286
Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
cosylanthocyanidin, ꢁ5:1). However, after 30 min spinosin had re-
acted completely, and no trace of the starting material could be de-
tected in the HPLC profile.
ker Ultrashield Plus AV-500 MHz instrument (Tables 1–4). All
experiments were recorded at 298 K unless otherwise noted.
Chemical shift values were set relative to the deuterio-methyl
13C signal and the residual 1H signal of the solvent; at d 49.0
and d 3.4 for CD3OD (containing CF3COOD), and at d 39.6 and d
2.49 for (CD3)2SO. NMR experiments of dissolved C-glycosylflav-
ones were recorded using S1 (5% CF3COOD in (CD3)2SO, v/v), S2
((CD3)2SO) or S3 (CD3OD), while C-glycosylanthocyanidins were
dissolved in S4 (5% CF3COOD in CD3OD, v/v), or S5 (20% CF3COOD
in (CD3)2SO, v/v).
3.3. Acid hydrolysis of anthocyanins
6,8-Di-C-b-glucosylapigeninidin, 8, and pelargonidin 3-O-b-glu-
coside (P1) (Nerdal et al, 1992) dissolved in MeOH were mixed
with aqueous HCl (2 M) (1:1, v/v) (Gao and Mazza, 1994). The mix-
ture was distributed into nine equal portions and subjected to
heating at 110 °C. After different time intervals: 0, 15, 30, 60, 90,
120, 180, 240 and 500 min, respectively, each sample was cooled
in an ice bath and monitored by HPLC (Fig. 3). In a similar way a
mixture of 9 and P1 were subjected to the same hydrolysis proce-
dure. The identity of pelargonidin (P2) was confirmed by its molec-
ular ion at m/z at 271.06 in the LC–MS spectra.
High-resolution LC–electrospray mass spectrometry (ESI+/TOF),
spectra were recorded using a JEOL AccuTOF JMS-T100LC in combi-
nation with an Agilent Technologies 1200 Series HPLC system. A
Zorbax SB-C18 (50 mm ꢀ 2.1 mm, length ꢀ i.d., 1.8
lm) column
was used for separation, and combinations of two solvents were
used for elution: A, H2O containing 0.5% TFA (v/v) and B, acetoni-
trile containing 0.5% TFA (v/v) (Table 5). The following solvent
composition was used: 0–1 min 5% B (isocratic), 1–3 min 5 to
13% B (linear gradient), 3–6 min 13% B (isocratic), 6–8 min 13 to
30% B (linear gradient), 8–14 min 30 to 40% B (linear gradient).
3.4. High performance liquid chromatography
Preparative HPLC (Gilson 305/306 pump equipped with an HP-
1040A detector) was performed using an Econosil C18 column
The flow rate was 0.4 ml minꢂ1
.
(250 mm ꢀ 22 mm; length ꢀ I.D., 10.0
lm), and combinations of
two solvents were used for elution: A, H2O–HCOOH (9:0.5, v/v)
and B, H2O–MeOH–HCOOH (4:5:0.5, v/v). See Rayyan et al.
(2005) for more experimental details.
Acknowledgements
This work is part of Project No. 157347/I20, which receives
financial support from the Norwegian Research Council of Norway.
The authors are grateful to Mr. Morten T. Olstad for assistance
regarding isolation of some of the flavones, as well as Mr. Terje Ly-
gre and Dr. Egil Nodland for technical support regarding recording
of LC–MS spectra.
Analytical HPLC was performed with an ODS-Hypersil column
(20 ꢀ 0.5 cm, length ꢀ i.d., 5
lm) using the solvents A, H2O con-
taining 0.5% TFA (v/v) and B, acetonitrile containing 0.5% TFA (v/
v). The following gradient was used: 10% B (isocratic) in 0–4 min,
10–40% B (linear gradient) from 4 to 21 min, 40% B (isocratic) from
21 to 28 min. The flow rate was 1.0 ml minꢂ1
.
References
3.5. Spectroscopy
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The NMR experiments (1 H, 1H–13
C C HSQC,
HMBC, 1H–13
1H–1H COSY, 1H–1H TOCSY, 1H–1H ROESY, 1H–1H NOESY and
CAPT were obtained at 600.13/500.13 and 150.90/125.76 MHz
for 1H and 13C, respectively, on a Bruker Biospin AV-600 MHz
instrument equipped with a TCI 1H–13C/15N CryoProbe and a Bru-