A new benzoic acid derivative isolated from Piper cf.

cumanense Kunth
(Piperaceae)
Jorge E. Parra *, Oscar J. Patino*, Juliet A. Prieto *, Wilman A. Delgado*, Luis E. Cuca *
Laboratorio de Investigacion en Productos Naturales Vegetales, Departamento de Qumica, Facultad de Ciencias, Universidad Nacional de Colombia, AA
14490, KR 30 45-03, Bogota, Colombia
1. Introduction
Currently, natural products research is focused on areas such as
pharmacy and agriculture among others, looking for contributions
to development new phytosanitary agents to control pests and
illnesses that affect many plants which are essential sources to get
food or to be used with industrial purposes. The indiscriminate and
constant use of agrochemicals has caused the emergence of
resistant plagues and phytophatogen microorganisms to the
current control methods (Regnault-Roger et al., 2004; Bakouri
et al., 2008). Many pathogens such as Fusarium oxysporum
(vascular wilt), Fusarium solani (fruit rot) and Botrytis cinerea
(fruit rot) cause many damage pre and post harvest (Bajpai et al.,
2008).
Piperaceae family is from tropical area of India and is composed
by 5 genera where Piper and Peperomia are the most important.
Peperomia species are used as decorative plant (Dias et al., 2001).
Traditionally, many species of Piper genus have been used as spices,
phytomedicines and pests control agents (Garca, 1992; Arnason
et al., 2005). To conrm these uses, phytochemical and biological
activity studies have been developed.
Those studies have allowed the isolation of different com-
pounds such as amides, avonoids, kavapyrones, lignans, neo-
lignans, piperolides, propenylphenols and terpenes (Parmar et al.,
1997), all of them characterized for their insecticide, antifungal
and/or antibacterial activity (Koroishi et al., 2008; Lago et al., 2004;
Celis et al., 2008; Quilez et al., 2010).
Piper genus includes shrubs and seldom trees, which grow in
wet and shaded places (Garca, 1992). About 2000 known species
are distributed in tropical areas around the world (Quijano-Abril
et al., 2006). In Colombia, the Herbario Nacional Colombiano
reports the presence of 312 species distributed in all country,
which correspond to the 30% of the existing Piper species in the
world. (ICN, 2011). P. cf. cumanense Kunth is a shrub that grows in
some American countries (Brazil, Costa Rica, Colombia and
Ecuador) (Global Biodiversity Information, 2010). Previous inves-
tigations on P. cf. cumanense reports that this species exhibited
antiparasitic (Garavito et al., 2006) and antifungal activities (Parra
et al., 2011; Svetaz et al., 2010). This paper describes the isolation
and characterization of a new benzoic acid derivative (1) from
inorescenses of P. cf. cumanense Kunth, along with ve known
compounds; also in this study we report the antifungal activity
against F. oxysporum f. sp. dianthi and B. cinerea of compound 1.
2. Results and discussion
Using some chromatographic methods over silica gel, the
ethanolic extract obtained from the air-dried and powdered
Phytochemistry Letters 6 (2013) 590592
A R T I C L E I N F O
Article history:
Received 27 June 2012
Received in revised form 5 July 2013
Accepted 19 July 2013
Available online 2 August 2013
Keywords:
Piper cf. cumanense Kunth
Piperaceae
Benzoic acid
Antifungal activity
Fusarium oxysporum f. sp. dianthi
Botrytis cinerea
A B S T R A C T
New benzoic acid derivative (1), together with ve known compounds has been isolated from the
inorescences of Piper cf. cumanense Kunth (Piperaceae). The structure was identied on basis of
spectroscopic analysis and comparison with literature data. The compound (1) showed antifungal
activity against Fusarium oxysporum f. sp. dianthi and Botrytis cinerea.
2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
* Corresponding authors. Tel.: +57 1 3165000x14476; fax: +57 1 3165220.
E-mail addresses: jeparraa@unal.edu.co (J.E. Parra), ojpatinol@unal.edu.co (O.J.
Patino), japrietor@unal.edu.co (J.A. Prieto), wadelgadoa@unal.edu.co (W.A. Del-
gado), lecucas@unal.edu.co (L.E. Cuca).
Contents lists available at ScienceDirect
Phytochemistry Letters
j o u r n al h omep ag e: ww w. el s evi er . co m/ l oc at e/ p hyt ol
1874-3900/$ see front matter 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
http://dx.doi.org/10.1016/j.phytol.2013.07.014
inorescences of P. cumanense was fractionated and puried to
yield cumenic acid 1 ((E)-3-(3,7-dimethyl-1-oxo-2,6-octadienyl)-
4-hydroxy-5-(3-methyl-2-butenyl)benzoic acid), a new preny-
lated benzoic acid derivative (Fig. 1).
In addition, were isolated ve known compounds, cumanensic
acid 2 previously isolated from the leaves, caryophyllene oxide 3,
b-cubebene 4, caryophyllene 5 and a-bergamotene 6.
Compound 1 was obtained as yellow needle with melting
point 112113 8C. The IR spectrum shows two intense signals for
carbonyl groups at 1689 cm
1
and 1635 cm
1
, and signals for
aromatic ring at 1500 cm
1
and 1442 cm
1
. The
1
H NMR
spectrum shows signals that integrated for 27 protons and the
13
C RMN spectrum presents signals for 22 carbon atoms. The
signals observed at d
H
8.47 (d, J = 1.97 Hz, 1H, H-2) and 8.03 (d,
J = 1.97 Hz, 1H, H-6) in
1
H RMNspectrum together with signals at
d
C
171.9 (C-7), 166.0 (C-4), 136.0 (C-6), 131.5 (C-5), 130.9 (C-2),
119.5 (C-3) and 118.8 (C-1) in
13
C RMN spectrumcorresponding
to a benzoic acid derivative where the aromatic ring is 1,3,4,5-
tetrasubstituted (Lago et al., 2004). The signals that appear at d
H
5.34 (m, 1H, H-2
00
), 3.39 (d, J = 7.26 Hz, 2H, H-1
00
), 1.77 (d,
J = 0.79 Hz, 3H, H-4
00
) and 1.73 (bs, 6H, H-5
00
) for
1
H together with
signals at d
C
134.0 (C-3
00
), 120.9 (C-2
00
), 27.6 (C-1
00
), 25, 8 (C-5
00
)
and 17.8 (C-4
00
) for
13
C are characteristic for an isoprenyl group
(Flores et al., 2009; Lago et al., 2004; Moreira et al., 1998). The
signals in
1
H RMNspectrum at d
H
6.85 (bs, 1H, H-2
0
), 5.14 (m, 1H,
H-6
0
), 2.33 (m, 2H, H-4
0
), 2.29 (m, 2H, H-5
0
), 2.23 (d, J = 1.0 Hz, 3H,
H-10
0
), 1.73 (bs, 6H, H-9
0
) and 1.65 (s, 3H, H-8
0
) and the signals in
13
C RMN spectrum at d
C
196.2 (C-1), 163.0 (C-3
0
), 133.0 (C-7
0
),
122.8 (C-6
0
), 119.1 (C-2
0
), 41.8 (C-4
0
), 26.2 (C-5
0
), 25.8 (C-9
0
), 20.3
(C-10
0
) and 17.7 (C-8
0
) are characteristic for an oxogeranyl group
(Moreira et al., 1998). Finally, the signal observed at d
H
13.81 (s,
1H) corresponds to chelated hydrogen of a hydroxyl group (OH)
on the aromatic ring (Flores et al., 2009). Each of the fragments
was conrmed by the correlations observed in the 2D experi-
ments COSY, HMQC and HMBC. To establish the location of
substituents on the aromatic ring and the assignment of
quaternary carbons was used HMBC experiment. The correlation
of the protons at d
H
3.39 (H-1
0
) with carbons at d
C
131.5 (C-5) and
166.0 (C-4) allowed to locate the isoprenyl group on the
quaternary carbon at d
C
131.5 (C-5) of the aromatic ring. The
oxogeranyl group was positioning on the C-3 of the aromatic ring
by the correlations of the proton at d
H
8.47 (H-2) with the ketone
carbonyl carbon at d
C
196.2 (C55O) and by the correlation
betweenthe protons at d
H
6.85 (H-2
0
) with the quaternary carbon
at d
C
163 (C-3
0
). The HRESIMS in negative mode showed a
pseudo-molecular ion peak [MH]

m/z 355.1999 (calc. for

C
22
H
28
O
4
356.4592) and the resulting molecular formula was
determined as C
22
H
28
O
4
, representing 9 degrees of unsaturation.
The compound 1 was denominated as (E)-3-(3,7-dimethyl-1-
oxo-2,6-octadienyl)-4-hydroxy-5-(3-methyl-2-butenyl), and
was denominated cumenic acid (Fig. 2).
The cumenic acid may has chemotaxonomic value for the genus
because these type of compounds have been found in other species
of the genus Piper, as in P. heterophyllum, P. aduncum, P.
lhotzkyanum and P. crassinervium (Flores et al., 2009; Yamaguchi
et al., 2006; Lago et al., 2004; Moreira et al., 1998; Baldoqui et al.,
1999; Kitamura et al., 2006), where is characteristic observe
substitution patterns like the exhibited by compound 1. The
literature describes the possible biosynthetic pathway of this type
of substances, showing that the prenylated benzoic acid deriva-
tives are biosynthetically related with chromenes, therefore is
possible to say that cumenic acid is biosynthetically related with
cumanensic acid also isolated from this species.
The known compounds 2 and 3 were identied by comparing
their spectral data with those reported in the literature. Compound
2 corresponds to cumanensic acid (Parra et al., 2011), previously
isolated and identied in the leaves of P. cf. cumanense Kunth, and 3
corresponds to caryophyllene oxide (Krebs et al., 1990).
The mixture M1 was analyzed by GC and GCMS. Four
sesquiterpenes accounting 30.3% of relative composition of the
mixture were identied as as b-cubebene 4 (5.1%), b-caryophyl-
lene 5 (15.7%), a-bergamotene 6 (5.0%) and caryophyllene oxide
(4.5%) by Nist 0.8 Mass spectral Library and by comparison of mass
spectra of each peak with the reported in literature (Adams, 1995).
The antifungal activity against F. oxysporum f. sp. dianthi and B.
cinerea of 1 was evaluated by direct bioautography in a TLC
bioassay (Patino and Cuca, 2010). The minimum amount of 1
required for the inhibition of fungal growth was appreciable at
1 mg for F. oxysporum f. sp. dianthi and at 10 mg for B. cinerea. The
compound 1 is promising as it has antifungal activity against F.
oxysporum f. sp. dianthi similar to that of the positive control
Benomyl (<1 mg) and of 10 mg for B. cinerea.
3. Experimental
3.1. General
IR spectrum was obtained on a Perkin Elmer FT-IR Panagon 500
series 1000 spectometer as a thin lm.
1
H and
13
C NMR spectra as
well as 2D spectra (COSY, HMQC and HMBC) were recorded on a
Bruker Avance 400 spectrometer operating at 400 MHz for
1
H and
100 MHz for
13
C using the solvent peaks as internal references, the
spectra were in CDCl
3
(d 7.26 in
1
H and d 77.0 in
13
C). HRMS were
determined on a Shimadzu LCMS-IT-TOF mass spectrometer
system with ESI in negative ion mode. GCMS analysis was
performed in a Agilent Technologies 7890A GC System using fused
capillary column (RTX-6 60 m 0.25 mm), He as carrier gas and
temperature programming from 50 to 140 8C (4 8C/min), from 160
to 220 8C (2.5 8C/min) and from 220 to 280 8C (8 8C/min). Flash
chromatography (FC) was carried out with silica gel (230400
mesh, Merck), and analytical chromatography was performed
using silica gel 60 PF254 (0.25 mm). The visualization of the
compounds was carried out with iodine vapor and UV light of 254
and 365 nm.
Fig. 1. Chemical structure of compound 1.
Fig. 2. HMBC correlations of 1.
J.E. Parra et al. / Phytochemistry Letters 6 (2013) 590592 591
3.2. Plant material
The inorescences of P. cf. cumanense Kunth were collected in
the town of Arbela ez, Cundinamarca department, Colombia, during
August 2010 by Wilman Delgado. The plant material was identied
by Adolfo Jara. A voucher specimen (COL 518185) has been
deposited at Herbario Nacional Colombiano, Instituto de Ciencias
Naturales, Universidad Nacional de Colombia.
3.3. Extraction and isolation
Air-dried and powdered inorescences of P. cf. cumanense
Kunth (107 g) was exhaustively extracted with 96% ethanol by
maceration at room temperature. The solvent was evaporated
under vacuum to afford 23 g of the crude extract. A part of this
extract (7 g) was fractionated by ash chromatography (FC) on
silica gel and eluted with CH
2
Cl
2
EtOAc (9:13:7) mixtures, to
give seven fractions (17). The fraction 1 (2061 mg) was
puried with CHCl
3
MeOH (99:1) to give four fractions (1A
4A). Fraction 1A (177 mg) was analyzed by GC/MS, corre-
sponding to a mixture (M1). The fraction 2A (201 mg)
correspond to compound 1 and the fraction 3A correspond
to compound 2.
The fraction 2 (1956 mg) was puried by successive FC eluting
with CH
2
Cl
2
EtOAc (95:57:3), hexaneacetone (7:34:6) and
hexaneEtOAc (9:14:6) mixtures to obtain 97 mg of compound 3.
3.3.1.1. (E)-3-(3,7-Dimethyl-1-oxo-2,6-octadienyl)-4-hydroxy-5-(3-
methyl-2-butenyl)benzoic acid (1)
Clear yellow crystals (CHCl
3
), melting point 112113 8C. IR
(lm) n
max
= 3394, 2970, 2924, 1689, 1635, 1604, 1500, 1442,
1280, 1026, 910 and 756 cm
1
.
1
H NMR spectral data (400 MHz,
CDCl3): d 13.81 (s, 1H, H-4), 8.47 (d, J = 1.97 Hz, 1H, H-2), 8.03 (d,
J = 1.97 Hz, 1H, H-6), 6.85 (s, 1H, H-2
0
), 5.34 (m, 1H, H-2
00
), 5.14
(m, 1H, H-6
0
), 3.39 (d, J = 7.26 Hz, 2H, H-1
00
), 2.33 (d, J = 6.46 Hz,
2H, H-4
0
), 2.29 (m, 2H, 5
0
), 2.23 (d, J = 1.04 Hz, 3H, H-10
0
), 1.77 (d,
J = 0.79 Hz, 3H, H-4
00
), 1.73 (s, 6H, H-9
0
, H-5
00
), 1.65 (s, 3H, H-8
0
).
13
C NMR (100 MHz, CDCl
3
): d 196.2 (RCOR, C-1
0
), 172.0 (COOH,
C-7), 166 (C, C-4), 163 (C, C-3
0
), 136 (CH, C-6), 134 (C, C-3
00
), 133
(C, C-7
0
), 131.5 (C, C-5), 130.9 (CH, C-2), 122.8 (CH, C-6
0
), 120.9
(CH, C-2
00
), 119.5 (C, C-3), 119.1 (CH, C-2
0
), 118.8 (C, C-1), 41.8
(CH
2
, C-4
0
), 27.6 (CH
2
, C-1
0
), 26.2 (CH
2
, C-5
0
), 25.8 (CH
3
, C-9
0
),
25.8 (CH
3
, C-5
00
), 20.3 (CH
3
, C-10
0
), 17.8 (CH
3
, C-4
00
), 17.7 (CH
3
, C-
8
0
). HRESIMS [MH]

m/z 355.1999 (calc. for C

22
H
28
O
4
356.4592).
3.4. Antifungal assay
The antifungal activity of the isolated compounds against F.
oxysporumf. sp. dianthi and B. cinerea was determined using the
bioautographic technique (Patin o and Cuca, 2010). The micro-
organisms used in the antifungal assay have been maintained at
the Universidad Nacional de Colombia Bogota (Laboratorio de
Investigacio n en Productos Naturales Vegetales, Departamento
de Qumica, Facultad de Ciencias). 10 ml of the solutions were
prepared, in different concentrations, corresponding to 100, 50,
25, 10, 5, 2 and 1 mg of pure compounds. The samples were
applied to TLC plates, and then were sprayed with a spore
suspension of fungi inglucose and salt solution and incubated for
72 h in the darkness in a moistened chamber at 25 8C. Exposure
of TLC plates to UV light (254 nm) and iodine vapours
signicantly enhanced contrast in order to detect inhibition
zones, indicating the minimal amount of compound required for
the inhibition of fungal growth. Benomyl was used as positive
control (1 mg), and the solvents used to dissolved the samples
were the negative controls.
Acknowledgements
The authors thank to Colciencias (1101-05-17783), Universidad
Nacional de Colombia for nancial support, to Universidad de
Cundinamarca (Fusagasuga). Also thank to NMR Laboratory and
LCMS Laboratory at Universidad Nacional de Colombia-Bogota for
recording of NMR spectra and HRESIMS, respectively.
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