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W. Hidalgo et al. / Phytochemistry 13 (2015) 68–73
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Table 1
Examples of phenylpropanoids and phenylphenalenones sharing the same substitution pattern of the phenyl ring.
Phenylpropanoic acids
Phenylphenalenones
Part A
R1
R2
H
H
H
OH
OCH3
Cinnamic acid
4-Coumaric acid
Caffeic acid
Anigorufone
R1
R2
OH
OH
OH
OH
Hydroxyanigorufone
Dihydroxyanigorufone
Musanolone F (3)
O
R1
R2
4'
3'
1
A
D
Ferulic acid (1)
9
B
C
HOOC
HOOC
7
Part B
R
H
CH3
4-Coumaric acid (+SAM)
40-Methoxy-cinnamic acid (2)
40-O-Methylirenolone
OR
OH
O
1
OCH3
4'
4
Part A: Moieties shown in bold are linked by confirmed biosynthetic precursor–product relationships (Schmitt et al., 2000). Part B: The precursor of 40-O-methylirenolone in
M. acuminata is uncertain. For details see text and Otálvaro et al. (2010).
SAM = S-adenosyl-L-methionine.
and phenylphenalenones with corresponding methoxy-substituted
phenyl ring substitutions. Here we report the 1H NMR and HPLC-
guided identification of phenylpropanoic acids using an isotope
dilution approach. In the second part of this work, we offer evi-
dence for a precursor–product relationship by administering the
13C-labeled phenylpropanoids then isolating the resulting phenyl-
phenalenones and analyzing its 13C enrichment by 13C NMR and
HRMS.
13C-labeled phenylpropanoic acids and their unlabeled native
counterparts in the crude root extracts of both cultures.
The fingerprint signals of H-2 of the E-configured double bonds
(JH2–H3 = 16 Hz) and their 13C satellite signals (JC2–H2 = 161 Hz)
were used to detect isotopologue mixtures of non-metabolized
[2-13C]-phenylpropanoids together with the unlabeled counter-
parts in the plant extracts. Using this approach, the natural
occurrence of free ferulic acid (1) was confirmed in root cultures
of A. preissii and detected for the first time in root cultures of
W. thyrsiflora (Fig. 1A and B). 40-Methoxycinnamic acid (2) was
found in A. preissii but not in W. thyrsiflora (data not shown).
Isoferulic acid and 30,40-methylenedioxy phenylpropanoic acid
were not detectable in either of the two root cultures.
2. Results and discussion
2.1. Detection of phenylpropanoic acids in plant root cultures
Moreover, the proportion of [2-13C]-labeled and unlabeled
phenylpropanoids was determined from the integral ratio of the
doublets of H-2 and the 13C satellite signals in the 1H NMR spectra
(Schneider et al., 2003). Since the feeding experiments with
A. preissii and W. thyrsiflora were performed under identical condi-
tions, the integral ratios suggested a lower endogenous level of free
ferulic acid (1) in W. thyrsiflora (ratio 13C-labeled: unlabeled 8.0:1)
compared to A. preissii (2.7:1). The isotopologue ratio of [2-13C]-2
to unlabeled 2 (natural abundance 13C contents) in A. preissii was
found to be 8.1:1 under the experimental conditions.
As ferulic acid (1) is a common cell wall lignin component
(Buanafina, 2009), the proportion of this phenylpropanoic acid
potentially available for the biosynthesis of phenylphenalenones
is difficult to estimate. Unlike ferulic acid (1), 40-methoxycinnamic
acid (2) seems not to be used as a precursor in major plant
metabolic pathways such as lignin formation. Therefore, all of
the endogenous pool found in A. preissii should be available for
the biosynthesis of phenylphenalenones. Hence, our search for
methoxyphenylphenalenones and their biosynthesis was focused
on A. preissii.
In vitro root cultures of A. preissii and W. thyrsiflora are rich
sources of phenylphenalenones (Hölscher and Schneider, 1997;
Opitz et al., 2002; Opitz and Schneider, 2003; Fang et al., 2011)
and therefore were selected for the present study. However, only
one compound of that type, 2-hydroxy-9-(40-hydroxy-30-methoxy-
phenyl)-1H-phenalen-1-one (musanolone F (3)), which has a
methoxylated lateral phenyl ring D, was reported from A. preissii
(Schmitt et al., 2000). Musanolone F (3) is biosynthetically derived
from ferulic acid (1), which is an abundant phenylpropanoic acid in
A. preissii (Schmitt and Schneider, 2001). Most of the phenylphena-
lenones occurring in W. thyrsiflora have an unsubstituted ring D
(Fang et al., 2011, 2012), and no compound with a methoxy
substituent in ring D has yet been reported from this plant.
Also no data about the occurrence of phenylpropanoic acids in
W. thyrsiflora are available.
We synthesized [2-13C]ferulic acid ([2-13C]-1), [2-13C]isoferulic
acid, [2-13C]40-methoxycinnamic acid ([2-13C]-2), and [2-13C]30,40-
methylenedioxycinnamic acid and used an isotope dilution
approach to analyze the two plant systems for the occurrence of
these phenylpropanoic acids. Although compound 1 is widely
distributed in plants (Kroon and Williamson, 1999) including
A. preissii (Schmitt and Schneider, 2001), isoferulic acid and
40-methoxycinnamic acid (2) are rarely found in plants (Liu
et al., 1999; Sobolev et al., 2006; Kuddus et al., 2010). Free
30,40-methylenedioxycinnamic acid has to our knowledge been
identified only in Piper philippinum (Chen et al., 2007) and M.
acuminata (Otálvaro et al., 2010). The four [2-13C]-labeled phenyl-
propanoids were separately administered to the two in vitro-root
cultures. 1H NMR and HPLC-guided isolation was used to identify
2.2. Detection and biosynthesis of methoxyphenylphenalenones in
plant root cultures
The administration of [2-13C]ferulic acid (1) and [2-13C]40-
methoxycinnamic acid (2) resulted in their successful incorpora-
tion into the corresponding phenylphenalenones from A. preissii,
whereas no incorporation of 1 into any phenylphenalenone was
detected in the case of W. thyrsiflora. Musanolone F (3) was the