Anthocyanins and proanthocyanidins from blueberry and blackberry fermented beverages to reduce inflammation and type-2 diabetes: a comprehensive in vitro and in vivo evaluation

Type-2 diabetes is a serious metabolic disease that currently affects 9.3% of the U.S. population and is aggravated by diets low in fruit and vegetable intake leading to postprandial oxidative stress and inflammation. Berries are one of the richest dietary sources of polyphenolic compounds associated with decreased markers of chronic inflammation and decreased risk for type-2 diabetes, specifically anthocyanins (ANC) and proanthocyanidins (PAC), polymerized forms of ANC and other phenolics. Fermentation is a feasible way to increase phenolic content of berry juice products, and fermented products may be more bioactive than their unfermented counterparts, thus increasing the potential for health benefits. Our long-term goal, as outlined in Chapter 1, was to fill the gap of knowledge in understanding the mechanisms by which dietary bioactives found in fermented berry beverages contribute to the management of type-2 diabetes using in vitro enzymatic kinetics, analytical assays, computational modeling, in vitro cell culture, and in vivo animal feeding trials. A review of the literature focusing on the role of berries to reduce diabetes is presented in Chapter 2. Our research focused on the evaluation and potential mechanisms of action of alcohol-free blueberry (Vaccinium corymbosum) and blackberry (Rubus spp.) fermented beverages and their bioactive ANC and PAC components to reduce inflammation and type-2 diabetes in vitro and in vivo through the aims introduced in Chapter 3. First we prepared blueberry and blackberry wines that were comparable to those commercially available (Chapter 4), and found that blueberries and their wine, especially when fermented at cold temperature, contain phenolic compounds that act as antioxidants and potential inhibitory factors of starch-cleaving enzymes that could be used to limit glucose absorption (Chapter 5). We then generated ANC- and PAC-enriched fractions and found that blueberry and blackberry ANC and PAC inhibited expression of proteins in the NF-κB-mediated pathway in macrophages, indicating potential to reduce chronic inflammation associated with hyperglycemia (Chapter 6). ANC were found to bind strongly within active pockets of dipeptidyl-peptidase IV (DPP-IV), an incretin-cleaving target of diabetes therapy, to effectively reduce its activity (IC50 = 4.0 μM) (Chapter 7). ANC were also able to reduce the inflammatory cross-talk between macrophages and adipocytes (Chapter 8). Further, ANC increased insulin secretion from pancreatic β-cells by 233 and 100 µIU insulin/mL directly and after epithelial transport, respectively, due to reduced DPP-IV expression and up-regulated insulin-receptor associated genes and proteins (Chapter 9). Lastly, in Chapter 10 we established the role of phenolic compounds in 30:70% blueberry-blackberry alcohol-free fermented beverage to reduce body weight (BW) gain (10.3 vs. 16.7 g), fat mass (18.0 vs. 31.3% BW), and fasting blood glucose (184 vs. 222 mg/dL), in diet-induced obese C57BL/6j mice consuming ANC beverage vs. water, (p < 0.05). There were some adverse effects noted at the highest dose, warranting future studies into the optimal dose of phenolic compounds extracted and concentrated from the alcohol-free fermented blueberry-blackberry beverage. These results, as summarized in Chapter 11, suggest that ANC in blueberry and blackberry fermented beverages are beneficial sources of antioxidants, decrease glucose absorption, and inhibit inflammation and DPP-IV, and therefore could be used in the management of type-2 diabetes