C-glycosylanthocyanidins synthesized from C-glycosylflavones

Article abstract of DOI:10.1016/j.phytochem.2008.12.012

Nine C-glycosyldeoxyanthocyanidins, 6-C-β-glucopyranosyl-7-O-methylapigeninidin, 6-C-β-glucopyranosyl-7-O-methylluteolinidin, 6-C-β-(2″-O-β-glucopyranosylglucopyranosyl)-7-O-methylap igeninidin, 6-C-β-(2″-O-β-glucopyranosylglucopyranosyl)-7,4′-d i-O-methy

Full text of DOI:10.1016/j.phytochem.2008.12.012

Phytochemistry 70 (2009) 278–287
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
C-glycosylanthocyanidins synthesized from C-glycosylflavones
*
Ørjan Bjorøy, Saleh Rayyan, Torgils Fossen, Kjersti Kalberg, Øyvind M. Andersen
Department of Chemistry, University of Bergen, Allégt. 41, N-5007 Bergen, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 August 2008
Received in revised form 14 November 2008
Available online 29 January 2009
Nine C-glycosyldeoxyanthocyanidins, 6-C-b-glucopyranosyl-7-O-methylapigeninidin, 6-C-b-glucopyran-
osyl-7-O-methylluteolinidin, 6-C-b-(200-O-b-glucopyranosylglucopyranosyl)-7-O-methylapigeninidin, 6-
C-b-(200-O-b-glucopyranosylglucopyranosyl)-7,40-di-O-methylapigeninidin, 8-C-b-glucopyranosylapige-
ninidin,
8-C-b-(200-O-
a
-rhamnopyranosylglucopyranosyl)apigeninidin,
8-C-b-(200-O- -(4000-O-acetyl-
a
rhamnopyranosyl)glucopyranosyl)apigeninidin, 6,8-di-C-b-glucopyranosylapigeninidin (8), 6,8-di-C-b-
glucopyranosyl-40-O-methylluteolinidin (9), have been synthesized from their respective C-glycosylflav-
ones (yields between 14% and 32%) by the Clemmensen reduction reaction using zinc-amalgam. The var-
ious precursors (C-glycosylflavones) of the C-glycosylanthocyanidins were isolated from either flowers of
Iris sibirica L., leaves of Hawthorn ‘Crataegi Folium Cum Flore’, or lemons and oranges. This is the first time
C-glycosylanthocyanidins have been synthesized. The structures of all flavonoids including the flavone
rotamers were elucidated by 2D NMR techniques and high-resolution electrospray MS. The distribution
of the various structural forms of 8 and 9 are different at pH 1.1, 4.5, and 7.0, however, the two pigments
undergoes similar structural transformations at the various pH values. Pigments 8 and 9 with C–C link-
ages between the sugar moieties and the aglycone, were found to be far more stable towards acid hydro-
lysis than pelargonidin 3-O-glucoside, which has the typical anthocyanidin C–O linkage between the
sugar and aglycone. This stability may extend the present use of anthocyanins as nutraceuticals, pharma-
ceuticals or colorants.
Keywords:
6-C-glycosyl-3-deoxyanthocyanidins
8-C-glycosyl-3-deoxyanthocyanidins
6,8-Di-C-glycosyl-3-deoxyanthocyanidins
C-glycosylflavones
Rotamers
Clemmensen reduction
Stability
pH effects
2D NMR
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ocyanidins (aglycones), which are significantly more unstable than
the intact anthocyanins. Loss of sugar units may also limit in vivo
The anthocyanins produce most red to blue colours in plants,
and nearly 600 natural anthocyanins have hitherto been reported
(Andersen and Jordheim, 2006). In recent decades there have been
considerable interests in the development of new anthocyanin-
based food colorants (Francis, 1989), including the use of deoxyan-
thocyanins (Awika et al., 2004). The motive of extended use is
increasing with their potential positive health effects (Clifford
and Brown, 2006), including for instance antioxidant activity
(Zheng and Wang, 2003; Prior and Wu, 2006), anticarcinogenic ef-
fects (Hou, 2003; Dreiseitel et al., 2008) and anti-viral activity
(Andersen et al., 1997; Knox et al., 2001). Their stabilities are af-
fected by a number of factors including temperature, light, oxygen,
enzymes, etc. (Francis, 1989; Matsufuji et al., 2007). A particular
problem is the pH influence on the anthocyanin properties; the
stability of most anthocyanins is particularly low under weakly
acidic and neutral conditions (Brouillard and Dangles, 1994; Cabri-
ta et al., 2000; Torskangerpoll and Andersen, 2004). In nearly all
previously reported natural anthocyanins the sugar moieties are
connected to the anthocyanidins through an O-linkage. When in-
gested the anthocyanidin O-glycoside may be hydrolysed to anth-
transportation.
The conversion of flavonoids to the corresponding anthocyanins
by the Clemmensen reduction using zinc-amalgam has previously
been performed on a limited number of flavonols (Iacobucci and
Sweeny, 1983). Elhabiri et al. have synthesized cyanidin and cyani-
din 3-rutinoside by reduction of the corresponding flavonols, quer-
cetin (1995a) and quercetin 3-rutinoside (1995b), respectively, and
converted synthetic 7,30,40-tri-O-methylquercetin 3-O-b-
D-gluco-
-glucoside to the corre-
side and 5,30-di-O-methylquercetin 3-O-b-
D
sponding anthocyanins (1995b). Recently, Oyama et al. (2007)
synthesized pelargonidin 3-O-(600-acetylglucoside) from the analo-
gous synthetic kaempferol-glucoside. Some flavanones and flavone
aglycones have been reduced with sodium-amalgam to their corre-
sponding benzopyrylium compounds (Asahina et al., 1929). The
flavone O-glucoside, apiin, and some flavanone O-glycosides like-
wise gave on reduction substances forming red to violet-red dyes.
However these latter products could not be isolated in pure form
(Asahina et al., 1929). It has also been reported that when the fla-
vone O-glucoside, toringin, was reduced using magnesium, the
acidic alcoholic solution turned pink (Shibata et al., 1919).
Although C-glycosylflavones are relatively common in nature
(Jay et al., 2006), only two C-glycosylanthocyanidins, 8-C-b-glucos-
ylcyanidin 3-O-b-(600-O-malonylglucoside) and 8-C-b-(6000-O-trans-
* Corresponding author. Tel.: +47 55 583 460; fax: +47 55 589 490.
E-mail address: oyvind.andersen@kj.uib.no (Ø.M. Andersen).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.12.012

Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
279
sinapoylglucosyl)cyanidin 3-O-b-(600-O-malonylglucoside), have
previously been reported (Saito et al., 2003; Tatsuzawa et al.,
2004). These anthocyanins were together with four common
cyanidin O-glycoside isolated from flowers of the toad lily (Tricyrtis
formosana). No C-glycosylanthocyanidin has previously been syn-
thesized, and C-glycosylflavones have as far as we know not been
used before as starting material for production of anthocyanins
in reductive synthesis.
In this paper we have extended the use of zinc-amalgam in the
Clemmensen reduction reaction to include the synthesis of nine C-
glycosylanthocyanidins (1–9) from their corresponding C-glyco-
sylflavones (F1–F9), which were isolated from various natural
sources. Flavones are lacking the 3-hydroxyl group, and pigments
1–9 synthesized from their precursors (F1–F9) are thus 3-deoxyan-
thocyanins. The reason for preparing the C-glycosyl-3-deoxyanth-
ocyanidins is founded on two characteristics, which we wanted
to combine in the new pigments; The relatively high colour stabil-
ity of 3-deoxyanthocyanidins compared with their anthocyanidin
analogues (Sweeny and Iacobucci, 1983), and the high stability of
C-glycosylflavonoids towards acid hydrolysis, relative to flavonoid
O-glycosides (Markham, 1982).
intermediates, to their corresponding 5,6-dihydroxyflavylium salts
under mildly acidic conditions. As verified by the HPLC profiles de-
tected at 475 nm ( 20 nm), no significant 6/8 isomerisation (1–7),
demethylation (1–4, 9), or loss of any acetyl moiety (7) nor termi-
nal O-glycoside (3–4, 6–7) occurred during the reduction reaction.
However the HPLC profiles detected at 280 nm ( 10 nm) revealed
that the reduction of the C-glycosylflavones gave several unidenti-
fied aromatic compounds in addition to the C-glycosyldeoxyanth-
ocyanidin pigments. The structures of 1–9 were elucidated by
means of NMR (Tables 3 and 4), on-line UV–vis spectroscopy (Table
5), and high-resolution electrospray mass spectrometry (HR-ESMS)
(Table 5), in a similar way as described in the next paragraph for 1.
C-glycosyl-3-deoxyanthocyanidins have previously not been made
or isolated from any source.
The anthocyanin yield of the Clemmensen reduction of 6-C-glu-
cosyl-7-O-methylapigenin (swertisin, F1) isolated from iris (Kaw-
ase, 1968; Asen et al., 1970) was 20% (Table 5). The aromatic
1
region of the H NMR spectrum of 1 revealed an AA0XX0 system
at d 8.48 (H-20,60) and d 7.18 (H-30,50), and 3 protons at d 9.26
(dd, 8.9 and 0.9 Hz, H-4), d 8.30 (d, 8.9 Hz, H-3) and d 7.47 (d,
0.9 Hz, H-8), respectively, corresponding to a 3-deoxyanthocyani-
din with a symmetrical B-ring (Table 3). A 3H singlet at d 4.21
(OMe) belonging to the aglycone was confirmed to be at the 7-po-
sition by the crosspeak at d 4.21/169.7 (OMe/C-7) in the long-range
1H-13C HMBC spectrum, in accordance with the deoxyanthocyani-
din 7-O-methyl-apigeninidin. All the sugar proton resonances were
assigned by the 2D 1H–1H DQF-COSY experiment, and the corre-
sponding 13C resonances (Table 4) were then identified by the 2D
1H–13C HSQC and 1D 13C CAPT experiments. The anomeric shift va-
lue d 5.16 (H-100) with a 3JHH = 9.8 Hz, together with the six 13C res-
onances between 61 ppm and 83 ppm were in accordance with a
b-glucopyranosyl attached to the aglycone by a C–C bond. The
crosspeaks at d 5.16/114.8 (H-100/C-6), d 5.16/169.7 (H-100/C-7)
and d 5.16/156.7 (H-100/C-5), in the HMBC spectrum of 1, confirmed
2. Results and discussion
Nine C-glycosyl-3-deoxyanthocyanidins (1–9) (Fig. 1) were
independently synthesized from their respective C-glycosylflavo-
nes, F1–F9, by the Clemmensen reduction reaction using zinc-
amalgam. Analysis of the reaction products by HPLC proved that
the reduction of F1–F7 gave the corresponding C-glycosyldeoxy-
anthocyanidins, 1–7, which individually during the purification
steps provided a mixture of the 6-C-glycosyldeoxyanthocyanidin
and the 8-C-glycosyldeoxyanthocyanidin. The mechanism for this
isomerisation may be similar to that given by Jurd (1962), where
5,8-dihydroxyflavylium salts isomerised, probably via chalcone
Fig. 1. Structures of the C-glycosyl-3-deoxyanthocyanidins obtained by Clemmensen reduction of corresponding C-glycosylflavones. 1: 6-C-b-glucopyranosyl-7-O-
methylapigeninidin, 2: 6-C-b-glucopyranosyl-7-O-methylluteolinidin, 3: 6-C-b-(200-O-b-glucopyranosylglucopyranosyl)-7-O-methylapigeninidin, 4: 6-C-b-(200-O-b-glucopyr-
anosylglucopyranosyl)-7,40-di-O-methylapigeninidin, 5: 8-C-b-glucopyranosylapigeninidin, 6: 8-C-b-(200-O- -rhamnopyranosylglucopyranosyl)apigeninidin, 7: 8-C-b-(200-O-
a
a
-(4000-O-acetylrhamnopyranosyl)glucopyranosyl)apigeninidin, 8: 6,8-di-C-b-glucopyranosylapigeninidin, 9: 6,8-di-C-b-glucopyranosyl-40-O-methylluteolinidin, F1: 6-C-
glucosyl-7-O-methylapigenin (swertisin), F2: 6-C-glucosyl-7-O-methylluteolin (swertiajaponin), F3: 6-C-sophorosyl-7-O-methylapigenin (flavoayamenin, spinosin), F4: 6-C-
sophorosyl-7,40-di-O-methylapigenin (embinoidin), F5: 8-C-glucosylapigenin (vitexin), F6: 8-C-neohesperidosylapigenin (200-O-rhamnosylvitexin), F7: 8-C-(4000-acetylneohe-
speridosyl)apigenin (4000-O-acetyl-200-O-rhamnosylvitexin), F8: 6,8-di-C-glucosylapigenin (vicenin), F9: 6,8-di-C-b-glucopyranosyl-40-O-methylluteolin (6,8-di-C-
glucosyldiosmetin).

280
Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
Table 1
1H NMR data for the C-glycosylflavones, F1–F9, dissolved in either S1 (5% CF3COOD in (CD3)2SO, v/v), S2 ((CD3)2SO) or S3 (CD3OD), at 25 °C. See Fig. 1 for structures.
F1 (S1)
F2 (S1)
F3 (S2)
F4 (S1)
F5 (S2)*
F6 (S3)*
F7 (S3)*
F8 (S1)
F9 (S1)
3
6
8
20
6.81 s
6.83 s
–
6.70 s
6.72 s
–
6.84 s
6.86 s
–
6.93 s
6.92 s
–
6.77 s
6.79 s
6.26 s
6.24 s
–
6.66 s
6.67 s
6.36 s
6.35 s
–
6.68 s
6.72 s
6.39 s
6.34 s
–
6.81 s
6.80 s
–
6.77 s
6.79 s
–
6.81 s
6.80 s
6.76 s
6.75 s
7.45 d 2.1
7.45 d 2.1
6.80 s
6.77 s
6.79 s
6.83 s
8.07 0d0
9.1
–
–
7.95 0d0 8.8
7.95 0d0 8.8
7.97 0d0 8.7
7.97 0d0 8.7
8.01 0d0 8.6
7.91 0d0 8.4
8.04 0d0 8.7
7.89 0d0 8.7
8.07 0d0 9.0
7.89 0d0 9.0
8.02 0d0 8.7
7.94 0d0 8.7
7.51 d 2.3
7.44 d 2.3
8.06 0d0
9.1
30
50
60
6.92 0d0 8.9
6.92 0d0 8.9
–
6.93 0d0 8.7
6.93 0d0 8.7
7.12 0d0
9.1
6.88 0d0 8.6
6.94 0d0 8.4
7.01 0d0 8.7
7.02 0d0 8.7
7.02 0d0 9.0
7.00 0d0 9.0
6.89 0d0 8.7
6.93 0d0 8.7
–
7.11 0d0
9.1
6.92 0d0 8.9
6.92 0d0 8.9
6.90 d 9.1
6.90 d 9.1
6.93 0d0 8.7
6.93 0d0 8.7
7.12 0d0
9.1
6.88 0d0 8.6
6.94 0d0 8.4
7.01 0d0 8.7
7.02 0d0 8.7
7.02 0d0 9.0
7.00 0d0 9.0
6.89 0d0 8.7
6.93 0d0 8.7
7.05 d 8.6
7.10 d 8.6
7.11 0d0
9.1
7.95 0d0 8.8
7.95 0d0 8.8
7.44 dd 9.1,
2.1
7.44 dd 9.1,
2.1
7.97 0d0 8.7
7.97 0d0 8.7
8.07 0d0
9.1
8.01 0d0 8.6
7.91 0d0 8.4
8.04 0d0 8.7
7.89 0d0 8.7
8.07 0d0 9.0
7.89 0d0 9.0
8.02 0d0 8.7
7.94 0d0 8.7
7.66 dd 8.6, 2.3
7.58 dd 8.6, 2.3
8.06 0d0
9.1
7-MeO
40-MeO
3.88 s
3.85 s
3.88 s
3.86 s
3.89 s
3.89 s
3.91 s
3.90 s
3.85 s
3.85 s
3.88 s
3.86 s
6-C-Glc
4.58 d 9.8
4.60 d 9.7
6-C-Glc
4.59 d 9.9
4.61 d 9.8
6-C-Glc
4.69 d 9.9
4.67 d 9.9
6-C-Glc
4.70 d
10.1
4.68 d
10.1
4.30 dd 10.1
8.9
4.47 dd 10.1
8.9
3.44 t 8.9
3.42 t 8.9
8-C-Glc
4.67 d 9.8
4.82 d 9.8
8-C-Glc
5.12 d 9.9
5.21 d 9.9
8-C-Glc
5.13 d 9.9
5.24 d 9.8
6-C-Glc
4.80 d 9.7
4.66 d 9.8
6-C-Glc
4.79 d 9.8
4.66 d 9.8
100
200
300
4.18 dd 9.8, 8.8
3.99 dd 9.7, 8.8
4.19 t br 9.2
4.00 t br 9.3
4.30 d 9.5
4.47 d 9.5
3.82 t 9.6
3.86 m
4.34 dd 9.9, 8.6
4.32 t 9.0
4.31 dd 9.9, 8.6
3.49 t br 9.3
3.98
3.49 t br 9.2
3.48
4.18 dd 9.8
8.9
3.77 dd 9.2
8.6
3.17 t 8.8
3.20 t 8.8
3.19 m
3.21 m
3.43 m
3.41 m
3.24 m
3.31 m
3.74 m
3.78 m
3.30 t 9.1
3.21
3.29 t 9.0
3.21
3.84 t 8.9
3.72 t 9.2
3.65 m
3.57 dd br 9.2
5.9
400
500
3.09 m
3.07 m
3.14 m
3.16 m
3.11 m
3.08 m
3.14 m
3.16 m
3.13 m
3.15 m
3.17 m
3.16 m
3.14 m
3.16 m
3.16 m
3.18 m
3.37 t 9.4
3.24 m
3.21 m
3.34 m
3.74 m
3.78 m
3.55 m
3.64 m
3.37 t 9.1
3.33
3.33 m
3.45
3.36 t 9.0
3.32 m
3.43
3.67 m
4.09 dd 12.3
1.4
4.04 dd 12.3
1.4
3.91 m
600A
600B
3.69 m
3.69 m
3.69 m
3.69 m
3.68 m
3.68 m
3.69 m
3.69 m
3.75 dd 11.6,
1.4
3.68 m
4.06 m
4.03 m
3.63 m
3.62 m
3.68
3.69
3.36 m
3.36 m
3.37 dd 11.8
6.0
3.37 m
3.37 m
3.38 dd 12.0
6.5
3.51 dd 11.6
6.7
3.89 m
3.88 m
3.63 m
3.62 m
3.91 m
3.68
3.69
3.37 dd 11.8
6.0
3.38 dd 12.0
6.5
3.48 m
200-O-Glc
4.17 d 8.0
4.15 d 8.0
2.84 m
200-O-Glc
4.19 d 7.9
4.16 d 7.9
2.85 m
200-O-Rha
5.19 d 1.8
5.29 d 1.8
3.94 dd 3.2, 1.8
3.88 dd 3.2, 1.8
200-O-Rha
5.39 d 1.8
5.50 d 1.7
3.91 m
3.80 dd 3.3
1.7
8-C-Glc
8-C-Glc
1000
2000
4.75 d 9.9
5.00 d 9.6
3.88 t br 9.3
3.56
4.74 d 9.8
4.98 d 10.0
3.86 t br 9.3
3.86
2.84 m
2.84 m
3000
4000
5000
3.06 m
3.05 m
3.07 t 9.0
3.04 t 8.9
3.49 dd 9.5, 3.2
3.16 m
3.57 dd 9.8
3.3
3.10 dd 9.8
3.3
4.70 t 9.8
4.63 t 9.8
3.27 t 9.1
3.39
3.27 m
3.40
2.96 m
3.01 m
2.95 t br
9.3
3.00 t br
9.1
2.77 dt 9.6, 3.5
2.56 ddd 9.3
3.4, 2.7
3.21 t 9.5
3.16 m
3.39 t 9.1
3.16
3.39 t 9.2
3.15
2.75 m
2.56 m
2.53 dd 9.5, 6.3
2.40 dd 6.3, 9.5
2.41 m
2.17 m
3.23 m
3.18
3.25 m
3.17
6000
6000
A
B
3.18 m
3.16 m
3.20 d 3.5
3.16 m
0.73 d 6.3
0.87 d 6.3
0.77 d 6.3
0.60 d 6.3
3.75 dd 12.0
1.8
3.69
3.50 dd 12.0
5.8
3.79 dd 12.0, 1.8
3.69
3.18 m
2.94 m
3.20 d 3.5
2.96 m
3.54 dd 12.0, 6.5
3.42
3.42
4
000-Ac
20000
2.11 s
2.02 s
Chemical shifts and coupling constants are in ppm and Hz, respectively. The two signals given for most positions correspond to two rotamers: major (top) and minor
(bottom).
*
Rayyan et al. (2005). s = Singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet, br = broad, Glc = glucoside, Rha = rhamnoside, Ac = acetyl.

Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
281
the C-C linkage between the sugar and the aglycone at the 6-posi-
tion. The HR-ESMS spectrum of 1 exhibited a [M]+ ion of m/z
431.1361, corresponding to the molecular formula C22H23O9 (calc.
431.1342), confirming the identification of 6-C-b-glucopyranosyl-
7-O-methylapigeninidin (6-C-b-glucopyranosyl-5,40-dihydroxy-7-
methoxy-2-phenylbenzopyrylium).
The NMR spectra revealed rotameric pairs of the 8-C-glycosyl-
anthocyanidins, 5–9, and their parent C-glycosylflavones, F5–F9,
Table 2
13C NMR data (in ppm) for the C-glycosylflavones, F1–F9, dissolved in either S1 (5% CF3COOD in (CD3)2SO, v/v), S2 ((CD3)2SO) or S3 (CD3OD), at 25 °C. See Fig. 1 for structures.
F1 (S1)
F2 (S1)
F3 (S2)
F4 (S1)
F5 (S2)*
F6 (S3)*
F7 (S3)*
F8 (S1)
F9 (S1)
2
163.9
163.9
103.1
103.1
182.3
182.1
160.4
159.6
109.8
109.7
163.9
165.0
90.2
164.1
164.1
103.2
103.2
182.3
182.0
160.4
160.1
109.8
109.8
163.8
165.0
90.2
163.89
164.04
103.20
103.30
182.51
182.19
160.75
159.88
108.79
108.74
164.04
165.27
90.50
163.2
163.3
103.6
103.6
181.9
182.2
159.6
160.4
108.5
108.5
165.1
163.8
90.7
164.02
163.81
102.52
102.72
182.18
181.53
160.46
160.79
98.20
166.53
166.74
103.53
103.46
183.99
166.66
165.95
103.58
104.21
184.11
163.9
163.6
102.5
102.6
182.2
182.2
158.6
163.6
163.2
103.1
103.0
182.1
182.1
158.4
159.9
107.3
108.9
160.6
161.6
105.2
102.9
154.8
153.7
103.8
103.1
123.1
122.8
113.4
113.0
146.6
146.6
151.0
151.0
111.5
111.9
119.0
118.7
3
4
5
162.56
162.47
99.81
162.82
163.20
99.77
6
107.3
160.7
105.2
154.9
99.46
101.06
164.04
164.35
105.52
105.41
157.77
156.60
105.89
105.86
123.42
123.35
129.99
101.32
164.12
164.34
105.75
105.61
157.80
156.34
105.98
104.74
123.36
123.32
130.13
129.52
117.01
117.15
162.79
7
162.63
162.96
104.68
104.57
156.06
156.03
104.12
103.89
121.68
121.62
129.05
128.57
115.87
116.10
161.22
161.35
115.87
116.10
129.05
128.57
8
91.0
91.0
90.96
90.2
9
156.8
157.0
104.8
104.2
121.1
121.1
128.5
128.5
116.0
116.0
161.4
161.4
116.0
116.0
128.5
128.5
56.5
156.9
156.9
104.8
104.3
121.5
121.5
113.6
113.6
145.7
145.7
149.9
149.9
116.1
116.1
119.1
119.1
56.5
157.16
157.28
104.63
104.38
121.27
121.23
128.76
128.74
116.19
116.19
161.48
161.48
116.19
116.19
128.76
128.74
56.73
157.0
156.9
104.1
104.4
122.6
122.6
128.3
128.3
114.4
114.4
162.3
162.3
114.4
114.4
128.3
128.3
56.1
10
103.8
103.2
121.4
121.3
129.0
128.7
115.7
115.9
161.2
161.2
115.7
115.9
129.0
128.7
10
20
30
116.91
40
162.56
162.47
116.91
50
117.01
117.15
130.13
129.52
60
129.99
7-MeO
40-MeO
56.2
56.3
56.32
56.4
55.5
55.5
55.7
55.5
6-C-Glc
72.9
72.6
69.6
70.3
79.1
79.1
70.9
70.9
81.7
81.9
61.7
61.7
6-C-Glc
73.0
72.7
69.7
70.4
79.1
79.1
70.8
71.0
81.7
81.9
61.8
61.8
6-C-Glc
71.24
70.91
81.45
80.99
78.89
78.49
70.65
70.65
82.15
81.85
61.66
61.66
6-C-Glc
70.6
70.9
81.1
80.6
78.6
78.3
70.3
70.3
81.6
81.8
61.3
61.3
8-C-Glc
73.45
74.35
70.89
71.09
78.70
78.72
70.25
70.60
81.74
81.93
61.27
61.35
8-C-Glc
73.59
74.86
78.04
77.88
81.48
81.17
72.12
71.56
82.68
82.75
63.01
62.55
8-C-Glc
73.77
75.30
76.09
76.29
81.80
81.45
72.51
71.50
82.99
83.00
63.13
62.45
6-C-Glc
74.0
73.1
71.9
70.2
77.7
78.8
68.9
68.9
80.8
81.2
59.7
59.9
6-C-Glc
73.8
73.0
71.7
71.6
77.6
78.7
68.8
69.1
80.7
81.2
59.6
59.9
100
200
300
400
500
600
200-O-Glc
105.66
105.48
74.92
74.75
76.58
76.54
69.65
69.33
76.88
76.62
60.79
60.24
200-O-Glc
105.2
105.3
74.5
74.5
76.2
76.2
69.3
69.0
76.6
200-O-Rha
102.37
200-O-Rha
101.15
101.19
72.09
71.80
70.02
8-C-Glc
73.2
74.9
70.8
71.8
78.7
78.0
70.4
70.3
81.8
81.7
61.1
61.1
8-C-Glc
73.1
74.8
70.6
70.6
77.8
78.8
70.5
70.2
82.0
81.4
61.4
61.0
1000
2000
3000
4000
5000
6000
72.37
72.02
71.84
71.88
73.43
73.14
69.84
69.91
17.97
17.90
75.16
67.27
67.03
17.84
17.95
76.3
60.5
59.9
4
000-Ac
10000
20000
172.54
21.00
21.03
Signals with two and one significant decimal are recorded from the 13C CAPT and the heteronuclear experiments, respectively. The two signals given for most positions
correspond to two rotamers: major (top) and minor (bottom).
*
Rayyan et al. (2005). Glc = glucoside, Rha = rhamnoside, Ac = acetyl.

282
Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
caused by restricted rotation around the linkage between the ano-
meric carbon (100-C; sp3) and the aglycone (8-C and possibly 6-C in
8, 9, F8 and F9; sp2) (Tables 1–4). The equilibrium between the two
rotamers of each of 5–9 was confirmed by observation of strong
exchange peaks between equivalent protons of each rotameric
pairs in the NOESY and ROESY spectra. Rotamers were not ob-
served for the 6-C-glycosylanthocyanidins, 1–4, dissolved in acidi-
fied deuterated methanol, however rotameric pairs were observed
for their parent C-glycosylflavones, F1–F4, dissolved in deuterated
DMSO. In this context one should keep in mind that distinct NMR
Table 3
1H NMR data for the C-glycosylanthocyanidins, 1–9, dissolved in either S4 (5% CF3COOD in CD3OD, v/v) or S5 (20% CF3COOD in (CD3)2SO, v/v) at 25 °C. See Fig. 1 for structures.
1 (S4)
2 (S4)
3 (S4)
4 (S4)
5 (S4)
6 (S4)
7 (S4)
8 (S5)
9 (S5)
3
4
6
8.30 d 8.9
8.23 d 8.9
8.23 d 8.9
8.34 d 8.7
8.13 d br 8.7
8.12 d 8.8
8.12 d 8.8
9.16 d 8.8
9.16 d 8.8
6.85 s
8.18 d 8.7
8.25 d 8.7
9.22 d 8.7
9.29 d 8.7
6.90 s
8.28 d 8.8
8.28
9.20 d 8.8
9.20
8.26 d 8.7
8.32 d 8.7a
9.19 d 8.7
9.21 br
–
9.26 dd 8.9,
0.9
–
9.20 dd 8.9,
0.9
–
9.24 d 8.9
9.34 dd 8.7,
0.8
9.20 d 8.7
6.86 s
–
–
6.84 s
6.85 s
8
7.47 d 0.9
7.40 d 0.7
7.89 d 2.4
7.37 s
7.45 d 0.8
–
–
–
–
–
20
8.48 0d0 9.1
8.34 0d0 8.9
8.56 0d0 br 9.1
8.49 0d0 br 8.0
8.43
8.48 0d0 9.1
8.37 0d0 9.0
7.17 0d0 8.9
7.14 0d0 9.0
7.17 0d0 8.9
7.14 0d0 9.0
8.48 0d0 9.1
8.37 0d0 9.0
8.54 0d0 8.9
8.42 0d0 8.9
7.19 0d0 8.9
7.22 0d0 8.9
7.19 0d0 8.9
7.22 0d0 8.9
8.54 0d0 8.9
8.42 0d0 8.9
8.45 0d0 8.8
8.40 0d0 br
7.06 0d0 8.8
7.10 0d0 br
7.06 0d0 8.8
7.10 0d0 br
8.45 0d0 8.8
8.40 0d0 br
7.86 d 1.7a
7.80 br
–
30
50
60
7.18 0d0 9.1
7.18 0d0 9.1
8.48 0d0 9.1
–
7.18 0d0 8.9
7.18 0d0 8.9
8.34 0d0 8.9
7.37 0d0 br 9.1
7.37 0d0 br 9.1
8.56 0d0 br 9.1
7.18 0d0 8.9
7.14 d 8.6
7.18 0d0 8.9
7.25 d 8.5
7.28 br
8.04 dd 8.6,
2.4
8.49 0d0 br 8.0
8.43
8.11 dd 8.5,
1.7a
8.09 br
7-MeO
40-MeO
4.21 s
4.20 s
4.18 s
4.22 s
4.10 s
3.98 s
6-C-Glc
5.16 d 9.8
6-C-Glc
5.15 d 9.8
6-C-Glc
5.24 d 10.0
6-C-Glc
5.26 d 9.9
8-C-Glc
5.15 d br
5.30
8-C-Glc
8-C-Glc
5.23 d 10.1
5.44 m
4.27 dd 10.1,
8.6
6-C-Glc
5.12 d 9.8
4.94 d br
3.46 m
3.70
6-C-Glc
5.12 d 9.7
4.93 d 9.7a
3.46 m
100
200
5.21 d 10.1
5.36 d 9.6
4.26 dd 10.1,
8.7
3.75 m
3.75
3.64
4.3 m
4.34 m
4.14 t br
3.70
4.39 t br 9.6
4.29 dd 9.9,
8.8
300
400
3.64 t 8.9
3.73 m
3.9 m
3.8 m
3.83 t 9.0
3.77 m
3.69
3.86
3.79 t 8.7
3.83 m
3.86 t br 9.4
3.70 m
3.81 t 8.8
3.90 t 8.8
3.87 t br 9.3
3.64 dd 9.8,
9.0
3.58 m
3.70 ddd 9.8,
5.4, 2.3
4.03 dd 12.2,
2.2
4.02 dd 11.9,
2.3
3.93 dd 12.2,
4.9
3.38 m
3.42
3.46 m
3.38 m
3.33
3.46 m
3.37
3.72 dd 9.8,
8.9
500
3.60 ddd 9.8,
3.7, 2.5
3.62
3.97
3.6 m
4.0 m
3.61 m
3.98 m
3.61
3.60 m
3.68 m
3.41 m
3.69 m
3.43 m
3.37
600A
3.98 d 3.7
3.96 d 2.5
4.04 dd br
4.05 m
4.04 m
3.70 m
3.7
600B
3.97
3.9 m
3.78 m
3.94
3.94 m
3.87 m
3.66 m
3.66 m
3.61
3.88
200-O-Glc
4.38 d 7.8
200-O-Glc
4.39 d 7.8
200-O-Rha
5.16 d 1.9
5.26 d 1.9
3.96 m
200-O-Rha
5.33 d 1.8
5.45 m
3.96 dd 3.3,
1.8
3.80 m
3.58 dd 9.8,
3.3
8-C-Glc
8-C-Glc
4.88 d 9.8
5.25 d 9.1a
3.83 m
1000
2000
4.88 d 10.0
5.30 d br
3.83 t br 9.3
3.52
3.1 m
3.3 m
3.06 dd 8.9,
7.8
3.85 m
3.55
3000
3.29 t 9.1
3.47 dd 9.6,
3.2
3.35 m
3.35
3.36 m
3.56
2.93 dd 9.6,
3.2
3.20 t 9.6
3.12 t 9.6
2.49 m
2.89 dd 9.8,
3.3
4000
5000
3.0 m
2.9 m
3.02 t 9.1
2.93 m
4.72 t 9.8
4.61 t 9.8
2.48 0dq0 9.8,
6.3
2.14 0dq0 9.8,
6.3
3.52 m
3.33 m
3.51 t 9.1
3.50
3.38 m
3.57
2.16 m
6
000A
3.3 m
3.1 m
3.26 m
3.07 m
0.58 d 6.3
0.82 d 6.3
0.47 d 6.3
0.75 d 6.3
3.74 m
3.59 m
3.82 m
3.79
3.63 m
3.70
6000
B
4
000-Ac
20000
2.10 s
2.02 s
Chemical shifts and coupling constants are in ppm and Hz, respectively. The two signals given for 5–9 correspond to two rotamers: major (top) and minor (bottom).
s = Singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet, br = broad, 0dq0 = double quartet, Glc = glucoside, Rha = rhamnoside, Ac = acetyl.
a
Coupling constant was determined at 292 K.

Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
283
signals of rotameric conformers of C-glycosylflavones seem to be
more observable in DMSO than in methanolic solutions (Rayyan
et al., 2005).
On the basis of examination of the UV–visible spectra of 8 and 9
in aqueous buffer solutions at various pH values it seems that both
pigments undergoes similar structural transformations (Fig. 2). At
pH 1.1 it is assumed that both pigments occur mainly on their
respective flavylium forms with visible kmax at 472 nm and
485 nm. At pH 4.5 the pigments have a local absorption maximum
around 400 nm and a broad absorption band around 500 nm with
Table 4
13C NMR data (in ppm) for the C-glycosylanthocyanidins, 1–9, dissolved in either S4 (5% CF3COOD in CD3OD, v/v) or S5 (20% CF3COOD in (CD3)2SO, v/v) at 25 °C. See Fig. 1 for
structures.
1 (S4)
2 (S4)
3 (S4)
4 (S4)
5 (S4)
6 (S4)
7 (S4)
8 (S5)
9 (S5)
2
173.62
173.4
173.20
172.7
172.6
173.07
173.0
173.41
173.9
170.72
170.22
3
112.36
149.58
156.68
114.82
169.72
93.40
112.6
149.0
156.5
114.7
169.3
93.2
112.00
149.88
156.7
111.9
150.0
156.7
114.8
170.4
93.1
110.0
149.3
159.5
110.38
110.89
149.53
150.03
160.05
160.00
102.73
102.56
170.39
170.3
107.22
106.17
157.82
157.21
114.05
113.46
121.53
121.00
133.94
133.58
118.46
118.56
167.51
167.58
118.46
118.56
133.94
133.58
110.57
110.98
149.68
149.9
160.14
160.0
102.37
103.93
170.24
170.7
107.41
106.36
157.90
157.0
110.67
110.7
149.03
149.0
110.89
111.32
148.97
149.1
4
5
155.06
155.12
6
114.6
102.0
103.5
170.3
113.25
166.74
107.43
155.53
112.92
120.19
113.43
167.38
107.38
155.75
113.5
7
170.1
8
93.4
106.4
157.8
113.8
121.2
133.5
118.2
167.3
118.2
133.5
9
159.25
114.19
120.97
133.92
118.66
168.19
118.66
133.92
58.27
159.1
113.9
121.3
116.4
148.3
157.3
118.1
126.1
58.2
159.7
159.8
115.0
122.4
132.9
116.8
168.0
116.8
132.9
10
10
20
30
40
50
60
113.66
121.29
133.66
118.53
167.8
113.95
113.4
121.46
121.2
121.99
134.19
133.56
118.55
118.80
167.84
168.0
118.55
118.80
134.19
133.56
133.26
133.2
117.54
117.6
115.58
115.0
147.91
165.98
155.51
118.53
133.66
58.14
117.54
117.6
133.26
133.2
113.12
113.2
124.21
124.5
7-MeO
40-MeO
58,0
56.6
56.66
6-C-Glc
76.51
6-C-Glc
76.5
6-C-Glc
75.11
6-C-Glc
74.8
8-C-Glc
74.7
8-C-Glc
73.36
74.19
78.65
77.85
81.31
81.05
72.06
72.1
82.98
83.0
62.48
62.70
8-C-Glc
73.39
74.32
77.36
76.94
81.46
81.13
72.08
71.55
83.10
83.02
62.41
62.49
6-C-Glc
75.38
74.4
73.01
71.4
77.64
77.5
68.90
6-C-Glc
75.29
74.58
73.02
71.8
77.66
78.2
68.92
69.67
81.39
81.7
100
200
300
400
500
600
74.07
79.14
70.84
82.74
61.58
74.1
79.1
70.8
82.9
61.5
79.0
80.2
70.3
82.7
61.3
81.2
78.8
70.2
82.4
62.9
72.7
79.8
71.8
82.7
62.3
81.38
59.68
59.69
59.94
200-O-Glc
105.2
200-O-Glc
104.9
200-O-Rha
102.85
102.5
72.12
72.04
71.92
72.27
73.29
73.05
70.18
70.11
18.12
17.97
200-O-Rha
101.95
101.7
71.99
71.6
8-C-Glc
73.71
75.0
71.17
72.4
78.52
77.1
70.46
8-C-Glc
73.71
75.2
71.05
72.6
78.60
77.51
70.94
70.8
82.52
81.6
1000
2000
3000
4000
5000
6000
75.8
77.7
71.0
77.2
62.2
75.6
77.6
70.9
77.4
62.0
70.05
70.0
75.12
75.02
67.68
67.38
17.91
17.91
82.20
61.04
61.71
61.7
4
000-Ac
10000
20000
172.17
172.0
20.95
20.9
Signals with two and one significant decimal are recorded from the 13C CAPT and the heteronuclear experiments, respectively. The two signals given for 5–9 correspond to
two rotamers: major (top) and minor (bottom). Glc = glucoside, Rha = rhamnoside, Ac = acetyl.

284
Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
shoulders on both sides. Based on these absorption characteristics,
it is assumed that at pH 4.5 each pigment occur mainly as a mix-
ture of chalcone form(s) (kmax around 400 nm), flavylium cation
(shoulder in absorption spectrum with kmax around 470 nm) and
quinonoidal form(s) (shoulder in absorption spectrum with kmax
around 530 nm) similar to the distribution of the transformed
forms of apigeninidin (Brouillard et al., 1982). At pH 7 the picture
is comparable to that at pH 4.5 for both 8 and 9, however, the pro-
portion(s) of the chalcone form(s) seem to increase on the expense
of the other forms. Possible colourless hemiketal forms are not
considered.
To compare the stability of the C–C linkage of C-glycosylantho-
cyanidins with the C–O linkage of anthocyanidin O-glycoside to-
wards acid, mixtures of nearly equal parts of 6,8-di-C-b-
glucosylapigeninidin (8), and pelargonidin 3-O-b-glucoside (P1),
and 6,8-di-C-b-glucopyranosyl-40-O-methylluteolinidin (9) and P1
were prepared. Aliquots of these solutions were subjected to acid
hydrolysis at 110 °C at different time intervals (15, 30, 60, 90,
120, 180, 240 and 500 min, respectively). The HPLC profiles
(Fig. 3) revealed that after 30 min hydrolysis more than half of
the amount of P1 was converted to the aglycone pelargonidin
(P2), after 60 min P1 was not detected at all, while most of P2
was degraded further after 500 min. Similar acid hydrolysis prod-
ucts were not observed for the C-glycosylanthocyanidins, 8 and
9, during the hydrolysis period. Thus, contrary to the 3-O linkage
of P1, the corresponding 8-C and 6-C linkages of 8 and 9, which
are not found in normal anthocyanidin O-glycosides, seemed to
be far more stable towards acid hydrolysis. This may encourage ex-
tended use of anthocyanins like the C-glycosylanthocyanidins, 1–9,
as nutraceuticals, pharmaceuticals or colorants in the future.
3. Experimental
3.1. Isolation of the C-glycosylflavones
Flowers of Iris sibirica L. (550 g) were extracted twice with
MeOH containing 0.5% trifluoroacetic acid (v/v), TFA, for 16 h at
4 °C. The combined extracts were concentrated under reduced
pressure, and purified by partition against equal portions of ethyl
acetate (EtOAc) (five times). The concentrated aqueous layer was
applied on an Amberlite XAD-7 column. The flavonoids adsorbed
on the column were washed with H2O before they were eluted
with MeOH containing 0.2% TFA. The flavonoids were further
purified by Sephadex LH-20 column chromatography using
MeOH:H2O (containing 0.5% TFA) (1:4, v/v) as eluent. After con-
centration of the Sephadex LH-20 fractions containing the vari-
ous flavones, individual flavones, F1–F4 (Kawase, 1968; Asen
et al., 1970; Hirose et al., 1981; Hilsenbeck and Mabry, 1983,
1990), were precipitated after storage at 4 °C for 24 h. F2 and
F4 were further purified by preparative HPLC. Embinoidin (F4),
6-C-sophorosyl-7,40-di-O-methylapigenin, has previously been re-
ported to occur only in the genus Siphonoglossa (Hilsenbeck and
Mabry, 1983, 1990).
The herbal drug ‘‘Crataegi folium cum flore” (dried hawthorn
leaves and flowers) was purchased from Norsk Medisinaldepot
ASA (Bergen, Norway). This drug has been specified for example
in the German, Swiss and French pharmacopeias (DAB 10, Ph. Helv.
VII, Ph Franc. X) (Sticher and Meier, 1998). Plant material (200 g)
was extracted three times with 20% aqueous MeOH at 4 °C. The ex-
tracts were after filtration concentrated under reduced pressure,
purified by partition against diethyl ether, and subjected to Amber-
lite XAD-7 column chromatography. The columns were washed
with distilled water before the flavonoids were eluted with meth-
anol. The individual flavonoids, including F5–F7 (Nikolov et al.,
1982; Rayyan et al., 2005), were separated by Sephadex LH-20 col-
umn chromatography using MeOH–H2O (40:60; v/v) to MeOH–
H2O (70:30; v/v) (stepwise gradient) as eluents.
Orange juice (20 l) was filtered prior to purification by parti-
tion against EtOAc. The aqueous layer was then concentrated un-
der reduced pressure, and applied to an Amberlite XAD-7
column. The column was washed with H2O before the adsorbed
flavonoids were eluted with MeOH containing 0.2% TFA (v/v).
The MeOH fraction was concentrated to a dark brown syrup,
and subjected to Sephadex LH-20 chomatography. The flavones
were separated using isocratic elution with MeOH–H2O (1:4, v/
v) containing 0.2% TFA. Flavonoids from lemon juice (20 l) were
treated according to a similar procedure, before the Sephadex
LH-20 fractions containing F8 and F9 from both juices were sep-
arately combined. F8 and F9 (Tomas et al., 1978; Kumamoto
et al., 1985) were then further purified by subjecting them indi-
vidually to a second Sephadex LH-20 column, using the same
separation conditions as the first Sephadex column, followed by
preparative HPLC. HPLC-fractions containing F8 were combined
and evaporated to dryness, and the solid was dissolved in a min-
imum amount of H2O containing 0.2% TFA (v/v). F8 was precipi-
tated after 3 days storage at 4 °C. HPLC-fractions containing F9
were also combined and evaporated to dryness, and the solid
was suspended into cold MeOH containing 0.2% TFA (v/v) before
the suspension was filtered giving F9.
The structures of F1–F9 including their rotamers were eluci-
dated by means of one- and two-dimensional NMR spectroscopic
techniques (Tables 1 and 2), UV–vis spectroscopy and high-resolu-
tion electrospray mass spectrometry.
Fig. 2. Effect of pH on the UV–visible absorption spectra of 6,8-di-C-b-glucopyr-
anosylapigeninidin (0.17 mM), 8 (top), and 6,8-di-C-b-glucopyranosyl-40-O-meth-
ylluteolinidin (0.21 mM), 9 (bottom) in aqueous buffer solutions measured minutes
after dissolution.

Ø. Bjorøy et al. / Phytochemistry 70 (2009) 278–287
285
Fig. 3. Left: Various HPLC profiles detected at 485 20 nm of the same mixture of 6,8-di-C-b-glucopyranosylapigeninidin (8) and pelargonidin 3-O-b-glucopyranoside (P1)
dissolved in MeOH–HCl (2 M) (1:1, v/v) subjected to 110 °C at different time intervals. Each chromatogram is scaled to its highest peak. Right: UV–visible spectra of 8, P1 and
P2. P2 = pelargonidin.
3.2. Synthesis of anthocyanidin C-glycosides
v) was then added to the zinc-amalgam. This latter procedure ap-
peared not to influence the yield of the product (9), however, the
use of HCl gas and dried anhydrous MeOH was positively pre-
vented. The increased reaction volume was also beneficial for dis-
solution of F9. The various crude products were first analysed by
analytical HPLC, and thereafter the C-glycosylanthocyanidins, 1–
9, were purified by preparative HPLC, or by a small column
(30 ꢀ 1 cm) packed with Sephadex LH-20 material. The yields ob-
tained during the various reductive syntheses are presented in Ta-
ble 5.
Prior to the main synthesis of the various C-glycosylanthocyani-
dins, a test synthesis with 20 mg F3 (spinosin) was performed. The
reaction proceeded for 30 min. Samples from the reaction mixture
were collected after every 5th min, respectively, and analysed by
analytical HPLC. After 5 min, most of the spinosin sample was still
intact (spinosin:C-glycosylanthocyanidin, ꢁ50:1). After 15 min, a
significant amount of the flavone was still present (spinosin:C-gly-
General procedure: Zinc-amalgam was prepared as described
by Elphimoff-Felkin and Sarda (1977). F1–F9 (70–200 mg) were
individually dissolved in 20 ml dry methanol containing 3% HCl
gas by weight (Vogel et al., 1989). The solutions were, if necessary,
gently heated to 40 °C to dissolve the compounds before being
chilled to room temperature on ice. Zinc-amalgam (3 g) was there-
after added, and after thorough stirring for 30 min the liquid
phases containing the products were separated from amalgam res-
idues by filtration through glass wool. The reaction time was nota-
bly influenced by the quality of the zinc-amalgam being as short as
12 min using fresh zinc. By using an alternative procedure, HgCl2
(1 g) and Zn (12.5 g) were mixed with 200 ml H2O–conc. HCl
(96:4, v/v) with stirring for 30 min. The liquid phase was removed
and the zinc-amalgam washed with ethanol followed by diethyl
ether. F9 (200 mg) dissolved in 250 ml MeOH–conc. HCl (97:3, v/
Table 5
Chromatographic (HPLC), UV–vis* and high-resolution electrospray MS data recorded for the C-glycosylanthocyanidins, 1–9. The yields give the quantitative amounts of the
various pigments formed after reductive synthesis of their respective C-glycosylflavones. See Fig. 1 for structures.
Pigment
tR (min)
kUV-max (nm)
kVIS-max (nm)
A440/AVIS-max (%)
[M]+ observed (m/z)
[M]+ calculated (m/z)
Molecular formula
Yielda
mg
mmol
%
1
2
3
4
5
6
7
8
9
5.35
4.74
5.70
12.13
4.05
3.86
5.15
3.57
3.63
279, 326
281, 325sA
280, 327
280, 328
279, 323
279, 325
280, 326
281, 326
283, 325sA
469
486
472
470
424s, 476
424s, 481
426s, 482
474
63
42
61
67
60
56
52
66
46
431.1361
447.1294
593.1865
607.1998
417.1212
563.1749
605.1848
579.1700
609.1790
431.1342
447.1291
593.1870
607.2027
417.1186
563.1765
605.1870
579.1714
609.1819
C22H23O9
C22H23O10
C28H33O14
C29H35O14
C21H21O9
C27H31O13
C29H33O14
C27H31O14
C28H33O15
24b
25b
35b
11b
13b
14b
36b
41
4.4 ꢀ 10ꢂ2
4.5 ꢀ 10ꢂ2
5.5 ꢀ 10ꢂ2
1.5 ꢀ 10ꢂ2
2.4 ꢀ 10ꢂ2
2.1 ꢀ 10ꢂ2
4.9 ꢀ 10ꢂ2
5.9 ꢀ 10ꢂ2
7.0 ꢀ 10ꢂ2
20
21
25
14
15
15
32
18
22
488
51
*
In MeCN–H2O (1:4) containing 0.5% TFA (v/v/v);
a
As trifluoroacetic acid salt.
b
Total anthocyanin yield, which includes the 6-C- and 8-C-glycosylanthocyanidin isomers gathered (see Section 2 for further details).
A
Weak shoulder.

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
.
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