Methods for the Metabolomic Standardization of Natural Health Products

ABSTRACT The present study establishes analytical methods for the metabolomic analysis of Natural Health Products (NHPs), exemplified with two highly complex mixtures, the essential oil (EO) of Eucalyptus citriodora (lemon-scented oil), and a hops total extract (Humulus lupulus L.). The first NHP, E. citriodora EO, was investigated for its chemical and biological profiles.The biochemometric analysis was applied in order to address the metabolomic complexity inherent to essential oils. The subsequent analysis by biochemometric, using the ‘r’ Pearson’s correlation factor to match the biopeaks in the anti-TB biochromatogram to the GC peaks related to a specific chemical entity or constituent of the EO fraction. Biochemometric analysis resulted in the identification of 25 anti-TB bioactives in E. citriodora EO, including the monoterpene compounds, citronellol, isopulegol, α-terpineol, and the sesquiterpene constituents spathulenol, ledol, viridiflorol, farnesol, β-eudesmol, and cubenol. The second NHP, Hops, has been known to contain a rich pool of prenylated phenols. Although, most reports are based on the biological activities exerted by the prenylated flavanones, isoxanthohumol (IX), 8-prenylnaringenin (8-PN), 6-prenylnaringenin (6-PN), and the prenylated chalcone, xanthohumol (XH). Therefore, in order to explore further the hops metabolome, the analytical approach, known as Chemical Subtraction, was applied. A series of extracts, known as Knock-Out Extracts (Ts-KOEs) were generated. These extracts were depleted of either XH, IX, 8-PN, 6-PN, or the four compounds simultaneously in these extracts. The generation of these Ts-KOEs was done with the application of CS and a combination of orthogonal solvent systems (SSs). The efficiency of the subtraction methods was analyzed by quantitative 1H-NMR (q1HNMR). Also, the residual complexity of the subtracted target metabolites was evaluated by more traditional methods such as UHPLC-UV. The biological evaluation of the Ts-KOEs leads to observe the correlation of the XH content and the decrease of quinone reductase 1 induction activity. Estrogenic activity, expressed as alkaline phosphatase induction, was also observed in the Ts-KOEs were the potent phytoestrogen, 8-PN, had been depleted. Therefore, there is evidence of other minor but possibly bioactive metabolites in the hops extracts, which can modulate specific biological activities, or work together synergistically along with other Hops constituents. The present study presents the findings of the application of biochemometrics and its analytical components, and the application of chemical subtraction to both bioactive and complex natural products. Identity of bioactive entities, chemical and biological profiles, as well as quantitative information is acquired and integrated through the implementation of these standardization methods.