Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NFkappaB and AP-1 activity

Abstract

Dietary freeze-dried black raspberries inhibit tumor induction by N-nitrosomethylbenzylamine in the rat esophagus, but the constituents responsible for this chemopreventive activity have not been identified. We fractionated freeze-dried black raspberries and used mouse epidermal JB6 Cl 41 cells stably transfected with either a nuclear factor kappa B (NFkappaB)- or an activator protein 1 (AP-1)-luciferase reporter, and treated with racemic anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), to assess the inhibitory effects of the fractions. The ethanol and water extracts of the freeze-dried black raspberries had inhibitory activity and these extracts were fractionated by HPLC to give several bioactive fractions. Further HPLC analysis yielded multiple subfractions, some of which inhibited BPDE-induced NFkappaB activity. Major constituents of the most active subfractions were identified by their spectral properties and in comparison with standards as cyanidin-3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside) and cyanidin 3-O-rutinoside. Analysis of freeze-dried black raspberries indicated that these three components comprised approximately 3.4% of the material by dry weight. Consistent with these results, standard cyanidin-3-O-glucoside and cyanidin chloride were also good inhibitors of BPDE-induced NFkappaB activity. The results of this study demonstrate that cyanidin glycosides of freeze-dried black raspberries are bioactive compounds which could account for at least some of the chemopreventive activity observed in animal models.

Conflict of interest statement

Conflict of Interest Statement: None declared.

Figures

Fig. 1

Summary of fractionation scheme used to identify constituents of freeze-dried black raspberries which could decrease BPDE-induced NFκB or AP-1 activity. Percent figures represent percentage by weight of the freeze-dried black raspberries.

Fig. 2

Ability of extracts of freeze-dried black raspberries to decrease BPDE (2 μM)-induced NFκB (A) and AP-1 (B) activity. Data are expressed as activity relative to the solvent control (0.1% DMSO, used to dissolve the extracts). The positive control is BPDE (2 μM, first point on x-axis). Closed circles, pentane extract; open circles, methylene chloride extract; closed triangles, ethanol extract; open triangles, water extract. Each point is the mean ± SD of three determinations.

Fig. 3

Chromatograms obtained upon HPLC analysis of (A) the ethanol extract and (B) the water extract of freeze-dried black raspberries.

Fig. 4

Chromatogram obtained upon HPLC analysis of (A) fraction 5 of the ethanol extract of freeze-dried black raspberries (see Figure 1); (B) fraction 6 of the ethanol extract: (C) fraction 7A of the ethanol extract; and (D) fraction 6 of the water extract. Subfractions were tested for their ability to decrease BPDE-induced NFκB activity and the results are shown in the insets as activity relative to control (0.1% DMSO) used to dissolve the extracts. Each bar represents the mean ± SD of three determinations.

Fig. 5

Structures of cyanidins and summary of evidence for their identification in bioactive fractions of freeze-dried black raspberries. Spectroscopic data were consistent with previously reported data (28).

Fig. 6

UV spectra of bioactive constituents and standards: 1, subfraction M of ethanol fraction 5; 2, standard cyanidin-3-O-glucoside; 3, subfraction JJ of ethanol fraction 6; 4, subfraction K of ethanol fraction 6; 5, standard cyanidin 3-O-rutinoside.

Fig. 7

Chromatogram obtained upon HPLC analysis of an acidic methanol extract of freeze-dried black raspberries with UV detection at 280 nm.

Fig. 8

Ability of standard cyanidin-3-O-glucoside (open triangles) and cyanidin chloride (closed triangles) to decrease 2 μM BPDE-induced NFκB activity. The positive control is 2 μM BPDE with no cyanidin added (first point on x-axis). The cyanidins themselves, without BPDE, had no effect: cyanidin-3-O-glucoside (open circles) and cyanidin chloride (closed circles). Data are expressed as NFκB activity relative to control (0.1% DMSO) used to dissolve the extracts. Each point is the mean ± SD of three determinations.