High-fat diet (HFD)-induced leaky gut syndrome combined with low-grade inflammation increase reactive oxygen species (ROS) in the intestine and may contribute to dysbiosis and metabolic syndrome (MetS). Poorly bioavailable and only partially metabolizable dietary polyphenols, such as proanthocyanidins (PACs), may exert their beneficial effects on metabolic health by scavenging intestinal ROS. To test this hypothesis, we developed and validated a novel, noninvasive, in situ method for visualizing intestinal ROS using orally administered ROS-sensitive indocyanine green (ICG) dye. C57BL/6J mice fed HFD for 10 weeks accumulated high levels of intestinal ROS compared to mice fed low-fat diet (LFD). Oral administration of poorly bioavailable grape polyphenol extract (GPE) and β-carotene decreased HFD-induced ROS in the gut to levels comparable to LFD-fed mice, while administration of more bioavailable dietary antioxidants (α-lipoic acid, vitamin C, vitamin E) did not. Forty percent of administered GPE antioxidant activity was measured in feces collected over 24 h, confirming poor bioavailability and persistence in the gut. The bloom of beneficial anaerobic gut bacteria, such as Akkermansia muciniphila, associated with improved metabolic status in rodents and humans may be directly linked to protective antioxidant activity of some dietary components. These findings suggest a possible mechanistic explanation for the beneficial effects of poorly bioavailable polyphenols on metabolic health.

15 High-fat diet (HFD)-induced leaky gut syndrome combined with low-grade inflammation increase 16 reactive oxygen species (ROS) in the intestine and may contribute to dysbiosis and metabolic 17 syndrome (MetS). Poorly bioavailable and only partially metabolizable dietary polyphenols, such 18 as proanthocyanidins (PACs), may exert their beneficial effects on metabolic health by 19 scavenging intestinal ROS. To test this hypothesis, we developed and validated a novel, 20 noninvasive, in situ method for visualizing intestinal ROS using orally administered ROS-sensitive 21 indocyanine green (ICG) dye. C57BL/6J mice fed HFD for 10 weeks accumulated high levels of 22 intestinal ROS compared to mice fed low-fat diet (LFD). Oral administration of poorly bioavailable 23 grape polyphenol extract (GPE) and -carotene decreased HFD-induced ROS in the gut to levels 24 comparable to LFD-fed mice, while administration of more bioavailable dietary antioxidants (α-25 lipoic acid, vitamin C, vitamin E) did not. Forty percent of administered GPE antioxidant activity 26 was measured in feces collected over 24 h, confirming poor bioavailability and persistence in the 27 gut. The bloom of beneficial anaerobic gut bacteria, such as Akkermansia muciniphila, associated 28 with improved metabolic status in rodents and humans may be directly linked to protective 29 antioxidant activity of some dietary components. These findings suggest a possible mechanistic 30 explanation for the beneficial effects of poorly bioavailable polyphenols on metabolic health. 31 32 33 34 35 36 37

Study of plant metabolome is a growing field of science that catalogs vast biochemical and functional diversity of phytochemicals. However, collecting and storing samples of plant metabolome, sharing these samples across the scientific community and making them compatible with bioactivity assays presents significant challenges to the advancement of metabolome research. We have developed a RApid Metabolome Extraction and Storage (RAMES) technology that allows efficient, highly compact, field-deployable collection and storage of libraries of plant metabolome. RAMES technology combines rapid extraction with immobilization of extracts on glass microfiber filter discs. Two grams of plant tissue extracted in ethanol, using a specially adapted Dremel® rotary tool, produces 25–35 replicas of 10 mm glass fiber discs impregnated with phytochemicals. These discs can be either eluted with solvents (such as 70% ethanol) to study the metabolomic profiles or used directly in a variety of functional assays. We have developed simple, non-sterile, anti-fungal, anti-bacterial, and anti-oxidant assays formatted for 24-multiwell plates directly compatible with RAMES discs placed inside the wells. Using these methods we confirmed activity in 30 out of 32 randomly selected anti-microbial medicinal plants and spices. Seven species scored the highest activity (total kill) in the anti-bacterial (bacteria from human saliva) and two anti-fungal screens (Fusarium spp. and Saccharomyces cerevisiae), providing functional validation of RAMES technology. RAMES libraries showed limited degradation of compounds after 12 months of storage at -20°C, while others remained stable. Fifty-eight percent of structures characterized in the extracts loaded onto RAMES discs could be eluted from the discs without significant losses. Miniaturized RAMES technology, as described and validated in this manuscript offers a labor, cost, and time-effective alternative to conventional collection of phytochemicals. RAMES technology enables creation of comprehensive metabolomic libraries from various ecosystems and geographical regions in a format compatible with further biochemical and functional studies.