In vitro investigation of anti-osteoporotic bioactivity of extracts from indigenous plants and investigating whether these induce an oestrogen growth factor response

Abstract

Aims: The main aim of the study is the production of an extract from a local indigenous plant that induces the differentiation of the osteoblast cell line MC3T3-E1 without inducing an estrogen growth factor response in the breast cancer cell line MCF-7. Methods: A wide range of different methodologies have been used. Methanol extraction has been utilized for the extraction process. Using the osteoblast cell line MC3T3-E1, titrations were performed so as to obtain the working concentrations for betaoestradiol, the extracts, and an already commercialized product (Lignan). To determine the cytotoxicity, XTT assays were performed on the different compounds tested. A chemiluminescence method has also been utilized to directly measure the osteogenesis rate by measuring the uptake of the stain Alizarin red for calcium mineralization (bone matrix). Western blotting is being used to measure the activation of the Estrogen Receptor (ER-alpha) by using a specific phospho-antibody. Real time-PCR will be utilized for expression studies of identified oestradiol response genes. Results: Using methanol reflux, two different extracts have been produced. The working concentrations of the test compounds have already been identified and XTT assays have showed us that none of the test compounds tested (beta-oestradiol, lignan, and carob pod extract) is cytotoxic. The osteogenesis rate has been quantified and after 15 days, a 3.6 fold increase in osteogenesis has been obtained with beta-oestradiol and a 7.6 fold increase in osteogenesis has been obtained with Lignan, when compared with a no factor control. Out of the 2 extracts produced, one of them did not produce any significant results. However, pilot studies conducted on the second extract have shown very promising results and the current experiments being performed will determine the ideal working concentration of this extract. We also confirmed the work done by Takamizawa et al (2004) where whilst ascorbic acid 2-phosphate stimulates osteogenesis, ascorbic acid showed a repressive effect depending on concentration used. Conclusions: To date we can conclude that both betaoestradiol, and Lignan increase the rate of osteogenesis. Although the primary extract did not produce any significant results, pilot studies on the secondary extract have shown us that the results are very promising. The effect of these test compounds on ER-alpha and on oestradiol response genes still needs to be determined.