Retrodihydrochalcones in Sorghum species: Key intermediates in the biosynthesis of 3-deoxyanthocyanidins?

Article abstract of DOI:10.1016/j.phytol.2011.12.004

Two new open chain flavonoids were isolated from the butanediol/ethanol extract of Sorghum bicolor (L.) Moench leaf sheaths by fractionation and purification processes. This work led to the structural characterization of the 3-(2,4,6-trihydroxyphenyl)-1-(

Full text of DOI:10.1016/j.phytol.2011.12.004

Phytochemistry Letters 5 (2012) 174–176
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Retrodihydrochalcones in Sorghum species: Key intermediates in the biosynthesis
of 3-deoxyanthocyanidins?
*
´
Ali Khalil, Raymonde Baltenweck-Guyot , Ruben Ocampo-Torres, Pierre Albrecht
Institut de Chimie, UMR 7177 CNRS/Universite´ de Strasbourg, 1 rue Blaise Pascal, 67000 Strasbourg, France
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new open chain flavonoids were isolated from the butanediol/ethanol extract of Sorghum bicolor (L.)
Moench leaf sheaths by fractionation and purification processes. This work led to the structural
characterization of the 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one (or 2,4,40,6-
tetrahydroxydihydrochalcone) 1, and 3-(2,6-dihydrox-4-methoxyphenyl)-1-(4-hydroxyphenyl)-pro-
pan-1-one (or 2,40,6-trihydroxy-4-methoxydihydro-chalcone) 2. The structures of these flavonoids
were determined by extensive spectroscopic analyses, including UV, ESIMS, HRESIMS, 1D and 2D NMR.
The chemical properties of 1 were similar to those earlier described in literature for apiforol, never fully
characterized. These results led us to re-question the real structure of this flavan-4-ol, which is often
described to be present in Sorghum and has even been considered as a key intermediate in the formation
of Sorghum 3-deoxyanthocyanidins.
Received 30 September 2011
Received in revised form 5 December 2011
Accepted 8 December 2011
Available online 20 December 2011
Keywords:
Sorghum bicolor (L.) Moench
Var. bicolor
Structural characterization
NMR
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
MS
Flavonoids
Retrodihydrochalcone
1. Introduction
The present work is based on an extract of Sorghum leaf sheaths
with the aim to increase our knowledge of Sorghum 3-deoxy-
Sorghum bicolor (L.) Moench, the fifth-ranking cereal global
production cultivated worldwide is rich in polyphenols belonging
to the flavonoid and phenolic acid families (Awika and Rooney,
2004). Flavonoids are common plant secondary metabolites that
may be implicated in plant growth and development processes,
including protection from UV irradiation as well as in insect
attraction for pollinization and seed dispersion. Some Sorghum
flavonoids like 3-deoxyanthocyanidins (Snyder and Nicholson,
1990; Lo et al., 1999) and, more recently, flavones apigenin and
luteolin (Du et al., 2010) have been identified as phytoalexins
produced as response to fungal attack. In addition, numerous
works on Sorghum plants mentioned the occurrence of red
pigments called phlobaphenes; their precise structures are not
defined, but they are proposed to arise as polymerization of
different products such as flavan-4-ols called apiforol and
luteoforol (Chopra et al., 2002; Schijlen et al., 2004). However, a
reliable structure of these flavan-4-ols has never been clearly
established.
flavonoids. The analysis by LC-UV-ESIMS of an extract of Sorghum
bicolor (L) Moench showed the presence of many polyphenols,
some of which had a molecular mass corresponding to unknown
compounds. Here we describe in particular the isolation and
structural characterization of two new flavonoids, retrodihydro-
chalcones 1 and 2, which may be key intermediates in the
formation of Sorghum 3-deoxyanthocyanidins.
2. Results and discussion
The butanediol/ethanol extract of Sorghum leaf sheaths was
initially concentrated to eliminate ethanol. It was then fractionated
using a C18 endcapped cartridge into three fractions (elution by
water, ethyl acetate and methanol) (Khalil et al., 2010). Subsequent
chromatographic separation of ethyl acetate fraction and further
purification of sub-fractions (Khalil et al., 2010) allowed the
isolation of 1 and 2. Structural assessment of these two compounds
was obtained by analysis of electrospray mass spectrometry, 1H
and 13C NMR spectroscopic data. Assignments of NMR signals were
obtained especially by COSY, NOESY, HSQC and HMBC experi-
ments.
´
* Corresponding author. Present address: Metabolisme Secondaire de la Vigne,
The UV spectrum (MeOH) of compound 1 showed maximum
absorption at 288 nm, similarly to flavanols and flavanones. The
pseudo molecular ion peak [M+Li]+ (ion Li+ from calibration of the
UMR 1131 INRA/Universite´ de Strasbourg, 28 rue de Herrlisheim, 68021 Colmar,
France. Tel.: +33 03 89 22 49 94; fax: +33 03 98 22 49 33.
E-mail address: baltenwe@unistra.fr (R. Baltenweck-Guyot).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.12.004

A. Khalil et al. / Phytochemistry Letters 5 (2012) 174–176
175
Table 1
1H and 13C NMR data for compounds 1 and 2 (500 and 125 MHz respectively, in
CD3OD), (ppm).
H
H3CO
H
H
H
OH
d
OH
H
Position
Compound 1
d1H [int., mult.,
Compound 2
d1H [int., mult.,
d13
C
d13
C
J (Hz)]
J (Hz)]
H
H
Noesy
OH
O
H
C5O
a
b
–
203.1
39.3
–
203.3
39.3
HMBC
3.09, 2H, t (7.5)
2.83, 2H, t (7.5)
3.10, 2H, t (7.4)
19.5
2.85, 2H, t (7.5)
19.8
1
107.3
157.7
95.6
–
108.6
158.1
94.2
Fig. 2. Major HMBC and NOE correlations observed on compound 2.
2,6
3,5
4
–
–
5.84, 2H, s
5.92, 2H, s
corresponding to protons of a methoxyl group. Moreover, these
hydrogens were spatially near to H-3/5 (NOE effect, see Fig. 2). The
long-range correlation observed in the HMBC spectrum between
–
158.3
129.9
132.1
116.1
163.8
–
–
160.8
130.0
132.3
116.3
164.0
55.2
10
–
–
20,60
30,50
40
7.90, 2H, d (8.6)
7.91, 2H, d (8.8)
6.80, 2H, d (8.7)
–
6.79, 2H, d (8.6)
the C-4 (d 160.8) and these protons (d 3.66) confirmed the presence
–
–
of a methoxyl group linked at C-4. Thus, compound 2 was
identified as the dihydrochalcone 3-(2,6-dihydrox-4-methoxy-
phenyl)-1-(4-hydroxyphenyl)-propan-1-one (or 2,40,6-trihydroxy-
4-methoxydihydrochalcone). It corresponds to an analogue of the
7-O-methyl-deoxyflavonoids (7-O-methylapigenin, 7-O-methyl-
luteolin and 7-O-methylapigeninidin) previously identified in the
Sorghum leaves (Muller-Harvey and Reed, 1992; Misra and
Seshadri, 1967; Pale et al., 1997).
These two dihydrochalcones 1 and 2 are named retrodihy-
drochalcones because the typical O-substitution patterns of the A-
and B-rings of flavonoid structures are apparently reversed and the
carbonyl group shifted from C-1 to C-3.
–OCH3
3.66, 3H, s
mass spectrometer) was observed for compound 1 at m/z 281.097,
obtained by HRESIMS, established its formula to be C15H14O5.
The 1H NMR spectrum (500 MHz in CD3OD, TMS) of 1 (Table 1)
showed three aromatic protons signals indicative of a flavonoid
skeleton. They corresponded to the H-20/60 equivalent protons
occurring at 7.90 ppm as a doublet (J = 8.6 Hz) and coupled to the
H-30/50 equivalent protons evident as a doublet (J = 8.6 Hz) at
6.79 ppm. An aromatic two protons singlet observed at 5.84 ppm
was assigned to H-3/5 equivalent protons. Two triplets, two
protons each, appearing at 3.09 (J = 7.5 Hz) and 2.83 ppm
Few plants containing retrodihydrochalcones are known: other
retrodihydrochalcones were earlier identified from Dracaena
loureiri (Meksuriyen and Cordell, 1988) and from the stem bark
of Uvaria mocoli (Fleicher et al., 1998).
(J = 7.5 Hz) were identified as vicinal aliphatic protons H-
H-
respectively. The 1H–1H COSY spectrum confirmed correla-
tions between aromatic protons H-20/60 and H-30/50 and between
aliphatic protons H- and H- . The major NOE effects were
a and
b
a
b
It is notable that new elucidated retrodihydrochalcone 1 has the
same molecular weight (MW 274), the same molecular formula
(C15H14O5) and a similar UV spectrum as those attributed in the
literature to apiforol (see structure in Fig. 3) (Watterson and
Buttler, 1988). However, even if the latter compound has never
been fully characterized in plants, the hypothetical structure of
apiforol has been proposed as a potential precursor of Sorghum 3-
deoxyanthocyanidins (Kambal and Bate-Smith, 1976; Styles and
Ceska, 1989). Therefore, the reliability of the apiforol structure
should be questioned. To compare the structure of apiforol with
that of retrodihydrochalcone 1, the latter was treated in the same
reaction conditions as used by Watterson and Buttler (1988) on the
proposal apiforol: a hot aqueous concentrated solution of HCl
(Bate-Smith reaction, Bate-Smith, 1969). As for apiforol detected
by Watterson and Buttler (1988), we observed the solution change
from colorless to yellow. Analysis of the yellow solution by
electrospray ionization mass spectrometry in positive mode and by
1H NMR spectroscopy indeed demonstrated the presence of
apigeninidin 3 by comparison with a reference sample (Fig. 3),
already reported in Sorghum, a result identical with that obtained
by Watterson and Buttler (1988) for apiforol.
observed between these aliphatic hydrogens and the H-20/60
phenolic protons. The carbon chemical shift values corresponding
to each carbon attached to respective heteronuclear hydrogens
were evidenced on the basis of the HSQC spectrum.
The long-range correlation observed in the HMBC spectrum
between the carbon of the carbonyl group (
protonsH-20/60 (
7.90)and aliphaticprotonsH-
2.83), confirmed the position of the carbonyl group next to the
phenyl ring B. Similarly, protons H-3/5 ( 5.84), H- 3.09) and H-
2.83) showed correlations to carbon C-1 ( 107.3), while protons
H-30/50 ( 3.09) showed correlations to C-10 (
6.79) and only H-
d
203.1) and the aromatic
d
a
(d
3.09)andH-b(d
d
a
(d
b
(d
d
d
a
(d
d
129.9). Thus, compound 1 was characterized as a novel open chain
flavonoid, the 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-
propan-1-one or 2,4,40,6-tetrahydroxydihydrochalcone.
The UV spectrum (MeOH) of compound 2 was similar to that of
compound 1. The pseudo molecular ion peak [MꢀH]ꢀ observed for
compound 2 at m/z 287.091, obtained by HRESIMS, established the
molecular formula of 2 to be C16H16O5. Consequently, compound 2
corresponded to the same molecular formula as 1 with one
additional CH2 methylene group. Fig. 1
NMR experiments showed that compound 2 had structural
similarity with 1: the chemical shifts of all aromatic and aliphatic
protons of 2 were analogous to those of compound 1. A notable
difference was the presence of a three proton singlet at 3.66 ppm,
Identical experiments were carried out on compound 2, and
similar results were obtained, i.e. the formation of 7-O-methyla-
pigeninidin 4 by the Bate-Smith reaction. The similarity between
our results and those of Watterson and Buttler (1988) on proposed
apiforol led us to question the real structure of the latter and its
5
3'
B
6'
RO
OH
OH
2'
6
4'
5'
4
3
OH
OH
A
1
1'
HO
O
RO
O
2
OH
O
apiforol
3, R = H
4, R = CH3
1, R = H
2, R = CH3
OH
OH OH
Fig. 1. Structure of compounds 1 and 2.
Fig. 3. Structure of apiforol, apigeninidin 3 and 7-O-methyl-apigeninidin 4.

176
A. Khalil et al. / Phytochemistry Letters 5 (2012) 174–176
real existence, as well as that of other flavan-4-ols in Sorghum
plant. The two novel retrodihydrochalcones could indeed be key
intermediates in the biosynthesis pathway of 3-deoxyanthocya-
nidins in Sorghum plants. This hypothesis is now subjected to
further investigations.
3.4.2. 3-(2,6-Dihydrox-4-methoxyphenyl)-1-(4-hydroxyphenyl)-
propan-1-one (2)
Pale yellow solid; UV (MeOH) l
max: 280 nm; 1H NMR (500 MHz,
CD3OD) and 13C NMR (125 MHz, CD3OD): see Table 1; ESIMS (+) m/
z 289.1 [M+H]+, 311.1 [M+Na]+, ESIMS (ꢀ) m/z 287.1 [MꢀH]ꢀ;
HRESIMS (ꢀ) m/z 287.091 [MꢀH]ꢀ (calcd. for C16H15O5ꢀ 287.091).
3. Experimental
3.5. Bate-Smith reaction
3.1. General experimental procedures
30
mg of each retrodihydrochalcone 1 and 2 were reacted
1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were
measured in CD3OD on a Bruker AMX500 spectrometer at 298 K and
with TMS as internal standard. 1H chemical shifts were assigned
using 1D and 2D 1H NMR (COSY and NOESY), whereas 13C
resonances were assigned using 2D NMR techniques (HMBC and
HSQC). AnEsquire3000+ and a MicroTOF(BrukerDaltonics, Bremen,
Germany) mass spectrometers equipped with an electrospray
source for ionization was used for the ESIMS and HRESIMS. Samples
were introduced into the spectrometers with a syringe pump
individually with 2–3 ml of conc. HCl (37%, 12 M). The mixture was
heated for 15 min in a water bath at 100 8C. The resulting 3-
deoxyanthocyanidins (apigeninidin 3 and 7-O-methylapigeninidin
4) were then analyzed by UV-visible spectroscopy, MS and NMR.
Acknowledgments
We wish to thank L’OREAL Research and Development, Aulnay-
sous-Bois, France for providing us with a sample of the Sorghum
extract; the Centre National de la Recherche Scientifique (CNRS)
and the Universite´ de Strasbourg (UdS) for financial support; Dr G.
Hussler, L’OREAL, for helpful discussion; Dr R. Graff for NMR
(200
ml/min). Spectra were recorded in positive and negative mode
between m/z 80 and 1000. The capillary voltage was ꢁ4 kV, the
capillary temperature 180 8C and the capillary exit 100 V.
`
measurements. One of us (A.K.) thanks the Ministere de
3.2. Plant material
´
l’Enseignement Superieur et de la Recherche (France) for a doctoral
fellowship.
The Sorghum extract was obtained by extraction of red Sorghum
leaf sheath [Sorghum bicolor var. bicolor (Moench)], cultivar Gervex
1296 (CIRAD) from Burkina Faso.
References
3.3. Extraction and isolation
Awika, J.M., Rooney, L.W., 2004. Sorghum phytochemicals and their potential
impact on human health. Phytochemistry 65, 1199–1221.
Bate-Smith, E.C., 1969. Luteoforol (30,4,40,5,7-pentahydroxyflavan) in Sorghum vul-
gare L. Phytochemistry 8, 1803–1810.
Chopra, S., Gevens, A., Svabek, C., Wood, K., Peterson, T., Nicholson, R., 2002. Excision
of the transposon from a hyper-mutable allele shows that the sorghum gene
controls the biosynthesis of both 3-deoxyanthocyanidin phytoalexins and
phlobaphene pigments. Physiol. Mol. Plant Pathol. 60, 321–330.
Du, Y., Chu, M., Wang, M., Chu, I., Lo, C., 2010. Identification of flavone phytoalexins
and a pathogen-inducible flavone synthase II gene (SbFNSII) in Sorghum. J. Exp.
Bot. 61, 983–994.
Fleicher, T., Waigh, R., Waterman, P., 1998. A novel retrodihydrochalcone from the
stem bark of Uvaria mocoli. Phytochemistry 47, 1387–1391.
Kambal, A.E., Bate-Smith, E.C., 1976. A genetic and biochemical study on pericarp
pigments in a cross between two cultivars of grain sorghum, Sorghum bicolor.
Heredity 37, 413–416.
Khalil, A., Baltenweck-Guyot, R., Ocampo, R., Albrecht, P., 2010. A novel symmetrical
pyrano-3-deoxyanthocyanidin from a Sorghum species. Phytochem. Lett. 3, 93–
95.
Lo, S.C., De Verdier, K., Nicholson, R.L., 1999. Accumulation of 3-deoxyanthocyani-
din phytoalexins and resistance to Colletotrichum sublineolum in Sorghum.
Physiol. Mol. Plant Pathol. 55, 263–273.
Meksuriyen, D., Cordell, G.A., 1988. Retrodihydrochalcones from Dracaena loureiri. J.
Nat. Prod. 51, 1129–1135.
Misra, K., Seshadri, T., 1967. Chemical components of Sorghum durra glumes. Indian
J. Chem. 5, 409–412.
Muller-Harvey, I., Reed, J., 1992. Identification of phenolic compounds and their
relationships to in vitro digestibility of Sorghum leaves from bird-resistant
varieties. J. Sci. Food Agric. 60, 179–196.
The Sorghum extract was obtained using a mixture of 1,3-
butanediol and ethanol (1:2) as solvent. Ethanol was then removed
by evaporation; the Sorghum extract contained 5% of dry total
extract by weight. 5 ml of the butanediol solution was diluted with
water (1:3) and separated in several fractions according to their
polarity, using a Macherey-Nagel Chromabond1 C18 endcapped
cartridge (C18 ec, 6 ml, 500 mg). Eluting with 10 ml of water
permits to eliminate salts and acids; the flavonoids were then
eluted with 15 ml of ethyl acetate which gave a pink fraction. The
latter was concentrated and then separated by HPLC (Waters,
Milford, MA, USA) used together with an autosampler (Waters 717
plus) on a reverse phase C18 Zorbax-SB semi preparative column
(250 ꢂ 9.6 mm, 5
mm, Agilent, Santa Clara, CA, USA). The detection
was carried out using a diode array detector (Waters 996) with a
flow of 4 ml/min. The eluents A (H2O) and B (CH3CN) were used
with a 45 min gradient to separate compounds 1 and 2: 0–15 min,
25–35% B; 15–33 min, 35–42% B, 33–40 min, 42–100% B, 40–
45 min, 100% B. Compounds 1 (retention time 14.1 min) and 2
(retention time 17.5 min) were collected separately and then
concentrated under vacuum to give ca. 0.5 mg of compound 1 and
ca. 0.4 mg of compound 2.
Pale, E., Koudas-Bonafos, M., Nacro, M., Vanhaelen, M., Vanhaelen-Fastre´, R., Ottin-
ger, R., 1997. 7-O-Methylapigeninidin, an anthocyanidin from Sorghum cauda-
tum. Phytochemistry 45, 1091–1092.
3.4. Compounds characterization
Schijlen, E., de Vos, R., van Tunen, A., Bovy, A., 2004. Modification of flavonoid
biosynthesis in crop plants. Phytochemistry 65, 2631–2648.
Snyder, B.A., Nicholson, R.L., 1990. Synthesis of phytoalexins in sorghum as a site-
specific response to fungal ingress. Science 248, 1637–1639.
Styles, E.D., Ceska, O., 1989. The genetic control of flavonoid synthesis in maize. Can.
J. Genet. Cytol. 19, 289–302.
Watterson, J., Buttler, L., 1988. Occurrence of an unusual leucoanthocyanidin
and absence of proanthocyanidins in sorghum leaves. J. Agric. Food Chem.
31, 41–45.
3.4.1. 3-(2,4,6-Trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-
one (1)
Pale yellow solid; UV (MeOH) l
max: 280 nm; 1H NMR (500 MHz,
CD3OD) and 13C NMR (125 MHz, CD3OD): see Table 1; ESIMS (+) m/
z 275.1 [M+H]+, 297.1 [M+Na]+, ESIMS (ꢀ) m/z 273.1 [MꢀH]ꢀ;
HRESIMS (+) m/z 281.097 [M+Li]+ (calcd. for C15H14O5Li+ 281.099).