Neo-angiogenesis is the critical step in the development and progression of most of the human cancers. Beyond the critical size of 1–2 mm, oxygen and nutrients have difficulty in diffusing to the core cells of the tumor, causing a state of cellular hypoxia. Under hypoxic conditions, cancer cells secrete several pro-angiogenic factors such as VEGF and bFGF, which cause recruitment of endothelial cells from the neighboring blood vessels 
. But the continuous and excessive presence of pro-angiogenic stimuli in the tumor microenvironment interferes with the normal maturation of vessel network, and as a result, vessels in the tumor area show abnormal morphology and physiology. Hence, it is possible to specifically target abnormal tumor angiogenesis by inhibiting endothelial cell recruitment as well as their proliferation in the tumor microenvironment. Notably, the results from the present study clearly suggest the strong efficacy of four pure flavonolignans from Milk Thistle extract, on these aspects, which signifies their angiopreventive efficacy against PCA. These findings are aslo transnationally noteworthy, as currently, anti-angiogenic strategies are extensively pursued towards preventing the progression of diagnosed pre-malignant tumors or for the therapeutic regression of the already advanced disease 
Milk thistle has been used for centuries to treat chronic liver disease, and to protect the liver against toxins 
. In recent years, Milk thistle research use has grown significantly with close to two dozen clinical trials underway or already completed evaluating its efficacy against variety of diseases including chronic hepatitis, diabetes, asthma, mushroom poisoning and various cancers (these studies are listed at ClinicalTrials.gov). In the past, there have been numerous efforts in isolating and purifying the individual constituents in Milk thistle to better exploit its clinical usefulness. Members of our team were the first to purify and elucidate seven distinct flavonolignans from Milk Thistle extract, namely: silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, and isosilychristin, and one flavonoid, taxifolin 
. Their biological effects as pure compounds were assessed on several anti-proliferative end points in human PCA cell lines, where isosilybin B ranked as the most potent flavonolignan for nearly all the end points, including the inhibition of cell growth, clonogenic potential, PSA and androgen receptor levels, and topo IIα promoter activity 
. Despite these advances in the Milk Thistle research, the in vivo
biological efficacy and related toxicity of these pure flavonolignans remained unknown because of insufficient compound quantities. In particular, isosilybin B was the most challenging of the major diastereoisomer to purify, due to its limited abundance in the natural extract 
and its relatively long retention time in most reverse-phase HPLC systems. The pre-clinical or future clinical use of these pure compounds needed advancement in their isolation; therefore, we developed a hybrid chromatographic/precipitative technique for gram scale purification of flavonolignan diastereoisomers from Milk Thistle extract 
. We believe that this is a major advancement towards the future translational significance of these pure flavonolignans.
The results from the present study adequately proved that the pure diastereoisomers from Milk Thistle have strong angiopreventive efficacy through targeting the pro-angiogenic signaling in PCA cells as well as in endothelial cells; the important component of PCA microenvironment (). Furthermore, based upon the overall in vitro and in vivo analyses, it is also clear that the comparative efficacy of isosilybin B occupies two opposite ends in terms of its effect on PCA cells and endothelial cells. Specifically, it is the lead agent in terms of its efficacy against PCA cells but it is the least effective agent against endothelial cells. On the contrary, silybin A appears to be the most promising agent in terms of its effects on endothelial cells. Therefore, it is prudent to suggest that a defined mixture of silybin A and isosilybin B should be tested, particularly in vivo, with an expectation of these two compounds acting in concert to exert maximum benefits via targeting both the tumor and the tumor microenvironment components.
Diastereoisomers exhibit anti-angiogenic effects through targeting signaling molecules in both prostate cancer cells and endothelial cells.
In summary, the present study, for the first time, report the anti-cancer efficacy of four flavonolignans from Milk Thistle extract in an in vivo system of advanced stage human PCA. These results further suggest the stereochemistry based differential efficacy of these flavonolignans towards cancer and endothelial cells. Furthermore, these results confirm the non-toxicity as well as PCA specific anti-angiogenic effects of the pure diastereoisomers, which suggest their usefulness in PCA angioprevention.