Polymethoxyflavones (PMFs), a unique class of flavonoids found in citrus fruits has shown beneficial bioactivities. Biological fate of PMFs in the gastrointestinal tract is critical for their bioactivities. PMFs can be biotransformed to produce various metabolites with different bioactivities, and PMFs can also interact with the food matrix, which in turn affects their bioactivities. Therefore, we investigated the biotransformation of two PMFs, namely nobiletin (NBT) and 5-hydroxylnobiletin (5HN) by the gut microbiome. Mice were fed with NBT or 5HN, and small intestinal contents were collected and subjected to anaerobic fermentation by gut microbiome isolated from mice and human volunteers. HPLC analysis demonstrated that the gut microbiome largely deconjugated glucuronide and sulfate conjugates of NBT, 5HN and their metabolites and caused demethylation at certain positions of flavonoid. Moreover, the colonic microbial metabolites showed much stronger anti-carcinogenic effect than those found in the small intestine. The second part of this dissertation focuses on characterizing different PMFs, and their interaction with dietary protein by surface enhanced Raman spectroscopy (SERS). 3¢-hydroxylnobiletin (3HN), 4¢-hydroxylnobiletin (4HN), and 5-hydroxylnobiletin (5HN) exhibited significantly different SERS behaviors after binding with silver dendrites. 5HN had the highest peak intensity, while 3HN had the lowest peak intensity. The HPLC analysis revealed that 36.13 ±1.06% of 5HN, 18.40 ± 3.31% of 4HN and 9.66 ± 0.94% of 3HN were bound to silver dendrites. We speculated that different positions of hydroxylation of PMFs were critical for different binding affinities. Furthermore, the molecular interaction of NBT and 5, 3’, 4’- trihydroxylnobiletin (THN), and native and thermal denatured kappa casein, were characterized by SERS and fluorescence spectroscopy. Both SERS and fluorescence quenching studies showed that increased binding of NBT to thermal denatured casein than native casein. However, binding affinity of THN to thermal denatured casein decreased. Our results suggested that NBT interacted with kappa casein through hydrophobic interaction. THN interacted with kappa casein through hydrogen bonding and hydrophobic interaction, and hydrogen binding was the driving force. This dissertation demonstrates the significance of the microbial biotransformation of PMFs in the colon, and the feasibility of SERS to characterize PMFs and their interactions with protein.