Antioxidant activity of prenylated hydroquinone and benzoic acid derivatives from Piper crassinervium Kunth

Article abstract of DOI:10.1016/j.phytochem.2006.03.001

Growing evidence suggests that RNOS (reactive nitrogen and oxygen species) are involved in the damage of biomolecules, contributing to the aetiology of several human diseases. Thus, the demand for antioxidants has stimulated the search for new compounds with potential use in this field. The in vitro antioxidant potential of prenylated hydroquinones and prenylated 4-hydroxy-benzoic acids from fruits of P. crassinervium was evaluated in terms of their capacity to suppress both DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and chemiluminescence produced from luminol, using 2,2′-azo-bis(2-amidinopropane) (ABAP) as a peroxyl radical source. The inhibition of lipid peroxidation was assessed using liposomes from phosphatidylcholine as a membrane model. The prenylated hydroquinones had higher antioxidant activity than the benzoic acids and, among the hydroquinones, the E isomer was more efficient than the Z isomer.

Full text of DOI:10.1016/j.phytochem.2006.03.001

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 1838–1843
www.elsevier.com/locate/phytochem
Antioxidant activity of prenylated hydroquinone and
benzoic acid derivatives from Piper crassinervium Kunth
a
b
Lydia F. Yamaguchi , Joao Henrique G. Lago b,c, Tatiane M. Tanizaki ,
˜
a
b,
*
Paolo Di Mascio , Massuo J. Kato
a
Departamento de Bioquı´mica, Instituto de Quimica, Universidade de Sa˜o Paulo, Cx Postal 26077, CEP 05599-970, Brazil
Departamento de Quı´mica Fundamental, Instituto de Quimica, Universidade de Sa˜o Paulo, Cx Postal 26077, CEP 05599-970, Brazil
ˆ
´
Faculdade de Ciencias Biologicas, Exatas e Experimentais, Universidade Presbiteriana Mackenzie, CEP 01302-907, Sa˜o Paulo, SP, Brazil
b
c
Received 20 July 2005; received in revised form 29 November 2005
Available online 18 April 2006
Dedicated to Professor Rod Croteau on the occasion of his 60th birthday.
Abstract
Growing evidence suggests that RNOS (reactive nitrogen and oxygen species) are involved in the damage of biomolecules, contrib-
uting to the aetiology of several human diseases. Thus, the demand for antioxidants has stimulated the search for new compounds with
potential use in this field. The in vitro antioxidant potential of prenylated hydroquinones and prenylated 4-hydroxy-benzoic acids from
fruits of P. crassinervium was evaluated in terms of their capacity to suppress both DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and
chemiluminescence produced from luminol, using 2,20-azo-bis(2-amidinopropane) (ABAP) as a peroxyl radical source. The inhibition
of lipid peroxidation was assessed using liposomes from phosphatidylcholine as a membrane model. The prenylated hydroquinones
had higher antioxidant activity than the benzoic acids and, among the hydroquinones, the E isomer was more efficient than the Z isomer.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Piper crassinervium; Piperaceae; Prenylated hydroquinones; Prenylated benzoic acids; Antioxidants; Lipoperoxidation; DPPH; Chemilumin-
scence
1. Introduction
Piper species have also been described to contain struc-
turally similar compounds to prenylated benzoic acids, and
in the case of P. aduncum (Baldoqui et al., 1999) antimicro-
bial and molluscicidal activities were evaluated (Okunade
et al., 1997; Orjala et al., 1993), P. arieianum, P. tabogatum
and P. dilatatum also contain further prenylated benzoic
acids with antifungal activity (Green et al., 1991; Roussis
et al., 1990; Terreaux et al., 1998).
The antioxidant potential of the compounds from
P. crassinervium (1–6) were evaluated through the capacity
to inhibit the luminescence from luminol induced by 2,20-
azo-bis (2-amidinopropane) (ABAP) radical liberation.
Additionally, the ability to scavenge 1,1-diphenyl-2-pic-
ryl-hydrazyl (DPPH), a stable radical was analyzed, as
well. Finally, protection against lipoperoxidation was eval-
uated using Fe3+/EDTA and ascorbic acid induced-perox-
idation in liposome from phosphatidylcholine.
Hydroquinones bearing prenyl moieties are natural
products common to marine organisms such as Ascidiaceae
(Aplidium sp.) and brown algae (Howard et al., 1979; Fadli
et al., 1991) and have rarely been found in other organisms.
The derivatives isolated from leaves of Piper crassinervium
are among the few cases of their occurrence in land plants
(Danelutte et al., 2003; Lago et al., 2004). The biological
activities for such compounds include antitumoral, antileu-
kemic and mitosis inhibition (Muller et al., 1985a,b).
Furthermore they have analgesic, relaxant (De Pasquale
et al., 1991) and antioxidant effects (Cotele et al., 1991).
*
Corresponding author. Tel.: +55 11 30913813; fax: +55 11 38155579.
E-mail address: majokato@iq.usp.br (M.J. Kato).
0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.03.001

L.F. Yamaguchi et al. / Phytochemistry 67 (2006) 1838–1843
1839
as a pale yellow amorphous solid. Its molecular formula
was determined as C17H24O2 by 13C NMR spectroscopy
and HREIMS, which showed a molecular ion peak at m/
z 260.1780. The 13C NMR spectrum revealed 17 carbon
signals, 10 of which were sp2 carbons [d 111.5 (CH),
112.5 (CH), 116.6 (CH), 121.9 (CH), 124.3 (CH), 131.6
(C), 131.4 (C), 136.5 (C), 149.3 (C), 151.6 (C)], and seven
aliphatic carbons [d 56.1 (CH3), 39.8 (CH2), 28.0 (CH2),
26.7 (CH2), 25.2 (CH3), 17.7 (CH3), 16.1 (CH3)]. These
data suggested a geranyl-hydroquinone derivative as for
compounds 2 and 3, previously isolated from leaves of
P. crassinervium (Danelutte et al., 2003; Baldoqui et al.,
1
1999). The H NMR spectrum exhibited three aromatic
proton resonances at d 6.62 (dd, J = 8.5 and 3.5 Hz),
6.65 (d, J = 3.5 Hz) and 6.72 (d, J = 8.5 Hz), which were
indicative of a 1,2,4-trisubstituted aromatic ring, and
two broad-triplets at d 5.27 (J = 7.2 Hz) and 5.09
(J = 6.8 Hz) assignable to olefinic protons in the side-
chain. This spectrum also showed two broad-singlets at
d 1.58 (3 H), 1.67 (6 H), characteristic of methyl groups
in a double bond, and a singlet at d 3.76 (3H) of an aro-
matic methoxyl group. The doublet at d 3.26 (J = 7.2 Hz),
associated to the absence of carbonyl groups absorptions
as revealed by IR spectrum, indicated the presence of a
geranyl moiety linked to the aromatic ring and not an
oxo-geranyl moiety, as observed in compounds 2–6
previously isolated from leaves of P. crassinervium (Lago
et al., 2004).
The positions of the substituents were confirmed
through analysis of the HMBC spectrum. The correlation
between the methoxyl signal at d 3.76 (s) and the carbon
at d 151.6 (C-1) associated to the cross-peaks between d
3.26 (H-10) and d 151.6 (C-1), 131.6 (C-2), 116.6 (C-3),
149.3 (C-4), and 136.5 (C-30), indicated that the methoxyl
group and geranyl moiety were connected to C-1 and C-2
positions in the aromatic ring, respectively. Other impor-
tant correlations are given in Table 1. Therefore, from
2. Results and discussion
2.1. Characterization of the compounds
The compounds 2–6 were previously isolated and char-
acterized as potent antifungal agents and were identified
by direct comparison with authentic standards (Danelutte
et al., 2003; Lago et al., 2004). Compound 1 was isolated
Table 1
1H and 13C NMR data (CDCl3) for the prenylated hydroquinone 1 from P. crassinervium
No.
Ca
H
DQ-COSY
HMBC (H ! C)
1
151.6 (C)
–
–
–
2
3
4
131.6 (C)
116.6 (CH)
149.3 (C)
–
–
H-5
–
–
6.65 d (3.5)
–
C-1, C-5, C-10
–
5
6
112.5 (CH)
111.5 (CH)
39.8 (CH2)
121.9 (CH)
136.5 (C)
28.0 (CH2)
26.7 (CH2)
124.3 (CH)
131.4 (C)
25.2 (CH3)
17.7 (CH3)
16.1 (CH3)
56.1 (CH3)
6.62 dd (8.5, 3.5)
6.72 d (8.5)
3.26 d (7.2)
5.27 br. t (7.2)
–
H-3, H-6
H-5
C-1, C-3
C-2, C-4
C-1, C-2, C-3, C-20, C-30
C-2, C-10, C-40, C-100
–
C-20, C-30, C-60, C-100
C-30, C-60, C-70
C-50, C-80, C-90
–
C-60, C-70, C-90
C-60, C-70, C-80
C-20, C-30, C-40
C-1
10
20
30
40
50
60
70
80
90
100
OMe
a
H-20
H-10, H-100
–
H-50, H-100
H-40, H-60
H-50, H-90
–
H-90
H-60, H-80
H-20
2.02 m
2.11 m
5.09 br.t (6.8)
–
1.58 br.s
1.67 br.s
1.67 br.s
3.76 s
–
Multiplicity determined by DEPT 135°.

1840
L.F. Yamaguchi et al. / Phytochemistry 67 (2006) 1838–1843
Table 2
the above evidence, the structure of 1 was determined as
the new 1-methoxy-4-hydroxy-2-(30,70-dimethyl)-20E, 60-
octadienylbenzene.
Antioxidant properties of the prenylated derivatives 1–6, hydroquinone
and TroloxÒ as determined by the quenching of the free-radical induced
luminescence of luminol, the DPPH radical assay and the inhibition of
lipoperoxidation
2.2. Antioxidant activity
Compounds
Inhibition of
DPPH radical Inhibition of
luminescencea assaya EC50
lipoperoxidationa
DPPH, a stable free radical, has been used to evaluate
free radical scavenging capacities of compounds (Blois,
1958). The scavenging activity of the compounds is shown
in Table 2. Addition of compounds 1–3, TroloxÒ or
hydroquinone to the DPPH radical cation solution elicited
a progressive decrease in the concentration of free radicals
(Fig. 1). The compound 2 (EC50 = 22.08 lM) had the
highest hydrogen-donating capacity. The compounds 1
and 3 were weakly active (EC50 > 40.00 lM), while
compounds 4, 5 and 6 of the benzoic acids derivatives
and even p-hydroxybenzoic acid were inefficient as
antioxidants.
Slope
(lM)
IC50 (lM)
1
2.16 0.04
2.34 0.04
1.15 0.04
0.09 0.11
0.05 0.15
0.05 0.12
–
>40.00
22.08 0.34
>40.00
–
–
–
–
14.48 0.38
26.43 0.43
63.11 0.45
69.54 0.40
81.16 0.39
89.74 0.47
–
2
3
4
5
6
p-Hydroxybenzoic
acid
Hydroquinone
Diacetoxyhy
droquinone
TroloxÒ
2.46 0.06
–
1.20
–
45.00 0.51
–
4.49 0.04
10.00 1.93
3.70 0.42
a
Mean values ( SD); n = 3.
The luminescence of luminol can be induced by alkyl-
peroxyl free radicals generated from pyrolysis of ABAP
(Lissi et al., 1992). The addition of the compounds to reac-
tion mixture (luminol and ABAP) can delay the emission of
luminescence. The dependence of induction time upon anti-
oxidants is shown in Fig. 2, and the relative efficiency of the
different antioxidants was obtained by slopes of the curve
(Table 2).
Prenylated hydroquinones (1–3) were found to be more
potent luminescence inhibitors as compared to benzoic
A
B
1
2
3
60
50
40
30
20
10
0
6
4
5
20
15
10
5
TroloxR
0
0
50
100
150
200
0
5
10
15
20
Concentration (μM)
Fig. 1. The effect of the prenylated hydroquinones 1–3, of the prenylated benzoic acids 4–6, and of TroloxÒ on the induction time of emission of the
radical-induced luminescence of luminol as a function of the concentration of additive.
A
B
120
100
80
60
40
20
0
100
80
60
40
20
0
TroloxR
6
4
5
TroloxR
1
2
3
0
10
20
30
40
0
10
20
30
40
Concentration (μM)
Fig. 2. Inhibition of the stable DPPH radical by different concentrations of the prenylated hydroquinones 1–3, of the prenylated benzoic acids 4–6, and of
TroloxÒ.

L.F. Yamaguchi et al. / Phytochemistry 67 (2006) 1838–1843
1841
acids (4–6) (Table 2, Figs. 2A and B). A preliminary struc-
ture–activity relationship indicated that compound 2, the E
isomer, has a higher activity than its Z isomer 3.
Silica gel (Merck 230–400 mesh) and Sephadex LH-20
(Sigma) were used for all CC separations, while silica gel
60 PF254 (Merck) was used for analytical (0.50 mm) and
preparative TLC (1.0 mm). 1H and 13C NMR spectra were
recorded at 500 and 125 MHz, respectively, using a Bruker
DRX-500 spectrometer, with CDCl3 (Aldrich, CA, USA)
as solvent and TMS as internal standard. Chemical shifts
were reported in d units (ppm) and coupling constants (J)
in Hz. LREIMS and HREIMS were measured at 70 eV,
respectively, on a HP 5990/5988 A and a VG Autospec
spectrometers. IR spectra were measured in KBr pellets
in a Perkin–Elmer Infrared Spectrometer model 1750.
UV spectra were recorded in a HP 8452 A spectrophotom-
eter using CHCl3 as solvent.
The ability of the compounds to suppress peroxidation
was evaluated using
a
Fe3+/EDTA, ascorbic acid-
induced, peroxidation in liposomes of phosphatidylcho-
line (Zhao and Jung, 1995). The concentration of an
antioxidant needed to decrease the lipoperoxidation by
50% (IC50) is a parameter used to measure antioxidant
activity. The IC50 of prenylated hydroquinone 1 (14.48
lM) was higher than others except for TroloxÒ (IC50
2.59 lM) (Table 2).
=
A broad evaluation in the structure–activity relationship
including hydroquinone, diacetoxyhydroquinone, and p-
hydroxybenzoic acid was carried out. Hydroquinone was
a very efficient suppressor of DPPH and chemilumines-
cence but was not as TroloxÒ or prenylated hydroquinone
to inhibit lipoperoxidation. The diacetoxyhydroquinone
and p-hydroxybenzoic acid were not able to act as
antioxidants.
4.2. Plant material
Fresh fruits of P. crassinervium (Kunth) were collected
in Iporanga city in the region of Vale do Ribeira, Atlantic
Forest (Sao Paulo State, Brazil) and identified by Dr. Guil-
˜
lermo E. Delgado (Universidad Nacional Pedro Ruiz
Gallo, Peru). The voucher specimen (Kato-0084) was
3. Conclusions
deposited in the Herbarium of Instituto de Botaˆnica, Sao
˜
Paulo - SP, Brazil.
The prenylated hydroquinones studied have consider-
able radical scavenger capacities in the DPPH, chemilumi-
nescence and lipoperoxidation assays as compared to
benzoic acids. The presence of the hydroxyl group at C-4
confers the antioxidant activity for compounds 1–6 since
the prenylated benzoic acids, diacetoxyhydroquinone and
p-hydroxybenzoic acid, did not display any activity in
chemiluminescence and DPPH assays. Compound 1 had
a higher activity for lipoperoxidation analyses. The higher
efficiency of prenylated hydroquinones 2 and 3 to inhibit
lipoperoxidation, as compared to hydroquinone, should
be due to the presence of the lipophylic prenylated group
that could be inserted into the liposome. The linear hydro-
quinone 2, with an E-configuration, showed higher activity
than 3, probably due to its better insertion in the liposome.
The benzoic acid derivatives 4–6 were not as efficient as
hydroquinone derivatives, and the E-geometries had a
higher capacity to suppress the chemiluminescence than
the Z-form, as suggested by higher conjugation in the
E-configuration.
4.3. Extraction and isolation
Dried fruits (50 g) were extracted with MeOH (three
times, 350 mL, at room temperature, for one day) to yield
a crude extract (4.16 g), which was partitioned between n-
hexane and MeOH/H2O (4:1). The n-hexane phase was
dried (anhydr. Na2SO4) and concentrated in vacuo to give
the corresponding extract (2.48 g). This fraction was sub-
jected to silica gel CC eluted with n-hexane:EtOAc of
increasing polarity to yield 10 fractions. Fraction 2
(121 mg) was applied to a silica gel column eluted with
increasing amounts of EtOAc in n-hexane to yield 6 sub-
fractions (I–VI). Subfraction V (23 mg) was purified by
prep. TLC (n-hexane:CH2Cl2:EtOAc 5:9:1) to give 1
(10 mg). Fraction 3 (381 mg) was submitted to TLC (n-hex-
ane:EtOAc 4:1, two elutions) affording compounds 2
(307 mg) and 3 (22 mg). Compound 4 (38 mg) was
obtained after purification by prep. TLC (n-hexane:EtOAc
7:3) of fraction 6 (144 mg). Fraction 7 (56 mg) was purified
by prep. TLC (CH2Cl2:EtOAc 7:3, two elutions) yielding 5
(6 mg). Fraction 9 (57 mg) was subjected to column chro-
matography on Sephadex LH-20 (n-hexane:CH2Cl2 1:4
and CH2Cl2:Me2CO 3:2) to yield 6 (8 mg).
4. Materials and methods
4.1. General experimental procedures
4.4. 1-Methoxy-4-hydroxy-2(30,70-dimethyl)-2E0,60-
octadienylbenzene (1)
MeOH, CH3CN, n-hexane, EtOH, BuOH, HCl, and
HClO4 were purchased from Merck (Darmstadt, Ger-
many). TroloxÒ, whereas sodium phosphate, Tris, EDTA,
thiobarbituric acid, DPPH, FeCl3, ascorbic acid, luminol,
glycine, NaOH, defeoxamine, p-hydroxybenzoic acid and
hydroquinone were from Sigma (CA, USA). ABAP was
purchased from Polyscience (PA, USA).
Amorphous solid. IR (KBr) mmax cmꢀ1: 3389, 2925, 1499,
1452, 1216, 1037, 804 cmꢀ1. UV kmax (MeOH) nm (loge):
262 (3.71), 320 (3.30) nm; LREIMS (70 eV) m/z. (rel. int.):
260(25), 217(7), 191(52), 176(15), 161(20), 149(10), 137(39),
123(59), 107(26), 91(14), 77(20), 69(53), 55(17), 41(100).

1842
L.F. Yamaguchi et al. / Phytochemistry 67 (2006) 1838–1843
´
HREIMS (70 eV) m/z: 260.1780 (calcd. for C17 H24O2
‘‘Conselho Nacional para o Desenvolvimento Cientıfico e
260.1777). For 1H and 13C NMR spectra: see Table 1.
Tecnologico’’, CNPq (Brazil), by ‘‘Programa de Apoio
´
aos Nu´cleos de Exceleˆncia’’, PRONEX/FINEP (Brazil),
´
4.5. Diacetoxyhydroquinone
‘‘Pro-Reitoria de Pesquisa da Universidade de Sao Paulo
˜
(Brazil)’’ and ‘‘John Simon Guggenheim Memorial Foun-
dation’’ (P.D.M. Fellowship). L. F.Y and J.H.G.L. are re-
cipient of FAPESP fellowship. We are grateful to Prof.
Luis Henrique Catalani for the availability of their
luminometer.
Hydroquinone (60 mg) was acetylated with Ac2O
(7.0 mL) and pyridine. The work-up, followed by silica-
gel chromatography of the crude product furnished the
diacetylated derivative (62 mg, yield 78 %) as a white amor-
phous solid, which was characterized by NMR spectro-
scopic analysis.
4.6. Suppression of luminol luminescence induced by 2,20-
azo-bis(2-amidinopropane) (ABAP) thermolysis
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