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A new benzoic acid derivative isolated from Piper cf. cumanense Kunth (Piperaceae)
Phytochemistry Letters 6 (2013) 590–592
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
A new benzoic acid derivative isolated from Piper cf. cumanense Kunth (Piperaceae) ˜ o *, Juliet A. Prieto *, Wilman A. Delgado *, Luis E. Cuca * Jorge E. Parra *, Oscar J. Patin Laboratorio de Investigacio´n en Productos Naturales Vegetales, Departamento de Quı´mica, 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
A B S T R A C T
Article history: Received 27 June 2012 Received in revised form 5 July 2013 Accepted 19 July 2013 Available online 2 August 2013
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. ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Piper cf. cumanense Kunth Piperaceae Benzoic acid Antifungal activity Fusarium oxysporum f. sp. dianthi Botrytis cinerea
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 (Garcı´a, 1992; Arnason et al., 2005). To confirm these uses, phytochemical and biological activity studies have been developed.
* Corresponding authors. Tel.: +57 1 3165000x14476; fax: +57 1 3165220. E-mail addresses: [email protected] (J.E. Parra), [email protected] (O.J. ˜ o), [email protected] (J.A. Prieto), [email protected] (W.A. DelPatin gado), [email protected] (L.E. Cuca).
Those studies have allowed the isolation of different compounds such as amides, flavonoids, kavapyrones, lignans, neolignans, 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 (Garcı´a, 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 investigations 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 inflorescenses of P. cf. cumanense Kunth, along with five 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
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2013.07.014
J.E. Parra et al. / Phytochemistry Letters 6 (2013) 590–592
591
Fig. 1. Chemical structure of compound 1. Fig. 2. HMBC correlations of 1.
inflorescences of P. cumanense was fractionated and purified 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 prenylated benzoic acid derivative (Fig. 1). In addition, were isolated five 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 112–113 8C. The IR spectrum shows two intense signals for carbonyl groups at 1689 cm1 and 1635 cm1, and signals for aromatic ring at 1500 cm1 and 1442 cm1. The 1H 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 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 at dC 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 corresponding to a benzoic acid derivative where the aromatic ring is 1,3,4,5tetrasubstituted (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 with signals 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). The signals 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 in 13 C 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 fragments was confirmed by the correlations observed in the 2D experiments 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 dH 3.39 (H-10 ) with carbons at dC 131.5 (C-5) and 166.0 (C-4) allowed to locate the isoprenyl group on the quaternary carbon at dC 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 dH 8.47 (H-2) with the ketone carbonyl carbon at dC 196.2 (C5 5O) and by the correlation between the protons at dH 6.85 (H-20 ) with the quaternary carbon at dC 163 (C-30 ). The HRESIMS in negative mode showed a pseudo-molecular ion peak [MH] m/z 355.1999 (calc. for C22H28O4 356.4592) and the resulting molecular formula was determined as C22H28O4, representing 9 degrees of unsaturation. The compound 1 was denominated as (E)-3-(3,7-dimethyl-1oxo-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 derivatives 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 identified by comparing their spectral data with those reported in the literature. Compound 2 corresponds to cumanensic acid (Parra et al., 2011), previously isolated and identified 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 GC–MS. Four sesquiterpenes accounting 30.3% of relative composition of the mixture were identified as as b-cubebene 4 (5.1%), b-caryophyllene 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 ˜ o and Cuca, 2010). The minimum amount of 1 bioassay (Patin 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 film. 1H and 13C NMR spectra as well as 2D spectra (COSY, HMQC and HMBC) were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz for 1H and 100 MHz for 13C using the solvent peaks as internal references, the spectra were in CDCl3 (d 7.26 in 1H and d 77.0 in 13C). HRMS were determined on a Shimadzu LCMS-IT-TOF mass spectrometer system with ESI in negative ion mode. GC–MS 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 (230–400 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.
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J.E. Parra et al. / Phytochemistry Letters 6 (2013) 590–592
3.2. Plant material
Acknowledgements
The inflorescences 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 identified by Adolfo Jara. A voucher specimen (COL 518185) has been deposited at Herbario Nacional Colombiano, Instituto de Ciencias Naturales, Universidad Nacional de Colombia.
The authors thank to Colciencias (1101-05-17783), Universidad Nacional de Colombia for financial 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.
3.3. Extraction and isolation
References
Air-dried and powdered inflorescences 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 flash chromatography (FC) on silica gel and eluted with CH2Cl2–EtOAc (9:1–3:7) mixtures, to give seven fractions (1–7). The fraction 1 (2061 mg) was purified with CHCl3–MeOH (99:1) to give four fractions (1A– 4A). Fraction 1A (177 mg) was analyzed by GC/MS, corresponding 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 purified by successive FC eluting with CH2Cl2–EtOAc (95:5–7:3), hexane–acetone (7:3–4:6) and hexane–EtOAc (9:1–4:6) mixtures to obtain 97 mg of compound 3.
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3.3.1.1. (E)-3-(3,7-Dimethyl-1-oxo-2,6-octadienyl)-4-hydroxy-5-(3methyl-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 cm1. 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 ). 13 C 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, C80 ). HRESIMS [MH] 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 the ˜ o and Cuca, 2010). The microbioautographic technique (Patin 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 Quı´mica, 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 in glucose 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 significantly 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.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
A new benzoic acid derivative isolated from Piper cf. cumanense Kunth (Piperaceae) ˜ o *, Juliet A. Prieto *, Wilman A. Delgado *, Luis E. Cuca * Jorge E. Parra *, Oscar J. Patin Laboratorio de Investigacio´n en Productos Naturales Vegetales, Departamento de Quı´mica, 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
A B S T R A C T
Article history: Received 27 June 2012 Received in revised form 5 July 2013 Accepted 19 July 2013 Available online 2 August 2013
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. ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Piper cf. cumanense Kunth Piperaceae Benzoic acid Antifungal activity Fusarium oxysporum f. sp. dianthi Botrytis cinerea
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 (Garcı´a, 1992; Arnason et al., 2005). To confirm these uses, phytochemical and biological activity studies have been developed.
* Corresponding authors. Tel.: +57 1 3165000x14476; fax: +57 1 3165220. E-mail addresses: [email protected] (J.E. Parra), [email protected] (O.J. ˜ o), [email protected] (J.A. Prieto), [email protected] (W.A. DelPatin gado), [email protected] (L.E. Cuca).
Those studies have allowed the isolation of different compounds such as amides, flavonoids, kavapyrones, lignans, neolignans, 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 (Garcı´a, 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 investigations 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 inflorescenses of P. cf. cumanense Kunth, along with five 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
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2013.07.014
J.E. Parra et al. / Phytochemistry Letters 6 (2013) 590–592
591
Fig. 1. Chemical structure of compound 1. Fig. 2. HMBC correlations of 1.
inflorescences of P. cumanense was fractionated and purified 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 prenylated benzoic acid derivative (Fig. 1). In addition, were isolated five 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 112–113 8C. The IR spectrum shows two intense signals for carbonyl groups at 1689 cm1 and 1635 cm1, and signals for aromatic ring at 1500 cm1 and 1442 cm1. The 1H 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 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 at dC 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 corresponding to a benzoic acid derivative where the aromatic ring is 1,3,4,5tetrasubstituted (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 with signals 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). The signals 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 in 13 C 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 fragments was confirmed by the correlations observed in the 2D experiments 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 dH 3.39 (H-10 ) with carbons at dC 131.5 (C-5) and 166.0 (C-4) allowed to locate the isoprenyl group on the quaternary carbon at dC 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 dH 8.47 (H-2) with the ketone carbonyl carbon at dC 196.2 (C5 5O) and by the correlation between the protons at dH 6.85 (H-20 ) with the quaternary carbon at dC 163 (C-30 ). The HRESIMS in negative mode showed a pseudo-molecular ion peak [MH] m/z 355.1999 (calc. for C22H28O4 356.4592) and the resulting molecular formula was determined as C22H28O4, representing 9 degrees of unsaturation. The compound 1 was denominated as (E)-3-(3,7-dimethyl-1oxo-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 derivatives 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 identified by comparing their spectral data with those reported in the literature. Compound 2 corresponds to cumanensic acid (Parra et al., 2011), previously isolated and identified 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 GC–MS. Four sesquiterpenes accounting 30.3% of relative composition of the mixture were identified as as b-cubebene 4 (5.1%), b-caryophyllene 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 ˜ o and Cuca, 2010). The minimum amount of 1 bioassay (Patin 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 film. 1H and 13C NMR spectra as well as 2D spectra (COSY, HMQC and HMBC) were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz for 1H and 100 MHz for 13C using the solvent peaks as internal references, the spectra were in CDCl3 (d 7.26 in 1H and d 77.0 in 13C). HRMS were determined on a Shimadzu LCMS-IT-TOF mass spectrometer system with ESI in negative ion mode. GC–MS 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 (230–400 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.
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3.2. Plant material
Acknowledgements
The inflorescences 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 identified by Adolfo Jara. A voucher specimen (COL 518185) has been deposited at Herbario Nacional Colombiano, Instituto de Ciencias Naturales, Universidad Nacional de Colombia.
The authors thank to Colciencias (1101-05-17783), Universidad Nacional de Colombia for financial 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.
3.3. Extraction and isolation
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Air-dried and powdered inflorescences 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 flash chromatography (FC) on silica gel and eluted with CH2Cl2–EtOAc (9:1–3:7) mixtures, to give seven fractions (1–7). The fraction 1 (2061 mg) was purified with CHCl3–MeOH (99:1) to give four fractions (1A– 4A). Fraction 1A (177 mg) was analyzed by GC/MS, corresponding 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 purified by successive FC eluting with CH2Cl2–EtOAc (95:5–7:3), hexane–acetone (7:3–4:6) and hexane–EtOAc (9:1–4:6) mixtures to obtain 97 mg of compound 3.
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3.3.1.1. (E)-3-(3,7-Dimethyl-1-oxo-2,6-octadienyl)-4-hydroxy-5-(3methyl-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 cm1. 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 ). 13 C 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, C80 ). HRESIMS [MH] 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 the ˜ o and Cuca, 2010). The microbioautographic technique (Patin 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 Quı´mica, 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 in glucose 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 significantly 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.