Nutrition and Health Sciences, Department of


Date of this Version

Summer 5-26-2016


Alharbi, M. (2016). Characterization of extraction methods to recover phenolic-rich extracts from black beans that inhibit alpha-amylase and alpha-glucosidase using response surface approaches. (M.S. Thesis).


A Thesis Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Nutrition, Under the Supervision of Professor Vicki Schlegel. Lincoln, Nebraska: May, 2016

Copyright © 2016 Mazen O. Alharbi


Black beans contain high phenolic contents that are considered potent antioxidants. Relatively little is known about their ability to inhibit the carbohydrate-hydrolyzing enzymes α-amylase and α-glucosidase from releasing glucose from starch and/or disaccharides. The objective of this project was to determine the optimum procedures for extracting total phenols (TP), total flavonoids (TF) and total condensed tannins (TCT) from black beans (Phaseolus vulgaris), and then to determine the ability of the phenolic rich extracts to inhibit α-amylase and α-glucosidase. Due to their high phenolic levels, it was hypothesized that black beans would be an effective inhibitor of α-amylase and α-glucosidase, which would have potent health benefits for diabetics. The rationale for this project is that extracts obtained from black beans have the potential of inhibiting these key carbohydrate-hydrolyzing enzymes due to the high levels and different types of phenols present in black beans. The chemical diversity of the phenols extracts used affected the results, which in turn was affected by the extraction procedures. Therefore, extractions procedures were characterized using three different solvents and adjusting for solvent:water ratio, solid:solvent ratio and mixing time using a face centered cubic response surface design. This experimental design resulted in 17 samples per solvent system, each of which were tested for total phenols (TP), total flavonoids (TF) and total condensed tannins (TCT) followed by their ability to inhibit α-amylase and α-glucosidase. The optimized factors for TP (3.82 mg/g) consisted of acetone: water ratio of 25:75, a solid:solvent ratio of 18 percent and mixing time of approximately 111 minutes. The optimal factors for TF (3.61 mg/g) were a 25:75 acetone: water, 30 percent solid:solvent ratio and a mixing time of approximately 143 minutes. Optimum levels of TCT (15.58 mg/g) were achieved with an 25:75 acetone: water ratio, a 14 percent solid:solvent and 60 minutes of mixing. The acetone solvent system produced the highest inhibition of α-amylase (36.65 % inhibition /mg of extraction) and α-glucosidase (34.10 % inhibition /mg). It should be noted that although the inhibition occurred from different extracts but but both used acetone as the solvent system. Inhibition of α-amylase decreased by ~3 fold using both ethanol and methanol at 10.29 % 10.84 % inhibition /mg extraction, respectively. However, for α-glucosidase, the inhibition by an ethanol extract was slightly higher (16.68 % inhibition/mg) than that exerted by methanol (6.16 % inhibition /mg). Yet, α-glucosidase inhibition for the acetone demonstrated the highest correlations with TP, TF and TCT with R values as follows: R=97, R=96, and R=91, respectively, indicating that total phenolic levels present in any given extract were responsible for the inhibitory effect. In summary, the significance of this project is that black bean extracts are capable of inhibiting key carbohydrate hydrolyzing enzymes, but this property depends on the extract and thus the extraction procedure used.

Advisor: Vicki Schlegel