- Email: [email protected]
Osteoblastic proliferative activity of Epimedium brevicornum Maxim.
ARTICLE IN PRESS
Phytomedicine 12 (2005) 189–193 www.elsevier.de/phymed
Osteoblastic proliferative activity of Epimedium brevicornum Maxim. F.-H. Menga, Y.-B. Lia, Z.-L. Xionga, Z.-M. Jiangb, F.-M. Lia, a
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China General Hospital of Shenyang Military Region, Shenyang, China
b
Received 13 January 2004; accepted 23 March 2004
Abstract The effect of the extracts of Epimedium brevicornum Maxim. was investigated on proliferative activity in vitro. The osteoblast-like UMR106 cells was employed as an osteoblast model. The EtOH extract and the n-butanol fraction from the crude extract were found to show proliferation stimulating activity. Three flavonoid compounds (icariin, epimedin B and epimedin C) were isolated from this fraction by activity-guided assay, and the effects on cell proliferation were studied. Icariin produced the most significant promoting effect on the proliferation in osteoblast-like UMR106 cells. The results suggested that E. brevicornum Maxim. extracts might have potential activity against osteoporosis, and flavonoids such as icariin might be the active constituents stimulating osteoblasts. r 2004 Elsevier GmbH. All rights reserved. Keywords: Epimedium brevicornum Maxim.; Osteoblast-like UMR 106 cells; Osteoblastic proliferative activity
Introduction The Herba Epimedii (family Berberidaceae) is a famous Chinese herbal medicine, made from the dried aerial parts of Epimedium brevicornum Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim, Epimedium wushanense T.S. Ying. and Epimedium koreanum Nakai (Chinese Pharmacopoeia Commission, 2000). They are commonly used in the treatment of cardiovascular diseases and other chronic illness (infertility, amnesia and asthenia, impotence and senile functional diseases) in China for over 2000 years (Zheng et al., 1998). Recently, Herba Epimedii is applied in many Chinese formulas for anti-osteoporosis (Gao et al., 1999). Pharmacological studies also showed that it had Corresponding author. Tel.: +86 24 2384 3711x3361;
fax: +86 24 8389 0024. E-mail address: [email protected] (F.-M. Li). 0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.03.007
potential activity against osteoporosis (Li et al., 1996; Ma et al., 2002). But it is not clear whether Herba Epimedii affects osteoblast proliferation and what the active constituents are for anti-osteoporosis. The aim of the study is to screen out active portions with anti-osteoporosis activity from the aerial parts of E. brevicornum Maxim., and isolate the active constituents by activity-guided assay in order to consider the standards of the quality control of Herba Epimedii and determine the active lead structures for anti-osteoporosis.
Materials and methods Materials Aerial parts of E. brevicornum Maxim. (including branches and leaves) were collected in a valley, located in Hanzhong, Shaanxi province, China, in September
ARTICLE IN PRESS 190
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2001. The raw material was identified by Qishi Sun, professor of pharmacognosy, Shenyang Pharmaceutical University, China. A reference specimen (voucher No. EB20010903) has been deposited in the Herbarium of the Shenyang Pharmaceutical University, China. Osteoblast-like UMR 106 cells were obtained from Beijing Medical University (of origin from the University of Melbourne Repatriation Hospital, Victoria, Australia). Minimum essential medium (MEM) was obtained from Gibco (USA) and fetal calf serum (FCS) from TBD Bio-engineering Co. (Tianjin, China). Trypsin was supplied by Diffco (USA) and tissue culture materials were provided by Nunc (Denmark). MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] was obtained from Sigma (USA). All other chemicals were of analytical grade.
Extraction and isolation The dried and powered aerial parts of E. brevicornum Maxim. (2000 g) was thoroughly extracted with 95% ethanol. The ethanol extract was drained out and concentrated under reduced pressure, the produced black residue (430 g) was suspended with hot distilled water and the suspension was successively extracted with petroleum ether (b.p. 60–90), ethyl acetate, and nbutanol. The organic solvent was evaporated and the residue was weighed. The activity assay was conducted for each of these extracts by co-culture with osteoblastlike UMR106 cells in vitro. The dark brown residue of nbutanol extract (20 g), which was found active, was then subjected to column chromatography on silica gel. The column was eluted gradiently with CHCl3:MeOH (100:1-1:1). The collected eluates were examined for purity by thin layer chromatography, and those with same Rf values were pooled. Five fractions were obtained by combination of some eluates and three compounds were isolated by activity-guided assay. Icariin (see Fig. 1) was from eluates 95 to 110 at gradient of 100:15, and recrystallization in methanol to
Me
OMe O OR1 OH
Preparation of test samples The crude extract of E. brevicornum Maxim. and its fractions were dissolved in ethanol or distilled water to give completely dissolved solutions (10 mg/ml, expressed in the weight of raw materials per ml). The compounds were dissolved in ethanol to give concentrations of 10 mmol/l, NaF was dissolved in distilled water to give concentrations of 10 mmol/l as positive control, and all samples were prepared duplicate. The solutions were sterilized with 0.2 mm aseptic filter (Gelmann Science) and stored at 4 1C, and all sample solutions were diluted with MEM to the required concentration before use. The blank controls contained untreated cells with MEM and the same proportion of ethanol or water as in the test samples.
The correlation between cell number and absorbance In order to validate the cell proliferation assay, the relationship between cell number and absorbance was examined. Cells were counted with a hemocytomer and diluted with MEM into eight levels of concentrations before seeded into 96-well culture plates. After attachment for 12 h at 37 1C and 5% CO2, MTT was added and the absorbance was determined. The result demonstrates a strong linear correlation in the range of 3.1 103–4.0 105 cell/ml between absorbance (Y ) and cell number (X ): Y ¼ 0:034 þ 2:03 106 X (r2 ¼ 0:9958).
The time course on cell proliferation
Me
RO
get 180 mg. Epimedin B was from eluates 120 to 128 at gradient of 100:20, purified by using silica TLC with ethyl acetate–methanol (10:3) as the developing solvent and than recrystallization in chloroform–methanol to get 110 mg. Epimedin C was from eluates 181 to 195 at gradient of 100:30, and recrystallization in chloroform– methanol to get 130 mg. The purity of these compounds was more than 99.0% tested by RP-HPLC with DAD, and their structures were identified by comparison their physical properties and spectral data with literature values (Guo et al., 1996; Kuroda et al., 2000; Sun et al., 1995).
O
Fig. 1. Formula of icariin. R=Glucosyl, R1=Rhamnosyl.
In order to established the optimal culture period required for mitogenic bioassay, the time course on proliferation was examined. Clinical studies have shown that sodium fluoride (NaF) is the most potent agent for increasing bone density in patients with osteoporosis (Farley et al., 1990). It was therefore employed as a positive control in this study. As shown in Fig. 2, there was a time lag of 12 h between the addition of NaF (concentration of 102 mmol/l) to the cell cultures and cell proliferation. It was then followed by cell proliferation for approximate 36 h. Afterwards, the cell number began to decrease. Consequently, a culture
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191
procedure as described previously (Li et al., 2001). After UMR106 cells were co-cultured with the prepared sample solutions for 48 h at 37 1C in a humidified atmosphere of 95% air and 5% CO2, the medium was removed, 50 ml MTT solution (1 mg MTT/ml PBS) was then added into the wells, and the incubation continued for another 4 h. Finally, MTT solution was removed and 150 ml DMSO (per well) was added. The absorbance was recorded on an enzyme immunoassay plate reader (BIO-RAD, USA) at wavelength of 595 nm with a reference at 655 nm.
period of 48 h was determined for this mitogenic bioassay.
Proliferation assay The stimulating proliferation activity of the solutions on osteoblast-like UMR106 cells was assayed in the same 40
Proliferation(%)
30 20
Statistical analysis
10
Data were expressed as the mean7standard deviation. Statistical significances were analyzed by using the Student’s t-test. A value of po0:05 was considered significant. Linear regression analysis was performed by the correlation coefficient. Growth stimulation ratios (GSR) were calculated using the following equation: GSR%=(AsampleAblank)/Ablank 100, where A is the average absorbance of six experiments with 6 replicates.
0 -10 0
20
40
60
80
Time (h)
Fig. 2. The time course on cell proliferation.
Table 1.
Effects of various extracts of Epimedium brevicornum Maxim. on UMR106 cell proliferation
Samples
Concentration
ABlank ðx¯ SDÞ
A595=655 nm ðx¯ SDÞ
Proliferation (%)
Crude extract (mg/ml)
1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101
0.18470.016
0.21270.015 0.23470.016 0.25770.021 0.21870.019 0.20170.014 0.19170.013 0.19670.015 0.19370.014 0.19070.013 0.18670.015 0.19370.017 0.20470.016 0.21870.017 0.23170.020 0.22270.019 0.21470.018 0.23970.016 0.26170.022 0.23170.021 0.20770.013 0.20370.017 0.19670.016 0.19370.015 0.19770.015 0.19570.014 0.19670.014 0.21170.018 0.24970.015 0.25770.013 0.19470.014
15.1 27.4* 40.1** 18.5 9.4 1.8 4.5 2.6 0.9 1.3 2.4 8.5 15.8 23.1* 18.3 11.1 23.6* 35.3** 19.7 7.2 5.3 1.7 1.2 2.3 0.8 5.3 13.6 30.7** 37.6** 4.0
Petroleum ether fraction (mg/ml)
Ethyl acetate fraction (mg/ml)
n-Butanol fraction (mg/ml)
Aqueous fraction (mg/ml)
NaF (mmol/l)
0.18870.019
0.18870.019
0.19370.020
0.19370.020
0.18670.017
*po0.05; **po0.01 significant as compared to blank using Student’s t-test.
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Effects of flavonoids on UMR106 cell proliferation
Table 2. Samples
Concentration (mmol/l)
A595=655 nm ðx¯ SDÞ
Proliferation (%)
Blank control Icariin
0 1.0 104 1.0 103 1.0 102 1.0 101 1.0 104 1.0 103 1.0 102 1.0 101 1.0 104 1.0 103 1.0 102 1.0 101 1.0 102
0.19570.015 0.20470.017 0.26270.018 0.28370.022 0.24470.019 0.21070.015 0.22270.017 0.24470.019 0.26670.020 0.25470.020 0.27670.021 0.24870.018 0.22570.016 0.27270.018
4.7 34.2** 45.1** 25.3* 7.9 13.8 24.9* 36.3** 30.4** 41.6** 27.0* 15.5 39.4**
Epimedin B
Epimedin C
NaF
po0:05;
po0:01 significant as compared to blank control using Student’s t-test.
Results As shown in Table 1, when the cells were cultured with 1.0 103 mg/ml crude extract, cell proliferation was significantly stimulated (an increase of 40.1% in osteoblastic proliferation). The effect is higher than that of NaF at a concentration of 102 mmol/l that produced an increase of 37.6%. The activities of fractions partitioned from the ethanol extract were examined and it was found that the n-butanol layer remained the similar stimulating activity as the crude extract. For the compounds isolated, icariin produced the most significant promoting effect on UMR106 cell proliferation (an increase of 45.1% in osteoblastic proliferation at a concentration of 102 mmol/l); epimedin B and epimedin C also exhibited significant stimulating activities in osteoblastic proliferation (Table 2).
produced the most significant promoting effect on UMR106 cell proliferation. Epimedin B and C also showed markedly stimulating activity, but they were less effective than icariin. Osteoblast-like UMR106 cells are characterized extensively as osteoblast in nature, and they have been widely used as a developed osteoblast model in studying the effect mechanism of anti-osteoporostic drugs on osteoblasts (Gray et al., 1987). The agents of stimulating proliferation of osteoblasts may have the promoting activity in the phase of bone formation, and they may be used for anti-osteoporosis. We have successfully developed a proliferation assay with UMR106 cells for screening anti-osteoporostic agents from traditional Chinese medicines (Gao et al., 2000; Li et al., 2001; Wang et al., 2001). Compared with animal experiment in vivo, this cell-culture model has advantages such as low dosage, short experiment period, and high reproducibility.
Discussion Herba Epimedii is a typical ‘‘kidney-tonifying’’ traditional Chinese medicine. According to traditional Chinese medicine theory, ‘‘kidney’’ controls bone. The ‘‘kidney-tonifying’’ action of traditional Chinese medicine might have relationship with bone formation. In addition to the reported effects of E. brevicornum Maxim. on the anti-osteoporosis activity in mouse (Li et al., 1996; Ma et al., 2002), our study provided evidence of a potential activity against osteoporosis on the osteoblast-like UMR106 cells. The results suggested that the EtOH extracts and its n-BuOH fraction might contain active constituents stimulating osteoblasts. Icariin, a major pharmacologically active substance,
Acknowledgements The authors are grateful to Dr. Qishi Sun of Shenyang Pharmaceutical University for the identification of Epimedium species.
References Chinese Pharmacopoeia Commission, 2000. Herba Epimedii. In: Chinese Pharmacopoeia Commission (Eds.), Chinese Pharmacopoeia, Part I. Chemical Industry Publisher, Beijing, pp. 267–268.
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Farley, S.M., Libanati, C.R., Mariano-Menez, M.R., TudtudHans, L.A., 1990. Fluoride therapy for osteoporosis promotes a progressive increase in spinal bone density. J. Bone Min. Res. 5 (Suppl. 1), S37–S42. Gao, S.Q., Fu, D.X., Zhang, H.M., 1999. Advances in the study on the treatment of osteoporosis with Herba Epimedii and its compound prescriptions. China J. Chin. Materia. Medica 24 (4), 249–251. Gao, X.Y., Wang, D.W., Li, F.M., Jiang, Z.M., 2000. The effect of the extracts of Achyranthes bidentata Bl. on proliferation of osteoblast-like UMR106 cells. J. Shenyang Pharm. Univ 17 (3), 210–213. Gray, T.K., Flynn, T.C., Gray, K.M., Nabell, L.M., 1987. 17 beta-estradiol acts directly on the clonal osteoblastic cell line UMR106. Proc. Natl. Acad. Sci. USA 84, 6267–6271. Guo, B.L., Yu, J.G., Xiao, P.G., 1996. Chemical studies of Epimedium brevicornum Maxim. China J. Chin. Materia. Medica 21 (5), 290–292. Kuroda, M., Mimaki, Y., Sashida, Y., Umegaki, E., Yamazaki, M., Chiba, K., Mohri, T., Kitahara, M., Yasuda, A., Naoi, N., Xu, Z.W., Li, M.R., 2000. Flavonol glycosides from Epimedium sagittatum and their neurite outgrowth activity on PC12 h cells. Planta Med. 66, 575–577.
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Li, Q.N., Liao, J.M., Wu, T., Huang, L.F., Liang, N.C., 1996. Epimedium sagittatum Maxim. preventing hormone-induced osteoporosis in rats. Chin. Pharm. J. 31 (8), 467–470. Li, F.M., Wang, D.W., Jiang, Z.M., Gao, X.Y., Zhao, H.Q., 2001. Activity stimulating osteoblast-like cells proliferation of some traditional Chinese medicinal drugs and other plants. Pharm. Biol. 39, 351–356. Ma, H.P., Jia, Z.P., Ge, X., He, X.Y., Chen, K.M., Bai, M.H., 2002. Studies on the theraputic effect of total flavonoids of Herba Epimedii on experimental osteoporosis in rats. Huaxi J. Pharm. 17 (3), 163–167. Sun, P.Y., Chen, Y.J., Wang, Z.X., 1995. Chemical studies of Epimedium koreanum Nakai. J. Shenyang Pharm. Univ. 12 (3), 234–236. Wang, D.W., Li, F.M., Gao, X.Y., Jiang, Z.M., 2001. Effect of Drynaria baronii rhizome extracts on the proliferation in osteoblast-like UMR 106 cells. Pharm. Biol. 39 (4), 259–262. Zheng, H.Z., Dong, Z.H., She, Q., 1998. Herba Epimedii. In: Zheng, H.Z., Dong, Z.H., She, Q. (Eds.), Modern Research and Application of Traditional Chinese Medicine. Xueyan press, Beijing, pp. 4225–4254.
Phytomedicine 12 (2005) 189–193 www.elsevier.de/phymed
Osteoblastic proliferative activity of Epimedium brevicornum Maxim. F.-H. Menga, Y.-B. Lia, Z.-L. Xionga, Z.-M. Jiangb, F.-M. Lia, a
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China General Hospital of Shenyang Military Region, Shenyang, China
b
Received 13 January 2004; accepted 23 March 2004
Abstract The effect of the extracts of Epimedium brevicornum Maxim. was investigated on proliferative activity in vitro. The osteoblast-like UMR106 cells was employed as an osteoblast model. The EtOH extract and the n-butanol fraction from the crude extract were found to show proliferation stimulating activity. Three flavonoid compounds (icariin, epimedin B and epimedin C) were isolated from this fraction by activity-guided assay, and the effects on cell proliferation were studied. Icariin produced the most significant promoting effect on the proliferation in osteoblast-like UMR106 cells. The results suggested that E. brevicornum Maxim. extracts might have potential activity against osteoporosis, and flavonoids such as icariin might be the active constituents stimulating osteoblasts. r 2004 Elsevier GmbH. All rights reserved. Keywords: Epimedium brevicornum Maxim.; Osteoblast-like UMR 106 cells; Osteoblastic proliferative activity
Introduction The Herba Epimedii (family Berberidaceae) is a famous Chinese herbal medicine, made from the dried aerial parts of Epimedium brevicornum Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim, Epimedium wushanense T.S. Ying. and Epimedium koreanum Nakai (Chinese Pharmacopoeia Commission, 2000). They are commonly used in the treatment of cardiovascular diseases and other chronic illness (infertility, amnesia and asthenia, impotence and senile functional diseases) in China for over 2000 years (Zheng et al., 1998). Recently, Herba Epimedii is applied in many Chinese formulas for anti-osteoporosis (Gao et al., 1999). Pharmacological studies also showed that it had Corresponding author. Tel.: +86 24 2384 3711x3361;
fax: +86 24 8389 0024. E-mail address: [email protected] (F.-M. Li). 0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.03.007
potential activity against osteoporosis (Li et al., 1996; Ma et al., 2002). But it is not clear whether Herba Epimedii affects osteoblast proliferation and what the active constituents are for anti-osteoporosis. The aim of the study is to screen out active portions with anti-osteoporosis activity from the aerial parts of E. brevicornum Maxim., and isolate the active constituents by activity-guided assay in order to consider the standards of the quality control of Herba Epimedii and determine the active lead structures for anti-osteoporosis.
Materials and methods Materials Aerial parts of E. brevicornum Maxim. (including branches and leaves) were collected in a valley, located in Hanzhong, Shaanxi province, China, in September
ARTICLE IN PRESS 190
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2001. The raw material was identified by Qishi Sun, professor of pharmacognosy, Shenyang Pharmaceutical University, China. A reference specimen (voucher No. EB20010903) has been deposited in the Herbarium of the Shenyang Pharmaceutical University, China. Osteoblast-like UMR 106 cells were obtained from Beijing Medical University (of origin from the University of Melbourne Repatriation Hospital, Victoria, Australia). Minimum essential medium (MEM) was obtained from Gibco (USA) and fetal calf serum (FCS) from TBD Bio-engineering Co. (Tianjin, China). Trypsin was supplied by Diffco (USA) and tissue culture materials were provided by Nunc (Denmark). MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] was obtained from Sigma (USA). All other chemicals were of analytical grade.
Extraction and isolation The dried and powered aerial parts of E. brevicornum Maxim. (2000 g) was thoroughly extracted with 95% ethanol. The ethanol extract was drained out and concentrated under reduced pressure, the produced black residue (430 g) was suspended with hot distilled water and the suspension was successively extracted with petroleum ether (b.p. 60–90), ethyl acetate, and nbutanol. The organic solvent was evaporated and the residue was weighed. The activity assay was conducted for each of these extracts by co-culture with osteoblastlike UMR106 cells in vitro. The dark brown residue of nbutanol extract (20 g), which was found active, was then subjected to column chromatography on silica gel. The column was eluted gradiently with CHCl3:MeOH (100:1-1:1). The collected eluates were examined for purity by thin layer chromatography, and those with same Rf values were pooled. Five fractions were obtained by combination of some eluates and three compounds were isolated by activity-guided assay. Icariin (see Fig. 1) was from eluates 95 to 110 at gradient of 100:15, and recrystallization in methanol to
Me
OMe O OR1 OH
Preparation of test samples The crude extract of E. brevicornum Maxim. and its fractions were dissolved in ethanol or distilled water to give completely dissolved solutions (10 mg/ml, expressed in the weight of raw materials per ml). The compounds were dissolved in ethanol to give concentrations of 10 mmol/l, NaF was dissolved in distilled water to give concentrations of 10 mmol/l as positive control, and all samples were prepared duplicate. The solutions were sterilized with 0.2 mm aseptic filter (Gelmann Science) and stored at 4 1C, and all sample solutions were diluted with MEM to the required concentration before use. The blank controls contained untreated cells with MEM and the same proportion of ethanol or water as in the test samples.
The correlation between cell number and absorbance In order to validate the cell proliferation assay, the relationship between cell number and absorbance was examined. Cells were counted with a hemocytomer and diluted with MEM into eight levels of concentrations before seeded into 96-well culture plates. After attachment for 12 h at 37 1C and 5% CO2, MTT was added and the absorbance was determined. The result demonstrates a strong linear correlation in the range of 3.1 103–4.0 105 cell/ml between absorbance (Y ) and cell number (X ): Y ¼ 0:034 þ 2:03 106 X (r2 ¼ 0:9958).
The time course on cell proliferation
Me
RO
get 180 mg. Epimedin B was from eluates 120 to 128 at gradient of 100:20, purified by using silica TLC with ethyl acetate–methanol (10:3) as the developing solvent and than recrystallization in chloroform–methanol to get 110 mg. Epimedin C was from eluates 181 to 195 at gradient of 100:30, and recrystallization in chloroform– methanol to get 130 mg. The purity of these compounds was more than 99.0% tested by RP-HPLC with DAD, and their structures were identified by comparison their physical properties and spectral data with literature values (Guo et al., 1996; Kuroda et al., 2000; Sun et al., 1995).
O
Fig. 1. Formula of icariin. R=Glucosyl, R1=Rhamnosyl.
In order to established the optimal culture period required for mitogenic bioassay, the time course on proliferation was examined. Clinical studies have shown that sodium fluoride (NaF) is the most potent agent for increasing bone density in patients with osteoporosis (Farley et al., 1990). It was therefore employed as a positive control in this study. As shown in Fig. 2, there was a time lag of 12 h between the addition of NaF (concentration of 102 mmol/l) to the cell cultures and cell proliferation. It was then followed by cell proliferation for approximate 36 h. Afterwards, the cell number began to decrease. Consequently, a culture
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191
procedure as described previously (Li et al., 2001). After UMR106 cells were co-cultured with the prepared sample solutions for 48 h at 37 1C in a humidified atmosphere of 95% air and 5% CO2, the medium was removed, 50 ml MTT solution (1 mg MTT/ml PBS) was then added into the wells, and the incubation continued for another 4 h. Finally, MTT solution was removed and 150 ml DMSO (per well) was added. The absorbance was recorded on an enzyme immunoassay plate reader (BIO-RAD, USA) at wavelength of 595 nm with a reference at 655 nm.
period of 48 h was determined for this mitogenic bioassay.
Proliferation assay The stimulating proliferation activity of the solutions on osteoblast-like UMR106 cells was assayed in the same 40
Proliferation(%)
30 20
Statistical analysis
10
Data were expressed as the mean7standard deviation. Statistical significances were analyzed by using the Student’s t-test. A value of po0:05 was considered significant. Linear regression analysis was performed by the correlation coefficient. Growth stimulation ratios (GSR) were calculated using the following equation: GSR%=(AsampleAblank)/Ablank 100, where A is the average absorbance of six experiments with 6 replicates.
0 -10 0
20
40
60
80
Time (h)
Fig. 2. The time course on cell proliferation.
Table 1.
Effects of various extracts of Epimedium brevicornum Maxim. on UMR106 cell proliferation
Samples
Concentration
ABlank ðx¯ SDÞ
A595=655 nm ðx¯ SDÞ
Proliferation (%)
Crude extract (mg/ml)
1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101 1.0 105 1.0 104 1.0 103 1.0 102 1.0 101
0.18470.016
0.21270.015 0.23470.016 0.25770.021 0.21870.019 0.20170.014 0.19170.013 0.19670.015 0.19370.014 0.19070.013 0.18670.015 0.19370.017 0.20470.016 0.21870.017 0.23170.020 0.22270.019 0.21470.018 0.23970.016 0.26170.022 0.23170.021 0.20770.013 0.20370.017 0.19670.016 0.19370.015 0.19770.015 0.19570.014 0.19670.014 0.21170.018 0.24970.015 0.25770.013 0.19470.014
15.1 27.4* 40.1** 18.5 9.4 1.8 4.5 2.6 0.9 1.3 2.4 8.5 15.8 23.1* 18.3 11.1 23.6* 35.3** 19.7 7.2 5.3 1.7 1.2 2.3 0.8 5.3 13.6 30.7** 37.6** 4.0
Petroleum ether fraction (mg/ml)
Ethyl acetate fraction (mg/ml)
n-Butanol fraction (mg/ml)
Aqueous fraction (mg/ml)
NaF (mmol/l)
0.18870.019
0.18870.019
0.19370.020
0.19370.020
0.18670.017
*po0.05; **po0.01 significant as compared to blank using Student’s t-test.
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Effects of flavonoids on UMR106 cell proliferation
Table 2. Samples
Concentration (mmol/l)
A595=655 nm ðx¯ SDÞ
Proliferation (%)
Blank control Icariin
0 1.0 104 1.0 103 1.0 102 1.0 101 1.0 104 1.0 103 1.0 102 1.0 101 1.0 104 1.0 103 1.0 102 1.0 101 1.0 102
0.19570.015 0.20470.017 0.26270.018 0.28370.022 0.24470.019 0.21070.015 0.22270.017 0.24470.019 0.26670.020 0.25470.020 0.27670.021 0.24870.018 0.22570.016 0.27270.018
4.7 34.2** 45.1** 25.3* 7.9 13.8 24.9* 36.3** 30.4** 41.6** 27.0* 15.5 39.4**
Epimedin B
Epimedin C
NaF
po0:05;
po0:01 significant as compared to blank control using Student’s t-test.
Results As shown in Table 1, when the cells were cultured with 1.0 103 mg/ml crude extract, cell proliferation was significantly stimulated (an increase of 40.1% in osteoblastic proliferation). The effect is higher than that of NaF at a concentration of 102 mmol/l that produced an increase of 37.6%. The activities of fractions partitioned from the ethanol extract were examined and it was found that the n-butanol layer remained the similar stimulating activity as the crude extract. For the compounds isolated, icariin produced the most significant promoting effect on UMR106 cell proliferation (an increase of 45.1% in osteoblastic proliferation at a concentration of 102 mmol/l); epimedin B and epimedin C also exhibited significant stimulating activities in osteoblastic proliferation (Table 2).
produced the most significant promoting effect on UMR106 cell proliferation. Epimedin B and C also showed markedly stimulating activity, but they were less effective than icariin. Osteoblast-like UMR106 cells are characterized extensively as osteoblast in nature, and they have been widely used as a developed osteoblast model in studying the effect mechanism of anti-osteoporostic drugs on osteoblasts (Gray et al., 1987). The agents of stimulating proliferation of osteoblasts may have the promoting activity in the phase of bone formation, and they may be used for anti-osteoporosis. We have successfully developed a proliferation assay with UMR106 cells for screening anti-osteoporostic agents from traditional Chinese medicines (Gao et al., 2000; Li et al., 2001; Wang et al., 2001). Compared with animal experiment in vivo, this cell-culture model has advantages such as low dosage, short experiment period, and high reproducibility.
Discussion Herba Epimedii is a typical ‘‘kidney-tonifying’’ traditional Chinese medicine. According to traditional Chinese medicine theory, ‘‘kidney’’ controls bone. The ‘‘kidney-tonifying’’ action of traditional Chinese medicine might have relationship with bone formation. In addition to the reported effects of E. brevicornum Maxim. on the anti-osteoporosis activity in mouse (Li et al., 1996; Ma et al., 2002), our study provided evidence of a potential activity against osteoporosis on the osteoblast-like UMR106 cells. The results suggested that the EtOH extracts and its n-BuOH fraction might contain active constituents stimulating osteoblasts. Icariin, a major pharmacologically active substance,
Acknowledgements The authors are grateful to Dr. Qishi Sun of Shenyang Pharmaceutical University for the identification of Epimedium species.
References Chinese Pharmacopoeia Commission, 2000. Herba Epimedii. In: Chinese Pharmacopoeia Commission (Eds.), Chinese Pharmacopoeia, Part I. Chemical Industry Publisher, Beijing, pp. 267–268.
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