The measurement of antioxidant capacity in fruits differs from that of other biological samples due to their low pH and very low lipophilic antioxidant capacity. In this report, we present a modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method for fruits and compare its performance with the other commonly used antioxidant methods of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The antioxidant capacity and reaction kinetics of four phenolic compounds, two antioxidant standards, and five fruits were also investigated. The modified ABTS method prepared at a pH of 4.5 with sodium acetate buffer is highly stable and easily applied to fruit samples as compared to the standard (pH 7.4) version. The measured antioxidant capacity of samples varied with the assay method used, pH, and time of reaction. Traditional antioxidant standards (trolox, ascorbic acid) displayed stable, simple reaction kinetics, which allowed end point analysis with all of assays. Of the phenolic compounds examined, chlorogenic and caffeic acids exhibited the most complex reaction kinetics and reaction rates that precluded end point analysis while gallic acid and quercetin reached stable end points. All fruit extracts exhibited complex and varied kinetics and required long reaction times to approach an end point. Because the antioxidant capacity of fruit extracts is a function of the array of individual antioxidants present, accurate comparisons among fruit samples require that reaction times be standardized and of sufficient length to reach steady state conditions and that more than one assay be used to describe the total antioxidant activity of fruit samples.

Anthocyanin constituents in black raspberries (Rubus occidentalis L.) were investigated by HPLC-DAD, and their involvement as potent, significant antioxidants in black raspberries was demonstrated by three common antioxidant assays (FRAP, DPPH, ABTS) in this study. Five anthocyanins were present in black raspberries: cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside. Their identities and structures, with particular emphasis on cyanidin 3-xylosylrutinoside, were confirmed by NMR spectroscopy. Two of these anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, predominated, comprising 24-40 and 49-58%, respectively, of the total anthocyanins in black raspberries. On the basis of both potency and concentration, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were found to be the significant contributors to the antioxidant systems of black raspberries. These findings indicate that these two anthocyanin compounds may function as the primary phenolic antioxidants in black raspberries. These two compounds exhibit potential biological activities that may be exploited in conjunction with other naturally occurring bioactive compounds in black raspberry fruit-based products used in clinical trials for the treatment of various types of cancer.

Black raspberries (Rubus occidentalis L.) are rich in phytonutrients that have demonstrated chemoprotective properties against certain degenerative diseases. To estimate variability in phytonutritional quality among sources of black raspberry, 19 samples representing four common midwestern cultivars obtained from eight production sites were assayed for their antioxidant capacity [2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing ability of plasma (FRAP) methods], total phenolic content (TP), total monomeric anthocyanin levels (TMA), and levels of cyanidin 3-rutinoside. The antioxidant potential among samples averaged 2.92 ± 0.29 and 4.62 ± 0.88 mmol TE·100 g−1 fresh weight by the DPPH and FRAP methods, respectively; TP, TMA, and cyanidin 3-rutinoside means averaged 449 ± 62, 336 ± 109, and 244 ± 84 mg·100 g−1 fresh weight, respectively. Levels of FRAP, TP, TMA, and cyanidin 3-rutinoside were strongly correlated (r = +0.85 to +0.96). Mean antioxidant capacities and phenolic constituent levels were similar among ‘Bristol’, ‘Jewel’, and ‘MacBlack’ samples; values for a single sample of ‘Haut’ were lower but comparable to levels found in individual samples of the other three cultivars. Black raspberry production site differences were statistically significant for FRAP, TMA, cyanidin 3-rutinoside, and titratable acidity (TA) levels. Inverse relationships (r = –0.65 to –0.74) among black raspberry samples for FRAP, TMA, or cyanidin 3-rutinoside levels versus levels of TA suggested that site differences may be partially attributable to fruit ripeness at harvest. Relationships among these parameters versus regional differences in soil temperatures were also significant but weak. Regardless of its environmental or physiological drivers, point-source variation in fruit phytonutrient contents may be a relevant concern in health-related studies or clinical applications. Moreover, it may impact the nutritional benefits to the consumer and affect the quality advantages associated with direct-marketed black raspberries.

A process was developed to ascertain the bioactive components of black raspberry (Rubus occidentalis L.) fruit extracts by relating chemical constituents determined by high-field nuclear magnetic resonance (NMR) spectroscopy to biological responses using partial least-squares regression analysis. To validate our approach, we outlined relationships between phenolic signals in NMR spectra and chemical data for total monomeric anthocyanin (TMA) content and antioxidant capacity by the ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. Anthocyanins, cyanidin 3-O-rutinoside (Cy 3-rut), cyanidin 3-O-(2(G))-xylosylrutinoside (Cy 3-xylrut), and cyanidin 3-O-glucoside (Cy 3-glc), were significant contributors to the variability in assay results, with the two most important NMR bins corresponding to the methyl peaks in Cy 3-rut (6''') and/or Cy 3-xylrut (6(IV)). Many statistically important bins were common among assay models, but differences in structure-activity relationships resulted in changes in bin ranking. The specificity of these results supported the application of the process to investigate relationships among health-beneficial natural products and potential biological activity.

Polyphenolic-rich berry fruits are known to activate redox-sensitive cellular signaling molecules such as phosphatidylinositol-3-kinase (PI3 kinase)/kinase B (Akt), resulting in a cascade of downstream signaling pathways. This study investigated the ability of strawberry (SB), wild blueberry (WBB), and cranberry (CB) extracts to induce the activation of PI3 kinase/Akt signaling in vitro in human umbilical endothelial cells (HUVECs) and whether this activation would enhance cell migration and angiogenesis. Anthocyanin profiles of the extracts were characterized using HPLC-ESI/MS, and Akt activation was investigated using the Alpha Screen SureFire assay. The total anthocyanin contents of SB, WBB, and CB extracts were 81.7, 82.5, and 83.0 mg/100 g fresh weight, respectively. SB, WBB, and CB extracts activated Akt in a dose-dependent manner via PI3 kinase and induced cell migration and angiogenesis in vitro in HUVECs. The results from this study suggest that polyphenolics in berry fruits may play a role in promoting vascular health.