Production of stilbenoids and phenolic acids by the peanut plant at early stages of growth

Article abstract of DOI:10.1021/jf0602673

The peanut plant (Arachis hypogaea) is known to produce stilbene phytoalexins as a defensive response to fungal invasion; however, the distribution of phytoalexins among different organs of the peanut plant at early stages of growth under axenic conditions has not been studied. Axenic plants produced a stilbenoid, resveratrol, as well as soluble bound and free phenolic acids, including 4-methoxycinnamic acid, which is reported in peanuts for the first time. Neither resveratrol nor phenolic acids were found in the root mucilage; the prenylated stilbenes were restricted to the mucilage and were not found in other organs of the peanut plant. These findings may lead to a better understanding of the defensive role of peanut stilbenes and phenolic acids.

Full text of DOI:10.1021/jf0602673

J. Agric. Food Chem. 2006, 54, 3505−3511 3505
Production of Stilbenoids and Phenolic Acids by the Peanut
Plant at Early Stages of Growth
,
†
†
‡
VICTOR S. SOBOLEV,* BRUCE W. HORN, THOMAS L. POTTER,
STEPHEN T. DEYRUP,§ AND JAMES B. GLOER§
National Peanut Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
P.O. Box 509, Dawson, Georgia 39842; Southeast Watershed Research Laboratory, Agricultural
Research Service, U.S. Department of Agriculture, P.O. Box 946, Tifton, Georgia 31793; and
Department of Chemistry, 305 Chemistry Building, The University of Iowa, Iowa City, Iowa 52242
The peanut plant (Arachis hypogaea) is known to produce stilbene phytoalexins as a defensive
response to fungal invasion; however, the distribution of phytoalexins among different organs of the
peanut plant at early stages of growth under axenic conditions has not been studied. Axenic plants
produced a stilbenoid, resveratrol, as well as soluble bound and free phenolic acids, including
4-methoxycinnamic acid, which is reported in peanuts for the first time. Neither resveratrol nor phenolic
acids were found in the root mucilage; the prenylated stilbenes were restricted to the mucilage and
were not found in other organs of the peanut plant. These findings may lead to a better understanding
of the defensive role of peanut stilbenes and phenolic acids.
KEYWORDS: Resveratrol; phytoalexins; mucilage; p-coumaric acid; ferulic acid; caffeic acid; 4-meth-
oxycinnamic acid; phenolic acids; peanuts; groundnuts; Arachis hypogaea; HPLC-MS
INTRODUCTION
EXPERIMENTAL PROCEDURES
Stilbenoids are produced by the peanut plant as a defensive
response to fungal invasion and other exogenous stimuli (1-
0). Peanut stilbenoids are suggested to serve as phytoalexins
Reagents, Materials, and Basic Apparatus. HPLC-grade solvents
used in the sample extraction and preparation of the mobile phase were
obtained from Fisher (Suwanee, GA). Celite 545, NaOH, and Na SO
2 4
1
(
were purchased from J. T. Baker (Phillipsburg, NJ). A 2% NaOCl
solution was prepared by dilution of household bleach (Clorox) with
distilled water (1:2, v/v). A high-speed blender (13000 rpm) with a
glass jar (General Electric) was used in the research. Medium for peanut
germination was composed of 1% agar (Difco) in sterile water; 25 mL
was placed in each 100 mm × 15 mm Petri dish.
5, 6, 8, 9, 11, 12) and possess antifungal activity against
Aspergillus flaVus, A. parasiticus, and other fungi (2, 7, 11, 13)
and play a key defensive role in peanuts when the water activity
of the plant tissues is sufficiently high (1). Phenolic acids are
aromatic secondary plant metabolites that are widespread
throughout the plant kingdom (14). Phenolic acids have been
connected with diverse functions, including nutrient uptake,
protein synthesis, enzyme activity, photosynthesis, dormancy,
and allelopathy; they are known to serve as structural compo-
nents and are suggested to play a role as defensive compounds
15-20). At least seven basic phenolic acids have been detected
in peanut kernels and are associated with peanut dormancy (15,
1). The biosynthetic origin of phenolic acids as well as stilbenes
is well-known (22-24); however, their particular role and sites
of accumulation in many plants, including peanuts, are not
known or understood. The purpose of this work was to
characterize the distribution and levels of stilbenes and phenolic
acids in peanut organs at early stages of growth under axenic
conditions.
Reference Compounds. trans-Resveratrol (∼99%) was purchased
from Sigma. p-Coumaric, ferulic, caffeic, and 4-methoxycinnamic acids
2
were purchased from Aldrich (Milwaukee, WI). ESI-MS/MS (MS )
and UV spectra were obtained for the above commercial compounds
to serve as comparison standards for identifying trans-resveratrol and
phenolic acids (Figure 1) extracted from peanuts. The values deter-
(
+
mined in this research are given in parentheses as [M + H] values
-
for trans-resveratrol and 4-methoxycinnamic acid, or as [M - H]
2
values for phenolic acids followed by UV absorption maxima: trans-
resveratrol (m/z 229; 305 and 317 nm); p-coumaric acid (m/z 163; 293
sh, 300 sh, and 308 nm); caffeic acid (m/z 179; 299 and 323 nm);
ferulic acid (m/z 193; 299 sh, and 322 nm); 4-methoxycinnamic acid
(m/z 179; 293 sh, 300 sh, and 308 nm).
Plant Material and Processing. Seeds of two runner peanut
cultivars, Georgia Green and Virugard, and a Valencia cultivar, GT-
01, were used in the present study. Mature sound peanut pods were
1
surface-sterilized with 2% NaOCl followed by drying at room tem-
perature on paper towels overnight (25). The pods were manually
cracked, the kernels were aseptically removed from the hulls, and
blemish-free kernels were selected for the experiments. The peanut
kernels (three per plate) were incubated on agar for 5 days at 30 °C
*
Author to whom correspondence should be addressed [telephone (229)
9
95-7446; fax (229) 995-7416; e-mail vsobolev@nprl.usda.gov].
†
National Peanut Research Laboratory.
‡
Southeast Watershed Research Laboratory.
§
University of Iowa.
1
0.1021/jf0602673 CCC: $33.50
© 2006 American Chemical Society
Published on Web 04/22/2006

3506 J. Agric. Food Chem., Vol. 54, No. 10, 2006
Sobolev et al.
analyzed by HPLC. Alternatively, the peanut organs were subjected to
acidic hydrolysis (20) followed by extraction with EtOAc as described
above.
Derivatization of p-Coumaric Acid. The methyl ester of p-coumaric
acid was obtained by treatment of ∼3 mg of the acid 3 (Figure 1)
3
with 1.5 mL of BCl in MeOH (10% w/v) (Alltech). The reaction
mixture was heated for 5 min at 55 °C. Equal amounts of water and
toluene (10 mL) were added to stop the reaction and to extract the
2 4
methyl ester. The toluene layer was dried over Na SO , filtered,
evaporated to dryness with a stream of N , redissolved in MeOH, and
2
subjected to HPLC-diode array detection-MS analysis.
Full methylation of p-coumaric acid to obtain methyl 4-methoxy-
cinnamate was accomplished by dissolving ∼1.5 mg of p-coumaric
acid 3 (Figure 1) in 0.6 mL of acetone and combining the solution
with 200 mg of K CO and 80 µL of dimethyl sulfate in a 4-mL clear
2 3
glass vial sealed with a Teflon-lined cap. The reaction mixture was
agitated with a magnetic stirring bar for 1 h at room temperature. The
Figure 1. Structures of trans-resveratrol (1), mucilagin A (2), p-coumaric
acid (3), caffeic acid (4), ferulic acid (5), and methoxycinnamic acid (6).
vial was then charged with 2 mL of 10% NH
and vigorously stirred for 20 min. The toluene layer was separated
with a Pasteur pipet, dried with anhydrous Na SO , and evaporated to
. The residue was dissolved in methanol
4
OH and 1 mL of toluene
2
4
dryness under a stream of N
for HPLC analysis.
2
4
-Methoxycinnamic acid (6; Figure 1) was obtained by hydrolysis
of methyl 4-methoxycinnamate with 2 N NaOH for 35 min at room
temperature as described above.
HPLC-Diode Array Detection-MS Analyses. Analyses were
performed using an HPLC system equipped with an LC-10ATvp pump
(
5
Shimadzu), an SPD-M10Avp diode array detector covering the 200-
00 nm range with Shimadzu Client/Server software, version 7.3, and
a model 717 plus autosampler (Waters). The separation was performed
on a 50 mm × 4.6 mm i.d., 2.5 µm XTerra MS C18 analytical column
2 2
(Waters). H O (A), MeOH (B), and 1% HCOOH in H O (C) were
Figure 2. Germinated peanut seed after 5 days of incubation: 1, mucilage;
combined in the following gradient: initial conditions, 95% A/0% B/5%
C, increased linearly to 0% A/95% B/5% C in 15 min, held isocratic
for 1 min, decreased to initial conditions in 0.01 min. The flow rate
was 1.2 mL/min. The column was maintained at 35 °C in a model 105
column heater (Timberline Instruments, Boulder, CO). The eluate from
the diode array detector was split with a T-unit (Upchurch Scientific,
Oak Harbor, WA) for optimal MS performance. The flow rate through
the ESI probe was set at 0.375 mL/min. MS analyses were performed
using a Finnigan LCQ Advantage MAX ion trap mass spectrometer
equipped with an ESI interface and operated with Xcalibur version
1.4 software (Thermo Electron Corp., San Jose, CA). The data were
acquired in the full-scan mode (MS) from m/z 100 to 2000. Heated
capillary temperature was 200 °C, sheath gas flow was 30 units,
2
, root tip (4
−5 mm); 3, root; 4, hypocotyl; 5, epicotyl/plumule; 6, cotyledon;
7, testa.
without light and then examined under the stereomicroscope to ensure
axenic conditions. Root mucilage was removed with a microspatula,
and the germinated seed was manually dissected into the following
parts: root tip (4-5 mm), root, hypocotyl, epicotyl, cotyledons, and
testa (Figure 2).
To determine the production of phenolic compounds by the kernels
in their transitional stage from dormancy to germination, peanut kernels
were placed into a beaker with sterile distilled water and left at room
temperature for 18 h to imbibe water (55% of the original kernel
weight). The kernels then were removed from the beaker, blotted with
a paper towel, and manually separated into the following parts: embryo,
cotyledons, and testa. Dry peanut kernels were frozen for ease of
separation into the same parts. In all experiments the plant parts were
frozen before analysis to stop possible enzymatic reactions.
Extraction and Hydrolysis. Root mucilage (0.2 g) was mechanically
removed from the root tips, mixed with an equal amount (w/w) of Celite
2
capillary voltage was 13 V, and source voltage was 4.5 kV. In MS
+
-
analyses, the [M + H] and [M - H] ions observed for each
chromatographic peak in full-scan analyses were isolated and subjected
to source collision-induced dissociation (CID) using He buffer gas. In
all CID analyses, the isolation width, normalized collision energy,
relative activation Q, and activation time were m/z 1.5, 25 or 30%,
2
and 0.25 and 30 ms, respectively. The results of MS experiments are
5
45, and ground in an agate mortar. The mixture was extracted with 1
represented throughout the text as follows: m/z aaa@bb f aaa, ccc,
ddd, where aaa is parent ion, bb is normalized collision energy (%),
and ccc and ddd are fragment ions. Concentrations of trans-resveratrol,
p-coumaric (3), caffeic (4), ferulic (5), and 4-methoxycinnamic (6) acids
mL of MeOH for 30 min with periodic shaking every 5 min. The extract
was filtered through a glass-fiber filter. From 5 to 30 µL of the filtrate
was analyzed by HPLC.
(Figure 1) were determined by reference to peak areas of corresponding
Soluble phenolic compounds were extracted from peanut organs with
standards. Because extinction coefficients for new mucilage stilbenes
could not be determined due to small quantities, we suggested them to
be close to the extinction coefficient of a similar known compound,
trans-3′-isopentadienyl-3,5,4′-trihydroxystilbene (7). For our calcula-
tions we used an extinction coefficient of log 4.34 for each stilbene at
the maximum absorption wavelength for that compound.
8
0% MeOH in a high-speed blender for 1 min. The blender was flushed
with N before the extraction. The blended extract was then agitated
by purging N into the extract for 1 h at room temperature. A filtered
2
2
aliquot of the extract was used for direct determination of free phenolic
acids. Bound soluble phenolic acids were analyzed as free acids after
basic hydrolysis with NaOH. For the hydrolysis, 1 mL of 2 or 4 N
NaOH solution was added to 1 mL of the MeOH extract of each peanut
NMR Characterization. 1H and 13C NMR experiments were
performed on a Bruker DRX-400.
organ, and the mixture was agitated by bubbling N gas for 10-15 s
2
to minimize the oxidation of phenolic acids. The mixture was allowed
to stand for 2 or 4 h at room temperature. The sample was then adjusted
RESULTS AND DISCUSSION
to pH 2-3 with 6 N HCl, and 10 mL of H
compounds were extracted with EtOAc (4 × 10 mL). Combined EtOAc
layers were dried over anhydrous Na SO , evaporated to dryness, and
redissolved in 1 mL of MeOH. From 5 to 30 µL of the extract was
2
O was added. Phenolic
Structure Elucidation. In addition to known phenolic acids
found in embryo, epicotyl/plumule, root, and hypocotyl extracts
after basic hydrolysis (Tables 1 and 2), an unidentified
2
4

Stilbenoids and Phenolic Acids in Peanuts
J. Agric. Food Chem., Vol. 54, No. 10, 2006 3507
Table 1. Presence of Stilbenoidsa and Majorb Phenolic Acids in Parts of Peanut Seeds and Seedlings and Control Seeds of Tested Peanut
Cultivarsc
resveratrol
mucilagin A
minor prenylated
p-coumaric acid
caffeic acid
ferulic acid
methoxycinnamic acid
d
e
e
e
e
plant part
dry seed
(1)
(2)
stilbenes
(3)
(4)
(5)
(6)
cotyledons
embryo
testa
imbibed seed
cotyledons
embryo
+
+
+
+
+
+
+
+
+
+
testa
seedling
cotyledons
epicotyl/plumule
hypocotyl
testa
+
+
+
+
+
+
+
+
+
+
+
+
root
+
+
+
+
root tip (4−5 mm)
mucilage
+
+
a
Stilbenoids were determined before the hydrolysis. b Totaling
c
g
90% (area percent by HPLC). Data from three replicates each of Georgia Green and Virugard runner
d
e
cultivars and a Valencia cultivar; “+” indicates presence of the metabolite. Sobolev et al. (27). As free acids after basic hydrolysis.
Table 2. Total Majora Free and Bound Soluble Phenolic Acids Content in Parts of Seeds and Seedlings of Different Peanut Varieties
p-coumaric acid (3), caffeic acid (4), ferulic acid (5), and 4-methoxycinnamic acid (6) contentb
(mean ± SD, µg/g of wet wt; n ) 3)
organ/section of plant
Georgia Green
Valencia
Virugard
dry seed
cotyledons
embryo
ndc
nd
nd
nd
nd
nd
nd
nd
nd
testa
imbibed seed
cotyledons
embryo
(3) 79.0
(3) 75.0
±
±
±
11.3
8.4
6.6
205.5
73.9
22.8
±
±
±
46.9
7.5
9.0
34.6 ± 21.4
251.5
67.8
±
±
48.8
20.7
(4) 19.7
testa
nd
nd
nd
seedling
cotyledons
(3) 321.9
5) 34.8
(3) 82.4
±
±
±
±
±
±
46.5
6.2
17.9
2.3
212.3
±
±
44.8
9.7
18.1
3.6
8.6
37.8
8.8
124.6
±
±
19.1
7.4
21.9
3.0
(
18.6
83.5
13.8
20.4
63.5
27.7
57.3
13.3
32.2
6.1
hypocotyl
root
±
±
(
(
4) 11.0
6) 22.5
±
±
±
±
±
±
±
7.7
8.8
7.4
(3) 136.8
4) 56.7
6) 109.6
22.7
11.5
18.9
88.9
75.3
26.1
8.3
±
89.0
63.6
81.2
45.2
21.6
25.8
(
±
±
±
(
±
±
±
±
±
±
9.5
root tip (4
−
5 mm)
(6) 127.3
(3) 708.3
no quantitative data
no quantitative data
epicotyl/plumule
856.2
258.7
75.7
±
132.3
1305.8
415.2
103.2
97.0
±
±
±
266.5
97.6
46.3
(4) 202.3
±
37.5
16.6
12.9
(
(
5) 72.1
6) 50.5
nd
±
9.8
63.7
±
±
45.3
testa
mucilage
nd
nd
nd
nd
nd
a
90% (area percent by HPLC). b Calculated as free phenolic acids after basic hydrolysis. c Not detected or a minor phenolic
e10%, area percent by HPLC; see footnote a).
Largest chromatographic peaks totaling
g
acid in the group of remaining compounds (
compound was detected in those same organs in concentrations
comparable to concentrations of the major phenolic acids. The
unknown compound was observed in root and hypocotyl extracts
before hydrolysis only in extremely low concentrations (Table
of the molecular ion was not assured. In contrast to other peanut
phenolic acids, negative ESI-MS did not produce an informative
spectrum. In addition, the unknown was not easily hydrolyzed,
which suggested a phenol derivative rather than an ester.
Negative ESI of the methyl ester of p-coumaric acid produced
3
), which suggested its bound nature. The UV spectrum of the
-
compound virtually matched that of p-coumaric acid (3),
suggesting a characteristic p-coumaroyl moiety (14, 19, 26).
The lower polarity of the unknown compound compared to other
peanut phenolic acids suggested substitution of the carboxyl and/
or phenolic hydroxy group of the p-coumaric acid with a
hydrophobic moiety. Positive ESI-MS produced a complex
spectrum (see Supporting Information); definite identification
an informative spectrum (m/z 177 [M - H] ), which did not
match that of the unknown compound. On the other hand, basic
hydrolysis of synthesized methyl 4-methoxycinnamate (positive
+
ESI, m/z 193@30 f 161 [M - OCH3] ; no informative MS
spectrum was produced by negative ESI) yielded a phenolic
acid identical to the unknown compound. The retention time
and UV and mass spectra of the acid matched those obtained

3508 J. Agric. Food Chem., Vol. 54, No. 10, 2006
Sobolev et al.
Table 3. Majora Free Phenolic Acids Content in Sections of the
Seedlings of Georgia Green Cultivar
Table 4. Resveratrol (1) Content in Parts of Seeds and Seedlings in
Different Peanut Varieties
mean
p-coumaric
acid (3)
±
SD,
µ
g/g of wet weight; n
)
3
resveratrol content
(
mean
± SD, µg/g; n ) 3)
caffeic
acid (4)
ferulic
4-methoxycinnamic
plant part
acid (5)
acid (6)
plant part
dry seed
cotyledons
embryo
Georgia Green
Valencia
Virugard
cotyledons
epicotyl/plumule
hypocotyl
testa
root
mucilage
18.2
±
±
±
2.7
15.0
0.4
ndb
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
1.6
nd
17.5
nd
384.7
3.0
nd
0.02
nd
1.93
±
0.01
<0.01
nd
<0.01
nd
a
±
±
0.7
3.7
testa
±
0.78
1.26
±
0.27
1.02
±
0.14
3.3
nd
±
0.3
imbibed seed
cotyledons
embryo
<0.01
nd
0.01
0.69
0.03
±
nd
±
0.06
0.31
0.02
<0.01
nd
a
testa
0.58
±
0.08
0.38
±
0.02
Largest free phenolic acids chromatographic peaks totaling
g90% (area percent
seedling
cotyledons
hypocotyl
root
by HPLC). b Not detected or a minor free phenolic acid in the group of remaining
free phenolic acids ( 10%, area percent by HPLC; see footnote a).
0.17
±
0.05
±
0.02
nd
nd
±
0.02
e
nd
−<0.01
nd−<0.01
−0.01
0.06
±
0.1
nd−<0.01
−
<0.01
for an authentic standard of 4-methoxycinnamic acid as well
as 4-methoxycinnamic acid (6) isolated from peanuts (see
Supporting Information).
root tip (4
epicotyl/plumule
testa
−
5 mm)
nd
nd
±
nd
nd
±
nd
nd
±
1.27
0.27
0.58
0.02
0.75
0.25
mucilage
nd
nd
nd
1
Analysis of the H NMR spectrum of the unknown compound
revealed the presence of an oxygenated para-disubstituted
a
Not detected.
1
3
aromatic ring, a trans-olefin, and a methoxy group. The
C
NMR spectrum showed resonances consistent with these units,
as well as a signal for an ester or acid carbonyl that must be
linked to the olefin group based on δC values. Comparison of
root tip, and epicotyl/plumule, as well as fast-forming mucilage
contained at most only traces of resveratrol (Table 4). None of
the known stilbenes or phenolic acids was detected in the
mucilage (Tables 1, 2, and 4); instead, several new prenylated
stilbenes (27) were accumulated at extremely high concentra-
tions. Concentrations of mucilagin A (2) and minor prenylated
stilbenes (in parentheses) were as follows: Georgia Green, 251.2
1
13
the H and C NMR spectra of the unknown compound with
literature data allowed for the assignment of the compound as
trans-4-methoxycinnamic acid.
1
trans-4-Methoxycinnamic acid: H NMR (CD3OD, 400
MHz) δ 7.63 (1H, d, J ) 16 Hz, H-7), 7.54 (2H, distorted d, J
(
113.8 (127.5 ( 9.0); Valencia, 339.3 ( 127.5 (311.6 ) 39.3);
)
8.8 Hz, H-2,6), 6.96 (2H, distorted d, J ) 8.8 Hz, H-3,5),
1
3
Virugard, 387.9 ( 31.6 (245.2 ( 44.1). In comparison,
resveratrol content in other parts of the peanut plant (Table 4)
did not exceed 1.93 µg/g (testa of Georgia Green seeds). None
of the mucilage stilbenes was found in the root tip (Table 1).
None of the known peanut stilbene phytoalexins, such as the
arachidins and trans-3′-isopentadienyl-3,5,4′-trihydroxystilbene
(5-8), were detected in any of the peanut organs.
6
.33 (1H, d, J ) 16 Hz, H-8), 3.83 (3H, s, MeO-4); C NMR
CD3OD, 100 MHz) δ171.0 (C-9), 163.2 (C-4), 146.3 (C-7),
31.0 (C-2,6), 128.6 (C-1), 116.8 (C-8), 115.6 (C-3,5), 56.0
(
1
(MeO-4).
Phenolic acids obtained after hydrolysis were characterized
2
by the following MS data: negative ESI-MS m/z 163@30 f
-
-
1
1
63 [M - H] , 119 [M - COOH] for p-coumaric acid;
-
-
Phenolic Acids. Phenolic acids were detected at higher levels
compared to resveratrol. Phenolic acids in free form were
observed in the extracts of cotyledons, hypocotyl, and root at
very low concentrations except in embryos (∼0.4 mg/g) (Table
3). p-Coumaric acid was detected in cotyledons, epicotyl/
plumule, hypocotyl, and root, whereas 4-methoxycinnamic acid
was found only in hypocotyl and root (Table 3). None of the
free acids was found in testa and root mucilage. Free caffeic
and ferulic acids were not detected in any of the organs.
79@30 f 179 [M - H] , 135 [M - COOH] for caffeic
-
-
acid; 193@25 f 193 [M - H] , 179 [M - CH3] , 149 [M -
-
2
COOH] for ferulic acid. Positive ESI-MS m/z 179@30 f
+
+
1
+
79 [M + H] , 161 [M - H2O + H] , 133 [M - H2O - CO
H] were observed for 4-methoxycinnamic acid. The data
for trans-resveratrol were as follows: positive ESI-MS m/z
+
2
+
+
2
29@30 f 229 [M + H] , 211 [M - H2O + H] , 135 [M -
+
C6H5OH + H] . The above data matched data obtained for
corresponding authentic standards.
Structure elucidation of the mucilage stilbenoids is described
by Sobolev et al. (27).
Much higher concentrations of phenolic acids were observed
in the extracts of organs after acidic or basic hydrolysis (Table
2), indicating the bound nature of the phenolic acids. The major
peanut phenolic acids in tested peanuts were identified as
p-coumaric (3), caffeic (4), ferulic (5), and 4-methoxycinnamic
(6) acids (Figure 1). Compared to dry seeds, embryos of the
imbibed seed produced p-coumaric and caffeic acids as major
acids and ferulic and 4-methoxycinnamic acids as minor
components. The epicotyl/plumule part of the seedlings pro-
duced all four phenolic acids as major acids (Tables 1 and 2).
The epicotyl/plumule in seedlings produced significantly higher
concentrations of ferulic and 4-methoxycinnamic acid compared
to imbibed seeds (Table 2). The most significant qualitative
and quantitative changes were observed in the seedlings (Tables
1, 2, and 4). Developing parts of the seedling such as hypocotyl,
root, and root tip produced 4-methoxycinnamic acid in addition
to p-coumaric and caffeic acids. Interestingly, the metabolite
profile of the 4-5-mm-long root tips did not match that of the
Metabolite Distributions. Figure 2 shows the typical ap-
pearance of germinating peanut seed. Metabolism in dormant
peanut seeds is extremely low at seed moisture levels of <10%
but increases rapidly during seed water absorption and hydration
of cell walls and protoplasm (28). Tables 1, 2, and 4 show that
the composition and concentrations of the metabolites changed
from dry to imbibed to germinated seeds.
Stilbenoids. Resveratrol 1 (Figure 1) was detected in all
peanut parts except embryo, epicotyl/plumule, root tip, and
mucilage (Tables 1 and 4). The content of resveratrol in all
parts was extremely low (Table 4); even in the testa, concentra-
tions were <2 µg/g (fresh weight). There were no significant
differences between the cultivars. Higher concentrations of
resveratrol in the dry seed testa can be attributed to lower water
content with a corresponding increase in resveratrol concentra-
tion. Fast-growing parts of the seedling, such as hypocotyl, root,

Stilbenoids and Phenolic Acids in Peanuts
J. Agric. Food Chem., Vol. 54, No. 10, 2006 3509
Figure 3. HPLC of peanut organ extracts after basic hydrolysis: A,
embryos; B, cotyledons; C, roots; D, hypocotyls; E, testae. Peaks:
coumaric acid (3); caffeic acid (4); ferulic acid (5); 4-methoxycinnamic
acid (6). The chromatograms are scaled for direct comparison of
concentrations of phenolic acids.
Figure 4. HPLC of embryo extract (Georgia Green cultivar) after basic
hydrolysis (A) and acidic hydrolysis (B). Peaks: p-coumaric acid (3); caffeic
acid (4); ferulic acid (5); 4-methoxycinnamic acid (6). Chromatogram B is
up-scaled compared to chromatogram A for a better visualization of minor
extract components.
whole root (Tables 1 and 2). The tips produced 4-methoxycin-
namic acid and did not produce other phenolic acids.
stilbenoids are labile and light-sensitive, and phenolic acids,
particularly ferulic acid, can be easily oxidized during prepara-
tion and analysis (4, 19). The acidic pH was chosen to keep
the stilbenes and phenolic acids in their neutral form, which
would provide predictable elution order and better separation
with improved peak shape. Recoveries of trans-resveratrol were
similar to those previously described (4) and exceeded 95% with
spike levels of >100 ng/g. The detection limit of trans-
resveratrol was 10 ng/g, and that for p-coumaric (3), caffeic
(4), ferulic (5), and 4-methoxycinnamic (6) acids ∼15 ng/g
(Figure 1), which was adequate for the quantitation of trace
amounts of the metabolites.
Peanut organs were subjected to acidic and basic hydrolysis
(Figure 4) to determine bound phenolic acids because only a
minor fraction of phenolic acids exists in plants as free acids
(14, 16, 19, 33, 34). Acidic hydrolysis of Georgia Green
embryos yielded 133.1 ( 15.3 µg/g of p-coumaric acid and 20.3
( 3.5 µg/g of caffeic acid. These values were significantly lower
than the values obtained after basic hydrolysis (708.3 ( 75.3
and 202.3 ( 26.1 µg/g, respectively) (Table 2). Phenolic acids
obtained by basic hydrolysis demonstrated more than satisfactory
stability under nitrogen at room temperature (19); therefore,
basic hydrolysis was chosen in this research.
Role of Metabolites. The most interesting results in this
research were obtained from the mucilage, which produced
extremely high concentrations of stilbenoids. The generally
accepted definition of phytoanticipins, antimicrobial compounds
that are present in plants before the plants are challenged by
microorganisms (35), suggests that mucilage stilbenoids repre-
sent phytoanticipins. In some instances, the same compound
may serve in the same plant as a phytoalexin and as a
phytoanticipin (35). Data on accumulation of preformed peanut
stilbenoids in axenic plants are lacking in the literature.
Uncertainty of the roles that stilbenoids play in the resistance
of plants to microbial challenge is due in part to insufficient
data on the antimicrobial properties of this group of compounds.
It is not clear why high concentrations of mucilagin A 2 (Figure
1) are produced by the mucilage under unchallenged conditions,
whereas other parts of the plant do not produce any measurable
The concentrations of major phenolic acids for three different
peanut genotypes are given in Table 2. Dry seed parts did not
accumulate detectable amounts of phenolic acids, whereas
cotyledons and particularly embryos of imbibed seeds produced
phenolic acids up to 0.25 mg/g (p-coumaric acid in the Virugard
cultivar). None of the acids was detected in the testa. Of the
seven parts of the seedling, four produced phenolic acids (in
descending order): epicotyl/plumule, cotyledons, root, and
hypocotyl. The epicotyl/plumule part accumulated high con-
centrations of p-coumaric acid (up to 1.3 mg/g in the Virugard
cultivar). Ferulic acid was produced only by the epicotyl/plumule
and cotyledons only in the seedling. Testa and mucilage of
experimental and control peanuts did not accumulate any of
the phenolic acids (Tables 1 and 2). In all of the experiments
phenolic acids were represented only by the cinnamates 3-6
(Figure 1). Concomitant distribution of phenolic acids and
resveratrol was observed in embryo, epicotyl/plumule, and testa
(Tables 1-4).
Experimental Rationale. The experiments were performed
with axenic seedlings grown under controlled conditions of
constant temperature and minimal light and nutrients. The
rationale for this experimental setup was dictated by the need
to exclude the complexities prevailing in the field (29, 30). For
example, peanut plants can be systemically invaded by A. flaVus
and A. parasiticus from soil during peanut seedling emergence
from the soil (31). Our research concentrated on the determi-
nation of two important groups of phenolic metabolites that may
play a role in young peanut plants as defensive compounds:
stilbenoids and phenolic acids.
Eighty percent MeOH was chosen as an effective solvent both
for free phenolic acids and for soluble bound acids (32). To
study the distribution of major phenolic metabolites in different
organs of the peanut plant, a gradient HPLC method was
developed. The method allowed for a baseline separation of all
major soluble metabolites of different polarities (Figure 3).
Organ extracts were analyzed by HPLC without any purification
to avoid possible loss of stilbenoids and phenolic acids. Peanut

3510 J. Agric. Food Chem., Vol. 54, No. 10, 2006
Sobolev et al.
amounts of stilbenes. The embryo/epicotyl, which is protected
by cotyledons, testa, and peanut shell, does not need to produce
any defensive chemicals at the expense of internal nutrients,
whereas the mucilaginous external layer produces such chemi-
cals in response to challenge by soil pathogenic microorganisms.
Extremely high concentrations of mucilage stilbenes suggest
that they may play a defensive role in the peanut plant because
several of the peanut stilbenes are known to be biologically
active (2, 6, 7, 11). Stilbenes likely possess high antimicrobial
activity because of the presence of lipophilic isoprenyl groups.
Isoprenylation increased the fungitoxicity of corresponding
nonprenylated stilbenes that had higher polarity and lower
activity (8). The interface between root and soil could be not
only a region of high metabolite activity as a result of plant-
microflora associations (36, 37) but also a region of antagonistic
plant-microflora interactions. Root mucilage of Virugard and
Valencia cultivars produced significantly higher levels of
stilbenoids than Georgia Green, which may indicate higher
resistance of Virugard and Valencia root systems to exogenous
infection.
genotypes produced the same set of stilbene phytoalexins and
phenolic acids. Root mucilage contained several new low-
polarity stilbene compounds and none of the known peanut
stilbenes or phenolic acids; the prenylated stilbenes were
restricted to the mucilage and were not found in other organs
of the peanut plant. Mucilagin A 2 (Figure 1) and other root
mucilage stilbenoids (27) may play a role as antimicrobial
compounds. A new low-polarity phenolic acid, 4-methoxycin-
namic acid, may possess properties of a phytoanticipin/phy-
toalexin on the basis of its production by the peanut root as
one of the major phenolic acids.
ABBREVIATIONS USED
ESI, electrospray ionization; HPLC, high-performance liquid
chromatography.
ACKNOWLEDGMENT
We express our gratitude to Dr. N. Puppala for Valencia peanuts.
Supporting Information Available: Positive ESI-MS spec-
3
Resveratrol 1 (Figure 1) has been found at low levels in
sound peanuts (38, 39); however, a lack of potential fungal
infection was not assured. In the present study all experiments
were performed under axenic conditions. Resveratrol does not
seem to serve as a phytoanticipin in axenic peanuts due to its
low concentrations in peanut organs. Damaged parts of the
peanut plant challenged by a fungus produced resveratrol and
other stilbenes in much higher concentrations (1-8). Whether
the above parts of an intact peanut seed and seedling in a fungal
environment are able to synthesize higher levels of resveratrol
was not addressed. Lack of known stilbenoids (5-8) in axenic
peanuts and their accumulation in challenged peanuts indicate
that they should be regarded as phytoalexins.
trum of 6 and positive ESI-MS spectrum of 6. This information
is available free of charge via the Internet at http://pubs.asc.org.
LITERATURE CITED
(
1) Dorner, J. W.; Cole, R. J.; Sanders, T. H.; Blankenship, P. D.
Interrelationship of kernel water activity, soil temperature,
maturity, and phytoalexin production in preharvest aflatoxin
contamination of drought-stressed peanuts. Mycopathologia
1989, 105, 117-128.
(2) Ingham, J. L. 3,5,4′-Trihydroxystilbene as a phytoalexin from
groundnuts (Arachis hypogaea). Phytochemistry 1976, 15, 1791-
1
793.
(
3) Paxton, J. D. Biosynthesis and accumulation of legume phy-
toalexins. In Mycotoxins and Phytoalexins; Sharma, R. P.,
Salunkhe, D. K., Eds.; CRC Press: Boca Raton, FL, 1991; pp
Occurrence of phenolic acids in different peanut organs and
their concentrations varied considerably (Table 2). The presence
of these acids in peanut organs in imbibed seeds and seedlings,
in which metabolism increased with increasing water activity
levels, but not in dry seed (Table 2), suggests that those acids
are associated with peanut dormancy or growth (28). Challenged
peanuts have been used to study phenolic acid elicitation and
phenolic acid antifungal activity (16, 21, 40). Ferulic acid, for
instance, possesses a wide spectrum of antiviral and antimicro-
bial activity (40). As an antioxidant, caffeic acid was a potent
anti-aflatoxigenic agent that completely inhibited production of
aflatoxins by a toxigenic A. flaVus strain at a concentration of
4
85-499.
(
(
(
(
4) Sobolev, V. S.; Cole, R. J.; Dorner, J. W.; Yagen, B. Isolation,
purification, and liquid chromatographic determination of stilbene
phytoalexins in peanuts. J. AOAC Int. 1995, 78, 1177-1182.
5) Arora, M. K.; Strange, R. N. Phytoalexin accumulation in
groundnuts in response to wounding. Plant Sci. 1991, 78, 157-
1
63.
6) Keen, N. T.; Ingham, J. L. New stilbene phytoalexins from
American cultivars of Arachis hypogaea. Phytochemistry 1976,
1
5, 1794-1795.
7) Cooksey, C. J.; Garratt, P. J.; Richards, S. E.; Strange, R. N. A
dienyl stilbene phytoalexin from Arachis hypogaea. Phytochem-
istry 1988, 27, 115-116.
0
.1% (18). At the same time caffeic acid did not have a visible
effect on fungal development at this concentration (18). Other
phenolic acids and stilbenoids that also possess antioxidative
properties may share a similar protective mechanism suggested
to be fungal “oxidative stress” (18). Ferulic and p-coumaric acids
at concentrations of 0.1 mM completely inhibited aflatoxin B1
biosynthesis by A. flaVus on day 4 of incubation (16). However,
a “phytoalexin-like” elicitation of p-coumaric and ferulic acids
in peanuts challenged by A. flaVus cannot be explained merely
by their antifungal properties (16). More likely, hydrophobic
phenolic acids, such as 4-methoxycinnamic acid 6 (Figure 1),
and an unknown low-polarity phenolic acid in peanuts (16) may
serve as antimicrobial agents. Hydrophobic moieties may
increase the antimicrobial activity of peanut preformed or
elicited cinnamates on the basis of their similarity to peanut
stilbenoids (8).
(8) Aguamah, G. A.; Langcake, P.; Leworthy, D. P.; Page, J. A.;
Pryce, R. J.; Strange, R. N. Two novel stilbene phytoalexins
from Arachis hypogaea. Phytochemistry 1981, 20, 1381-1383.
9) Edwards, C.; Strange, R. N. Separation and identification of
phytoalexins from leaves of groundnut (Arachis hypogaea) and
development of a method for their determination by reversed-
phase high-performance liquid chromatography. J. Chromatogr.
(
1
991, 547, 185-193.
(
10) Strange, R. N.; Ingham, J. L.; Cole, D. L.; Cavill, M. E.; Edwards,
C.; Cooksey, C. J.; Garratt, P. J. Isolation of the phytoalexin
medicarpin from leaflets of Arachis hypogaea and related species
of the Tribe Aeschynomeneae. Z. Naturforsch. 1985, 40C, 313-
3
16.
(
11) Wotton, H. R.; Strange, R. N. Circumstantial evidence for
phytoalexin involvement in the resistance of peanuts to Aspergil-
lus flaVus. J. Gen. Microbiol. 1985, 131, 487-494.
12) Chung, I.-M.; Parck, M. R.; Chun, J. C.; Yun, S. J. Resveratrol
accumulation and resveratrol synthase gene expression in
response to abiotic stress and hormones in peanut plants. Plant
Sci. 2003, 164, 103-109.
(
This research demonstrated that seven parts of the peanut
plant had different abilities to produce stilbene phytoalexins and
phenolic acids under axenic conditions. Three different peanut

Stilbenoids and Phenolic Acids in Peanuts
J. Agric. Food Chem., Vol. 54, No. 10, 2006 3511
(
13) Keen, N. T. The isolation of phytoalexins from germinating seeds
of Cicer arietinum, Vigna sinensis, Arachis hypogaea, and other
plants. Phytopathology 1975, 65, 91-92.
(27) Sobolev, V. S.; Potter, T. L.; Horn, B. W. Prenylated stilbenes
from peanut root mucilage. Phytochem. Anal. 2006, in press.
(28) Ketring, D. L.; Brown, R. H.; Sullivan, G. A.; Johnson, B. B.
Growth physiology. In Peanut Science and Technology; Pattee,
H. E., Young, C. T., Eds.; APRES: Yoakum, TX, 1982; 825
pp.
(29) McCully, M. E. Roots in soil: unearthing the complexities of
roots and their rhizospheres. Annu. ReV. Plant. Physiol. Plant.
Mol. Biol. 1999, 50, 695-718.
(30) Muraoka, Y.; Hamakawa, E.; Toyota, K.; Kimura, M. Observa-
tion of microbial colonization on the surface of rice roots along
with their development. Soil Sci. Plant Nutr. 2000, 46, 491-
502.
(31) Pitt, J. I.; Dyer, S. K.; McCammon, S. Systemic invasion of
developing peanut plants by Aspergillus flaVus. Lett. Appl.
Microbiol. 1991, 13, 16-20.
(32) Yu, J.; Ahmedna, M.; Goktepe, I. Effects of processing methods
and extraction solvents on concentration and antioxidant activity
of peanut skins phenolics. Food Chem. 2005, 90, 199-206.
(33) Talcott, S. T.; Duncan, C. E.; Del Pozo-Insfran, D.; Gorbet, D.
W. Polyphenolic and antioxidant changes during storage of
normal, mid, and high oleic acid peanuts. Food Chem. 2005,
89, 77-84.
(34) Dabrowski, K. J.; Sosulski, F. W. Composition of free and
hydrolyzable phenolic acids in defatted flours of 10 oilseeds. J.
Agric. Food Chem. 1984, 32, 128-130.
(35) VanEtten, H. D.; Mansfield, J. W.; Bailey, J. A.; Farmer, E. E.
Two classes of plant antibiotics: phytoalexins versus “phyto-
anticipins”. Plant Cell 1994, 6, 1191-1192.
(36) Leppard, G. G. Rhizoplane Fibris in wheat: demonstration and
derivation. Science 1974, 185, 1066-1067.
(
14) Robbins, R. J. Phenolic acids in foods: an overview of analytical
methodology. J. Agric. Food Chem. 2003, 51, 2866-2887.
15) Sanders, T. H.; Schubert, A. M.; Pattee, H. E. Maturity
methodology and postharvest physiology. In Peanut Science and
Technology; Pattee, H. E., Young, C. T., Eds.; APRES: Yoakum,
TX, 1982; 825 pp.
(
(16) Fajardo, J. E.; Waniska, R. D.; Cuero, R. G.; Pettit, R. E. Phenolic
compounds in peanut seeds: enhanced elicitation by chitosan
and effects on growth and aflatoxin B production by Aspergillus
1
flaVus. Food Biotechnol. 1994, 8, 191-211.
(
17) Hua, S. S.; Grosjean, O. K.; Baker, J. L. Inhibition of aflatoxin
biosynthesis by phenolic compounds. Lett. Appl. Microbiol. 1999,
2
9, 289-291.
(
18) Kim, J. H.; Campbell, B. C.; Yu, J.; Mahoney, N.; Chan, K. L.;
Molyneux, R. J.; Bhatnagar, D.; Cleveland, T. E. Examination
of fungal stress response genes using Saccharomyces cereVisiae
as a model system: targeting genes affecting aflatoxin biosyn-
thesis by Aspergillus flaVus Link. Appl. Microbiol. Biotechnol.
2
005, 67, 807-815.
(
19) Antolovich, M.; Prenzler, P.; Robards, K.; Ryan, D. Sample
preparation in the determination of phenolic compounds in fruits.
Analyst 2000, 125, 989-1009.
(
20) H a¨ kkinen, S.; Auriola, S. High-performance liquid chromatog-
raphy with electrospray ionization mass spectrometry and diode
array ultraviolet detection in the identification of flavonol
aglycones and glycosides in berries. J. Chromatogr. A 1998, 829,
9
1-100.
(
21) Devi, M. C.; Reddy, M. N. Phenolic acid metabolism of
groundnut (Arachis hypogaea L.) plants inoculated with VAM
fungus and Rhizobium. Plant Growth Regul. 2002, 37, 151-
(37) Bowen, G. D.; Rovira, A. D. Microbial colonization of plant
roots. Annu. ReV. Phytopathol. 1976, 14, 121-144.
(38) Sobolev, V. S.; Cole, R. J. trans-Resveratrol content in com-
mercial peanuts and peanut products. J. Agric. Food Chem. 1999,
47, 1435-1439.
(39) Sanders, T. H.; McMichael, R. W.; Hendrix, K. W. Occurrence
of resveratrol in edible peanuts. J. Agric. Food Chem. 2000, 48,
1243-1246.
1
56.
(
22) Herrmann, K. M. The shikimate pathway: early steps in the
biosynthesis of aromatic compounds. Plant Cell 1995, 7, 907-
9
19.
(
23) Hart, J. H. Role of phytostilbenes in decay and disease resistance.
Annu. ReV. Phytopathol. 1981, 19, 437-458.
(
24) Hahlbrook, K.; Scheel, D. Physiology and molecular biology of
phenylpropanoid metabolism. Annu. ReV. Plant Physiol. Plant
Mol. Biol. 1989, 40, 347-369.
(40) Ou, S.; Kwok, K.-C. Ferulic acid: pharmaceutical functions,
preparation and applications in foods. J. Sci. Food Agric. 2004,
84, 1261-1269.
(
25) Horn, B. W. Colonization of wounded peanut seeds by soil
fungi: selectivity for species from Aspergillus section FlaVi.
Mycologia 2005, 97, 205-220.
Received for review January 27, 2006. Revised manuscript received
March 23, 2006. Accepted March 23, 2006.
(
26) Goetz, G.; Fkyerat, A.; M e´ tais, N.; Kunz, M.; Tabacchi, R.; Pezet,
R.; Pont, V. Resistance factors to grey mould in grape berries:
identification of some phenolics inhibitors of Botrytis cinerea
stilbene oxidase. Phytochemistry 1999, 52, 759-767.
JF0602673