Metabolomic Methodology for Botanical Discovery and Standardization of Red Clover and Grape Seed Extracts

Metabolomic mining and metabolomic standardization are the two key areas in natural product research (NPR) applied towards human health. The application of NPR to drug discovery and development begins with metabolomic mining. In 2014, about 20-25% of all the drugs approved by the FDA or similar regulatory agencies belonged to the category of unmodified natural products, botanical natural products (mixtures) or natural product derived agents (semi-synthetically modifying the natural product). For instance, out of the total number of small molecule anti-cancer drugs approved between 1940s and 2014, about 40% were natural products or natural product derived agents. Following the discovery of active agents, the next important step is their chemical and biological standardization to ensure safety, efficacy, and reproducibility. Chemical standardization becomes increasingly important as natural product mixtures are also being accepted as approved therapeutic agents. Unlike the single chemical entities, NP mixtures reflect the metabolomic complexity of the source organism, which logically requires metabolomic standardization. In addition to the FDA approved drugs, a significant portion of the U.S. population uses non-vitamin and non-mineral NP derived dietary supplements as complementary and alternative therapy. Metabolomic standardization is indispensable in order to ensure the integrity of botanical dietary supplements, which in turn contributes to their quality and safety. In the present study, metabolomic mining was demonstrated through the discovery of oligomeric proanthocyanidins (OPAC) as dentin bioactive agents and their development into dental restorative biomaterials. A total of 21 PACs were isolated from grape seed extract (GSE), and 11 were evaluated as dentin biomodifying agents. A new and scalable chromatographic method using centrifugal partition chromatography (CPC) was developed for the preparation of DESIGNER biomaterial from grape seed extract. The DESIGNER material consisted of PACs specifically enriched in trimers and tetramers. This study resulted in four key conclusions: (i) galloyl moieties enhance the dentin biomodification potential of PACs; (ii) a degree of polymerization (DP) of 3-4 was found to be the most efficient for optimum collagen-PAC crosslinking; (iii) dimeric PACs with relatively rare ent-catechin and ent-epicatechin flavan-3-ol constituent units were identified from the GSE; and (iv) trimeric PACs with 4-aryl-2-flavanylbenzopyran moieties were identified for the first time from GSE, although it is speculated that they might be an artifact from the extraction process. These key conclusions support the hypothesis that grape seed PACs are a sustainable source of potent dentin biomodifying agents that could be clinically used as dental biomaterials for extending the lifespan of composite based dental restorations. In parallel to metabolomic mining, an advancement of the metabolomic standardization aspect of NPR was demonstrated through the development of new qHNMR based methodology for multi-target quantitation of isoflavones in a red clover extract (RCE). As a part of collaborative research performed at the UIC/NIH Botanical Center, a key aim of the present study was the evaluation of the long-term chemical stability of RCE used in clinical trials. Orthogonal to the previous HPLC-UV/MS based analyses, two mechanistically different qHNMR methods were evaluated. The first qHNMR approach was based on peak-fitting (PF-qHNMR) using global spectral deconvolution. The second and more "NMR aware" deconvolution approach used the quantum mechanical spin simulation performed iteratively (QM-qHNMR) to achieve a perfect match between the experimental and calculated NMR spectrum. Both strategies were employed to replace the traditional and more error-prone integration methods. A total of seven isoflavones in RCE were simultaneously quantified using both PF-qHNMR and QM-qHNMR methods resulting in a higher quantitative accuracy when using the QM-qHNMR method. In addition to the individual isoflavone content, qHNMR enabled standardization of RCE to a total isoflavone content (TIfCo) of 34.5 – 36.5 %w/w using the isoflavone fingerprint region. Finally, both the qHNMR methods confirmed the chemical stability of the RCE which was crucial for performing further biological evaluations and contributes to the quality assurance methods for botanical NPs used in dietary supplements.