Abstract:
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.
