Silke Hillebrand

Application of high-speed countercurrent chromatography to the large-scale isolation of anthocyanins

The paper reports the use of anthocyanins as natural food colorants and their potential health benefits regarding coronary heart disease and cancer prevention. The principles of countercurrent chromatography are described and several applications dealing with the isolation of anthocyanins from Tradescantia pallida leaves, purple corn, elderberry juice, red wine and blackberries are presented. Several hundred milligrams of pure anthocyanins were obtained within a single CCC run. Isolated pigments include monoglycosylated, acylated and highly glycosylated derivatives of anthocyanins. Purity and identity of the isolated anthocyanins were confirmed by HPLC with diode array detection, HPLC-electrospray ionization multiple mass spectrometry and nuclear magnetic resonance spectroscopy.

Anthocyanin Composition of Black Carrot (Daucus carota ssp. sativus var. atrorubens Alef.) Cultivars Antonina, Beta Sweet, Deep Purple, and Purple Haze

This study aimed to identify the pigment composition of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) cultivars Antonina, Beta Sweet, Deep Purple, and Purple Haze. Cyanidin 3-xylosyl(glucosyl)galactosides acylated with sinapic acid, ferulic acid, and coumaric acid were detected as major anthocyanins by high-performance liquid chromatography with diode array detection (HPLC-DAD) and with electrospray ionization multiple mass spectrometry (HPLC-ESI-MS(n)) analyses. The preparative isolation of these pigments was carried out by means of high-speed countercurrent chromatography (HSCCC). The color activity concept was applied to the isolated anthocyanins at three pH values. Cyanidin 3-xylosyl(sinapoylglucosyl)galactoside was found to exhibit a lower visual detection threshold and a higher pH stability than cyanidin 3-xylosyl(feruloylglucosyl)galactoside and cyanidin 3-xylosyl(coumaroylglucosyl)galactoside. The color parameters of the fresh roots of the four cultivars were described by the CIELab coordinates L* (lightness), C* (chroma), and h(ab) (hue angles). Total phenolics varied among the cultivars and ranged from 17.9 to 97.9 mg gallic acid equivalents (GAE)/100 g fresh weight (fw). For the content of monomeric anthocyanins, values between 1.5 and 17.7 mg/100 g fw were determined.

Microencapsulation by spray-drying of anthocyanin pigments from corozo (Bactris guineensis) fruit.

The anthocyanins of Bactris guineensis fruit were isolated with the aid of high-speed countercurrent chromatography (HSCCC) and preparative HPLC, and their chemical structures were elucidated by using spectroscopic methods. Among the identified pigments, cyanidin-3-rutinoside and cyanidin-3-glucoside were characterized as major constituents (87.9%). Peonidin-3-rutinoside, peonidin-3-glucoside, cyanidin-3-(6-O-malonyl)glucoside, and cyanidin-3-sambubioside were present in minor amounts. Four anthocyanin ethanolic extracts (AEEs) were obtained by osmotic dehydration and Soxhlet extraction and physicochemically characterized. The composition of anthocyanins was monitored by HPLC-PDA. The extracts with the highest anthocyanin content were subjected to the spray-drying process with maltodextrin. The so-obtained spray-dried powders were analyzed by scanning electron microscopy (SEM) and found to consist of spherical particles <50 microm in size. The anthocyanin composition was similar to that of the fruit. The microencapsulated powders were analyzed by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), revealing that they are quite stable until 100 degrees C. Storage stability tests of microcapsules showed that the release of anthocyanin pigments follows pseudo-first-order kinetics and that the process rate is increased by temperature and humidity. The most suitable conditions for storage were below 37 degrees C and <76% relative humidity, respectively.

Soluble and Bound Phenolic Compounds in Different Bolivian Purple Corn (Zea mays L.) Cultivars

In nine Bolivian purple corn ( Zea mays L.) varieties the content of phenolic compounds as well as the anthocyanin composition has been determined. The phenotypes under investigation included four red and five blue varieties (Kulli, Ayzuma, Paru, Tuimuru, Oke, Huaca Songo, Colorado, Huillcaparu, and Checchi). In purple corn, phenolic compounds were highly concentrated in cell walls. Thus, simultaneous determination of soluble and bound-form phenolics is essential for analysis, extraction, and quantification. The present study reports the determination of soluble and insoluble-bound fraction of phenolic compounds by HPLC-DAD and HPLC-ESI-MS(n) in Bolivian purple corn varieties. Enzymatic, thermal, and alkaline hydrolyses were used to obtain the cell wall-linked phenolic compounds. Ferulic acid values ranged from 132.9 to 298.4 mg/100 g, and p-coumaric acid contents varied between 251.8 and 607.5 mg/100 g dry weight (DW), respectively, and were identified as the main nonanthocyanin phenolics. The total content of phenolic compounds ranged from 311.0 to 817.6 mg gallic acid equivalents (GAE)/100 g DW, and the percentage contribution of bound to total phenolics varied from 62.1 to 86.6%. The total monomeric anthocyanin content ranged from 1.9 to 71.7 mg cyanidin-3-glucoside equivalents/100 g DW. Anthocyanin profiles are almost the same among the different samples. Differences are observed only in the relative percentage of each anthocyanin. Cyanidin-3-glucoside and its malonated derivative were detected as major anthocyanins. Several dimalonylated monoglucosides of cyanidin, peonidin, and pelargonidin were present as minor constituents.