Malonyl-conjugates of isoflavones: Structure, Bioavailability and Chemical Modifications during Processing

Soy isoflavones are often associated with prevention of cancer, cardiovascular diseases, osteoporosis, and postmenopausal symptoms. However, the demonstration of theses physiological effects is highly inconsistent. Not all soy foods deliver the same isoflavone-associated benefits. Inconsistency in isoflavone research is partly attributed to the inadequate profiling of isoflavones, lack of standardization of the source of isoflavones, and lack of standard analytical methods for profiling and quantifying isoflavones present in different soy matrices. We are convinced that inconsistent results are due to differences in the bioavailability of the different isoflavone forms consumed. Since isoflavones in soy foods differ in their forms (e.g. conjugated and non-conjugated), large differences may exist in their bioavailability. Therefore, it is crucial to adequately profile the administered isoflavones and study the effect of their conjugation on their bioavailability. Additionally, isomerization of different isoflavone forms occurs upon thermal processing. Complete structural elucidation of the isomers and determination of their thermal stability in soy systems are important for understanding their physiological relevance. Therefore, the overall objective of this study was to determine effect of processing on the chemical modifications of isoflavones and to detect all biologically relevant forms, together with providing adequate and reliable bioavailability data for each of the most abundant isoflavone forms. Isoflavones were extracted from soy grits and were separated and isolated using semi-preparative liquid chromatography. Identification of the different isoflavones forms and isomers was accomplished based on UV wave scan, mass spectrometry, and nuclear magnetic resonance (NMR) analysis. Effect of thermal processing on isomer stability was determined by subjecting soymilk to thermal treatment at 100°C for time intervals ranging from 1 to 60 min. A rapid analytical procedure was developed to quantify isoflavones in biological fluids using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The developed method was sensitive, selective, precise and accurate. Bioavailability of malonylglucosides and their respective non-conjugated glucosides was determined in a model rat system. Rats were gavaged with an assigned isoflavone form. Blood and urine samples were collected at different time intervals. Different isoflavone metabolites in plasma were determined using the developed SID-LCMS method. Bioavailability was determined by calculating pharmokinetic parameters, assuming first order disposition kinetics. NMR characterization of the malonylglucoside isomers revealed its structure to be 4"-O-malonylglucosides, suggesting a malonyl migration from the glucose-6-position to the glucose-4-position. The malonylgenistin isomer represented 6-9 % of the total calculated genistein content in soymilk heated at 100°C for various periods of time. Based on rat peak plasma and urine levels and area under the curve (AUC) of the aglycone post ingestion of the respective isoflavones, it was quite evident that the malonylglucosides were significantly (P ≤ 0.05) less bioavailable than their non-conjugated counterparts. The present work provided full elucidation of the chemical structure of malonylglucoside isomers. We demonstrated for the first time that the formation of the malonylisomers is governed by thermal processing time in a soymilk system. Disregarding the isomer formation upon heating can result in overestimation of loss in total isoflavone content and misinterpretation of the biological contributions. Additionally, this work provided a validated analytical SID-LC/MS method to detect natural and known synthetic selective estrogen receptor modulators (SERMs) in a single analytical assay. Finally, this work differentiated for the first time the bioavailability of malonylglucosides as compared to their non-conjugated counterparts. The observed differences explained to a significant extent the controversy in isoflavone research. We believe that the results of this work will help streamline the experimental approach undertaken by various researchers to achieve consistent clinical conclusions and to optimize the processing parameters that result in the most bioavailable isoflavone profile, thus maximizing their health benefits.