Phytochemistry Letters 6 (2013) 590–592

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 Quımica, Facultad de Ciencias, Universidad Nacional de Colombia, AA

14490, KR 30 45-03, Bogota, Colombia

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 five known compounds has been isolated from the

inflorescences of Piper cf. cumanense Kunth (Piperaceae). The structure was identified 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.

Contents lists available at ScienceDirect

Phytochemistry Letters

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol

1. Introduction

Currently, natural products research is focused on areas such aspharmacy and agriculture among others, looking for contributionsto development new phytosanitary agents to control pests andillnesses that affect many plants which are essential sources to getfood or to be used with industrial purposes. The indiscriminate andconstant use of agrochemicals has caused the emergence ofresistant plagues and phytophatogen microorganisms to thecurrent control methods (Regnault-Roger et al., 2004; Bakouriet 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 composedby 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 (Garcıa, 1992; Arnasonet al., 2005). To confirm these uses, phytochemical and biologicalactivity studies have been developed.

* 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).

1874-3900/$ – see front matter � 2013 Published by Elsevier B.V. on behalf of Phytoc

http://dx.doi.org/10.1016/j.phytol.2013.07.014

Those studies have allowed the isolation of different com-pounds such as amides, flavonoids, kavapyrones, lignans, neo-lignans, piperolides, propenylphenols and terpenes (Parmar et al.,1997), all of them characterized for their insecticide, antifungaland/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 inwet and shaded places (Garcıa, 1992). About 2000 known speciesare distributed in tropical areas around the world (Quijano-Abrilet al., 2006). In Colombia, the Herbario Nacional Colombianoreports the presence of 312 species distributed in all country,which correspond to the 30% of the existing Piper species in theworld. (ICN, 2011). P. cf. cumanense Kunth is a shrub that grows insome American countries (Brazil, Costa Rica, Colombia andEcuador) (Global Biodiversity Information, 2010). Previous inves-tigations on P. cf. cumanense reports that this species exhibitedantiparasitic (Garavito et al., 2006) and antifungal activities (Parraet al., 2011; Svetaz et al., 2010). This paper describes the isolationand characterization of a new benzoic acid derivative (1) frominflorescenses of P. cf. cumanense Kunth, along with five knowncompounds; also in this study we report the antifungal activityagainst F. oxysporum f. sp. dianthi and B. cinerea of compound 1.

2. Results and discussion

Using some chromatographic methods over silica gel, theethanolic extract obtained from the air-dried and powdered

hemical Society of Europe.

Fig. 1. Chemical structure of compound 1.

Fig. 2. HMBC correlations of 1.

J.E. Parra et al. / Phytochemistry Letters 6 (2013) 590–592 591

inflorescences of P. cumanense was fractionated and purified toyield 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 five known compounds, cumanensicacid 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 meltingpoint 112–113 8C. The IR spectrum shows two intense signals forcarbonyl groups at 1689 cm�1 and 1635 cm�1, and signals foraromatic ring at 1500 cm�1 and 1442 cm�1. The 1H NMRspectrum shows signals that integrated for 27 protons and the13C RMN spectrum presents signals for 22 carbon atoms. Thesignals observed at dH 8.47 (d, J = 1.97 Hz, 1H, H-2) and 8.03 (d,J = 1.97 Hz, 1H, H-6) in 1H RMN spectrum together with signals atdC 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 13C RMN spectrum correspondingto a benzoic acid derivative where the aromatic ring is 1,3,4,5-tetrasubstituted (Lago et al., 2004). The signals that appear at dH

5.34 (m, 1H, H-200), 3.39 (d, J = 7.26 Hz, 2H, H-100), 1.77 (d,J = 0.79 Hz, 3H, H-400) and 1.73 (bs, 6H, H-500) for 1H together withsignals at dC 134.0 (C-300), 120.9 (C-200), 27.6 (C-100), 25, 8 (C-500)and 17.8 (C-400) for 13C are characteristic for an isoprenyl group(Flores et al., 2009; Lago et al., 2004; Moreira et al., 1998). Thesignals in 1H RMN spectrum at dH 6.85 (bs, 1H, H-20), 5.14 (m, 1H,H-60), 2.33 (m, 2H, H-40), 2.29 (m, 2H, H-50), 2.23 (d, J = 1.0 Hz, 3H,H-100), 1.73 (bs, 6H, H-90) and 1.65 (s, 3H, H-80) and the signals in13C RMN spectrum at dC 196.2 (C-1), 163.0 (C-30), 133.0 (C-70),122.8 (C-60), 119.1 (C-20), 41.8 (C-40), 26.2 (C-50), 25.8 (C-90), 20.3(C-100) and 17.7 (C-80) are characteristic for an oxogeranyl group(Moreira et al., 1998). Finally, the signal observed at dH 13.81 (s,1H) corresponds to chelated hydrogen of a hydroxyl group (O–H)on the aromatic ring (Flores et al., 2009). Each of the fragmentswas confirmed by the correlations observed in the 2D experi-ments COSY, HMQC and HMBC. To establish the location ofsubstituents on the aromatic ring and the assignment ofquaternary carbons was used HMBC experiment. The correlationof the protons at dH 3.39 (H-10) with carbons at dC 131.5 (C-5) and166.0 (C-4) allowed to locate the isoprenyl group on thequaternary carbon at dC 131.5 (C-5) of the aromatic ring. Theoxogeranyl group was positioning on the C-3 of the aromatic ringby the correlations of the proton at dH 8.47 (H-2) with the ketonecarbonyl carbon at dC 196.2 (C55O) and by the correlationbetween the protons at dH 6.85 (H-20) with the quaternary carbonat dC 163 (C-30). The HRESIMS in negative mode showed apseudo-molecular ion peak [M�H]� m/z 355.1999 (calc. forC22H28O4 356.4592) and the resulting molecular formula wasdetermined as C22H28O4, 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), andwas denominated cumenic acid (Fig. 2).

The cumenic acid may has chemotaxonomic value for the genusbecause 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; Yamaguchiet al., 2006; Lago et al., 2004; Moreira et al., 1998; Baldoqui et al.,1999; Kitamura et al., 2006), where is characteristic observesubstitution patterns like the exhibited by compound 1. Theliterature describes the possible biosynthetic pathway of this typeof substances, showing that the prenylated benzoic acid deriva-tives are biosynthetically related with chromenes, therefore ispossible to say that cumenic acid is biosynthetically related withcumanensic acid also isolated from this species.

The known compounds 2 and 3 were identified by comparingtheir spectral data with those reported in the literature. Compound2 corresponds to cumanensic acid (Parra et al., 2011), previouslyisolated and identified in the leaves of P. cf. cumanense Kunth, and 3corresponds to caryophyllene oxide (Krebs et al., 1990).

The mixture M1 was analyzed by GC and GC–MS. Foursesquiterpenes accounting 30.3% of relative composition of themixture were identified 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 massspectra 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 TLCbioassay (Patino and Cuca, 2010). The minimum amount of 1required for the inhibition of fungal growth was appreciable at1 mg for F. oxysporum f. sp. dianthi and at 10 mg for B. cinerea. Thecompound 1 is promising as it has antifungal activity against F.

oxysporum f. sp. dianthi similar to that of the positive controlBenomyl (<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 500series 1000 spectometer as a thin film. 1H and 13C NMR spectra aswell as 2D spectra (COSY, HMQC and HMBC) were recorded on aBruker Avance 400 spectrometer operating at 400 MHz for 1H and100 MHz for 13C using the solvent peaks as internal references, thespectra were in CDCl3 (d 7.26 in 1H and d 77.0 in 13C). HRMS weredetermined on a Shimadzu LCMS-IT-TOF mass spectrometersystem with ESI in negative ion mode. GC–MS analysis wasperformed in a Agilent Technologies 7890A GC System using fusedcapillary column (RTX-6 60 m � 0.25 mm), He as carrier gas andtemperature programming from 50 to 140 8C (4 8C/min), from 160to 220 8C (2.5 8C/min) and from 220 to 280 8C (8 8C/min). Flashchromatography (FC) was carried out with silica gel (230–400mesh, Merck), and analytical chromatography was performedusing silica gel 60 PF254 (0.25 mm). The visualization of thecompounds was carried out with iodine vapor and UV light of 254and 365 nm.

J.E. Parra et al. / Phytochemistry Letters 6 (2013) 590–592592

3.2. Plant material

The inflorescences of P. cf. cumanense Kunth were collected inthe town of Arbelaez, Cundinamarca department, Colombia, duringAugust 2010 by Wilman Delgado. The plant material was identifiedby Adolfo Jara. A voucher specimen (COL 518185) has beendeposited at Herbario Nacional Colombiano, Instituto de CienciasNaturales, Universidad Nacional de Colombia.

3.3. Extraction and isolation

Air-dried and powdered inflorescences of P. cf. cumanense

Kunth (107 g) was exhaustively extracted with 96% ethanol bymaceration at room temperature. The solvent was evaporatedunder vacuum to afford 23 g of the crude extract. A part of thisextract (7 g) was fractionated by flash chromatography (FC) onsilica gel and eluted with CH2Cl2–EtOAc (9:1–3:7) mixtures, togive seven fractions (1–7). The fraction 1 (2061 mg) waspurified with CHCl3–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 correspondto compound 2.

The fraction 2 (1956 mg) was purified by successive FC elutingwith CH2Cl2–EtOAc (95:5–7:3), hexane–acetone (7:3–4:6) andhexane–EtOAc (9:1–4:6) 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 (CHCl3), melting point 112–113 8C. IR(film) nmax = 3394, 2970, 2924, 1689, 1635, 1604, 1500, 1442,1280, 1026, 910 and 756 cm�1. 1H 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-20), 5.34 (m, 1H, H-200), 5.14(m, 1H, H-60), 3.39 (d, J = 7.26 Hz, 2H, H-100), 2.33 (d, J = 6.46 Hz,2H, H-40), 2.29 (m, 2H, 50), 2.23 (d, J = 1.04 Hz, 3H, H-100), 1.77 (d,J = 0.79 Hz, 3H, H-400), 1.73 (s, 6H, H-90, H-500), 1.65 (s, 3H, H-80).13C NMR (100 MHz, CDCl3): d 196.2 (RCOR, C-10), 172.0 (COOH,C-7), 166 (C, C-4), 163 (C, C-30), 136 (CH, C-6), 134 (C, C-300), 133(C, C-70), 131.5 (C, C-5), 130.9 (CH, C-2), 122.8 (CH, C-60), 120.9(CH, C-200), 119.5 (C, C-3), 119.1 (CH, C-20), 118.8 (C, C-1), 41.8(CH2, C-40), 27.6 (CH2, C-10), 26.2 (CH2, C-50), 25.8 (CH3, C-90),25.8 (CH3, C-500), 20.3 (CH3, C-100), 17.8 (CH3, C-400), 17.7 (CH3, C-80). HRESIMS [M�H]� m/z 355.1999 (calc. for C22H28O4

356.4592).

3.4. Antifungal assay

The antifungal activity of the isolated compounds against F.

oxysporum f. sp. dianthi and B. cinerea was determined using thebioautographic technique (Patino and Cuca, 2010). The micro-organisms used in the antifungal assay have been maintained atthe Universidad Nacional de Colombia – Bogota (Laboratorio deInvestigacion en Productos Naturales Vegetales, Departamentode Quımica, Facultad de Ciencias). 10 ml of the solutions wereprepared, in different concentrations, corresponding to 100, 50,25, 10, 5, 2 and 1 mg of pure compounds. The samples wereapplied to TLC plates, and then were sprayed with a sporesuspension of fungi in glucose and salt solution and incubated for72 h in the darkness in a moistened chamber at 25 8C. Exposureof TLC plates to UV light (254 nm) and iodine vapourssignificantly enhanced contrast in order to detect inhibitionzones, indicating the minimal amount of compound required forthe inhibition of fungal growth. Benomyl was used as positivecontrol (1 mg), and the solvents used to dissolved the sampleswere the negative controls.

Acknowledgements

The authors thank to Colciencias (1101-05-17783), UniversidadNacional de Colombia for financial support, to Universidad deCundinamarca (Fusagasuga). Also thank to NMR Laboratory andLCMS Laboratory at Universidad Nacional de Colombia-Bogota forrecording of NMR spectra and HRESIMS, respectively.

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