Postprandial glycemic and lipidemic effects of black rice anthocyanin extract fortification in foods of varying macronutrient compositions and matrices

Energy-dense diets rich in rapidly digestible carbohydrates and fats contribute to hyperglycemia and obesity, key drivers of type 2 diabetes (T2D), cardiovascular disease (CVD), and related metabolic complications. Anthocyanins (ACNs), plant-derived polyphenols, have been associated with beneficial effects on glucose and lipid metabolism through inhibition of digestive enzymes (α-amylase, α-glucosidase, lipase), modulation of metabolic gene expression, and improvements in in vivo glycemic and lipidemic profiles. Black rice is a culturally relevant, ACN-rich source in Asia, with major ACNs cyanidin-3-glucoside (C3G), cyanidin-3-rutinoside (C3R), and peonidin-3-glucoside (P3G). Prior work has often examined ACNs in vitro or in long-term or compromised-metabolism populations, leaving a gap in end-to-end assessments that link food fortification, matrix interactions, and acute human postprandial responses in healthy individuals. Food matrices may alter ACN accessibility and bioactivity, and postprandial responses are increasingly recognized as predictors of metabolic disease. Research question: Does fortifying commonly consumed foods with black rice ACN extract (BRAE) modulate in vitro digestibility and in vivo postprandial glycemic and lipidemic responses in healthy individuals, and how do effects differ between a single high-carbohydrate food versus a mixed high-carbohydrate/high-fat meal matrix?

Evidence indicates ACNs acutely inhibit α-amylase, α-glucosidase, and lipase in vitro, and modulate carbohydrate and lipid metabolism pathways, with reported improvements in glucose and lipid profiles in vivo. Black rice ACNs (C3G, C3R, P3G) have shown anti-glycemic and anti-lipidemic effects in vitro and in animal studies. Fortification studies have largely focused on in vitro characterization of ACN-rich extracts in starch-based foods to reduce digestibility; human trials often involve long-term intake or participants with metabolic disorders. Interactions between ACNs and food matrices (proteins, lipids) can impact ACN bioaccessibility and bioavailability, but clinical evidence is limited and mixed. There is a paucity of acute human feeding studies assessing postprandial effects of ACN-fortified foods in healthy individuals, particularly within mixed-nutrient meal contexts.

Design: Two randomized, crossover RCTs with integrated in vitro assays. Each trial enrolled 24 healthy adults. Ethical approval obtained; trials registered (NCT03989674, NCT04063137). - Bread trial (single high-carbohydrate food): 6 visits over 2 h each with 2-day washouts. Three reference 50-g oral glucose challenges and three bread interventions: CON (no BRAE), 2-BB (2% BRAE/100 g flour), 4-BB (4% BRAE/100 g flour). Blood sampled at fasting, 15, 30, 45, 60, 90, 120 min for glucose and insulin. GI determined per ISO 26642:2010. ACN bioavailability (C3G, C3R, P3G) and metabolites (protocatechuic, vanillic, ferulic acids) assessed at 60 and 120 min by HPLC-DAD after SPE. - Burger trial (HC/HF mixed meal): 2 visits over 4 h each, 2-day washout. Randomized to CONBgr (burger with unfortified bread) or 4-BBgr (burger with 4% BRAE bread). Meal provided 50 g available carbohydrate, 33 g protein, 43.8 g fat. Blood sampled at fasting, 15, 30, 45, 60, 90, 120, 180, 240 min for glucose, insulin, lipid panel (TG, TC, HDL-c, LDL-c). Lipoprotein particles, subfractions, sizes, and lipid compositions quantified via Nightingale NMR platform. In vitro assays: - Starch hydrolase inhibition of major ACNs (C3G, C3R, P3G) vs acarbose; IC50 against α-amylase and α-glucosidase determined by turbidity-based assay using gelatinized wheat starch; % inhibition from AUC changes. - Simulated gastrointestinal digestion (oral, gastric, intestinal phases) for breads (CON, 2-BB, 4-BB) and burgers (CONBgr, 4-BBgr); quantify glucose release; fit first-order model C_t = C_∞ + (C_0 – C_∞)(1 – e^(-kt)); compute IAUC and kinetic constant k. - Pancreatic lipase inhibition in burger matrix using 4-methylumbelliferyl oleate (4-MUO); fluorescence over 0–240 min; IAUC and % inhibition. Test meal composition and ACN content: Breads standardized to 50 g available carbohydrates; 2-BB ~60 mg and 4-BB ~127 mg total ACNs per 100 g bread (C3G equivalents) by pH differential method and HPLC profiling. Participants: Healthy Asian Chinese adults (21–65 y), BMI 17–28 kg/m², normotensive, normal fasting glucose; additional lipid criteria for Burger trial (TG <1.7 mmol/L, LDL-c <2.6 mmol/L). Exclusions: smoking, pregnancy, medication use, relevant medical history. Standard pre-visit controls: 10 h fast; avoidance of fat- and ACN-rich foods (≥36 h), strenuous exercise and alcohol (24 h). Outcomes and statistics: Primary outcomes—Bread trial: GI, postprandial glucose/insulin; Burger trial: postprandial glucose/insulin, lipid panel; secondary: ACN bioavailability (Bread), lipoprotein particles/subfractions and lipid composition (Burger). Analyses: One-way ANOVA with Tukey (in vitro breads; Bread trial repeated measures); unpaired t tests (in vitro burger); paired t tests (Burger trial). Two-way ANOVA for time × intervention interactions. Significance p < 0.05. Power calculations targeted ≥12–15 completers; 24 recruited per trial.

In vitro: - Major black rice ACNs inhibited carbohydrases and lipase: significant inhibitory activities on starch hydrolases (p = 0.0004), pancreatic lipase (p = 0.0002), and reduced starch digestibility (p < 0.0001). - IC50 (µM) for α-amylase/α-glucosidase: Acarbose 6.0/2.9; C3G 364/1116; P3G 649/1343; C3R 483/904. C3G most potent vs α-amylase; C3R most potent vs α-glucosidase. - Simulated digestion (bread): BRAE reduced digestibility dose-dependently. IAUC (g/100 g*min): CON 810 vs 4-BB 710 (−12.3%), p < 0.0001; k: 0.00830 vs 0.00662 (−12.6%), p = 0.004. - Simulated digestion (burger): 4-BBgr vs CONBgr reduced IAUC 582 → 483 (−17.0%), p < 0.0001; k 0.00589 → 0.00425 (−16.1%), p = 0.030. Lipase activity IAUC reduced 12517 → 4713 RFU*min (−62.3%), p = 0.0002. Bread trial (n = 22 analyzed): - Postprandial glycemia/insulinemia over 2 h showed attenuation trends but limited significance. Mean glucose differed across groups (p = 0.050); 4-BB had lower mean glucose vs 2-BB and delayed T_max vs CON (mean difference 13.0 min, 95% CI 0.443, 25.5; p = 0.0416). IAUC for glucose not significantly different. - Mean insulin differed across groups (p = 0.0031); pairwise: 2-BB vs CON mean insulin −8.09 mU/L (95% CI −14.1, −2.09), p = 0.0144; IAUC not significantly different. - GI decreased: CON 95.1, 2-BB 94.1, 4-BB 68.2 (≈27-point reduction with 4-BB), shifting GI category from high to medium; GL reduced (47.6 → 34.1). - ACN bioavailability: Plasma peaks at ~60 min; metabolites exceeded parent ACNs—PCA accounted for 93.9% of total polyphenols at 60 min. Not dose-dependent; significant 60→120 min declines in VA (p = 0.0143) and FA (p = 0.0241) in 4-BB. Burger trial (n = 24): - Postprandial glucose/insulin over 4 h: no significant differences between CONBgr and 4-BBgr; insulin T_max delayed (55.6 → 77.5 min), p = 0.0386. - Lipid panel: Significant improvements with BRAE fortification—mean HDL-c over 0–240 min less decreased (−0.036 → −0.022 mmol/L), p = 0.0028; Apo-A1 (−0.045 → −0.015 mmol/L), p = 0.0203; Apo-B (−0.011 → −0.003 mmol/L), p = 0.0185; Apo-B:Apo-A1 ratio changed (0.458 → 0.467), p = 0.0008. TG, TC, LDL-c changes were not significantly different. - Lipoprotein particles/subfractions: Mean particle concentrations differed—VLDL (p ≈ 0.039–0.042), LDL (p = 0.0203), HDL (p = 0.0203). Subfractional changes significant for XS-VLDL, L-LDL, M-LDL, S-LDL, XL-HDL, L-HDL, M-HDL, S-HDL (p < 0.05). Lipid composition in LDL and HDL subfractions showed attenuation of total lipids, PL, TC, CE, FC with BRAE; TG changes minimal. Particle sizes did not differ significantly.

The study addressed whether ACN fortification of common foods modulates postprandial metabolism and how food matrices influence efficacy. Incorporation of BRAE into a single high-carbohydrate food modestly attenuated postprandial glucose and insulin responses acutely and substantially reduced GI (by ~27 points at 4% fortification), supporting potential benefits in glycemic control through lower-GI reformulations. In a mixed high-carbohydrate/high-fat meal, matrix complexity appeared to blunt glycemic effects despite equivalent available carbohydrate, aligning with the hypothesis that protein and fat components alter ACN accessibility and function. Nonetheless, BRAE fortification favorably modulated acute lipidemia, improving HDL-c and apolipoproteins (Apo-A1 and Apo-B) and remodeling LDL and HDL particle/subfraction profiles and lipid distributions, suggesting potential enhancement of reverse cholesterol transport and hepatic lipid clearance. Mechanistically, findings support dual actions: direct digestive enzyme inhibition (carbohydrases, lipase) reducing nutrient hydrolysis rates, and post-absorptive effects likely mediated by ACN metabolites (notably PCA) influencing lipoprotein metabolism, possibly via CETP modulation and upregulation of cholesterol efflux pathways (ABCG1, SR-B1). High inter- and intra-individual variability among healthy participants likely limited detection of some statistical differences, particularly in glycemia. Overall, the results emphasize that ACN efficacy is food-matrix dependent, with stronger lipoprotein-related benefits observed in mixed meals and clearer glycemic benefits (GI lowering) in single carbohydrate foods.

This end-to-end investigation demonstrates that fortifying wheat bread with black rice anthocyanin extract reduces in vitro starch digestibility and lowers the bread’s glycemic index by approximately 27 points at 4% fortification, with modest acute effects on postprandial glucose and insulin in healthy individuals. In a mixed high-carbohydrate/high-fat burger meal, glycemic responses were unaffected, but BRAE fortification significantly improved HDL-c and apolipoproteins and altered LDL/HDL particle and subfraction profiles and lipid compositions acutely. ACN metabolites were more bioavailable than parent compounds, indicating that downstream metabolic effects may be mediated predominantly by metabolites. Collectively, ACN fortification emerges as a promising, food-matrix–specific dietary strategy to enhance cardiometabolic profiles, particularly via lipoprotein remodeling in mixed meals and GI reduction in carbohydrate-rich foods. Future work should include longer-term interventions, larger and more diverse cohorts, controlled dietary run-in phases, and mechanistic studies to delineate pathways (e.g., CETP activity, cholesterol efflux, hepatic lipoprotein kinetics) and to optimize formulation and matrix considerations for maximal efficacy.

- Acute study design limits inference on long-term metabolic effects. - Healthy, relatively young sample with normal baseline metabolism may exhibit high inter- and intra-individual variability, reducing power to detect differences in postprandial glycemia. - Limited dietary control outside avoidance of ACN-rich and fat-rich foods before visits; residual dietary variability may have contributed to variability in phenolic and metabolic responses. - Matrix effects (protein/lipid interactions with ACNs) were not directly quantified; mechanisms remain inferential. - Only one fortification dose (4%) tested in the mixed-meal trial; dose–response in composite meals remains unclear. - Bioavailability assessed only up to 120 min and for selected ACNs/metabolites; broader metabolite profiling and longer time windows may provide additional insight.