Abstract
The osteoblast-like UMR106 cells were employed as an osteoblast model, and the effect of the aerial parts of Epimedium koreanum. Nakai extracts on cell proliferation was studied. The ethyl acetate fraction from the crude extract was found to have the maximal stimulating activity. Three flavonoid compounds (icariside II, icariin, and epimedin B) were isolated from this fraction by activity-guided assay, and their effects on cell proliferation was investigated. Icariin produced the most significant promoting effect on UMR106 cell proliferation. The results suggested that Epimedium koreanum. extracts might have potential activity against osteoporosis, and its ethyl acetate fraction might contain active constituents stimulating osteoblasts.
Introduction
The genus Epimedii. is widespread in Asia, Europe, and the Middle and Far East; it comprises more than 40 species found throughout the world. According to the Chinese Pharmacopoeia. (Chinese Pharmacopoeia Commission, 2000), the aerial parts of Epimedium koreanum. Nakai have been used as a material for Epimedium Herb, as well as E. brevicornum., E. sagittatum., E. pubescens., and E. wushanense.. In China, it has been reputed to be an effective therapy for impotence, asthenia, amnesia, or corresponding symptoms. Recently, the traditional Chinese medicine Herba Epimedii has been applied in many Chinese formulas for anti-osteoporosis (Gao et al., Citation1999). Pharmacological studies also show that it had potential activity against osteoporosis (Li et al., Citation1996; Ma et al., Citation2002).
However, it is not clear whether Herba Epimedii affects osteoblast proliferation and what the active constituents are for anti-osteoporosis. In this paper, the effect of Epimedium koreanum. extracts on the proliferation of osteoblast-like UMR106 cells was studied. Three flavonoid compounds (icariside II, icariin, epimedin B) were isolated from the aerial parts of Epimedium koreanum. by activity-guided assay, and their activities are reported.
Materials and Methods
Materials
Osteoblast-like UMR106 cells were obtained from Beijing Medical University (originating from the Massachusetts General Hospital, Boston, MA, USA). Minimum essential medium (MEM) was obtained from Gibco (Grand Island, NY, USA) and fetal calf serum (FCS) from TBD Bio-engineering Co. (Tianjin, China). Trypsin was supplied by Difco (Livonia, ML, USA), Nunc (Roskilde, Denmark) provided the tissue culture materials, and MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] was obtained from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. Raw materials of Epimedium koreanum. were purchased from Tianyitang Chinese drug store (Shenyang, China) and identified by Qishi Sun, Shenyang Pharmaceutical University, China.
Extraction and isolation
The powdered aerial parts of Epimedium koreanum. (1000 g) were refluxed for 2 h with 80% ethanol (2000 ml, 3 times). The ethanol solution was concentrated to dryness under reduced pressure to afford a dark brown mass (210 g). A part (200 g) of the mass was suspended with hot water and successively fractionated with petroleum ether (b.p. 60–90°C), chloroform, ethyl acetate, and butanol. The solvents and residual water were evaporated to give the petroleum ether fraction (11 g), chloroform fraction (2 g), ethyl acetate fraction (18 g), butanol fraction (20 g), and residual aqueous fraction (145 g), respectively. The ethyl acetate fraction (10 g) was chromatographed on a silica gel column (8 100 cm), eluted with CHCl3:MeOH by a stepwise manner, from 100:1, 100:5, 100:10, 100:15, … to 100:50, 500 ml for each step. Three compounds were isolated by activity-guided assay. Icariside II (18 mg) was isolated from 100:5, icariin (25 mg) was isolated from 100:20, and epimedin B (12 mg) was isolated from 100:35. Their structures were identified by comparison of their physical properties and IR and NMR spectral data with literature values (Li et al., Citation1995; Sun et al., Citation1995).
Preparation of test samples
The crude extract of Epimedium koreanum. Nakai 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 milliliter). The compounds were dissolved in ethanol to give concentrations of 10 mmol/l, and NaF was dissolved in distilled water to give concentrations of 10 mmol/l as positive control. The solutions were sterilized with a 0.2 µm aseptic filter (Gelmann Science, Ann Arbor, MI, USA) and stored at 4°C, and all sample solutions were diluted with MEM to the required concentration before use. The blank controls contained MEM and the same proportion of ethanol as in the test samples.
Proliferation assay
The stimulating proliferation activity of the solutions on osteoblast-like UMR106 cells was assayed in the same procedure as described previously (Wang et al., Citation2001). After UMR106 cells were cocultured with the prepared sample solutions for 48 h at 37°C in a humidified atmosphere of 95% air and 5% CO2, the medium was removed, 50 µL 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 µl DMSO (per well) was added. The absorbance was recorded on an enzyme immunoassay plate reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 595 nm with a reference at 655 nm.
Statistical analysis
Data are expressed as the mean ± standard deviation. Statistical significances were assessed by using the Student's t.-test. A value of p < 0.05 was considered significant. Linear regression analysis was performed by the correlation coefficient. Growth stimulation ratios (GSR) were calculated using the following equation: GSR% = (Asample − Ablank)/Ablank × 100, where A is the average absorbance of six wells.
Results
As shown in , when the cells were cultured with 1.0 × 10−3 mg/ml crude extract, cell proliferation was significantly stimulated (an increase of 35.5% in osteoblastic proliferation). The effect is similar to that of NaF at a concentration of 10−2 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 ethyl acetate layer retained the same stimulating activity as the crude extract. For the compounds isolated, icariin produced the most significant promoting effect on UMR106 cell proliferation; epimedin B and icariside II also exhibited significant stimulating activities in osteoblastic proliferation ().
Table 1.. The effects of various extracts of Epimedium koreanum. Nakai on UMR106 cell proliferation.
Table 2.. The effects of flavonoids on UMR106 cell proliferation.
Discussion
Osteoblasts play a key role in bone formation and remodeling. Agents stimulating osteoblast activity may represent a potential drug for osteoporosis or some other bone diseases. The UMR106 cell line, originating from a rat osteogenic osteosarcoma, is an osteoblast model, and it has stable phenotype and biochemical characteristics (Gray et al., Citation1987). It has preserved many properties of osteoblast, including cAMP responsive to parathyroid hormone (PTH), high alkaline phosphatase (ALP) activity, and synthesis of bone collagen (Patridge et al., 1983). Therefore, the UMR106 cell line has widely been used as an osteoblast model to study various hormones and factors acting on bone. With this in vitro. model, active components stimulating bone formation and potentially anti-osteoporostic may be isolated from traditional Chinese medicine. Compared with animal experiment in vivo., this cell-culture model has advantages such as low dosage, short experiment period, and high reproducibility.
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 a relationship with bone formation. In China, Herba Epimedii has frequently been used in many Chinese formulas for clinical anti-osteoporosis (Gao et al., Citation1999). Our study showed that the extract of Epimedium koreanum. markedly stimulated proliferation of osteoblast-like UMR106 cells. The results suggested that Epimedium koreanum. Nakai extracts and its ethyl acetate fraction might contain active constituents stimulating osteoblasts. Icariin, a major pharmacologically active substance, produced the most significant promoting effect on UMR106 cell proliferation. It might have potential activity against osteoporosis.
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