Antifungal piperolides from Piper malacophyllum (Prels) C. DC.

Abstracts

Bioactivity-guided fractionation of the CH2Cl2 extract from leaves of Piper malacophyllum using bioautography assay against fungi, led to the isolation of two known bioactive piperolides: 4,6-dimethoxy-5-E-phenylbutenolide (1) and 4,6-dimethoxy-5-Z-phenylbutenolide (2). IR, MS and NMR spectroscopic data were used for the identification of these compounds including comparison with previously reported data. The E configuration is associated to higher activity for compound 1, since the minimum amount required for the growth inhibition of Cladosporium cladosporioides and C. sphaerospermum were 1.0/1.0 mug (1) and 5.0/10.0 mug (2), respectively.

Piper malacophyllum; piperolides; antifungal activity

O estudo fitoquímico biomonitorado do extrato em CH2Cl2 das folhas de Piper malacophyllum através da técnica de bioautografia frente a fungos, resultou no isolamento de dois piperolidos bioativos: 4,6-dimetóxi-5-E-fenilbutenolido (1) e 4,6-dimetóxi-5-Z-fenilbutenolido (2). Essas substâncias foram identificadas com base na análise dos espectros de IV, EM e RMN, incluindo técnicas bidimensionais, e comparação com dados descritos na literatura. A configuração E da ligação dupla confere maior atividade para 1 considerando-se que as quantidades mínimas de 1 e 2 necessária para inibir o crescimento dos fungos Cladosporium cladosporioides e C. sphaerospermum foram de 1,0/1,0 mig (1) e de 5,0/10,0 mig (2), respectivamente.

ARTICLE

Antifungal piperolides from Piper malacophyllum (Prels) C. DC.

João Henrique G. Lago^{I, II}; Tatiane M. Tanizaki^I; Maria Claudia M. Young^III; Elsie F. Guimarães^IV; Massuo J. Kato^*, I

^IInstituto de Química, Universidade de São Paulo, CP 26077, 05599-970 São Paulo - SP, Brazil

^IIFaculdade de Ciências Biológicas, Exatas e Experimentais, Universidade Presbiteriana Mackenzie, 01302-970 São Paulo - SP, Brazil

^IIISeção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, CP 4005, 10051 São Paulo - SP, Brazil

^IVInstituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão, 915, 22460-030 Rio de Janeiro - RJ, Brazil

ABSTRACT

Bioactivity-guided fractionation of the CH₂Cl₂ extract from leaves of Piper malacophyllum using bioautography assay against fungi, led to the isolation of two known bioactive piperolides: 4,6-dimethoxy-5-E-phenylbutenolide (1) and 4,6-dimethoxy-5-Z-phenylbutenolide (2). IR, MS and NMR spectroscopic data were used for the identification of these compounds including comparison with previously reported data. The E configuration is associated to higher activity for compound 1, since the minimum amount required for the growth inhibition of Cladosporium cladosporioides and C. sphaerospermum were 1.0/1.0 mg (1) and 5.0/10.0 mg (2), respectively.

Keywords:Piper malacophyllum, piperolides, antifungal activity

RESUMO

O estudo fitoquímico biomonitorado do extrato em CH₂Cl₂ das folhas de Piper malacophyllum através da técnica de bioautografia frente a fungos, resultou no isolamento de dois piperolidos bioativos: 4,6-dimetóxi-5-E-fenilbutenolido (1) e 4,6-dimetóxi-5-Z-fenilbutenolido (2). Essas substâncias foram identificadas com base na análise dos espectros de IV, EM e RMN, incluindo técnicas bidimensionais, e comparação com dados descritos na literatura. A configuração E da ligação dupla confere maior atividade para 1 considerando-se que as quantidades mínimas de 1 e 2 necessária para inibir o crescimento dos fungos Cladosporium cladosporioides e C. sphaerospermum foram de 1,0/1,0 mg (1) e de 5,0/10,0 mg (2), respectivamente.

Introduction

The genus Piper is a source of several classes of bioactive secondary metabolites including alkaloids, amides, flavonoids, benzoic acid derivatives, terpenes, and cyclopentanediones.^1-3 In the course of our search aiming to unravel new antifungal metabolites from plants in Brazilian Atlantic Forest and Cerrado,^4,5 the crude CH₂Cl₂ extract from leaves of Piper malacophyllum was selected for bioactivity-guided phytochemical investigation due to the strong activity observed against Cladosporium cladosporioides and C. sphaerospermum. After several chromatographic steps, this extract yielded two structurally related piperolides, which had their structures identified by analysis of spectrometric data.

Results and Discussion

Bioactivity-guided fractionation of the CH₂Cl₂ extract from leaves of P. malacophyllum by silica gel and Sephadex LH-20 chromatography yielded two butenolides of the piperolide series.

Compound 1 had its molecular formula indicated as C₁₃H₁₂O₄ by analysis of LREIMS (70 eV, [M]^+. at m/z 232), and ¹³C NMR (BBD and DEPT 135º). The IR spectrum exhibited bands at 1748 (conjugated lactone carbonyl group) and at 1592, 1493, and 1446 (aromatic) cm^-1. The ¹H NMR (Table 1) spectrum of 1 showed multiplets at d 7.43-7.45 (2H) and d 7.73 (3H), a pattern of a monosubstituted aromatic ring. This spectrum also exhibited two methoxyl signals at d 3.99 (s) and d 3.65 (s) besides one singlet at d 5.27 (1H). These information, associated to the ¹³C NMR resonances at d 171.7 (C), 168.1 (C=O), 135.0 (C), 88.8 (CH) indicated a piperolide type structure containing one methoxyl group at C-4.⁶ The resonance signals at d 135.0 (C) and d 144.6 (C) are indicative of a tetrasubstituted double bound, and thus the second methoxyl group was placed at C-6. Finally, the signals at d 129.0 (CH), 128.6 (CH), and 131.2 (C) were assigned to C-8/C-12, C-9/C-11, and C-7 carbon atoms of the aromatic ring, respectively. The HMBC spectrum of 1 showed long-range ¹H-¹³C correlations between d 3.99 (OCH₃) and d 171.7 (C-4) and between d 3.65 (OCH₃) and d 144.6 (C-6) confirming the assignments of the methoxyl groups at C-4 and C-6. Additionally, the correlations between H-3 (d 5.27) and C-2 (d 168.1) / C-5 (d 135.0) and between H-8/H-12 (d 7.73) and C-6 (d 144.6) confirmed the piperolide moiety and its connectivity with the aromatic ring as well. The configuration of the double bond was determined based on nOe experiments. Irradiation of d 5.27 (H-3) gave a nOe enhancement for the hydrogens at d 3.99, confirming the positioning of this methoxyl group at C-4. On the other hand, irradiation at d 3.99 (4-OCH₃) gave a positive nOe for the hydrogens at d 3.65 (6-OCH₃) and d 5.27 (H-3), indicating the E configuration to double bond. Therefore, the compound 1 was identified as 4,6-dimethoxy-5-E-phenylbutenolide (5,6-E-fadyenolide), previously isolated from roots of Piper fadyenii. ^6,7

The LREIMS spectrum of 1 and 2 showed the same molecular ion-peak and fragment ions, suggesting similar structures. The IR spectrum with bands at 1745, 1592, 1493, and 1443 cm^-1 confirming the presence of conjugated lactone group and aromatic ring in the structure of compound 2. The ¹³C NMR spectra (Table 1) exhibited signals of two methoxyl groups (d 58.9 / 58.8), two olefinic carbons [d 144.0 (C) and 130.7 (C)], four carbons of the butenolide ring [d 170.8 (C), 167.8 (C=O), 129.0 (C), 87.5 (CH)], and six aromatic carbons [d 130.7 (C), 130.1 (2 CH), 130.0 (CH), 128.0 (2 CH)] similarly to those signals observed to the piperolide 1. The ¹H NMR spectrum showed a multiplet at d 7.37-7.42 (5H), indicative of a monosubstituted aromatic ring, and three singlets at d 5.11 (1H), 3.59 (3H) and 3.74 (3H), which confirmed the structural similarity between 1 and 2. The cross-peaks between d 3.59 (OCH₃) / 170.8 (C-4) and d 3.74 (OCH₃) / 144.0 (C-6) in its HMBC spectrum determined the position of the methoxyl groups at C-4 and C-6, as depicted to compound 1. This spectrum showed also cross peaks between H-3 (d 5.11) and C-2 (d 167.8) / C-5 (d 129.0) and between H-8/H-12 (d 7.37-7.42) and C-6 (d 144.0). Thus the connectivity between the butenolide and aromatic rings and the same planar structure of 1 was confirmed. However, irradiation of signal at d 3.59 (4-OCH₃) gave a nOe enhancement for the hydrogens at d 5.11 (H-3) and d 7.42 (H-8/H-12) while the signal at d 3.74 (6-OCH₃) were not affected, indicating the Z configuration for the double bound. Therefore, the compound 2 was identified as 4,6-dimethoxy-5-Z-phenylbutenolide, as previously defined to 5,6-Z-fadyenolide, also isolated from roots and aerial parts of Piper fadyenii.^6,7

There are only few report of piperolides in Piperaceae species and to date they have been described from Piper fadyenii^6,7 and P. sanctum^8,9 which occur from Jamaica and Mexico, respectively. In such reports⁸ the NMR data of compounds 1 and 2 were assigned only to the butenolide moiety. Thus herein we present an unambiguous assignment of their NMR data based on several 2D spectra including HMBC experiment (Table 1).

Both compounds were isolated in a bioactivity-guided fractionation procedure. The antifungal activity of compounds 1 and 2 was determined by means of direct bioautography on TLC plate¹¹. The detection limits of these compounds required to inhibit growth of the fungus C. cladosporioides and C. sphaerospermum (Table 2) were obtained according to methodology described elsewhere.^5,11,12 Compound 1 presented stronger antifungal activity than that observed for compound 2, suggesting that the configuration of the double bond in the butenolide ring could be associated to their fungitoxic potential. There was no previous report describing the fungitoxic potential of these compounds, mainly 1 which showed a similar activity of the positive control (nystatin and miconazole).

Experimental

Instrumental and chromatography materials

Silica gel (Merck 230-400 mesh) and Sephadex LH-20 (Pharmacia) were used for column chromatographic separation. Silica gel PF₂₅₄ (Merck) was used to TLC preparative purification. The NMR spectra were recorded on Bruker DRX-500 operating at 500 MHz to ¹H and at 125 MHz to ¹³C, in CDCl₃ with TMS as internal standard. IR spectra were obtained on a FT-IT 510 Nicolet spectrometer. LREIMS were measured at 70 eV on a HP 5990/5988A spectrometer. UV spectra were recorded on a UV/Visible Hitachi U-3000 spectrophotometer.

Plant material

The leaves of Piper malacophyllum (Prels) C. DC. was collected in December 2000 in the Parque Estadual Intervales, São Paulo State, Brazil and was identified by Dr. Elsie F. Guimarães (Jardim Botânico – Rio de Janeiro). The voucher specimen (Kato-0100) has been deposited at Herbarium of Instituto de Botânica, SMA, São Paulo, SP, Brazil.

Antifungal assays

The microorganisms used in the antifungal assays C. sphaerospermum (Penzig) SPC 491 and C. cladosporioides (Fresen) de Vries SPC 140 have been maintained at the Instituto de Botânica, São Paulo, SP, Brazil. Ten microliters of the solutions of the crude extracts, fractions and pure compounds were prepared, in different concentrations, corresponding to 20, 10, 5 and 1 mg for pure compounds and 100 mg for the crude extracts or fractions. The samples were applied to TLC plates, these being eluted with CHCl₃-MeOH 99:1 followed by complete removal of the solvent at room temperature. The chromatographic plates were sprayed with spores suspension of C. sphaerospermum and C. cladosporioides in a nutritive medium (glucose and salt solution¹¹) and incubated for 48 h and 37 ºC. After incubation, clear inhibition zones appeared against a dark background chromatogram. Nystatin and miconazole were used as positive controls whereas ampicillin and chloramphenicol were used as negative controls.^5,11

Extraction and isolation of constituents

The dried and powdered leaves of P. malacophyllum (200g) were extracted with CH₂Cl₂ (3 × 1.5 L), for two days, at room temperature. The resulting extract was filtered and concentrated in vacuum to afford 11.3 g of the crude extract. Bioactivity-guided fractionation of this extract by flash silica gel column chromatography using hexane (300 mL), hexane-CH₂Cl₂ (1:1 - 300 mL), CH₂Cl₂ (450 mL), CH₂Cl₂-EtOAc (1:1 - 300 mL), EtOAc (150 mL), EtOAc-MeOH (1:1 - 200 mL) and MeOH (200 mL) as eluents, afforded thirteen fractions (150 mL each), whose bioactivity was detected only in two of them (5 and 7). Fraction 5 (380 mg) was submitted to Sephadex LH-20 exclusion chromatography using hexane-CH₂Cl₂ (1:4 – 150 mL), CH₂Cl₂-Me₂CO (3:2 and 1:4 – 150 mL each) as eluent to give twelve sub-fractions (25 mL each). Antifungal assay on all these sub-fractions indicated that the bioactivity was concentrated on the sub-fractions 3 and 4. These sub-fractions were pooled (70 mg) and submitted to preparative silica-gel TLC purification, using hexane-EtOAc 4:1 as eluent. This purification procedure yielded 10 mg of 1 (yielding 0.09%). Fraction 7 (238 mg) was subjected to Sephadex LH-20 exclusion chromatography, using hexane-CH₂Cl₂ (1:4 – 100 mL), CH₂Cl₂-Me₂CO (3:2 and 1:4 – 100 mL each) as eluent, to give four sub-fractions (50 mL each). Compound 2 (8 mg – yielding 0.07%) was isolated from the bioactive sub-fraction 2 (112 mg), by further purification over column chromatography on silica-gel eluted with hexane-EtOAc 7:3 (150 mL).

4,6-Dimethoxy-5-E-phenylbutenolide (5,6-E-fadyenolide - 1)

Colorless crystals. mp 129-131ºC. IR(film) n_max / cm^-1: 2936, 1748, 1592, 1493, 1446, 1378, 1216, 1135; UV l_max MeOH nm (log e): 248 (4.05), 320 (4.06); LREIMS m/z (rel. int.): 232 (58) [M^+•], 217 (7), 202 (4), 189 (11), 161 (14), 145 (3), 131 (2), 115 (7), 105 (100), 91 (4), 77 (51), 69 (26), 51 (25); ¹H and ¹³C NMR spectra (see Table 1).

4,6-Dimethoxy-5-Z-phenylbutenolide (5,6-Z-fadyenolide - 2)

Colorless crystals. mp 130-132ºC. IR (film) n_max / cm^-1: 2941, 1745, 1592, 1493, 1443, 1385, 1218, 1135; UV l_max MeOH nm (log e): 245 (2.72), 313 (3.15); LREIMS m/z (rel. int.): 232 (67) [M^+•] 217 (7), 202 (4), 189 (14), 161 (13), 145 (4), 131 (3), 115 (8), 105 (100), 91 (5), 77 (53), 69 (27), 51 (26); ¹H and ¹³C NMR spectra (see Table 1).

Acknowledgments

This work was funded by grants provided by FAPESP (Biota Program, 98/05074-0) and CNPq/PADCT. T.M.T. thanks to PIBIC/CNPq for providing a scholarship.

Received: April 6, 2004

Published on the web: February 17, 2005

References

• 1. Parmar, V.S.; Jain, S.C.; Bisht, K.S.; Jain, R.; Taneja, P.; Jha, A.; Tyagi, O.D.; Prasad, A.K.; Wengel, J.; Olsen, C.; Boll, P.M.; Phytochemistry 1997, 46, 597.
• 2. Orjala, J.; Erdelmeier, C.A.J.; Wright, A.D.; Rali, T.; Sticher, O.; Planta Med 1993, 59, 546.
• 3. Facundo, V. A.; Sa, L. A.; Silva, S.A.; Morais, S. M.; Matos, C.R.R.; Braz-Filho, R. J. Braz. Chem. Soc. 2004, 15, 140.
• 4. Navickiene, H.M.B.; Alécio, A.C.; Kato, M.J.; Bolzani, V. da S.; Young, M.C.M.; Cavalheiro, A.J.; Furlan, M.; Phytochemistry 2000, 55, 621.
• 5. Danelutte, A.P.; Lago, J.H.G.; Young, M.C.M.; Kato, M.J.; Phytochemistry 2003, 64, 555.
• 6. Pelter, A.; Al-Bayati, R.; Hänsel, R.; Dinter, H.; Burke, B.; Tetrahedron Lett. 1981, 22, 1545.
• 7. Nair, M.G.; Mansingh, A.P.; Burke, B.A.; Agric. Biol. Chem 1986, 50, 3053.
• 8. Ganzera, M.; Khan, I.A.; Forst. Chromatographia 1999, 50, 649.
• 9. Ranjith, H.; Dharmaratne, W.; Nanayakkara, N.P.D.; Khan, I.A.; Phytochemistry 2002, 59, 429.
• 10. Singh, Y.M.; Blumenthal, M.; Herbalgram 1997, 39, 33.
• 11. Homans, A.L.; Fuchs, A.; J. Chromatogr 1970, 51, 327.
• 12. Rahalison, L.; Hamburger, M.; Monod, M.; Frenk, E.; Hostettmann, K.; Planta Med 1994, 60, 41.

Publication Dates

• Publication in this collection
  24 May 2005
• Date of issue
  Apr 2005

History

• Accepted
  17 Feb 2005
• Received
  06 Apr 2004