Energy-dense diets high in rapidly digestible carbohydrates and fats contribute to hyperglycemia and obesity, increasing the risk of type 2 diabetes (T2D), cardiovascular disease (CVD), and other metabolic complications. Dietary modifications, such as increasing anthocyanin (ACN) consumption from plant-derived foods, are considered a valuable strategy for mitigating these risks. ACNs, particularly those found in black rice, exhibit in-vitro inhibitory effects on α-amylase, α-glucosidase, and lipase, enzymes involved in carbohydrate and lipid hydrolysis. Studies have demonstrated in-vivo improvements in glucose and lipid profiles with ACN supplementation. Black rice, widely consumed in Asia, presents a readily available and popular source of ACNs. Previous ACN-food fortification research has primarily focused on in-vitro characterization or on individuals with pre-existing metabolic conditions. This study aims to address the gap in knowledge regarding the effects of ACN fortification in common composite foods on the postprandial metabolic responses of healthy individuals, considering the influence of various food matrices and macronutrients on ACN bioactivity and bioavailability. Postprandial metabolic responses, reflecting the non-fasted state, are increasingly recognized as crucial predictors of metabolic disorders.

The literature review extensively explores the existing research on the role of anthocyanins in mitigating cardiometabolic risks. Numerous studies highlight the in-vitro inhibitory effects of ACNs on key enzymes involved in carbohydrate and lipid metabolism. In-vivo studies, primarily animal models and human intervention trials focusing on specific populations (those with compromised metabolism), show improved glycemic and lipidemic profiles with ACN supplementation or consumption of ACN-rich foods. However, inconsistencies exist regarding the effects of food matrices and the acute postprandial impact of ACN-fortified foods on healthy populations. This necessitates a comprehensive investigation bridging in-vitro findings to human metabolic responses, considering the influence of different food matrices and macronutrient compositions.

This study employed two randomized controlled crossover trials (RCTs). The first RCT (Bread trial) assessed the effects of BRAE fortification on the glycemic index (GI) and postprandial glycemia of a starch-rich single food (wheat bread). Twenty-four healthy participants consumed three types of bread: control (CON), 2% BRAE-fortified (2-BB), and 4% BRAE-fortified (4-BB). Postprandial blood samples were collected for glucose and insulin analysis over 2 hours. The second RCT (Burger trial) examined the impact of BRAE fortification on postprandial glycemia and lipidemia using a starch- and fat-rich composite meal (beef burger) incorporating CON or 4-BB (4% BRAE-fortified bread). Twenty-four participants consumed both meals, with blood samples collected for glucose, insulin, and lipid panel analysis over 4 hours. In-vitro assays were performed to confirm the enzyme inhibitory activities of black rice ACNs (on α-amylase, α-glucosidase, and lipase) and the effect of BRAE fortification on starch digestibility and lipase activity in simulated gastrointestinal digestion models for both bread and burger. Anthocyanin bioavailability was assessed using HPLC analysis of plasma samples from the Bread trial. Lipoprotein particle and subfraction analysis was done via NMR spectroscopy in the Burger trial. Statistical analysis involved one-way and repeated measures ANOVA, paired t-tests, and Tukey's or Bonferroni's post-hoc tests, as appropriate.

In-vitro studies confirmed the inhibitory activity of major black rice ACNs on carbohydrases (p = 0.0004), lipases (p = 0.0002), and starch digestibility (p < 0.0001). The Bread trial showed a 27-point reduction in the GI of wheat bread with 4% BRAE fortification, although the postprandial glycemic response wasn't significantly altered. However, there was a significant delay in time to reach peak glucose concentrations in the 4-BB group compared to CON (p=0.0416). The Burger trial, while showing no significant changes in postprandial glycemia, revealed significant improvements in HDL-c ([0.0140 mmol/L, 95% CI: (0.00639, 0.0216)], p = 0.0028), Apo-A1 ([0.0296 mmol/L, 95% CI: (0.00757, 0.0515)], p = 0.0203), and Apo-B ([0.00880 mmol/L, 95% CI: (0.00243, 0.0152)], p = 0.0185) levels with BRAE fortification. Furthermore, significant modifications were observed in LDL and HDL subfractions (p < 0.05) and remodeled lipid distributions within HDL and LDL particles. Anthocyanin bioavailability analysis revealed higher plasma concentrations of ACN metabolites compared to parent ACNs, with protocatechuic acid being the most abundant polyphenol. In-vitro analyses of the burger confirmed the BRAE's effect on suppressing starch digestibility and pancreatic lipase activity.

The findings suggest that BRAE fortification might offer a viable approach to improve postprandial metabolic outcomes, particularly in terms of lipid profiles. The significant GI reduction in the bread trial underscores the potential of BRAE-fortified foods to improve glycemic control. The lack of a significant effect on postprandial glycemia in the burger trial might be attributed to the complexity of the food matrix, potentially interfering with ACN bioavailability and bioactivity. The contrasting effects on postprandial glucose and lipid responses highlight the importance of food matrix interactions and the need for further research into these interactions. The observed improvements in HDL-c and apolipoproteins suggest a positive effect on reverse cholesterol transport, although the increased Apo-B requires further investigation. The significant modifications in lipoprotein subfractions and lipid compositions point towards a modulatory role of BRAE on lipoprotein metabolism. The higher bioavailability of ACN metabolites supports the hypothesis that both the direct enzyme inhibitory actions of ACNs and the metabolic effects of its metabolites contribute to the overall beneficial impact.

This study provides evidence for the potential benefits of black rice anthocyanin extract fortification in improving postprandial glycemic and lipidemic profiles, particularly by reducing the GI of foods and positively influencing lipoprotein metabolism. However, the effects are food-matrix dependent, highlighting the need for further research to optimize the delivery systems for maximizing the benefits of anthocyanins. Future studies could focus on exploring the long-term effects of BRAE fortification, investigating the mechanisms underlying the observed lipoprotein modifications, and examining the influence of various dietary factors on ACN bioactivity and bioavailability.

The high inter-individual variability observed in the postprandial responses may have masked some of the effects of BRAE fortification, particularly in the glycemic response. The study's acute design may not fully reflect long-term effects. The lack of strict dietary control during the study could have influenced the results, especially the variability in polyphenolic concentrations. Further research is required to better understand the complex interactions between anthocyanins, food matrices, and other dietary factors.