Metabolite extraction methods have been shown to be a critical consideration for pharmacometabolomics studies and, as such, optimization and development of new extraction methods are crucial. In the current study, an organic solvent-free method, namely, pressurised hot water extraction (PHWE), was used to extract pharmacologically important metabolites from dried Moringa oleifera leaves. Here, the temperature of the extraction solvent (pure water) was altered while keeping other factors constant using a homemade PHWE system. Samples extracted at different temperatures (50, 100, and 150°C) were assayed for antioxidant activities and the effect of the temperature on the extraction process was evaluated. The samples were further analysed by mass spectrometry to elucidate their metabolite compositions. Principal component analysis (PCA) evaluation of the UPLC-MS data showed distinctive differential metabolite patterns. Here, temperature changes during PHWE were shown to affect the levels of metabolites with known pharmacological activities, such as chlorogenic acids and flavonoids. Our overall findings suggest that, if not well optimised, the extraction temperature could compromise the “pharmacological potency” of the extracts. The use of MS in combination with PCA was furthermore shown to be an excellent approach to evaluate the quality and content of pharmacologically important extracts.

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Pressurized hot water extraction (PHWE) has been classified as a green extraction technology, which is used for the extraction of bioactive compounds from plants. However, very few optimization strategies for the efficient extraction of bioactive compounds have been done. In the current study, optimization of PHWE of flavonoids from Momordica foetida was attempted with the aid of ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-qTOF-MS) and multivariate chemometric models. Plant metabolite extraction was achieved by altering the extraction temperature (100-300°C) during PHWE. UHPLCqTOF-MS analysis of the extracts revealed temperatures between 150 and 250°C to be optimal for the extraction of flavonoid molecules. Furthermore, analysis of the data using multivariate models such as principal component analysis (PCA) revealed various flavonoid distribution patterns between extracts obtained using these various temperatures. In addition, isomeric flavonoids with similar structural core conformations were also seen to be differentially extracted at the various temperatures. The use of UHPLC-MS and highdimensional chemometric models allowed for comprehensive optimization of the PHWE. The use of MS in combination with PCA is an excellent approach to evaluate the quality and content of pharmacologically relevant metabolites from subcritical fluid plant extracts.

Vernonia fastigiata is a multi-purpose nutraceutical plant with interesting biological properties. However, very little is known about its phytochemical composition and, thus the need for its phytochemical characterization. In the current study, an environmentally friendly method, pressurized hot water extraction (PHWE), was used to extract metabolites from the leaves of V. fastigiata at various temperatures (50 °C, 100 °C, 150 °C and 200 °C). Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-qTOF-MS) analysis in combination with chemometric methods, particularly principal component analysis (PCA) and liquid/gas chromatography mass spectrometry (XCMS) cloud plots, were used to descriptively visualize the data and identify significant metabolites extracted at various temperatures. A total of 25 different metabolites, including hydroxycinnamic acid derivatives, clovamide, deoxy-clovamide and flavonoids, were noted for the first time in this plant. Overall, an increase in extraction temperature resulted in an increase in metabolite extraction during PHWE. This study is the first scientific report on the phytochemical composition of V. fastigiata, providing insight into the components of the chemo-diversity of this important plant.