Occurrence of Benzoic Acid Esters as Putative Catabolites of Prunasin in Senescent Leaves of Prunus laurocerasus

Article abstract of DOI:10.1021/acs.jnatprod.5b01090

Prunus laurocerasus is an evergreen shrub containing large quantities of the cyanogenic glycoside prunasin (1) in its leaves, which decomposes to prunasin amide (2) or glucose-1-benzoate (4) when the leaves become chlorotic as a result of senescence or pseudosenescence. This study was aimed at the systematic identification of senescence-associated metabolites to contribute further insight into the catabolism of 1. LC-ESIMS profiles of senescent and green leaves were analyzed by principal component analysis. In senescent leaves, the concentrations of 36 compounds were increased significantly including several benzoic acid derivatives, of which prunasin amide-6′-benzoate (5) and prunasin acid-6′-benzoate (6) were isolated and identified. The observed metabolic changes were also induced by treatment of P. laurocerasus shrubs with exogenous ethylene. The data presented support an oxidative catabolism of 1 without release of hydrogen cyanide and the remobilization of its nitrogen in the course of senescence. The results are discussed in the context of functional diversification and drug discovery, where senescent plant material represents a widely unexplored source for the discovery of natural products.

Full text of DOI:10.1021/acs.jnatprod.5b01090

Article
pubs.acs.org/jnp
Occurrence of Benzoic Acid Esters as Putative Catabolites of Prunasin
in Senescent Leaves of Prunus laurocerasus
Jandirk Sendker,* Therese Ellendorff, and Aljoscha Holzenbein
̈
̈ ̈
Institute of Pharmaceutical Biology and Phytochemistry, University of Munster, Correnstraße 48, D-48149 Munster, Germany
*
S Supporting Information
ABSTRACT: Prunus laurocerasus is an evergreen shrub
containing large quantities of the cyanogenic glycoside
prunasin (1) in its leaves, which decomposes to prunasin
amide (2) or glucose-1-benzoate (4) when the leaves become
chlorotic as a result of senescence or pseudosenescence. This
study was aimed at the systematic identification of senescence-
associated metabolites to contribute further insight into the
catabolism of 1. LC-ESIMS profiles of senescent and green
leaves were analyzed by principal component analysis. In
senescent leaves, the concentrations of 36 compounds were
increased significantly including several benzoic acid deriva-
tives, of which prunasin amide-6′-benzoate (5) and prunasin
acid-6′-benzoate (6) were isolated and identified. The observed metabolic changes were also induced by treatment of P.
laurocerasus shrubs with exogenous ethylene. The data presented support an oxidative catabolism of 1 without release of
hydrogen cyanide and the remobilization of its nitrogen in the course of senescence. The results are discussed in the context of
functional diversification and drug discovery, where senescent plant material represents a widely unexplored source for the
discovery of natural products.
herry laurel (Prunus laurocerasus L., Rosaceae) is an
evergreen shrub originating from Asia Minor, where its
suggested to accumulate under conditions of senescence, as
leaves containing this compound as the main constituent are
very easily detachable, thus indicating the formation of a
petiolar abscission layer, which is a well-known indication for
C
fruits are a popular food and both the fruits and leaves are
1
utilized as traditional herbal medicines. More than 200 μmol/g
4
leaf senescence.
of the cyanogenic glycoside prunasin (1) occurs in its freeze-
dried or fresh green leaves, whereas similar molar amounts of
prunasin amide (2) and glucose-1-benzoate (4) replace 1 in
naturally occurring chlorotic leaves (Figure 1). It has been
shown that 2 results from decomposition of 1 in the
nonenzymatic, hydrogen-peroxide-consuming Radziszewski
reaction, especially when strong illumination has bleached
The present study aimed at providing further insight into the
decomposition of 1 in the leaves of P. laurocerasus by
identifying possible catabolites and providing additional
evidence for the interrelation of leaf senescence with the
accumulation of 4.
RESULTS AND DISCUSSION
2
,3
■
fresh green leaves during air drying. In contrast, 4 has been
In order to identify senescence-associated metabolites, leaves
from six different P. laurocerasus shrubs were collected and
immediately freeze-dried. The sequential mode of leaf
senescence of this evergreen plant allowed for the collection
of green and senescent leaves at the same time, and thereby leaf
metabolomes could be compared unbiased by temporal
variation. Chlorotic leaves considered as senescent rather than
5
pseudosenescent detached with a force of 6 newtons or less,
whereas it always took more than 10 newtons to detach green
leaves.
LC-MS fingerprints of all samples were analyzed by principal
component analysis (PCA). The fingerprints of senescent
leaves were discriminated readily from the fingerprints of green
Figure 1. Structures of prunasin and related compounds identified in
leaves of Prunus laurocerasus.
Received: December 10, 2015
©
XXXX American Chemical Society and
American Society of Pharmacognosy
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leaves along principal component 1 (PC1) in the score plot.
Consequently, the phytochemical differences between senes-
cent and green leaves clearly outclassed those among different
shrubs. According to previous observations, the molecular
features with the most pronounced loadings on PC1 were
glycosylated derivatives and anitrile glycosides partake in a
recycling pathway for the turnover of cyanogenic glycosides.
Further peaks were found with the exact masses of their
respective sodium adduct ions matching those of prunasin
anitrile (one peak, reduced in senescent leaves), prunasin
pentoside (one peak, unchanged), or prunasin anitrile pento-
2
attributed to 1 for green and to 4 for senescent leaves (Figure
2). For identifying further senescence-associated metabolites,
side (three peaks, increased in senescent leaves), each described
9
by Picm
̌
anova
́
et al. Owing to the low abundance of these
peaks, no mass spectrometric fragmentation nor UV data gave
further evidence of their identity.
It was noted that several of those compounds displaying the
largest differences between senescent and green leaf samples
showed fragments at m/z 105 and m/z 123, which were
10
attributed to benzoic acid derivatives (Figure 3). Searching
Figure 2. PCA score plot (upper panel) that discriminated LC-MS
profiles of green (○) from those of senescent (△) leaf samples of
Prunus laurocerasus along PC1. Labels in the PCA loadings plot (lower
panel) refer to m/z data of prunasin (1) and glucose-1-benzoate (4).
Figure 3. EICs indicative of benzoic acid derivatives of senescent and
normal green leaf samples of Prunus laurocerasus. The peak labeling
refers to Table S1, Supporting Information.
the LC-MS data for these fragments with EICs revealed that,
despite small but consistent amounts of 4, green leaves were
free of benzoic acid derivatives. As benzoic acid has been
suggested to be a catabolite of 1 in P. laurocerasus and Olinia
molecular features with strong negative loadings on PC1 were
selected and validated by tracing back to the original LC-MS
data, comparing the extracted ion chromatogram (EIC) peak
area of each compound’s most abundant pseudomolecular ion
with the Student’s t-test. Senescent leaf samples featured 36
peaks that were increased significantly (p ≤ 0.05) and at least 2-
fold when compared with green leaf samples (Table S1,
Supporting Information). High-resolution MS and, if available,
MS/MS spectra of these peaks were obtained from a
representative LC-MS data set that was analyzed using the
Dissect Compounds algorithm of Data Analysis 4.0 (Bruker
Daltonics). The algorithm detected 251 compounds and
effectively subtracted background MS signals as well as MS
signals of overlapping compounds from their mass spectra. Two
prominent groups of senescence-related metabolites were
identified tentatively on the basis of these data: (i) abscisic
acid-related megastigmane glycosides; such compounds pre-
viously have been detected in several plant species, including
2
ventosa (L.) Cuf., compounds 5 and 6 were selected for
isolation. From their fragment signals, these compounds were
also expected to comprise mandelic acid derivatives. The
compounds were isolated from naturally occurring senescent
leaves and readily identified as benzoyl derivatives of 2 and 3.
1
Both compounds showed in their H NMR spectra distinct
signals for two protons downfield of the anomeric proton (δ
4.44, 4.68 and 4.45, 4.67 ppm, respectively). From the HSQC
spectrum of 6, these signals were attributed to the protons at C-
6 of the glucose moiety. The linkage of this position to the
carbonyl carbon of benzoic acid was established using the
HMBC spectrum. The presence of a mandelic acid moiety in 6
and a mandelic acid amide moiety in 5 was supported by the
MS data; namely, an exact neutral fragment loss between [M +
+
H] and its respective fragment at m/z 267 was 152.0466 amu
(C H O ) for 6 and 151.0646 amu (C H NO ) for 5. Further
8
8
3
8
9
2
6
−8
Prunus spp. (Figure S1, Supporting Information);
(ii) the
support arose from characteristic CO stretching and N−H
concentrations of compounds related to 1 [prunasin amide (2),
prunasin acid (3), and especially 4] were increased strongly in
senescent leaves (Figure 1). The consistent detection of 2 and
deformation vibrations that occurred in their IR spectra (1719
−
1
−1
11
cm for 6 and 1670, 1602 cm for 5). The sugar moiety was
identified as D-glucose by capillary electrophoresis of a
hydrolysate after derivatization with (S)-(−)-1-phenylethyl-
3
also in freeze-dried green leaves is in accordance with a study
9
12
on Prunus dulcis (Mill.) D. A. Webb. These previous authors
amine. Circular dichroism (CD) spectroscopy was applied to
have suggested that these compounds along with their higher
determine the absolute configuration of the mandelic acid parts,
B
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Figure 4. Proposed decomposition of 1 in Prunus laurocerasus under conditions of senescence and pseudosenescence.
using (R)- and (S)-mandelic acid and authenticated 1 and 2
amidase to yield 3. This step has also been discussed to occur in
the recycling pathway proposed by Picmanova et al. based on
(both R-configurated mandelic acid derivatives) as a reference
̌
́
9
(Figure S2, Supporting Information). In agreement with
data from developing plants. It seems reasonable to suppose
that the putative amidase would be upregulated during
senescence, as pseudosenescence caused by abiotic stress
published data for different α-substituted phenylacetic acid
1
3
derivatives, only (S)-mandelic acid showed a positive Cotton
effect at 222 nm, whereas (R)-mandelic acid as well as any
other tested mandelic acid derivative showed a negative Cotton
effect between 210 and 227 nm. Consequently, 6 was identified
unambiguously as prunasin acid-6′-benzoate (prupersin B) and
2
results in accumulation of 2. Compound 4, and in turn
other benzoic acid derivatives, may then derive from 3 without
cleavage of the glucose moiety by oxidation with singlet oxygen,
with or without enzymatic participation (Figure 4). Addition-
ally, 1-O-acylglucose derivatives such as 4 have also been
reported as acyl donor molecules for the acylation of plant
secondary metabolites. Specifically, 4 has been described as an
acyl donor for the benzoylation of glucosinolates in Arabidopsis
5
as prunasin amide-6′-benzoate (persicaside) (Figure 1).
Previously, both compounds have been isolated from the seeds
of Prunus persica L.
14,15
Further compounds with fragments indicative of benzoic acid
were identified tentatively or characterized by their fragmenta-
tion patterns. In particular, these data strongly suggested the
presence of prupersin D (1,6′-dibenzoyl gentiobioside), also
10
thaliana. The reaction is catalyzed by the serine-carbox-
ypeptidase-like acyltransferase SCPL17, which also accepts 1-O-
22
sinapoyl-β-glucose as an acyl donor. Such an enzyme, using 4
as an acyl donor, might also catalyze the formation of 5, 6, and
the tentatively identified molecules prupersin D and
benzoylmalic acid in senescent leaves of P. laurocerasus.
Acylation may modulate the transport and storage of plant
15
known from P. persica, and benzoylmalic acid, which was
identified tentatively from a neutral loss of 134.0223 amu
+
(
C H O ) between [M + H] and the benzoic acid fragment at
4
6
5
1
0
m/z 105.0331. Notably, metabolic profiling of the seeds,
roots, stems, or leaves of up to 12-week-old P. dulcis plants did
not reveal any benzoic acid derivatives. This difference may be
22
secondary metabolites. Therefore, 6′-O-benzoylation may
prepare 2 for further breakdown, and compound 5 rather than
2 could be the substrate for a putative amidase. The observed
presence of 3 with the simultaneous absence of free benzoic
acid, however, supports a simpler breakdown, as shown in
Figure 4.
9
attributed to the different Prunus species or to the
developmental state of senescence. The latter hypothesis is
supported by earlier work where benzoic acid released from
senescent leaves has been suggested as an allelopathic
16,17
mechanism of Prunus serotina Ehrh.
To strengthen the hypothesis that the observed changes in
the metabolic profiles were factually attributable to senescence
and not to chlorosis by nonphysiological bleaching referred to
as pseudosenescence, two small shrubs of P. laurocerasus were
fumigated with ethylene in a commercial banana-ripening
room, and a third shrub was kept as a control. Ethylene-treated
shrubs showed a 4-fold to 8-fold increased number of senescing
leaves after 7 days when compared with the control (Figure 5).
LC-MS analysis of representative leaves sampled after 7 days
showed that, with regard to their contents of benzoic acid
derivatives, (i) senescent leaves differed strongly from young
green leaves and therefore mirrored the aforementioned
phytochemical characteristics. However, there was no difference
between the senescent leaves of control and ethylene-treated
shrubs, which supported the link of 4 with senescence. (ii)
Young green leaves showed considerably smaller amounts of 4
when compared with full-grown green leaves, and (iii) in
contrast to the control, older green leaves from the ethylene-
treated shrubs resembled senescent leaves in their significant
amounts of 5 and 6 and in their detachment force of less than
10 newtons (Figure 6). The latter observation suggests that the
phytochemical changes had begun before chlorosis became
conspicuous.
A relevant question for the apparent metabolic relationship
between the mandelic acid derivative glycosides and 4 was
whether the glucose moiety had been maintained or instead
cleaved at some point. The latter could be true if benzoic acid
originated from benzaldehyde after cyanogenic decomposition
of 1 and was glucosylated afterward. The presence of free
mandelic acid, mandelic acid amide, benzaldehyde, or benzoic
acid would support this hypothesis. These compounds were not
detectable in this study. In order to test if 4 could result from
some kind of oxidative reaction, compounds 1−3 were
incubated with H O at pH 6. Small amounts of 4 were
2
2
detected by LC-MS when 3, but not 1 or 2, was incubated for
0 h.
Oxidative decarboxylation may have decomposed 3 to 4
under consumption of singlet oxygen that evolved sponta-
2
18
neously from the disproportionation of hydrogen peroxide.
Singlet oxygen evolution has been described to increase during
19,20
leaf senescence and also to affect secondary metabolites.
A
comparable decarboxylation reaction has been described to
decompose indole-3-acetic acid to indole-3-carbaldehyde in the
21
presence of singlet oxygen. It may be suggested that under
conditions of senescence, compound 1 decomposes to benzoic
acid derivatives via an oxidative pathway, beginning with the2
formation of 2 by a nonenzymatic Radziszewski reaction,
followed by enzymatic hydrolysis of the amide group by an
A simple but important conclusion from these results is that
senescent leaves may yield significant amounts of natural
products that are hardly detectable in green leaves. With regard
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24
2
-fold amounts when compared with the green leaves.
Comparable to the formation of 2 and possibly 4 in P.
laurocerasus, the formation of artemisinin from its precursor
dihydroartemisinic acid depends on nonenzymatic oxidation.
This is obviously related to an oxidative burst, which is a
19,20,24
prominent and well-known feature of leaf senescence.
Besides their well-established deterrent properties against
herbivores, there is increasing evidence for multiple functions of
25,26
cyanogenic glycosides.
Multifunctionality of 1 in leaves of
P. laurocerasus seems reasonable, as its high concentration levels
clearly outweigh the needs for defense. The suggested
catabolism of 1 during senescence supports this concept with
regard to nitrogen storage, allelopathy, and the regulation of
abiotic stress and reactive oxygen species. Accumulation of 4 in
senescent leaves indicates that the nitrile nitrogen of 1 is
remobilized before leaf abscission, which is in accordance with
Figure 5. Development of new senescent leaves after fumigation of
Prunus laurocerasus shrubs with ethylene. At days 0, 7, and 10, any
chlorotic leaves present were counted and removed: (solid bars)
control shrub; (hatched bars) treated shrub 1; (white bars) treated
shrub 2.
27
the nutrient recycling described for senescent leaves. Benzoic
acid released from 4 has an allelopathic effect on red maple
17
seedlings. The apparent consumption of reactive oxygen
species during the breakdown of 1 indicates a possible
protection of plant tissue from reactive oxygen species that
evolve excessively from senescing and stressed plant
to P. laurocerasus, this applies for 5, 6, and several tentatively
identified benzoic acid and megastigmane derivatives (Figure
S1, Supporting Information), which also occur after ethylene
treatment of cherry laurel leaves in an industrial-scale fruit-
ripening chamber. Although different plant parts vary strongly
in sensitivity, the induction of senescence by exogenously
applied ethylene is regarded as a general feature of higher
19,25,28
parts.
of cyanogenic glycosides: (i) Lotaustralin in leaves of Hevea
brasiliensis (Willd. ex. Juss.) Mull. Arg. follows a circadian
rhythm with decreasing concentrations under illumination and
Further studies support a stress-protective effect
̈
2
3
plants. Consequently, new natural products discovered in
senescent leaves can be expected to be accessible biotechno-
29
recovery in darkness; (ii) the so-called stay-green cultivars of
sorghum contain 3 to 6 times more cyanogenic glycosides than
medium stay-green and senescent types (N. Bodapati, personal
communication), and (iii) drought-stressed sorghum plants
23
logically by a well-established technology, also in larger scale.
Currently, senescent plant material remains a widely unex-
plored source for drug discovery. Whether or not senescence-
associated plant metabolites are likely to show biological
activities remains to be demonstrated. Studies on Artemisia
annua L. provide promising evidence, since the antimalarial
constituent artemisinin is present in senescent leaves in nearly
30
show an increased HCN potential.
Future studies should elucidate the interesting difference in
catabolism of 1 to benzoic acid derivatives in senescent leaves
of P. laurocerasus or to anitrile glycosides in germinating seeds
Figure 6. Overlayed EICs for compounds 4−6 in different leaves from ethylene-treated shrubs and control (overlay of EICs m/z 285.0969,
40.1324, 441.1165 ± 0.005). The forces [newtons] needed to detach the leaves are given in the upper right corner of each chromatogram.
4
D
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9
and newly developed leaves of bitter almonds. However, the
fate of nitrile nitrogen and the role of enzymes in the proposed
catabolism need to be resolved.
Analysis of UHPLC-ESI-qTOF-MS Data. PCA was performed
using Profile Analysis 2.0 with raw data that had been calibrated using
Data Analysis 4.0. Chromatographic peaks were detected using the
Find Molecular Features algorithm (s/n threshold 5, correlation
coefficient threshold 0.7, minimum compound length 5 spectra,
smoothing width 5) on line data after spectral background subtraction
in the time range of 0.5 to 10 min and in the m/z range of 150 to
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using methanol solutions with an Autopol automatic
polarimeter (Rudolph Research Analytic). CD and UV spectra were
recorded using methanol solutions on a JASCO J-815 CD
spectrometer. IR spectra were recorded from pure substance on a
Nicolet 4700 IR spectrometer with an FMIR insert. NMR experiments
were performed on a Varian AS 400 Mercuryplus NMR spectrometer
in deuterated methanol. The preparative HPLC system consisted of
two Waters 515 pumps and a Latek A4080 UV detector (210 nm).
Fast centrifugal partition chromatography (FCPC) was carried out
with a Kromaton FCPC semipreparative instrument. The absolute
configuration of D-glucose was established by capillary electrophoresis
after hydrolysis (2 M TFA, 120 °C, 1 h) and derivatization of the
1
000. A full cross validation was performed with the data set and
resulted in a model containing no outliers. Molecular features were
considered as senescence-associated if the average peak areas obtained
from EICs were significantly different between green and senescent
leaves (p ≤ 0.05) and if the average peak area of senescent leaves was
at least 2-fold greater than the average peak area of green leaves.
2
Full-scan MS- and MS spectra containing the molecular features of
interest were extracted by using the Dissect Compounds function of
Compass DataAnalysis 4.0 SP1. Exact molecular weights were
determined using an in-house VBA script. Fragment ions were
obtained from MS2 experiments of protonated molecules or
ammonium adduct ions or from the Dissect spectra if no suitable
2
12
MS experiment was triggered. Sum formulas were determined using
hydrolysate with S-(−)-1-phenylethylamine.
the Smart Formula tool of Data Analysis. MetFrag was applied to
compare the observed fragments with fragments predicted for
compounds with the same sum formula, using ChemSpider as
Plant Material. Green and senescent leaves of P. laurocerasus were
collected from April to June 2010 from specified plants at 51.965,
.605 ± 100 m (vouchers PBMS 237, 238, 239.1-6, plants identified by
7
31
upstream database.
J.S.). Senescent and green leaves were collected from the plants at the
same time. Senescent leaves were identified by their color and by the
force needed for their detachment. Detachment force was charac-
terized by a spring balance (1 to 10 N) that was attached to the petiole
of a leaf using a wool string. The spring balance was then carefully
pulled while monitoring the scale until the leaf detached or the scale
ended. Green leaves could never be detached within the 10-newton
scale, whereas senescent leaves never needed more than 6 newtons or
even detached manually while trying to fix the wool string.
For PCA, both senescent and green leaves were collected from six
individual shrubs in triplicate and freeze-dried immediately after
harvest. Senescent leaves for the isolation of 5 and 6 were collected
from the same plants and several further shrubs of P. laurocerasus to
afford a sufficient amount of material.
Extraction and Isolation. Freeze-dried senescent leaves (200 g)
of P. laurocerasus were extracted three times with a 10-fold amount of
acetone−water (7:3) in an ultrasonic bath. The extract was freeze-
dried to yield 60 g of dry matter, of which 10 g was resuspended in 20
mL of water and poured into a glass column filled with 170 g of
Extrelut. The column was successively eluted with 2 L of dichloro-
methane and 2 L of 1-butanol, yielding 1.1 and 7.6 g of dried material,
respectively. The 1-butanol fraction was subjected to Sephadex LH-20
chromatography using ethanol as eluant to give 20 fractions.
Compound 6 (26 mg) was isolated from fraction 9 (0.24 g) by
preparative HPLC (Hypersil ODS, 5 μm, 250 × 16 mm eluted with
4
2% methanol in 0.1% TFA at 10 mL/min). Compound 5 was purified
from fraction 8 (2.4 g) by FCPC using ethyl acetate−1-butanol−water
10:3:10; 4 mL/min) for separation in a 200 mL coil at 1000 rpm; the
(
Ethylene Treatment of Whole Shrubs. Three comparable
shrubs of P. laurocerasus (about 50 cm height) were purchased in April
fraction eluting between 92 and 124 mL (0.042 g) was further
subjected to preparative HPLC (see above, but with 26% acetonitrile
in 0.1% TFA as eluant) to yield 3.2 mg of 5.
2
011 in a local market garden. Two of these were treated with ethylene
by placing them in a commercial banana-ripening room (Fruchtimport
vanWylick GmbH) (two fumigation cycles were applied as used for
bananas; details may be obtained on request from the senior author).
The third shrub was used as an untreated control. The development of
new senescent leaves was monitored for each shrub. Representative
leaves were analyzed for their detachment force and the presence of
compounds characteristic for naturally occurring senescent leaves.
UHPLC-ESI-qTOF-MS. Chromatographic separations were per-
formed on a Dionex Ultimate 3000 RS liquid chromatography system
on a Dionex Acclaim RSLC 120 C18 column (2.1 × 100 mm, 2.2 μm)
with a binary gradient (A: water with 0.1% formic acid; B: acetonitrile
with 0.1% formic acid) at 0.8 mL/min: 0 to 9.5 min, linear from 5% B
to 100%B; 9.5 to 12.5 min, isocratic 100% B; 12.5 to 12.6 min, linear
from 100% B to 5% B; 12.6 to 15 min, isocratic 5% B. The injection
volume was 2 μL. Eluted compounds were detected using a Dionex
Ultimate DAD-3000 RS over a wavelength range of 200−400 nm and
a Bruker Daltonics micrOTOF-QII time-of-flight mass spectrometer
equipped with an Apollo electrospray ionization source in positive
mode at 2 Hz over a mass range of m/z 50−1000 using the following
instrument settings: nebulizer gas nitrogen, 5 bar; dry gas nitrogen, 9
L/min, 220 °C; capillary voltage 4500 V; end plate offset −500 V;
transfer time 70 μs; collision gas nitrogen; collision energy 8 eV;
collision RF settings were combined with each single spectrum of 2500
summations as follows: 1250 summations with 130 Vpp + 1250
summations with 500 Vpp. Internal data set calibration (HPC mode)
was performed for each analysis using the mass spectrum of a 10 mM
solution of sodium formiate in 50% 2-propanol that was infused during
LC re-equilibration using a divert valve equipped with a 20 μL sample
loop.
Incubation of 1−3 with H O . Compound 1 was isolated from
2
2
2
1
fresh green leaves of P. laurocerasus as described previously.
1
Compound 2 was obtained from 1 based on a prior investigation.
Compound 3 was obtained as a mixture of 2S- and 2R-epimers after
boiling 2 for 1 h in 0.1 M sodium hydroxide, acidifying with 10%
trifluoroacetic acid to pH 2−3, and repeatedly extracting this solution
with a water-saturated mixture of two parts 1-butanol and one part 2-
propanol. Compounds 1−3 were each dissolved in water to a
concentration of 1 mg/mL (stock solutions). Of each stock solution,
100 μL was mixed in 1.5 mL Eppendorf tubes with 500 μL of
phosphate buffer pH 6 and either 100 μL of 0.5% H O (sample) or
2
2
water (control). Each vial was filled up to a final volume of 1000 μL
and incubated for 20 h at ambient temperature.
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b01090.
Structural formulas of compounds identified tentatively
by MS fragmentation data, CD spectra of compounds 5
and 6, table with MS fragmentation data (PDF)
Tabulated LC-MS data: monoisotopic m/z signals and
m/z signal intensities of all detected peaks of a senescent
leaf sample, ordered by retention time, using tab as data
separator and period as decimal separator (TXT)
E
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Article
(
24) Yang, R. Y.; Zeng, X. M.; Lu, Y. Y.; Lu, W. J.; Feng, L. L.; Yang,
AUTHOR INFORMATION
Corresponding Author
Tel: +49-251-83-33379. Fax: +49-251-83-38341. E-mail:
jandirk.sendker@uni-muenster.de.
■
X. Q.; Zeng, Q. P. Planta Med. 2010, 76, 734−742.
(
(
25) Møller, B. L. Curr. Opin. Plant Biol. 2010, 13, 338−347.
26) Neilson, E. H.; Goodger, J. Q. D.; Woodrow, I. E.; Møller, B. L.
*
Trends Plant Sci. 2013, 18, 250−258.
27) Avila-Ospina, L.; Moison, M.; Yoshimoto, K.; Masclaux-
Daubresse, C. J. Exp. Bot. 2014, 65, 3799−3811.
28) Selmar, D.; Kleinwachter, M. Ind. Crops Prod. 2013, 42, 558−
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(29) Kongsawadworakul, P.; Viboonjun, U.; Romruensukharom, P.;
(
Notes
The authors declare no competing financial interest.
(
5
̈
ACKNOWLEDGMENTS
■
We gratefully acknowledge The Official Medicines Control
Laboratory (OMCL) of the Institute for Public Health
Nordrhein-Westfalen for their support in measuring ATR-IR
spectra and optical rotation, T. J. Schmidt for supporting the
measurement of CD spectra, D. Arifin-Abidin and S. van
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̈
Bommel-Wegmann for in vitro experiments, H. Sheridan for
linguistic support, and Fruchtimport vanWylick GmbH for
providing the facilities for ethylene fumigation. Financial
support of this work by the graduate program of the Faculty
̈
of Chemistry and Pharmacy of the University of Munster is
gratefully acknowledged.
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DOI: 10.1021/acs.jnatprod.5b01090
J. Nat. Prod. XXXX, XXX, XXX−XXX