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Promoting osteoblast differentiation by the flavanes from Huangshan Maofeng tea is linked to a reduction of oxidative stress.

ABSTRACT

Epidemiological evidence has shown an association between tea consumption and the prevention of bone loss in the elderly. Previous studies indicated that green tea exerted osteoprotective effect in vivo. This study aims to investigate the constituents in Huangshan Maofeng tea and systemically evaluate their antioxidative and osteogenic effects in vitro. Five flavanes, isolated from Huangshan Maofeng tea, showed effects on proliferation of osteoblastic cells and ameliorated [H.sub.2][O.sub.2]-induced C2C12 mouse myoblast cell apoptosis at 3.125-50 [micro]g/ml. (-)-Epicatechin observably increased alkaline phosphatase (ALP) activity and hydroxyproline content. (-)-Epiafzelechin at 25 [micro]g/ml significantly increased the area of mineralized bone nodules. The activities of flavanes in promoting osteblastic proliferation and differentiation are positively correlated with activities in protecting against apoptosis in C2C12 cells. It indicates that antiosteoporosis effect of these flavanes may be linked to their antioxidative activity. The observed effects of these flavanes suggest that these flavanes may have beneficial effects on bone health.

Keywords:

Flavane

Osteoporosis

Oxidative stress

Green tea

Introduction

Osteoporosis is a disease characterized by the loss of bone mass and degeneration of bone microstructure, resulting in an increased risk of fracture. Osteoporosis, called postmenopausal osteoporosis, is common in women after menopause (Ozgocmen et al., 2007). It may also develop in men, especially in the aged man, which is called age-related bone loss (Overton and Basu, 1999). Osteoporosis may significantly affect life expectancy and quality of life in humans. Bone integrity requires a tight coupling between the activity of bone-forming osteoblasts and bone-resorbing osteoclasts (Riggs and Melton, 1992). During bone formation, osteoblasts undergo a cascade of complex events that might include three phases: proliferation, osteogenic differentiation, and mineralization of extracellular matrix (Zhang et al., 2008). The phenotype of mature osteoblasts is characterized by their ability to synthesize and secrete molecules of the extracellular matrix. Osteoblasts regulate them mineralization of the formed matrix by producing alkaline phosphatase (ALP) (Aubin, 1998). This enzyme hydrolyzes phosphate esters to increase the local phosphate concentration and enhance mineralization of the extracellular matrix (Lian et al., 1999). One of the characteristics of a mature osteoblast phenotype is the ability of the cells to synthesize ALP, which is considered an early marker of osteoblast differentiation (Tong et al., 1999). Collagen type I is an marker of osteogenic mature, and bone is a matrix with collagen type I (Sakano et al., 1993).

Oxidative stress, resulting from excessive formation of reactive oxygen species (ROS) or dysfunction of antioxidant defense system, represents a major cause of age-associated pathological conditions including aging (Linnane and Eastwood, 2006) and postmenopausal bone loss (Sendur et al., 2009). ROS are involved in osteogenesis including bone formation and resorption, which are associated with the aging process and may result in osteoporosis (Nohl, 1993; Basu et al., 2001; Muthusami et al., 2005; Isomura et al., 2004; Yalin et al., 2006). Oxidative stress is a pivotal pathogenic factor for age-related bone loss in mice, leading to an increase in osteoblast and osteocyte apoptosis and a decrease in osteoblast number and the rate of bone formation. Oxidative stress is involved in the osteoporosis from analysis of bone metabolism in iron-overloaded rats (Isomura et al., 2004). On the other hand, evidence suggests that ROS is also involved in bone resorption with a direct contribution of osteoclast-generated superoxide to bone degradation (Yang et al., 2001; Sontakke and Tare, 2002). Oxidative stress increases differentiation and function of osteoclasts (Sontakke and Tare, 2002), and inhibits osteoblastic differentiation (Mody et al., 2001; Fatokun et al., 2008).

Camellia sinensis, of the genus Camellia, is an evergreen plant that grows mainly in tropical and subtropical climates. Its leaves and leaf buds are used to produce the popular beverage tea. Huangshan Maofeng tea is a green tea produced in Anhui province of China. The tea is grown near Huangshan Mountain at altitudes of over 700 m. What distinguishes Huangshan Maofeng tea from other Maofeng teas is the color of its leaves. The tea liquor is jade green in color and has a light flowery fragrance. The tea is one of the most famous teas in China and can always be found on the China famous tea list. Green tea, unlike the preparation of black tea in which the leaves are fermented and oxidized, is produced by steaming the leaves, which preserves their polyphenolic content. The polyphenols comprise about 30-40% of the solid extract of leaves and are mostly categorized as catechins (Brown, 1999). Epidemiological studies have shown an association between a reduced risk of osteoporosis and the consumption of tea, including green tea (Shen et al., 2009a; Park et al., 2012; Shen et al., 2009b, 2010a, b; Hamdi Kara et al., 2007; Muraki et al., 2007; Hegarty et al., 2000; Wu et al., 2002; Devine et al., 2007; Yang and Landau, 2000). In previous study, green tea and green tea polyphenols (GTPs) benefit for bone health in ovariectomized (OVX) mice and old mice (Shen et al., 2009a; Shao et al., 2011; Shen et al., 2010a, b, 2009b, 2011a; Nakamura et al., 2010; Shen et al., 2011b). These evidences showed the therapeutic effects of the green tea on prevention of osteoporosis, however, most of these studies were focused on the crude extract or green tea polyphenols.

The present study is designed to investigate the chemical components of Huangshan Maofeng tea and evaluate potential osteogenic effects of these five flavanes on the proliferation, differentiation and mineralization of osteoblastic cells. To clarify the underlying mechanisms of flavanes, we investigated whether protection against osteoporosis by flavanes is linked to a reduction of oxidative stress.

Materials and methods

General

Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan) automatic digital polarimeter. NMR spectra were recorded on a Bruker DPX-400 spectrometer (400 MHz for [sup.1]H NMR, Karlsruhe, Germany) using standard Bruker pulse programs. Chemical shifts were showed as the [delta]-value with reference to tetramethylsilane (TMS)as an internal standard. And ES1-MS data were obtained on an Agilent 1200 HPLC/6410B TripleQuad mass spectrometer. Diaion D-101 macroporus resin was the product of Xi'an Lanxiao Resin Corporation Ltd. (Xi'an, China). Sephadex LH-20 (Pharmacia, Sweden), silica gel (Qingdao Ocean Chemical Co., Ltd., Qingdao, China), and octadecylsilanized (ODS) silica gel (Macherey-Nagel, Duren, Germany) were used for column chromatography. TLC was carried out on Silica gel 60 [F.sub.254] (0.25 mm, Merck, Darmstadt, Germany), and RP-18 [F.sub.254] (0.25 mm, Merck, Darmstadt, Germany) plates, and spots were visualized by spraying with 15% [H.sub.2]S[O.sub.4] followed by heating. HPLC was performed using an octadecylsilanized (ODS) silica gel column (XTerra 10 [micro]m, 19 mm x 250 mm. Waters). RPM1-1640 medium, fetal bovine serum (FBS), and trypsin-EDTA solution (1x) were obtained from Hyclone (Logan, UT). AnnexinV/PI Apoptosis Detection Kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Hydroxyproline Assay Kit was purchased from Nanjingjiancheng Bioengineering Institute (Nanjing, China). All other chemicals were analytical or HPLC grade and obtained from Shanghai Chemical Reagents Co., Ltd. (Shanghai, China).

Extraction and isolation

Huangshan Maofeng tea were purchased in Huangshan, Anhui Province, China, and subjected to taxonomic identification with Voucher specimens (No. 120708) deposited at the herbarium of Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical College, China. The tea (2.5 kg) was extracted three times with 70% ethanol. The solvent was removed under vacuum to yield the crude extract (700 g). A suspension of the extract in [H.sub.2]O was centrifuged and then applied to a D-101 macroresin column (80 mm x 1300 mm) and eluted with [H.sub.2]O (101), 10% ethanol (101), 30% ethanol (101), 50% ethanol (101), 70% ethanol (101), and 95% ethanol (101) successively. Each eluent was concentrated and dried to yield 220.3 g, 100.0 g, 89.0 g, 150.0 g, 67.6 g, 6.5 g of dried elutes, respectively.

Bioactivity-guided fractionation was used for the isolation work. On the basis of the bioactive results of the extracts, the 70% ethanol eluent with the most potential activity was fractionated over a silica gel (200-300 mesh) column eluting with a gradually amount of MeOH in CH[Cl.sub.3] to give 15 fractions. The CH[Cl.sub.3]-MeOH (25:1) elution was further purified by silica gel column, Sephadex LH-20, together with preparative HPLC, and got compounds 4 (152.33 mg), and 5 (70.56 mg), respectively. The CH[Cl.sub.3]-MeOH (15:1) elution was subjected to an octadecylsilanized (ODS) silica gel column, followed by a preparative HPLC with 20% methanol (containing 0.1% C[F.sub.3]COOH, pH 3.0) to give compounds 1 (65.56) and 2 (41.34 mg). The CH[Cl.sub.3]-MeOH (15:1) elution was separated over an ODS column, followed by preparative Rp-HPLC with 18.5% methanol (containing 0.1% C[F.sub.3]COOH, pH 3.0) to give compound 3 (33.47 mg).

HPLC

HPLC was performed on Phenomenex [C.sub.18] column (0 250 mm x 4.6 mm) in Agilent series 1100 (USA) to analyze the compounds in Huangshan Maofeng tea under following conditions: mobile phase: (A) [H.sub.2]O and 0.1% C[F.sub.3]COOH, (B) MeOH; elution program: linear gradient from 10% B to 30% B in 30 min, 30% B to 50% B in 20 min, 50% B to 80% B in 10 min and then 100% B maintained for 20 min; flow rate: 0.80 ml/min; detection wavelength: 254nm; injection volume: 10[micro]l; and oven temperature: 24[degrees]C.

Culture of rat osteoblast cells

Osteoblastic cells were enzymatically isolated from newborn rat calvaria by the method of Declercq et al. (Declercq et al., 2004). With some minor modifications. The bone pieces were digested sequentially in a trypsin II-S (25 mg)-collagenase IA (70 mg) in 15 ml PBS solution at 37[degrees]C for 30 min. Rat osteoblastic cells obtained from the last three digestion steps were pooled and plated together in a T25 tissue culture dish at a concentration of 50,000cells/[cm.sup.2]. And cultured in DMEM supplemented with 10% FBS and 1% antibiotic (100 units/ml penicillin and 100 [micro]g/ml streptomycin) at 37[degrees]C in a humid atmosphere containing 5% C[O.sub.2]. The medium was changed every 3 days to remove the non-adherent cells.

C2C12 cell culture

The C2C12 cell line was purchased from the American Type Culture Collection. Cells were cultured in basal medium constituted with Dulbecco's modified Eagle's medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (100 units/ml penicillin and 100[micro]g/ml streptomycin) for incubation at 37[degrees]C in a 5% C[O.sub.2] humidified atmosphere.

Cell viability assay

Cells were seeded in a 96-well plate at a density of 3 x [10.sup.3] cells/well. The total volume was adjusted to 100 pi with growth medium.

24 h after the seeding, the cells were exposed to flavanes or resveratrol (positive drug) of different concentrations. Cell viability was examined after 48 h using a standard MTT method (He and Liu, 2007). Drug effect was expressed as percentage relative to the controls.

ALP activity

Cells were cultured in DMEM supplemented with 10% FBS and 1 % antibiotic (100 units/ml penicillin and 100 [micro]g/ml streptomycin) at 37[degrees]C in a humid atmosphere containing 5% C[O.sub.2]. After 1 ml of 5 x [10.sup.4] cells/well of osteoblastic cells was seeded in 48-well plates for 24 h. The culture medium with five flavanes or resveratrol (positive drug) at concentrations of 0, 3.125, 6.25, 12.5, 25 and 50 [micro]g/ml was added in.

7 day after treatment, cells seeded in 48-well plates were washed twice with 50 mM PBS (pH 7.4) and kept in 0.1% Triton X-100 lysis buffer overnight at -20[degrees]C. The cells were later thawed (Rao et al., 2003). The ALP activity of the samples was determined by a colorimetric assay using an ALP reagent of p-nitrophenyl phosphate. 300 [micro]l of substrate buffer (6.7 mmol/1 disodium p-nitrophenylphosphate hexahydrate, 25 mmol/l diethanolamine and 1 mmol/l Mg[Cl.sub.2]) was added in. After the mixtures incubated at 37[degrees]C for 30 min, we measured the absorbance at 405 nm.

Hydroxyproline assay

After 2 mL of 1 x [10.sup.5] cells/well of osteoblastic cells was seeded in 12-well plates for 24 h, the culture medium with five flavanes or resveratrol (positive drug) at concentrations of 0, 3.125, 6.25, 12.5, 25 and 50 [micro]g/ml was added in.

21 day after treatment, cell samples were hydrolyzed in 6N HC1 (final concentration) for 12 h at 110[degrees]C. The samples were filtered and vacuum dried. The residue was dissolved in 200 [micro]l water. The hydroxyproline content was assayed according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Determination and quantification of mineralized bone nodules

After 2 ml of 1 x [10.sup.5] cells/well of osteoblastic cells was seeded in 12-well plates for 24 h, osteogenic medium (10 mM L-glycerophosphate, 50 [micro]g/ml ascorbic acid) with flavanes (1-5) or resveratrol (positive drug) was added in.

On day 20, cells seeded in 24-well plates were fixed in 95% ethanol for 15 min and the mineralized bone nodules were visualized by alizarin red staining techniques (Hale et al., 2000). We stained cells with alizarin red (40 mM, pH 7.2) for 10 min and then rinsed them with PBS. Nodules were visualized using an inverted microscope. And the area of mineralized nodules were quantified by the Image-Pro Plus 6.0 Software.

Flow cytometric analysis for apoptosis

To investigate the protective effects of flavanes on [H.sub.2][O.sub.2]induced cytotoxicity, C2C12 mouse myoblast cells were cultured in the presence of 100 [micro]M [H.sub.2][O.sub.2] with or without treatment with flavanes or quercetin (positive drug). Apoptosis was examined by Annexin V-fluorescein isothiocyanate staining (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's instructions. Cells were seeded on 6-well plates. 2 days after treatment, the cells were harvested by trypsinization, rinsed twice with PBS, and suspended in 500 [micro]l of binding buffer. The suspended cells were incubated for 15 min at 4[degrees]C with 5 [micro]l Annexin V-FITC solution, and incubated for another 5 min at 4[degrees]C after adding 10 [micro]l of PI solution, the FITC fluorescence intensity of 10,000 cells was measured with a flow cytometer (Beckman-Coulter, Inc., Indianapolis, IN). The apoptosis was expressed as the ratio of apoptotic cell count to total cell count.

Statistical analysis

All data were expressed as mean [+ or -] S.D. from at least three independent experiments, each performed in quintuplicate. Statistical significance was determined by analysis of variance and subsequently applying the Dunnett's t-test (p < 0.05).

Results

Flavanes identified from the extracts of Huangshan Maofeng tea

Five flavanes were isolated from the extracts of Huangshan Maofeng tea by bioassay-guided fractionation. Their structures were identified using chemical evidence, spectral analysis and comparison with literature data, namely (+)-catechin (1) (Shen et al., 1993), (-)-epiafzelechin (2) (Waterman and Faulkner, 1979), (-)-catechin (3) (Shen et al., 1993), (-)-epicatechin (4) (Shen et al., 1993), and (-)-afzelechin (5) (Hsu et al., 1993). And the structures of flavanes 1-5 were shown in Fig. 1. The HPLC fingerprint analysis of the extracts is shown in Fig. 2.

Effect of flavanes (1-5) in osteoblast viability in vitro

When cultured different concentration of flavanes (1-5) with rat osteoblast cells, flavanes (1-5) at concentration from 3.125 [micro]g/ml to 50 [micro]g/ml stimulated rat osteoblast cell growth and proliferation, this effect appeared in a dose-dependent manner. The effect of these five flavanes on osteoblast cells is shown in Fig. 3. It found that (-)-epicatechin at the concentration of 50 [micro]g/ml showed the highest promoting proliferative activity with about 3.83-fold when compared with the negative control, which exhibited more potential promiting activity than positive control-resveratrol.

Effects of flavanes (1-5) on ALP activity of rat osteoblast

To determine whether these flavanes could stimulate osteogenic differentiation, the effects of flavanes (1-5) on bone formation early marker, ALP activity, were detected. Our data illustrated that treatment of rat osteoblast cells with flavanes (1-5) for 7 days stimulated ALP activity in a dose-dependent manner. As shown in Fig. 4, all five flavanes at most of their treated concentrations increased the ALP activity compared with the control, where they exhibited comparable activity level compared with positive control. (-)-Epiafzelechin demonstrated a dose-dependent increase in ALP activity by 11.9%, 42.9%, 50.7%, 69.7% and 19.6% at the concentrations of 3.125, 6.25, 12.5, 25 and 50[micro]g/ml, as compared to the control. (-)-Catechin showed a dose-dependent increase in ALP activity by 8.5%, 24.5%, 29.7%, 61.7% and 46.7% at the concentrations of 3.125, 6.25, 12.5, 25 and 50[micro]g/ml, respectively, as compared to the control. (-)-Epicatechin significantly increased the ALP activity by 68.1%, 64.0%, 96.7% and 93.1% at lower concentrations of 6.25, 12.5, 25 and 50 [micro]g/ml, as compared to the control. (-)-Afzelechin demonstrated a dose-dependent effect on promoting ALP activity of rat osteoblast cells. It significantly increased the ALP activity by 6.1%, 10.0%, 2.7%, 17.3% and 14.9% at the concentrations of 3.125, 6.25, 12.5, 25 and 50[micro]g/ml, respectively, as compared to the control. (+)-Catechin, with a significant increase in ALP activity by 11.6%, 22.8% and 12.4% at the concentrations of 12.5,25 and 50 [micro]g/ml, respectively, as compared to the control, while 3.125 and 6.25 [micro]g/ml of (+)-catechin did not show promoting effect on the ALP activity. At 25 [micro]g/ml, the ALP activity of (-)-epicatechin was significantly increased by about 0.97-fold when compared with the negative control. And its osteogenic effect was stronger than the positive control resveratrol at 25[micro]g/ml. These results suggested that flavanes in Huangshan Maofeng tea could

promote intermediate-term marker, collagen, were determined. Our data illustrated that treatment of rat osteoblast cells with flavanes (1-5) for 21 days stimulated collagen secreting in a dose-dependent manner. As shown in Fig. 5, (-)-epiafzelechin demonstrated a dose-dependent increase in collagen content by 24.5%, 29.5%, 40.1% and 56.1% at the concentrations of 6.25, 12.5, 25 and 50 [micro]g/ml, as compared to the control. (-)-Catechin showed a dose-dependent increase in collagen content by 27.5%, 27.0%, 16.8% and 11.0% at the concentrations of 3.125, 6.25, 12.5 and 25 [micro]g/ml, respectively, as compared to the control. (-)-Epicatechin significantly increased in collagen content by 42.5%, 37.6% and 74.9% at the concentrations of 12.5,25 and 50 [micro]g/ml, as compared to the control. (-)-Afzelechin demonstrated a dose-dependent effect on promoting collagen content of rat osteoblast cells. It significantly increased the collagen content by 5.1%, 25.4% and 37.3% at the concentrations of 12.5,25 and 50 [micro]g/ml, respectively, as compared to the control. (+)-Catechin, with a significant increase in collagen content by 19.5%, 24.4% and 12.7% at the concentrations of 6.25, 12.5, and 25[micro]g/ml, respectively, as compared to the control, while 3.125 [micro]g/ml of (+)-catechin, (-)-epiafzelechin, (-)-epicatechin and (-)-afzelechin did not show promoting effect on the collagen content. Treated with 50[micro]g/ml (-)-epicatechin, the collagen content was significantly increased by about 0.75-fold when compared with the negative control. Its osteogenic effect was stronger than the positive control resveratrol at 50[micro]g/ml. These results suggested that flavanes in Huangshan Maofeng tea could promote intermediate-term osteogenic differentiation of rat osteoblast cells.

Effects of the flavanes (1-5) on the formation of mineralized bone nodules

The mineralized bone nodules formed by rat osteoblast cells were observed after 20 days of treatment. The mineralized bone nodules could be visualized by the naked eye as red-purple spots after staining with alizarin red (Fig. 6A). The addition of (-)-epicatechin, (-)-epiafzelechin, and (-)-catechin at concentrations as low as 3.125 [micro]g/ml to the culture media increased the formation of mineralized nodules compared with control. (-)-Epicatechin, (-)-epiafzelechin, and (-)-catechin caused a dose-dependent increase in the area of mineralized bone nodules as quantified using a Image-Pro Plus 6.0 software. Analysis of the results from alizarin red staining showed that at 6.25 [micro]g/ml, (-)-epiafzelechin increased the area of nodules by about 7.26-fold (Fig. 6B). Analysis of the results from alizarin red staining demonstrated that 25 [micro]g/ml (-)-epicatechin resulted in an about 5.44-fold increase in the area of nodules (Fig. 6B). A similar increase in area of nodules was also seen in cultures treated with 6.25 [micro]g/ml (-)-afzelechin (Fig. 6B).

Protective effects of flavanes (1-5) against [H.sub.2][O.sub.2]-induced cell injury in C2C12 mouse myoblast cells

To investigate the protective effects of flavanes (1-5) on [H.sub.2][O.sub.2]-induced cytotoxicity, C2C12 mouse myoblast cells were cultured in the presence of 100[micro]M [H.sub.2][O.sub.2] with or without treatment with flavanes (1-5). Treatment with flavanes (1-5) increased the cell viability of C2C12 mouse myoblast cells compared to the [H.sub.2][O.sub.2]-entreated control (Fig. 7A). Treatment with flavanes (1-5) decreased the number of apoptotic cells compared to the [H.sub.2][O.sub.2]-treated control group (Fig. 7B). From the results, all of the flavanes isolated from Huangshan Maofeng tea showed potential protective effects against [H.sub.2][O.sub.2]-induced cell injury in C2C12 mouse myoblast cells. Among them, (-)-epicatechin showed the highest protective activity, which exhibited more potential antioxidant activity than positive control-quercetin.

Discussion

This study reported for the first time to investigate the osteogenic effects of the chemical constituents of Huangshan Maofeng tea. These five identified flavanes showed effects on enhancing proliferation of rat osteoblast cells at the tested concentrations (3.125-50 [micro]g/ml). All the five flavanes at majority of the tested concentrations (3.125-50 [micro]g/ml) increased the osteogenic differentiation. Among them, (-)-epicatechin at the concentration of 25[micro]g/ml increased the ALP activity to the highest level by 96.7%, compared to the control, while (+)-catechin and (-)afzelechin did not alter ALP activity of rat osteoblast cells at the concentrations of 3.125-6.25 [micro]g/ml and mildly increased at the concentration of 12.5-50 [micro]g/ml. Collagen content of the rat osteoblastic cells was determined by hydroxyproline assay. Treated with 50[micro]g/ml (-)-epicatechin, the hydroxyproline content was significantly increased by about 0.75-fold when compared with the negative control. Its osteogenic effect was stronger than the positive control resveratrol at 50[micro]g/ml. (-)-Epiafzelechin, (-)-catechin, (-)-epicatechin, and (-)-afzelechin promoted mineralization of rat osteoblastic cells, while (+)-catechin did not show the positive effects on cell mineralization in osteoblastic cells.

The findings of the present study showed that (-)-epiafzelechin, (-)-catechin, (-)-epicatechin, and (-)-afzelechin in Huangshan Maofeng tea was able to activate simultaneously the rat osteoblastic cells at three phases. (-)-Epiafzelechin and (-)-epicatechin significantly promoted osteogenic proliferation, differentiation, and mineralization. It was also shown that flavanes, the most abundant constituents found in green tea, increases the formation of mineralized bone nodules from rat osteoblast cells by increasing osteoblastic differentiation. Different effects found between flavanes (1-5) might be explained by difference in configuration and hydroxy.

The present study evaluated the underlying mechanisms of flavanes (1-5) in association with their osteogenic effects where we showed that flavanes [(-)-epiafzelechin and (-)-epicatechin] promoted osteogenic effects through reduction of oxidative stress. Similar to an early study, osteogenic effects of green tea and green tea polyphenols were reported to be mediated by reducing oxidative stress (Shen et al., 2008, 2009c; Shao et al., 2011; Shen et al., 2009a, 2011c). Green tea and green tea polyphenols inhibited osteoclast genesis and osteoclast bone resorption (Nakamura et al., 2010), a new hypothesis was emerged that flavanes in green tea might be the phyto-molecules that promoted osteogenic genesis and inhibited osteoclast bone resorption by reducing oxidative stress. The present study showed that the activities of these flavanes in promoting osteblastic proliferation and differentiation are positively correlated with the activities of those flavanes in protecting against [H.sub.2][O.sub.2]-induced apoptosis in C2C12 myogenic cells. And it indicated that anti-osteoporosis effect of this kind flavane might be linked to a reduction of oxidative stress. Further investigations are desirable to confirm this hypothesis based on findings of our current in vitro study. Our data provide some cellular evidence in support of the epidemiological associations between green tea consumption and a reduced risk of osteoporosis.

http://dx.doi.org/10.1016/j.phymed.2013.08.026

ARTICLE INFO

Article history:

Received 25 May 2013

Received in revised form 24 July 2013

Accepted 23 August 2013

Acknowledgements

This research was supported by the Science 8t Technology Innovation Fund of Guangdong Medical College (STIF 201104) and National Natural Science Foundation of China (NSFC 81273518).

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Xiaobin Zeng (a), *, (1), Jun Tian (b,1), Kangyong Cai (c), Xin Wu (a), Yang Wang (d), Yayuan Zheng (a), Yanjie Su (a), Liao Cui (a), *

(a) Guangdong Key Laboratory for Research and Development of Natural Drugs, Department of Pharmacology, Guangdong Medical College, Zhanjiang 524023, Guangdong, China

(b) College of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, China

(c) Analysis Center of Guangdong Medical College, Guangdong Medical College, Zhanjiang 524023, Guangdong, China

(d) Shenzhen Xinpeng Shengwu Gongcheng Co. Ltd., Shenzhen 518055, Guangdong, China

* Corresponding authors. Tel.: +86 0759 2388405; fax: +86 0759 2388305.

E-mail addresses: 214210597@qq.com (X. Zeng), cuiliao@163.com (L. Cui).

(1) These authors contributed equally to this work.
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Author:Zeng, Xiaobin; Tian, Jun; Cai, Kangyong; Wu, Xin; Wang, Yang; Zheng, Yayuan; Su, Yanjie; Cui, Liao
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Feb 15, 2014
Words:5736
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