Anthocyanins represent a class of natural pigments that produce an extensive array of colors in plant tissues, and have been associated with the ability to elicit health beneficial properties related to their antioxidant ability. There has been increased interest in expanding the use of anthocyanins as natural food colorants, in response to legislative action and negative consumer perception in association with synthetic dyes. Researchers in our lab recently developed a patent-pending, rapid, economical, and high throughput solid-phase anthocyanin extraction (SPE) method, utilizing the Oasis® MCX mixed-mode cation-exchange SPE cartridge. The new method outperformed currently used SPE techniques for anthocyanin isolation at bench top scale (single run), and may prove applicable for the commercial production of large volumes of highly pure anthocyanin mixtures to be used as natural colorants or for research purposes. The objective of this study was to explore the potential for commercial application of the patent-pending mixed-mode anthocyanin purification method. In order to achieve this goal the robustness of the method was assessed with previously examined and new pigment sources, its compatibility with other commercially available mixed-mode SPE resins was investigated, sorbent capacity was quantified, and cartridge longevity was analyzed. Anthocyanin isolate purity was also evaluated via molar absorptivity calculations, utilizing Lambert-Beer’s law.
Various quantities of several anthocyanin containing crude extracts were purified by the proposed technique in order to validate the method, and assess sorbent holding capacity. Sequential purification procedures (up to 20) were conducted on fresh and used cartridges with newly prepared, as well as frozen crude extracts, to assess cartridge longevity and commercial applicability. Purified anthocyanin extracts as well as phenolic constituents were analyzed via high-performance liquid chromatography (HPLC) coupled to a photodiode array (PDA) detector. UV-Visible spectrophotometry was conducted for the determination of total phenolics, total monomeric anthocyanins, and anthocyanin isolate purity, utilizing Folin-Ciocalteau (FC) colorimetry, the pH differential method, and molar absorptivity calculations, respectively.
The mixed-mode anthocyanin purification method showed robustness under the conditions used, producing extracts of extremely high purity (some > 99%, based on HPLC-PDA percent area under the curve) regardless of the cartridge utilized (P>0.05). Neither cartridge significantly (P>0.05) outperformed the other in terms of capacity (0.54mg anthocyanins/500mg sorbent) or longevity, indicating the compatibility of the method with different cation-exchange/reversed-phase sorbents, and showing promise for commercial application. Significantly lower (P<0.01) isolate purities were calculated by spectrophotometric analysis utilizing molar absorptivities, indicating the presence of other materials that do not absorb in the UV-visible range and cannot be detected by the PDA.
The proposed method allowed for the simple isolation of high-purity anthocyanin extracts. The increased availability of high-purity anthocyanin mixtures may promote their incorporation in food. The mixed-mode sorbents, regardless of manufacturer, showed the ability to contribute to numerous purifications while maintaining excellent isolation ability, further strengthening the potential for application on a commercial scale. A scale-up of the cartridge size may benefit the food colorant and nutraceutical industries, as well as the cartridge manufacturers, by promoting the production of high-purity anthocyanin mixtures.