Two new chromone glycosides from Drynaria fortunei

Fitoterapia 84 (2013) 130–134

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Two new chromone glycosides from Drynaria fortunei Zhen-Ping Shang a, b, Jing-Jing Meng b, Qing-Chun Zhao a,⁎, Ming-Zhi Su b, Zhou Luo b, Lin Yang b, Jing-Jing Tan b a b

Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China

a r t i c l e

i n f o

Article history: Received 6 August 2012 Accepted in revised form 4 November 2012 Available online 15 November 2012 Keywords: Chromone glycosides Drynaria fortunei Drynachromoside A Drynachromoside B MC3T3-E1 cells MTT

a b s t r a c t Two new chromone glycosides, drynachromoside A (1), drynachromoside B (2), along with three known flavanones, 5,7,3′,5′-tetrahydroxy-flavanone (3), 5,7,3′,5′-tetrahydroxy-flavanone-7-Oβ-D-glucopyranoside (4), and 5,7,3′,5′-tetrahydroxy-flavanone-7-O-neohesperidoside (5), were isolated from the dry rhizomes of Drynaria fortunei by means of bio-active screening. The two former compounds were elucidated on the basis of physico-chemical property and spectroscopic data. The osteoblastic proliferation activities of these flavonoids were evaluated by the method of MTT. The results showed that compound 1 exhibited the biochemical effects on the proliferation of MC3T3-E1 cells, while Compound 2 showed inhibitory effects against MC3T3-E1 cells. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Osteoporosis is defined as “a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk.” It is becoming an increasingly important public health issue, and there is an urgent need for effective treatment to prevent bone fragility [1,2]. Traditional Chinese medicine has shown beneficial clinical effects on the prevention and treatment of osteoporosis [3]. Drynaria fortunei (Kunze) J. Sm, belonging to the family polypodiaceae, has been widely used for thousands of years as a folk medicine to treat osteoporosis [4] and other bone diseases. Xie et al. reported that osteopractic total flavone had an effect on bone mineral density and bone histomorphometry in ovariectomized rats [5]. In addition, Liu et al. showed that the water extract of Gusuibu can protect rat calvarial osteoblasts from hydrogen peroxide-induced insults [6]. In an animal

⁎ Corresponding author. Tel./fax: +86 2428856205. E-mail address: [email protected] (Q.-C. Zhao). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.11.001

study, crude extracts from Gusuibu were also shown to have systemic effects on bone formation in mice [7]. The therapeutic effects of the rhizomes of Drynaria fortunei on osteoporosis and bone fracture had been already reported in many studies. However, most of these studies were focused on the crude extract or total flavonoids. Few phytochemical studies have been conducted on the active constituents leading to the osteoprotective effects of D. fortunei [8]. In present study, the n-BuOH fraction (DFC) obtained from 60% ethanol extract of the rhizomes of D. fortunei (DFE) was found to stimulate the proliferation of MC3T3-E1 cells. Five flavonoids were isolated from the active fraction (DFC) by means of bioactive screening. At the same time, the effects of these flavonoids on the cell growth of MC3T3-E1 cells were described. 2. Experimental part 2.1. General UV spectra were taken with UV-2501PC spectrometer. IR spectra were measured with a Brucker IFS 55 spectrometer. HR-ESI-MS were measured on a Micromass Q-TOF mass spectrometer. NMR experiments were performed on Bruker

Z.-P. Shang et al. / Fitoterapia 84 (2013) 130–134

ARX-300 and Bruker AV-600 spectrometers in DMSO-d6 using TMS as an internal standard. Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Marine chemical company (Qingdao, P. R. China). Sephadex LH-20 was a product of Amersham Co. RP-18 (15–30 um) silica gel was purchased from Merk chemical Ltd. Preparative HPLC was carried out on a JASCO PU-2087 apparatus with an ODS column (YMC-Pack ODS-A, 150× 10 mm). All reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Tissue culture media, reagents and fetal bovine serum(FBS) were purchased from Gibco (Chagrin Falls, OH, USA). 2.2. Plant material The dry rhizomes of Drynaria fortunei were collected from Guang Xi, China, in October, 2008. Samples were authenticated and deposited at General Hospital of Shenyang Military Area Command, China. 2.3. Extraction and isolation The dry rhizomes of Drynaria fortunei (3.0 Kg) were extracted with 60% EtOH for 3 times (2 h each time). The ethanol extract was filtered and partitioned with petroleum ether, EtOAc and n-BuOH, respectively. The n-BuOH extract (50.0 g) was chromatographed on a silica gel (200–300 mesh 300 g, 10 × 60 cm) column and eluted with a gradient CH2Cl2/ MeOH (from 100:0 to 60:40, v/v) to yield six fractions (B1–B6). Fraction B4 (10.1 g) was subjected to silica gel column chromatography (200–300 mesh 150 g, 5 × 60 cm) with CH2Cl2/MeOH (from 100:0 to 50:50, v/v) to yield five fractions

Table 1 1 H-NMR and No.

13

131

(B41–B45). Fraction B42 (1.58 g) was separated on a silica gel (200–300 mesh 30 g, 4 × 30 cm) column with CH2Cl2/MeOH (100:0 to 60:40, v/v) to yield three fractions (B421–B423). Fraction B422 (480 mg) was subjected to Sephadex LH-20 column chromatography (φ 2 × 60 cm) with MeOH/H2O (1:1, v/v) and then purified by RP-HPLC to yield compounds 3 (15 mg). Fraction B43 (930 mg) was chromatographed on MPLC on ODS (φ 2 × 20 cm) with MeOH/H2O (from 10:90 to 100:0, v/v) to yield two fractions (B431–B432). Fraction B432 (380 mg) was subjected to Sephadex LH-20 column chromatography (φ 2 × 60 cm) with MeOH/H2O (1:1, v/v) to yield three fractions (B4321–B4323) and then subjected to preparative RP-HPLC eluted with MeOH/H2O (35:65, v/v) to yield compounds 4 (12 mg, Rt = 37 min), 5 (11 mg, Rt = 45 min), respectively. Fraction B5 (860 mg) was subjected to Sephadex LH-20 with CH2Cl2–MeOH (1:1, v/v) to yield three major fractions (B51–B53). Fraction B52 (240 mg) was separated on MPLC on ODS with MeOH–H2O (from 10:90 to 100:0, v/v) to yield three fractions (B521–B523). Finally, B521 (70 mg) and B523 (68 mg) were purified by RP-HPLC with MeOH–H2O (25:75, v/v) to yield compounds 1 (9.0 mg, Rt = 50 min), 2 (12.0 mg, Rt = 41 min), respectively. Drynachromoside A (1)white amorphous powder (CH2CL2MeOH); UV (MeOH) λmax ( ε): 285 (2.85), 312 (2.67) nm; IR (KBr) νmax 3406, 1665, 1385 cm−1; 1H-NMR (600 MHz, DMSOd6) and 13C-NMR (150 MHz, DMSO-d6) data, see Table 1; HR-TOF-MS: m/z 499.1447 [M-H] − (calcd for C22H27O13, 499.1452). Drynachromoside B (2) yellow powder (MeOH); UV (MeOH) λmax ( ε): 295 (2.93) nm; IR (KBr) νmax 3348, 1585, 1519, 1662 cm −1. 1H-NMR (600 MHz, DMSO-d6) and

C-NMR spectral data and significant HMBC correlations of compounds 1 and 2 in DMSO-d6 (150 MHz for 1 δC

2 3 4 5 5-OH 6 7 7-OH 8 9 10 11 1' 2' 3' 4' 5' 6'

168.5 108.4 182.1 161.3

1'' 2'' 3'' 4'' 5'' 6''

104.4 74.5 77.1 70.1 76.7 61.2

99.6 161.6 94.7 157.5 105.2 20.1 98.1 69.5 70.3 81.4 68.5 17.9

13

C, 600 MHz for 1H).

2 δH (J, Hz)

HMBC

6.25 s

2, 10, 11

12.73 s 6.45 d (2.1)

6, 10 7, 8, 10

6.68 d (2.1)

6, 7, 9, 10

2.38 5.55 3.88 3.87 3.57 3.39 1.20

s s m m m m d (5.7)

2, 3 7 3'

4.43 2.91 3.01 3.10 3.14 3.62 3.37

d (7.8) m m m m m m

4'

3', 5', 6', 1'' 4', 6' 4', 5'

3", 5" 2" 4"

δC 166.7 107.0 181.9 161.7 99.0 164.4 94.0 157.6 104.0 65.4 102.5 73.5 77.2 70.1 76.6 61.2

δH (J, Hz)

HMBC

6.48 s

2, 4, 10, 11

12.73 s 6.18 d (1.8)

6, 10 5, 7, 8, 10

10.92 s 6.33 d (1.8)

6, 8 4, 6, 7, 9, 10

4.68 4.28 3.05 3.13 3.11 3.16 3.65 3.42

2, 3, 1′` 11, 2', 3' 1', 3' 4', 6' 6' 4' 4' 5'

d (15.6), 4.57 d (15.6) d (7.8) m m m m dd (4.2,11.4) dd (4.2,11.4)

132

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13 C-NMR (150 MHz, DMSO-d6) data, see Table 1; HR-TOF-MS m/z: 369.0842 [M-H]− (calcd for C16H27O10, 369.0822).

2.4. Acid hydrolysis of compounds 1–2 Acid hydrolysis of compounds 1 and 2 was performed using the method of Hara et al. [9] to determine the absolute configuration of the monosaccharide. Compounds 1 and 2 (each 2 mg) were hydrolyzed with 1 N HCl (2 ml) for 2 h in a boiling water bath. The mixture was evaporated to dryness under vacuum, and then the residues were dissolved in water and extracted with EtOAc. After separating the organic layer, the aqueous phase was neutralized with Na2CO3 and evaporated to dryness. The released sugars were identified as glucose and rhamnose by comparison with authentic sugar on silica gel with CHCl3-MeOH-H2O-HOAC (7:3:0.5:1) using 5% H2SO4 as spraying reagent. L-cysteine methyl ester hydrochloride (1.0 mg) was added to the residues dissolved in pyridine, and then the reaction mixture was heated for 2 h at 60 °C. After drying with N2, trimethylsilyl imidazole was added to the residues, followed by heating at 60 °C for 1 h. The residues were extracted with hexane and H2O, and the organic layer was analyzed using GC chromatograph: column, HP-5 (30 m × 0.25 mm, 0.25 μm); detector, FID; column temperature: 160 °C/230 °C; programmed increase, 15 °C/min; carrier gas: N2 (1 ml/min); injection temperature: 270 °C and detector temperature: 280 °C; injection volume: 2.0 μl, split ratio: 1/20. The derivatives of D-glucose and L-rhamnose were detected, tR: 20.65 min (D-glucose derivative) and 22.02 min (L-rhamnose derivative). 2.5. Cell culture The Mouse osteoblast (MC3T3-E1) was obtained from American Type Culture Collection (Rockville, MD, U.S.A.). The cells were cultured in α-MEM medium supplemented with 10% FBS, 2 mM L-glutamine (Gibco, Grand Island, NY, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml), and they were maintained at 37 °C with 5% CO2 in a humidified atmosphere.

3. Results and discussion 3.1. Effects of 60% ethanol extract of the rhizomes of D. fortunei (DFE) and three fractions extracted with petroleum ether, EtOAC, and n-BuOH on the proliferation of MC3T3-E1 cells The MC3T3-E1 cell line is a mouse osteoblastic cell line with the capacity to differentiate into osteoblasts and osteocytes. N-acetyl-cysteine (NAC) is a putative antioxidant that can stimulate the proliferation of MC3T3-E1 cells. It was used as a positive control in the present study. The 60% ethanol extract of the rhizomes of D. fortunei (DFE) showed the cell proliferation of MC3T3-E1 cells at concentrations of 6.25 μg/ml and 12.5 μg/ml, respectively. DFE was extracted with petroleum ether (DFA), EtOAC (DFB) and n-BuOH (DFC). Table 2 showed the effects of DFE and fractions DFA, DFB, and DFC on the cell growth of MC3T3-E1 cells. In Table 2, DFC stimulated the proliferation of MC3T3-E1 cells by 28.9% (p b 0.001), and it showed stronger proliferation activity than DFB. Low doses of DFA showed mild effects on the proliferation of MC3T3-E1 cells, but higher doses of DFA showed strongest cytotoxicity in MC3T3-E1 cells. In summary, DFC exhibited significant activity on the proliferation of MC3T3-E1 cells. Therefore, activity-guided isolation from DFC was carried out subsequently. 3.2. Activity-guided isolation from DFC Five flavonoids were isolated from the active fraction (DFC) by means of repeated silica gel column chromatography, sephadex LH-20, PHPLC and recrystallization. The structures of two new chromone glycosides, drynachromoside A (1), drynachromoside B (2), along with three known flavanones,

Table 2 Effects of DFE and fractions DFA, DFB, and DFC on the cell growth of MC3T3-E1 cells. Samples

Concentration (μg/ml)

Acontrol at (mean ± S.D.)

Asample at (mean ± S.D.)

Proliferation (%)

DFE

3.125 6.25 12.5 25 50 100 3.125 6.25 12.5 25 50 100 3.125 6.25 12.5 25 50 100 3.125 6.25 12.5 25 50 100 3.125

0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.695 ± 0.019 0.695 ± 0.019 0.695 ± 0.019 0.695 ± 0.019 0.695 ± 0.019 0.695 ± 0.019 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006 0.694 ± 0.006

0.711 ± 0.005 0.767 ± 0.013 0.793 ± 0.061 0.742 ± 0.017 0.720 ± 0.065 0.695 ± 0.003 0.773 ± 0.048 0.738 ± 0.024 0.661 ± 0.016 0.622 ± 0.012 0.341 ± 0.04 0.172 ± 0.061 0.727 ± 0.008 0.788 ± 0.033 0.760 ± 0.036 0.744 ± 0.014 0.720 ± 0.026 0.702 ± 0.076 0.732 ± 0.02 0.780 ± 0.017 0.818 ± 0.014 0.894 ± 0.025 0.756 ± 0.007 0.679 ± 0.011 1.047 ± 0.047

2.4 10.5*** 14.3* 6.9** 3.8 0.1 11.4* 6.3 −4.7 −10.3*** −50.9*** −75.2*** 4.5* 13.4** 9.3* 7.0* 3.5 0.9 5.5* 12.4*** 17.9*** 28.9*** 8.9*** −2.2 50.5***

2.6. Cell proliferation assay Cell proliferation of MC3T3-E1 cells was determined in the colorimetric MTT cell proliferation assay [10]. In brief, exponentially growing cells were seeded (1× 104 cells) into 96-well plates and then were preincubated for 24 h so that they would undergo cell attachment. The medium was then aspirated and fresh medium (200 μL) containing various concentrations (3.125, 6.25, 12.5, 25, 50, 100 μg/ml) of the test fractions and compounds were added to the cultures. The cells were incubated in the presence of the test fractions and compounds at 37 °C for 24 h. MTT (25 μL/well) was added to each well, and plates were incubated at 37 °C for 4 h. The colored formazan product was then dissolved using 150 μL of DMSO. The plates were read using the microtiter plate reader at a wavelength of 492 nm. The percentage of cell proliferation was calculated according to the following formula. proliferation ratio (%) = [A492 (compounds) − A492 (control)/A492 (control)]× 100%. All of the assays were performed at least in triplicate.

DFA

DFB

DFC

NAC

*p b 0.05 vs control, **p b 0.01 vs control, ***pb 0.001 vs control.

Z.-P. Shang et al. / Fitoterapia 84 (2013) 130–134

5,7,3′,5′-tetrahydroxy-flavanone (3), [11] 5,7,3′,5′-tetrahydroxyflavanone-7-O-β-D-glucopyranoside (4), and 5,7,3′,5′tetrahydroxy-flavanone-7-O-neohesperidoside (5) were elucidated from the spectral analysis. Compound 1 was isolated as white amorphous powder. The molecular formula was determined to be C22H28O13 by HRTOF-MS at m/z 499.1447 [M-H]- (calcd for C22H27O13, 499.1452) and m/z 501.1603 [M+ H]+ (calcd for C22H29O13, 501.1608). The IR spectrum showed absorption bands at 3406 (OH), 1665 (C= O), and 1385 cm−1 (CH3). Its UV absorption maxima was displayed at λ 285, 312 nm. The 1H-NMR spectrum (Table 1) exhibited a strongly chelated hydroxy group at δ 12.73 (1H, s, 5-OH), a pair of meta-coupled doublets proton signals at δ 6.68 (1H, d, J = 2.1 Hz, H-8) and 6.45 (1H, d, J = 2.1 Hz, H-6), an olefinic proton signals at δ 6.25 (1H, s, H-3), and a methyl group at δ 2.38 (s). The 13C-NMR spectrum exhibited a carbonyl carbon signal at δc 182.1 (C-4), five sp 2 quaternary carbon signals at δc 168.5 (C-2), 161.6 (C-7), 161.3 (C-5), 157.5 (C-9), 105.2 (C-10), three sp2 methines at δc 108.4 (C-3), 99.6 (C-6), 94.7 (C-8), and a methyl carbon signals at δc 20.1 (C-11). Among them, four carbon signals were highly deshielded, implying that these carbons were attached to oxygen atoms. These data were proof of the presence of 5-hydroxy-7-O-substituted methylchromone structure [12,13], which were further confirmed by the HMBC spectrum (Table 1). HMBC correlations of δH 6.25 (1H, s, H-3) with C-2, C-10, C-11 and HMBC correlations from δH 2.38 (3H, s, H-11) to C-2, C-3 confirmed the presence of 5-hydroxy-7-O-substituted 2-methylchromone structure. Moreover, combination of NMR spectrum and molecular formula showed a saccharide chain contained two sugar units besides the presence of methylchromone nucleus. The anomeric proton signals at δH 5.55 (1H, s, H-1′) from 1H-NMR data, and carbon signals at δc 98.1 (C-1′), 81.4 (C-4′), 70.3 (C-3′), 69.5 (C-2′), 68.5 (C-5′) and 17.9 (C-6′) from 13C-NMR data, showed the presence of α-rhamnosyl moiety. Signals at δc 104.4 (C-1′′), 77.1 (C-3′′), 76.7(C-5′′), 74.5(C-2′′), 70.1 (C-4′′), 61.2 (C-6′′) from 13C-NMR spectrum and δH 4.43 (1H, d, J = 7.8 Hz, H-1′′) from 1H-NMR data were assigned to β-glucosyl moiety. Acid hydrolysis of 1 gave L-rhamnose and D-glucose. The sequences of the protons and carbons of the rhamnose and glucose were determined using HMBC and TOCSY experiments. In the HMBC spectrum of Compound 1 (Table 1), long-range connectivity was observed in the correlation peaks between C-7 and δH 5.55 (1H, s, H-1′) (Fig. 1), the rhamnose C-4′ at δC 81.4 (Table 1) and δH 4.43 (1H, d, J = 7.8 Hz, H-1′′) (Fig. 1). On the basis of the above evidence, the structure of compound 1 was identified as a new chromone glycoside, 5-hydroxy-2-methyl-4H-benzopyran4-one-7-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside, named drynachromoside A. Compound 2 was obtained as yellow powder and had the molecular formula C16H18O10 based on ion peak at m/z 369.0842 [M-H]- (calcd for C16H17O10; 369.0822) in HR-TOFMS. Its UV absorption maxima was observed at 295 nm in CH3OH. Its IR spectrum showed absorption bands of a hydroxyl group (3348 cm−1), aromatic groups (1585. 1519 cm−1), and a conjugated carbonyl group (1662 cm−1). In the 1H-NMR spectrum (Table 1), an olefinic proton signals at δ 6.48 (1H, s, H-3), a pair of meta-coupled aromatic proton signals at δ 6.33 (1H, d, J = 1.8 Hz, H-8) and 6.18 (1H, d, J = 1.8 Hz, H-6), a strongly chelated hydroxy group at δ 12.73 (1H, s, 5-OH), a

133

O 7

8 9 O 2

6'' 5''O

1' HO 6' 5' O 4 6 HO 4'' 1'' O 4' 2' 10 5 3' HO 2'' HO OH 3'' OH OH O

11

3

1 HO 7

8

6 5

11

9 O 2 10

OH

4

HO O 1'

3

2' 3' OH 4' OH O 5' 6' OH

O

2 Fig. 1. The structures of compounds 1 and 2.

hydroxy group at δ 10.92 (1H, s, 7-OH), and two methylene proton signals at 4.68 (1H, d, J = 15.6 Hz, –CH2OH), 4.57 (1H, d, J = 15.6 Hz, –CH2OH) were due to the aglycone, which were confirmed by the HMBC spectrum (Table 1). The anomeric protons signal at δ 4.28 (1H, d, J = 7.5 Hz, H-1′) and carbon signals at δ 102.5 (C-1′), 77.2 (C-3′), 76.6 (C-5′), 73.5 (C-2′), 70.1 (C-4′), 61.2 (C-6′) showed the presence of a β-glucose moiety, and then acid hydrolysis of 2 yielded D-glucose. The HMBC correlation signals (Table 1) found at H-1′ (δH 4.28) and C-11 (δC 65.4) suggested that the glucosyl group was linked to C-11. The 1H- and 13C-NMR spectra were similar to those of saikochromoside A [14], except for C7–OH. Therefore, compound 2 was identified as 2-hydroxymethyl-5,7-dihydroxy4H-benzopyran-4-one-1′-O-β-D-glucoside (Fig. 1), named drynachromoside B. The structures of the three known flavonoids (3–5) were identified as 5,7,3′,5′-tetrahydroxy-flavanone (3), 5,7,3′,5′tetrahydroxy-flavanone 7-O-β-D-glucopyranoside (4), and 5,7,3′,5′-tetrahydroxy-flavanone-7-O-neohesperidoside (5), respectively, by comparison of their spectroscopic data with

Table 3 Effects of two new compounds (1 and 2) on proliferation of the MC3T3-E1 cells. Samples

Concentration (μg/ml)

Acontrol at 492 nm (mean±S.D.)

Asample at 492 nm (mean±S.D.)

Proliferation ratio (%)

1

3.125 6.25 12.5 25 50 100 3.125 6.25 12.5 25 50 100 3.125

0.577 ± 0.009 0.577 ± 0.009 0.577 ± 0.009 0.577 ± 0.009 0.577 ± 0.009 0.577 ± 0.009 0.576 ± 0.003 0.576 ± 0.003 0.576 ± 0.003 0.576 ± 0.003 0.576 ± 0.003 0.576 ± 0.003 0.576 ± 0.003

0.583 ± 0.002 0.605 ± 0.008 0.612 ± 0.015 0.635 ± 0.011 0.624 ± 0.009 0.632 ± 0.016 0.521 ± 0.003 0.514 ± 0.002 0.511 ± 0.007 0.501 ± 0.013 0.512 ± 0.006 0.509 ± 0.003 0.892 ± 0.010

1.1 4.9 6.1* 10.1** 8.2** 9.5** −9.4*** −10.7*** −11.3*** −13.1*** −11.1*** −11.6*** 54.8***

2

NAC

*p b 0.05 vs control, **p b 0.01 vs control, ***pb 0.001 vs control.

134

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literature data, and they had been already reported from the rhizomes of D. fortunei [8]. 3.3. Effects of five flavonoids from rhizomes of D. fortunei on proliferation of MC3T3-E1 cells The MC3T3-E1 cells were treated with indicated concentrations of five flavonoids and N-acetyl-cysteine (NAC) used as a positive control for 24 h. The proliferative effects of compounds 1 and 2 on MC3T3-E1 cells were shown in Table 3. Compound 1 showed proliferative activity. However, Compound 2 exhibited mild inhibitory activity against MC3T3-E1 cells at the concentration ranged from 3.125 to 100 μg/ml. The others had no effects on proliferation of MC3T3-E1 at the concentrations of 3.125–100 μg/ml, and the results were not given. Acknowledgements We wish to gratefully acknowledge Jian-Mei Gao of Liaoning University of Traditional Chinese Medicine for bioactive tests, and we are grateful to Professor Jin hui Wang of Shi He Zi University for the HR-TOF-MS measurements, thanks are also given to Senior Engineer Wen Li and Yi Sha of Shenyang Pharmaceutical University for the measurements of NMR spectra. References [1] Lee J, Vasikaran S. Current recommendations for laboratory testing and use of bone turnover markers in management of osteoporosis. Ann Lab Med 2012;32(2):105-12.

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