1H nuclear magnetic resonance (NMR) based metabolomics on black raspberry (Rubus occidentalis L.) fruits and complex mixture analysis by multidimensional NMR and liquid chromatography-mass spectrometry (LC-MS) on a variety of berry fruits are focused to profile bioactive compounds that are effective in preventing the proliferation of cancer cells, notably with respect to colon and oral cancers.
Seeking to better understand health benefits from berry fruits, a NMR-based metabolomics model was initially developed to discern the relationships between constituents of black raspberries with respect to total monomeric-anthocyanin content (TMA), ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) using partial least square (PLS) regression analysis. The model allowed ascertaining active components and to analyze their contribution with regard to assay type and sample variation. Results showed that anthocyanins, cyanidin 3-O-rutinoside, cyanidin 3-O-(2(G))-xylosylrutinoside and cyanidin 3-O-glucoside were significant contributors to the variability in assay results. Many other statistically important bins from minor components were common among assay models indicating further investigation using more rigorous model systems.
HT-29 colon cancer cell line proliferation assay based metabolomics model was also developed to aid in and elaborate the initial model for discerning biological activities of minor nonanthocyanin components of black raspberries. In agreement with the initial model, cyanidin glycosides, particularly cyanidin 3-rutinoside and cyanidin 3-xylosylrutionoside, were shown to be the predominant components contributing to the bioactivity of black raspberries. In addition, this model allowed ascertaining biological activities of minor nonanthocyanin components. Among the nonanthocyanin components, citrate type moiety, salicylic acid glucosyl ester, m/p-hydroxybenzoic acid glucosyl ester, benzoic acid glucosyl ester, p-alkylphenyl glucoside, salicylic acid derivatives without attached sugar, quercetin 3-glucoside, quercetin 3-rutinoside, p-coumaric acid, epicatechin, trans-piceid, and methyl ellagic acid acetylpentose were found to be important contributors to the antiproliferative activity of black raspberries.
To unequivocally identify known and hitherto unknown compounds black raspberry non-anthocyanin phenolic compounds fractions were collected by semi-preparative HPLC then subjected to advanced 1H-NMR, 2D-NMR, HPLC-ESI-MS and MS/MS analysis. When results from the analysis of these fractions were combined with those from metabolomics, a variety of non-anthocyanin phenolic compounds were identified, including benzoic acids, hydroxybenzoic acids, hydroxycinnamic acids, quercetin, dihydrokaempferol, myricetin, ellagic acid, phloretin, resveratrols, terretonin and their glycosylated, acylated and methoxylated derivatives. These studies report the most complete analysis of black raspberry non-anthocyanin phenolics to date.
Finally LC-MS and detailed NMR analysis of Sambucus peruviana elderberry fractions were conducted to elucidate structures of major phenolic compounds of the berry. These techniques provided complete structures of cyanidin 3-glucoside, cyanidin 3-sambubioside and cyanidin 3-galactoside. In addition, the partial structure of an indole-3-lactic acid derivative was also solved.