provided another example of mevalonate pathway blockage in the cholesterol production pathway by HMG-CoA reductase inhibition will increase the bone cell activities.

Wong RW, Rabie AB
J. Orthop. Res. Nov 2006
PMID: 16944474

Naringin and Neoeriocitrin Stimulate Osteoblasts In Vitro

Abstract

Stimulative activity of Drynaria fortunei (Kunze) J. Sm. extracts and two of its flavonoids on the proliferation of osteoblastic like cells.

The osteoblastic activity of extracts of Drynaria fortunei (Kunze) J. Sm. rhizome was assayed in the UMR106 cell line cultured in vitro. An ethanol extract and its fractions were added to the cell culture at different concentrations. Osteoblastic proliferation stimulating activity was determined using the MTT method. The ethanol extract, and its ethyl acetate and n-butanol fractions exhibited stimulating activity. Two active constituents were isolated from n-butanol fraction by bioassay-directed isolation, and identified as naringin and neoeriocitrin. The latter is reported for the first time from this herbal medicine.

Li F, Meng F, Xiong Z, Li Y…
Pharmazie Nov 2006
PMID: 17152991

Naringin Enhances Proliferation of Human Bone Cells In Vitro

Abstract

Effects of naringin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cell.

Rhizoma drynariae is used commonly in the treatment of osteoporosis and bone nonunion in traditional Chinese medicine. Modern pharmacological research indicates that naringin is the main effective component of rhizoma drynariae, which can induce the expression of the osteogenic marker in the osteoblast cell line. However, no former study has described its effect on bone mesenchymal stem cells (BMSCs). In our experiment, we co-cultured human BMSCs with different concentrations of naringin solution, then the osteogenic differentiation markers and proliferation ability were analyzed. The results indicated that a certain concentration (1-100 microg/ml) of the naringin solution may enhance the proliferation and osteogenic differentiation of human BMSCs. Also, our research explains excellently the anti-osteoporotic and bone nonunion treatment mechanism of rhizoma drynariae, thus contributing to the exploration of osteogenic differentiation agents from Chinese herbs.

Zhang P, Peng-Zhang , Dai KR, Yan SG…
Eur. J. Pharmacol. Apr 2009
PMID: 19326565

Neoeriocitrin More Active Than Naringin in Bone Cells

Abstract

Comparison of neoeriocitrin and naringin on proliferation and osteogenic differentiation in MC3T3-E1.

Naringin is considered the main effective compound of Drynaria Rhizome, which is used commonly in the treatment of osteoporosis in traditional Chinese medicine. However, we found neoeriocitrin, a new compound isolated from Drynaria Rhizome, showed a better activity than naringin on proliferation and osteogenic differentiation in MC3T3-E1. Both neoeriocitrin and naringin exhibited the best effect on proliferation and osteogenic differentiation at concentration of 2μg/ml. Neoeriocitrin more significantly improved proliferation and alkaline phosphatase (ALP) activity as well as up-regulated Runx2, COLI and OCN expression by 56%, 37% and 14% respectively than naringin. Furthermore, neoeriocitrin could rescue the inhibition effect of cell differentiation induced by PD98059 to some degree. Therefore, neoeriocitrin may be a new promising candidate drug for treatment of osteoporosis.

Li L, Zeng Z, Cai G
Phytomedicine Aug 2011
PMID: 21741227

Quercetin and Rutin Inhibit Resorption by Their Estrogen Receptor Proteins

Abstract

Modulation of osteoclastogenesis in porcine bone marrow cultures by quercetin and rutin.

Flavonols, in contrast to soybean isoflavones, are the most abundant phytoestrogens in western diets, being present in onions, beans, fruits, red wine, and tea. They may protect against atherosclerosis, inhibit certain cancer cell types, and reduce bone resorption. The most widely distributed flavonol is quercetin, which occurs mainly as its glycoside, rutin, but data are very scarce regarding the precise mechanism of action of these compounds on bone-resorbing cells at concentrations similar to those detected in human plasma. We have therefore investigated the effects of nanomolar concentrations of quercetin and rutin on the development and activity of osteoclasts in vitro compared with the effects of 17beta-estradiol. Nonadherent porcine bone marrow cells were cultured on dentine slices in the presence of 10 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), with or without 10 nM quercetin, 10 nM rutin or 10 nM 17beta-estradiol for 11 days. Multinuclear TRAP+ cells that resorbed dentine (osteoclasts) developed in the presence of 1,25(OH)2D3, but their number was significantly reduced by quercetin, rutin, and 17beta-estradiol (P < 0.05). Like 17beta-estradiol, both flavonols also significantly reduced resorption (P<0.05) as assessed by the size of pits resorbed on dentine slices. Osteoclasts and osteoclast progenitors contained estrogen receptor alpha (ERalpha), ERbeta, and RANK proteins. Both flavonols increased nuclear ERbeta protein and decreased ERalpha protein of osteoclast progenitors. Moreover, rutin reduced RANK protein, whereas 17beta-oestradiol and quercetin promoted apoptosis by cleavage of caspase-8 and caspase-3. All the effects of flavonols were reversed by 1 microM ICI 182,780, an estrogen antagonist. Thus, the anti-resorbing properties of flavonols are mainly mediated by ER proteins through the inhibition of RANK protein or the activation of caspases.

Rassi CM, Lieberherr M, Chaumaz G, Pointillart A…
Cell Tissue Res. Mar 2005
PMID: 15688188

Resveratrol Analogues Show No Effect on Bones In Rats

Abstract

Potential of resveratrol analogues as antagonists of osteoclasts and promoters of osteoblasts.

The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations. In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss could not be detected. Based on their powerful antiresorptive activity in vitro, resveratrol analogues might be attractive modulators of bone remodeling. However, further studies are required to establish their efficacy in vivo.

Kupisiewicz K, Boissy P, Abdallah BM, Hansen FD…
Calcif. Tissue Int. Nov 2010
PMID: 20842496 | Free Full Text

Review: Resveratrol Osteogenic Effects In Vitro

Abstract

Osteogenic effects of resveratrol in vitro: potential for the prevention and treatment of osteoporosis.

There are a number of pharmacological agents for the treatment of bone mineral loss and osteoporosis. Hormone replacement therapy (HRT) with estrogen is an established treatment, but it has several adverse side effects and can increase the risk of cancer, heart disease, and stroke. There is increasing interest in nutritional factors and naturally occurring phytochemical compounds with the potential for preventing age-related and postmenopausal bone loss. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic phytoestrogen with osteogenic and osteoinductive properties. It can modify the metabolism of bone cells and has the capacity to modulate bone turnover. This paper provides an overview of current research on resveratrol and its effects on bone cells in vitro, highlighting the challenges and opportunities facing this area of research, especially in the context of providing nutritional support for postmenopausal women who may not benefit from HRT and older patients with various forms of arthritis, metabolic bone disease, and osteoporosis.

Mobasheri A, Shakibaei M
Ann. N. Y. Acad. Sci. Jul 2013
PMID: 23855466

Resveratrol Stimulates Osteocalcin in Rat Cells

Abstract

Estradiol and resveratrol stimulating effect on osteocalcin, but not osteonectin and collagen-1alpha gene expression in primary culture of rat calvarial osteoblast-like cells.

Evidence is available that some endocrine disruptors, acting as selective estrogen receptor modulators (SERMs), interfere with osteoblast differentiation and function. Therefore, we investigated whether 17beta-estradiol, bisphenol-A (BSP), silymarin, genistein, resveratrol, procymidone, linurone and benzophenone-3 (BP3) modulate differentiation of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. Disruptors were added at day 18 of culture and cells were harvested 48 h later. Real time-PCR revealed that estradiol and resveratrol enhanced osteocalcin mRNA expression in ROB cells, while other disruptors were ineffective. The expression of osteonectin and collagen-1alpha was not affected by any disruptor. Estradiol, resveratrol, genistein and BSP stimulated the proliferative activity of ROB cells. In contrast, procymidone and linurone inhibited the proliferative activity, and silymarin and BP3 were ineffective. The conclusion is drawn that i) only resveratrol is able, like estradiol, to stimulate the specialized functions of ROB cells, and ii) the proliferative activity of ROB cells is more sensitive to endocrine disruptors, some of which could probably act via a mechanism independent of their SERM activity.

Rucinski M, Ziolkowska A, Hochol A, Pucher A…
Int. J. Mol. Med. Oct 2006
PMID: 16964405

Rutin Effects Likely from Quercetin

Abstract

[Comparative study on effects of rutin and quercetin on metabolism in osteoblast cells].

To investigate the mechanism of rutin and its aglycone (quercetin) in preventing and treating osteoporosis and their effects on stimulating bone formation were studied comparatively in osteoblast cells.
The effects of rutin and quercetin on the proliferation, differentiation and mineralization were studied respectively by using tetrazolium (MTT), alkaline phosphatase (ALP) and mineralized nodules assays in newborn rat calvarial osteoblast (ROB) cells.
Quercetin showed significant stimulatory effects on proliferation and mineralization in ROB cells, so it could promote bone formation. However, rutin could not improve stimulation of bone formation because it increased proliferation and inhibited differentation and had no remarkable influence on ROB cells in vitro.
Rutin showed less activity than quercetin on bone formation in ROB cells in vitro. Therefore the mechanism that some foods and traditional Chinese medicine containing rutin can prevent and treat osteoporosis is that its metaboite in vivo can promote bone formation in ROB cells.

Yang YJ, Yang ZL, Wang DC, Xiao XC…
Zhong Yao Cai May 2006
PMID: 16981462

Quercetin Actions In Vitro

Abstract

Quercetin, a potent suppressor of NF-κB and Smad activation in osteoblasts.

Osteoclasts, the bone resorbing cells of the body, form when osteoclast precursors are exposed to the key osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL), a process requiring induction of NF-κB signaling. Quercetin is a ubiquitous plant-derived flavonoid with well documented anti-inflammatory properties, in part, a consequence of its capacity to downmodulate the NF-κB signal transduction pathway. Consistent with this mechanism of action quercetin is reported to suppress osteoclastogenesis in vitro and prevent bone loss in ovariectomized mice in vivo. By contrast, the effect of quercetin on osteoblasts, the cells responsible for bone formation, is contradictory with conflicting reports of inhibition as well as stimulation. Given our previous reports that NF-κB antagonists promote osteoblast differentiation and activity, we compared the effects of quercetin on osteoclast and osteoblast differentiation and on NF-κB signal transduction in vitro. As expected, quercetin potently suppressed osteoclastogenesis and NF-κB activation induced by RANKL in osteoclast precursors. However, the same doses of quercetin had no effect on osteoblast mineralization, and failed to significantly alleviate the inhibitory effect of NF-κB-induced by TNFα, even though quercetin potently suppressed NF-κB activation in these cells. This apparent contradiction was explained by the fact that addition to its anti-NF-κB activity, quercetin also potently antagonized both TGFβ and BMP-2-induced Smad activation in osteoblast precursors. Taken together our data suggest that multiple competing actions of quercetin mediate both stimulatory and inhibitory actions on osteoblasts with the final physiological effect likely a function of the net balance between these stimulatory and inhibitory effects.

Yamaguchi M, Weitzmann MN
Int. J. Mol. Med. Oct 2011
PMID: 21769418