Saponins are bioactive compounds found in many plant materials, including legumes. The objectives of the present work were to isolate, fractionate, and purify saponins from soybeans, known as soyasaponins; to determine their potential in vitro colon anti-carcinogenic activity; and to establish a structure-activity relationship.; Because of the absence of commercially available purified soyasaponins, researchers have always had to isolate the soyasaponins themselves, limiting research on their bioactivity. The methods developed here exploited simple and inexpensive technologies, hopefully encouraging more research interest. Isolation of soyasaponins was achieved using precipitation with ammonium sulphate, solid phase extraction and reverse phase low pressure liquid chromatography. Toxic solvents typically used in saponin chemistry, chloroform, acetonitrile and butanol, were avoided and instead ethanol, methanol and acetone were used. An analytical method named direct densitometry was developed, validated, and used, along with thin layer chromatography to routinely quantitate and evaluate the purity of isolated soyasaponins. Four soyasaponin mixtures, free of non-saponin contaminants were isolated: acetylated and deacetylated group A soyasaponins, group B soyasaponins, and a soyasaponin A2-rich fraction. Six soyasaponins, purified to greater than 80% one compound, were isolated: soyasaponin A 1, soyasaponin I, soyasaponin III, soyasapogenol B monoglucuronide, soyasapogenol A and soyasapogenol B. The identity of each of these fractions was confirmed by NMR spectroscopy.; To investigate the role of colonic microflora, soyasaponins were incubated under anaerobic conditions with human microflora. The soyasaponins were converted to soyasapogenol A and soyasapogenol B.; The purified soyasaponins were tested for their ability to suppress the growth of HT-29 colon cancer cells. The most potent compounds were the aglycones, soyasapogenol A and B, which demonstrated a dose dependent and statistically significant suppression of cell growth. The glycosides, soyasaponin A 1, soyasaponin A2, soyasaponin I, soyasaponin III, soyasapogenol B monoglucuronide, group B soyasaponins and deacetylated and acetylated group A fractions, on the other hand were largely inactive. These results suggest a structure-activity relationship: the polar glycosides are inactive while the lipophilic sapogenins are bioactive. Furthermore, in vitro fermentation experiments indicated that colonic microflora readily converted the soyasaponins to the aglycone form. These observations, taken together, suggest that the soyasaponins, after alteration by microflora, may be potential chemopreventive agents against colon cancer.
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