Characterization of low molecular weight chemical fractions of dry bean (Phaseolus vulgaris) for bioactivity using Caenorhabditis elegans longevity and metabolite fingerprinting

Abstract

Dry bean consumption has been reported to be associated with reduced risk for a number of chronic diseases including cancer. The extent to which these benefits are associated with primary versus secondary plant metabolites is not known. The work reported herein focuses on low molecular weight secondary metabolites and uses longevity extension of wild-type Caenorhabditis elegans nematodes as a surrogate marker for human health benefits. A modified Bligh and Dyer technique was used to extract freeze-dried bean, and the resulting fractions were evaluated for longevity extension and metabolite fingerprinting using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Dry bean extracts extended adult C. elegans lifespan by as much as 16%. Hydrophilic fractions increased lifespan, whereas the hydrophobic fraction induced longevity reduction. Metabolite fingerprinting revealed distinguishing spectral differences among the four chemical fractions evaluated and demonstrated that within each fraction chemical composition differed significantly based on dry bean genetic heritage.

Figure 1. Kaplan-Meier survival curve of C. elegans

exposed to methanol extracts of navy bean or white kidney bean or DMSO control solvent. A commercial mix of navy or white kidney beans was extracted with 65% methanol at neutral pH. C. elegans were treated with dry bean extract in liquid culture and supplemented with FUDR (5-Fluoro-2´-deoxyuridine) and OP50 E. coli.

Figure 2. Kaplan-Meier survival curves of C. elegans

exposed to (A) acidic, (B) neutral, (C) basic, and (D) chloroform extracts of navy bean (cv Seahawk), white kidney bean (cv Lassen), or control. Extracts were prepared using a modified Bligh and Dyer method. C. elegans treated with DMSO control solution or dry bean extract in liquid culture with FUDR (5-Fluoro-2´-deoxyuridine) and OP50 E. coli. (E) Feeding rates of C. elegans fed bean extracts used in survival analyses monitored on solid NGM-agar plates seeded with OP50 E. coli. Values are means ± SD.

Figure 3. Metabolite fingerprinting of dry bean market classes

Comparison of 65% methanol extracts of white kidney bean and navy bean metabolite profiles based on positive ionization mode. Percentage of variability explained by each component is indicated on axes. Each symbol represents an independent extraction and analysis of the same freeze dried material Ten replicate extractions were carried out. Two navy bean extracts were statistical outliers and were therefore excluded in PC analysis.

Figure 4. Variation between Bligh and Dyer extract fractions of dry bean as analyzed using LC-MS in positive ES mode

(A) Principal components analysis scores plots based on positive ionization mode for hydrophobic and hydrophilic extracts of white kidney and navy beans. (B) Dendrogram illustrating Euclidian distances resulting from agglomerative hierarchical cluster analysis using an average linkage clustering algorithm.

Figure 5. Comparison of features within hydrophobic and hydrophilic Bligh and Dyer dry bean extracts detected using LC-MS in positive ES mode

(A) Principal components analysis of acidic basic and neutral hydrophilic fractions of navy beans and white kidney beans. (B) Principal components analysis of hydrophobic features of navy beans and white kidney beans. Percentage of variability explained by each component is indicated on axes.

Figure 6. Classification of chromatographic features by mass

(A) Features in hydrophobic navy and white kidney bean extracts separated into 100 m/z increments. (B) Features in hydrophilic navy and white kidney bean extracts separated into 100 m/z increments.