xtraction, Isolation and Preliminary Characterization of Fenugreek (Trigonella foenum graecum L.) Saponins and Their Biological Activity Against Tilapia Aromatase
Fida Sammer Malek
فداء سمير مالك
Fenugreek (Trigonella foenum-graecum L.), is widely distributed throughout the world and belongs to the Fabacecae family. Recent researchers have identified fenugreek as a valuable medicinal plant with potential multipurpose uses and also as a source for preparing raw materials in pharmaceutical industry, especially steroidal hormones. Early studies have shown that saponins are the main attributer behind its medicinal uses and could therefore make a tangible contribution to the pharmaceutical industry income. Saponins are heterosides (substances that contain in their structure one or more sugar molecule) of plant origin which derive their name from their ability to form stable, soap-like foam in aqueous solutions. In plants, saponins play a role as secondary metabolites and are assigned for defense mechanism. In chemical terms, saponins contain sugar moieties attached to a triterpenoid or steroid aglycone. Saponins mixtures have a very complex structure and they are structurally related which make their exact separation, purification and identification a challenge. To determine the trigonella saponins (Ts) other beneficial effects rather that reported, we tested the inhibitory activities of fenugreek seeds methanol (MeOH) extract on aromatase enzyme. The study was also extended to characterize some saponins existed in fenugreek seeds and their effect on aromatase which is a group of cytochrome P450 enzymes responsible for the biosynthesis of estrogen, and establishing alternative methods based on saponins natural extracts to produce monosex culture of Nile tilapia (Oreochromis niloticus) which is considered as the only economically viable form of fish production worldwide. It is worth to notice that the use of synthetic hormones for achieving monosex populations is not sustainable and not environmentally friendly. In this work, we developed a new HPLC method coupled with evaporative light scattering detector (ELSD) and photodiode array (PDA) for the simultaneous determination of major saponins in fenugreek. Simultaneous separation of these saponins was achieved on a C18 analytical column. The mobile phase consisted of a gradient of water and acetonitrile. The method was validated for linearity, precision, accuracy, limit of detection and quantification. V Five saponins-rich extracts (20, 40, 60, 80 and 90% methanol) fractions from fenugreek were evaluated for their anti-aromatase activities. We tested the inhibitory activities of MeOH saponins glycosides extract on the Nile tilapia aromatase enzyme in vitro. To increase the understanding of the interaction of saponins molecules with the enzyme, part of the study focused on the use of Ts fractions as in vitro substrate for aromatase enzyme and compared the activity with the synthetic hormone (methyltesterone). The 90% methanol fraction (Ts 90%) was found to possess a maximum aromatase inhibitor activity, so it was subjected to further purification and sub-fractionation to different clusters by starting from analytical HPLC-PDA-ELSD reverse phase chromatography and passing by scaling up via semi preparative and preparative modes. Three main clusters were collected and the active fraction was called cluster III. The active cluster was subjected to further gradient preparative chromatography in attempt to collect pure saponins. The concentration of the active cluster was so minute to carry full characterization studies. However, ten milligrams of each pure saponin from the less active cluster II was isolated and tested in vitro. Full scans UPLC-ESI-MS and some MS-MS in the MRM mode was investigated and structures were suggested. The structure of saponins II (fraction IV) was fully characterized by using one dimensional 1H-NMR and 2D-NMR (1H-1H correlation COSY and 1H-13H correlation HSQC).