Red radish (Raphanus sativus L.) anthocyanins were extracted from liquid nitrogen powdered epidermal tissue using acetone and chloroform and isolated using C-18 resin. Chemical structure and conformation of major pigments were elucidated by 1-D and 2-D NMR, Mass Spectroscopy, HPLC, and spectral analyses. Two novel di-acylated anthocyanins, pelargonidin 3-0-[2-0-(β-D-glucopyranosyl)-(6-0- trans-p-coumaroyl- β-D-glucopyranoside)]-5-0-(6-0-malonyl-β-D-glucopyranoside)and pelargonidin 3-0-[2-0-(β-D-glucopyranosyl)-(6-0-trans-feruloyl-β-D-glucopyranoside)]- 5-0-(6-0-malonyl-β-D-glucopyranoside) represented 70% of total pigment. Two mono-acylated anthocyanins (20%) were pelargonidin 3-0-[2-0-(β-D-glucopyranosyl)-(6-0-trans-p-coumaroyl-β-D-glucopyranoside)]-5-0-(β-D-glucopyranoside) and pelargonidin 3-0-[2-0-(β-D-glucopyranosyl)-(6-0-trans- feruloyl-β-D-glucopyranoside)]-5-0-(β-D-glucopyranoside). NOESY revealed folding of the molecule. Electrospray (ES-MS) and tandem mass spectroscopy (MS-MS) were tested as tools for anthocyanin characterization. Anthocyanins were semi-purified using C-18 resin, washed with acidified water and ethyl acetate, and recovered with acidified methanol. Samples were injected into a mass spectrometer in aqueous or methanolic solutions. Charged character of anthocyanins favored the fast and effective detection of intact molecular ions requiring minimal sample preparation and with little interference. MS-MS provided clear and characteristic fragmentation patterns. Qualitative and quantitative anthocyanin pigment content of radish cultivars grown at 2 locations (Corvallis and Hermiston, OR) and harvested at 2 maturity stages, were evaluated. Pigment content depended on cultivar, root weight and location, with higher amounts obtained at Hermiston. Spring cultivars (n=22) had pigmentation in the skin, ranging from 39.3 to 185 mg anthocyanin/100g skin. Red-fleshed Winter cultivars (n=5) possessed from 12.2 to 53 mg anthocyanin/100g root. The major pigments were pelargonidin-3-sophoroside-5-glucoside, mono- or di-acylated with cinnamic and malonic acids; individual proportions varied among cultivars. Estimated pigment yields ranged from 1.3 to 15.8 kg/ha. Acylated and non-aeylated pelargonidin were isolated using semi-preparative HPLC to evaluate the effect of glycosylation and acylation in spectral characteristics, molar absorptivity and color. Molar absorptivity ranged from 15,600 to 39,590 for pelargonidin-3-glucoside and pg-3-rutinoside-5-glucoside acylated with p-coumaric acid, respectively. An hypsochromic shift on λmax was observed in presence of glycosylation. Pelargonidin-3,5-diglucoside and 3,5-triglucoside showed yellow-orange hue angle (>40°) in pH 1.0 buffer, higher than other pg-derivatives. Cinnamic acid acylation caused a bathochromic shift. Malonic acid acylation had little effect on color and no effect on λmax. The solvent system affected molar absorptivity and visual color characteristic of the pigments. Acylation increased pigment resistance to acid hydrolysis. / Graduation date: 1999

A method for separation and characterization of individual anthocyanins was developed. High Performance Liquid Chromatography (HPLC) with a polymer based reversed-phase column was used to separate the pigments while on-line Photodiode Array Detection (PDA) was employed to record the UV and Visible spectrum of the individual peaks. Spectral information obtained from on-line PDA detection provided information about: 1) the nature of the aglycone, 2) the sugar substitution pattern and 3) the presence or absence of hydroxy aromatic organic acids. The nature of the glycosidation can be determined from the HPLC retention characteristics. The HPLC/PDA methods were employed to characterize the anthocyanin profiles of the pigments in cranberry, roselle, cherry, bilberry, grape, red cabbage, black raspberry, blackberry, elderberry, plum, blackcurrant and strawberry. The anthocyanidin profiles were also determined for the samples for purposes of confirmation of the anthocyanin data. In addition to the anthocyanin and anthocyanidin profiles, the general coloring properties for most of the samples were also determined. Included were Hunter L, a, b values, total anthocyanin pigment concentration, wavelength maxima, percent tannin measurements, tinctoral strength, pH measurements and titratable acidity. / Graduation date: 1988

The behaviour of pigments in red wine, namely anthocyanins and anthocyaninderived pigments, was investigated at natural wine pH, at low pH and after addition of SO2, namely SO2 bleaching. An examination of current literature demonstrated absences in wine pigment research. Firstly, few researchers have published the colour properties of a particular wine pigment at different pH values and post-SO2 bleaching. This was demonstrated using the CIELab colours of two individual anthocyanin-derived wine pigments (4-vinylcatechol and 4-vinylsyringol adducts to malvidin 3-glucoside), and an anthocyanin, malvidin 3-glucoside. The colours of the anthocyanin-derived pigments and their resistance to pH change and SO2 bleaching were compared to malvidin 3-glucoside which was affected by media. Generally, in the literature, wine pigments are characterized as individual components. But many pigments contribute to wine colour. So, two novel methods were created and demonstrated using red wines: Shiraz wines from four regions in Australia, and Cabernet Sauvignon wines made using two different strains, Saccharomyces cerevisiae (SC) or Saccharomyces bayanus (SB). The first method can be used to determine the CIELab colour of chromatographically separated wine pigments and allows their colours to be re-created, regardless of their identity. Thus objective measurement of pigment colour at its natural concentration in wine is now possible. An additional method, the “post-column adjustment method” to pH-adjust and SO2 bleach HPLC-separated wine pigments was created. The concentration and colour of HPLC-separated wine pigments at low pH, at wine pH and post-SO2 bleaching can be measured. The method has highlighted the importance of the pH value when quantifying a wine pigment. For example, from low pH to wine pH, the apparent anthocyanin and pigmented polymer concentration was reduced, but the Vitisin A concentration was unchanged. SO2 bleaching resulted in negligible anthocyanin concentration and a further reduction in pigmented polymer concentration, with Vitisin A unaffected. Relative quantities of wine pigments in both SC and SB Cabernet Sauvignon wines were not affected by pH change or SO2 bleaching. Also, using the Shiraz wines and Cabernet Sauvignon red wines, existing and improved colour measurement techniques were discussed. For the Australian Shiraz wines, grape origin was found to influence red wine colour, CIELab values provided enhanced colour measurements, and high wine colour (at natural wine pH) cannot be attributed to individual monomeric anthocyanins (measured by HPLC analysis at low pH). Vitisin A was not responsible for differences in wine colour. SO2-stable wine colour was related to regional differences. The percentage of SO2 non-bleachable pigments was independent of wine region. Chemical index (ii) values indicated that the colour at 520 nm was attributable to pH-dependent wine pigments. Vitisin A and pigmented polymer concentrations correlated well with SO2-stable wine colour. Pigmented polymer concentration may be the driving force behind wine colour density. Copigmentation was of no importance in the young red wine samples studied. With the Cabernet Sauvignon red wines, the yeast strains used for fermentation affected wine colour and SO2-stable wine colour. The change in wine colour density was not related to change in total red pigment colour or anthocyanin concentration. Pigmented polymer concentration, SO2-stable wine colour and the percentage of SO2 non-bleachable pigments were consistently higher in the SB wines. The pH value was important when determining the colour of a wine or pigment. At low pH, the SC wines were more coloured than the SB wines. However, at real wine pH, the converse was true. For both wines, at low pH, the anthocyanin concentration was greater than the pigmented polymer concentration, indicating the importance of anthocyanins to wine colour only at low pH. But, at wine pH, the apparent anthocyanin concentration was much lower in both wines (for example, malvidin 3- glucoside provided more colour at low pH than at wine pH) than the apparent pigmented polymer concentration. Therefore, at wine pH, anthocyanins were less important to wine colour than pigmented polymers. The concentrations of Vitisin A were similar in all three media, but colour losses were observed at wine pH and post-SO2 bleaching. SB Vitisin A was more coloured. At low pH and at wine pH, Vitisin A was more coloured than malvidin 3-glucoside in both wines, even though the apparent Vitisin A concentration was lower. Differences in the colours of the SC and SB pigmented polymers peaks were observed at low pH, at wine pH and following SO2 bleaching. The SB pigmented polymers were darker and more colourful, exhibited more colour absorbance and a slight bathochromic shift of lmax value. From low pH to wine pH and following SO2 bleaching, pigmented polymers become lighter, whilst retaining orange-red hues. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277727 / Thesis (Ph.D.) -- School of Agriculture, Food and Wine, 2007

Copies of author's previously published works inserted. Addendum enclosed in pocket on back end paper. Bibliography: leaves 192-221.

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 210-219). Also available for download via the World Wide Web; free to University of Oregon users.

Copies of author's previously published works inserted. / Addendum enclosed in pocket on back end paper. / Bibliography: leaves 192-221. / x, 221 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1998?

Includes bibliographical references (leaves 218-227). / xviii, 227 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study concerns the structures, equilibrium distributions and formation of pigments found in red wine... A new method was developed for the isolation of C4-substituted pigments from the wine and grape marc extracts which was based on the reactivity of anthocyanins with bisulphite ion to form anionic bisulphite addition products. / Thesis (Ph.D.(Ag.Sc.))--Adelaide University, Dept. of Horticulture, Viticulture and Oenology, 2001

Thesis (Ph. D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1998? / Copies of author's previously published works inserted. Addendum enclosed in pocket on back end paper. Includes bibliographical references (leaves 192-221).

Thesis (Ph. D.)--Oregon State University, 1992. / Includes mounted photogrpahs. Typescript (photocopy). Includes bibliographical references (leaves 110-125). Also available online.

Thesis (M.S.) in Food Science and Human Nutrition--University of Maine, 2007. / Includes vita. Includes bibliographical references (leaves 69-74).