Antifungal iridoids, triterpenes and phenol compounds from Alibertia myrciifolia Sprunge Ex. Schum

ARTIGO

Antifungal iridoids, triterpenes and phenol compounds from Alibertia myrciifolia Sprunge Ex. Schum

João Henrique S. Luciano^I; Mary Anne S. LimaI, ^*; Edilberto Rocha Silveira^I; Ilka Maria Vasconcelos^II; Georgia Sampaio Fernandes^II; Elnatan Bezerra de Souza^III

^IDepartamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, CP 12200, 60451-970 Fortaleza - CE, Brasil

^IIDepartamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, 60451-970 Fortaleza - CE, Brasil

^IIICoordenação de Biologia, Universidade do Vale do Acaraú, 62040-370 Sobral - CE, Brasil

ABSTRACT

The new iridoid glucoside 10-O-vanilloyl-geniposidic acid has been isolated from the aerial parts of Alibertia myrciifolia along with hydroxyhopanone, 3α,22-dihydroxyhopane, ursolic acid, luteolin-3´,4´-dimethyl ether, caffeic acid and geniposidic acid. The structures of the isolated compounds were determined by means of mass spectrometry and nuclear magnetic resonance spectral analyses. The antifungal activities of the iridoids 10-O-vanilloyl-geniposidic acid and geniposidic acid were evaluated against the phytopathogenic fungi strains Colletotrichum gloeosporioides, Fusarium solani and Aspergillus niger.

Keywords:Alibertia myrciifolia; triterpenes; iridoids.

INTRODUCTION

Species of Rubiaceae have widespread occurrence in Brazil and are well known by their economic and therapeutic importance; however, plants belonging to the genus Alibertia have been limited to a few species. Previous reports reveal these species as rich sources of iridoids, flavonoids, caffeic acid and its esters, and pentacyclic triterpenes.^1,2

Alibertia myrciifolia Sprunge ex. Schum (Rubiaceae) is a small tree growing abundantly in the Brazilian Northeast region, specially in Araripe´s Plateau, where is known as "café bravo" (port. lit.: wild coffee). In our previous work we have isolated D-mannitol, scopoletin, pomolic acid, methyl esters of ursolic and oleanolic acids and the cytotoxic flavonoids corymbosin, lethedocin, apometzgerin, acacetin and apigenin.²

In continuation of our chemical investigation of the aerial parts of this plant, the present study deals with the isolation and structural determination of the new iridoid 10-O-vanniloyl-geniposidic acid (1) along with the known geniposidic acid (2),³ hydroxyhopanone (3),⁴ 3α,22-dihydroxyhopane (4),⁴ ursolic acid (5),⁵ luteolin-3´,4´-dimethyl ether (6)⁶ and caffeic acid (7).⁷ The structures of the known compounds were established by ¹H and ¹³C NMR spectral data and comparison with those reported in the literature. The antifungal activity of both iridoids 1 and 2 was evaluated against Colletotrichum gloeosporioides, Fusarium solani and Aspergillus niger.

EXPERIMENTAL

General procedures

IR spectra were recorded using a Perkin Elmer 1000 FT-IR spectrophotometer. Optical rotations were measured on a Perkin Elmer 341 polarimeter. The mass spectra were obtained on a Shimadzu QP 5000 DI-50 mass spectrometer by electron impact ionization (70 eV). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for ¹H and 125 MHz for ¹³C). Chromatography columns were carried out on silica gel 60 (Merck, 230-400 mesh), Waters RP-18 and Sephadex LH-20, and silica gel 60 (Merck, 60 F₂₅₄, 0.2 mm) was used for analytical TLC. All compounds were visualized on TLC by spraying with vanillin/perchloric acid/EtOH followed by heating.

Plant material

Aerial parts of Alibertia myrciifolia Spruce ex K. Schum. were collected at Chapada do Araripe, Crato, Ceará State. A voucher specimen (31016) has been identified by Dr. E. B. de Souza of the Universidade Estadual do Vale do Acaraú, Sobral, Ceará State, and deposited at the Herbário Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil.

Extraction and isolation

The air dried aerial parts (790.0 g) of A. myrciifolia were pulverized and extracted with hexane at room temperature. The solvent was removed under reduced pressure yielding a greenish resinous extract (6.6 g). The marc obtained after hexane extraction was extracted with EtOH to yield a dark brown solid (67.0 g), after evaporation under vacuum. The hexane extract (6.6 g) was coarsely fractionated on a silica gel column by elution with hexane, CHCl₃, EtOAc and MeOH. Chromatography of the CHCl₃ fraction (1.4 g) on silica gel using hexane/EtOAc mixtures with increasing polarity yielded 31 fractions (10 mL). The sub-fraction F-22 yielded β-sistosterol (25.0 mg). Successive flash chromatography of the EtOAc fraction (3.2 g) using 0-100% hexane/EtOAc mixture yielded 23 fractions (20 mL), that were pooled together in 10 fractions after TLC analysis. Flash chromatography of the EtOAc sub-fraction F-2 (119.5 mg) using hexane/EtOAc 85:15 mixture yielded hydroxyhopanone (3) (41.0 mg). Chromatography of the EtOAc sub-fraction F(4-5) (331.4 mg) using hexane/EtOAc 9:1 mixture yielded 3α,22-dihydroxyhopane (4) (28.2 mg).

The EtOH extract (67.0 g) was redissolved in a mixture of MeOH:H₂O (1:1 v/v) and submitted to liquid-liquid partition with hexane, CHCl₃, EtOAc and n-BuOH to give four fractions. The CHCl₃ fraction (15.4 g) was re-chromatographed on Sephadex LH-20 to give 12 sub-fractions (50 mL), which were combined in 5 sub-fractions according to TLC analysis. From the CHCl₃ sub-fraction (8-9) luteolin-3´,4´-dimethyl ether (6) (1.5 mg) was obtained. The EtOAc fraction (7.6 g) was further purified over Sephadex LH-20 by elution with MeOH to afford 20 sub-fractions (50 mL), which were combined in eight sub-fractions according to TLC. Flash chromatography of the EtOAc sub-fraction F(7-11) (4.7 g) using CHCl₃/EtOAc mixtures with increasing polarity, afforded ursolic acid (5) (9.1 mg). The EtOAc F(12-13) sub-fraction (180.0 mg) was further purified by flash chromatography after elution with a mixture of CH₂Cl₂:MeOH:AcOH (88:10:2) to yield caffeic acid (7) (16.4 mg). The n-BuOH fraction (9.4 g) was re-chromatographed on Sephadex LH-20, to give 11 sub-fractions (50 mL) which were combined in 5 according to TLC analysis. Chromatography of the n-BuOH sub-fraction F(7-8) (5.8 g) using Waters RP-18 column and the MeOH:H₂O (1:1) as eluent yielded geniposidic acid (2) (10.5 mg). By the same procedure, the n-BuOH F(9-11) (544.5 mg) sub-fraction was rechromatographed on a Waters RP18 column to afford 10-O-vanilloyl geniposidic acid (1) (3.5 mg).

10-O-Vanilloyl geniposidic acid (1): yellow resin [α]²⁰_D + 23.7º (CHCl_3;c 0,1). IR (film) ν_{max /}cm^-1: 3382, 2920, 2855, 1692, 1645, 1589, 1551, 1537, 1450, 1402, 1353, 1282, 1219, 1183, 1107, 1077, 1042, 908, 880. EI-MS, m/z 524 (10 %) 503 (0,5%), 484 (0,5%), 471 (0,5%), 445 (0,5%), 430 (0,5%), 413 (0,5%), 385 (0,5%), 368 (0,5%), 342 (0,5%), 318 (1%), 290 (1%), 274 (3%), 260 (1%), 245 (2%), 230 (7%), 202 (10%), 168 (47%), 153 (33%), 124 (79%), 109 (100%), 97 (20%), 81 (53%), 60 (65%), 44 (98%). ¹H and ¹³C NMR data are given in Table 1.

Antifungal assay

The filamentous fungus Colletotrichum gloeosporioides was obtained from the Departamento de Biologia, Universidade Federal do Ceará, Brazil, whereas Fusarium solani (URM 3708) and Aspergillus niger (URM 3292) were provided by the Departamento de Micologia, Universidade Federal Rural de Pernambuco, Brazil.

The assay for antifungal activity toward Colletotrichum gloeosporioides, Fusarium solani and Aspergillus niger was carried out in 100 x 15 mm Petri plates containing 25 mL of potato dextrose agar. After the mycelial colony had developed, sterile filter paper disks (0.6 cm in diameter, Whatman) were placed at a distance of 0.5 cm away from the rim of the mycelial colony. Aliquots (75 μL) of the iridoids 1 and 2 (solutions of 122.5 μg of 1 and 150 μg of 2 in 0.1% dimethyl sulfoxide) were added to the disks. Nistatin (2 μg) and DMSO were used as positive and negative control, respectively. The plates were incubated at 27 °C for 72 h until mycelial growth had enveloped the disks containing the negative control and had formed crescents halos of inhibition around disks containing samples with antifungal activity.⁸

RESULTS AND DISCUSSION

Compound 1 was obtained as a yellowish resin after isolation and purification. The IR spectrum showed absorption bands at 3382 cm^-1 for hydroxyl; at 1645 and 1692 cm^-1 for conjugated carbonyl groups, and at 1402 cm^-1 for C-O-H, respectively. The low resolution EIMS of 1 showed a molecular ion at m/z 524, in agreement with the molecular formula C₂₄H₂₈O₁₃.

The ¹H NMR spectrum of 1 showed a very similar feature to those described for geniposidic acid (2), by the signals of two olefinic hydrogens at δ 7.23 (d, J = 0.9 Hz, H-3) and 5.86 (s, H-7), two diastereotopic methylene groups at δ 4.99 (d, J = 14.0 Hz, H-10eq) and 4.87 (d, J = 14.0 Hz, H-10ax), and 2.89 (m, H-6) and 2.12 (m, H-6), and the β-D-glucopyranosyl moiety at δ 4.71 (d, J = 7.7 Hz, H-1´), 3.38 (m, H-5´), 3.33 (m, H-4´), 3.29 (m, H-3´), 3.25 (m, H-2´), 3.66 (dd, J = 12.1, 5.3 Hz, H-6´´) and 3.85 (d, J = 12.1 Hz, H-6´´). In addition, were also observed one methoxyl at δ 3.81 (s, OCH₃-3´´) and three aromatic hydrogens on an AMX spin system at δ 7.48 (dd, J = 8.4, 2.1 Hz, H-6''), 7.40 (d, J = 2.1 Hz) and 6.59 (d, J = 8.4 Hz) relative to the presence of an additional vanilloyl moiety.

The unequivocal NMR assignments of compound 1 shown in Table 1 was established by analysis from COSY, HMQC, HMBC and NOESY experiments. The site of the vanilloyl ester linkage at C-10 was confirmed by the ¹H,¹H scalar correlations observed from the COSY analysis, that revealed the correlations between the methylene group at δ 2.89 and 2.12 (2H-6) with the methine hydrogen at δ 3.37 (H-5) and the olefinic at δ 5.86 (H-7), which showed long-range coupling with the allylic methylene at δ 4.99 and 4.87 (2H-10). Although no correlation of the olefinic hydrogen at δ 5.86 (H-7) with the carbon of the methylene group at C-10 was observed in the HMBC spectrum, cross-peak correlations of this hydrogen were found with the methine carbons at δ 48.4 (C-9) and 37.7 (C-5) and with the methylene at δ 40.4 (C-6), and the exact location of the double bond between the carbons atoms C-7 and C-8 was deduced. The relative stereochemistry of 1 was established by the NOESY experiment and revealed by the diagnostic NOE cross-peak between the hydrogen H-5 at δ 3.30 with the H-9 at δ 2.77, that in turn showed correlation with the H-1 at δ 5.15, in agreement with the relative stereochemistry related to the geniposidic acid.³ Therefore, the structure of compound 1 was elucidated to be the new iridoid 10-O-vanilloyl-geniposidic acid.

Geniposidic acid (2), but not 10-O-vanniloyl geniposidic acid (1), was inhibitory to the mycelial mass development of C. gloeosporioides at 150 μg/mL (Figure 1S, supplementary material). In contrast, both compounds were inactive toward A. niger and F. solani.

SUPPLEMENTARY MATERIAL

The fungal growth inhibitory assay of compounds 1 and 2, and ¹H NMR, ¹³C NMR, COSY, HMQC, HMBC, IR and EIMS spectra of compound 1 are available free of charge at quimicanova sbq.org.br, as PDF file.

ACKNOWLEDGMENTS

The authors are grateful to CNPQ/CAPES/PRONEX/FUNCAP for the fellowships and financial support.

REFERENCES

1. Olea, R. S. G.; Roque, N. F.; Bolzani, V. S.; J. Braz. Chem. Soc. 1997, 8, 257; Silva, V. C.; Bolzani, V. S.; Young, M. C. M.; Lopes, M. N.; J. Braz. Chem. Soc. 2007, 18, 1405; Brochini, C. B.; Martins, D.; Roque, N. F.; Bolzani, V. S.; Phytochemistry 1994, 36, 1293; Young, M. C. M.; Braga, M. R.; Dietrich, S. M. C.; Gottlieb, H. E.; Trevisan, L. M. V.; Phytochemistry 1992, 31, 3433.

2. Luciano, J. H. S.; Lima, M. A. S.; Souza, E. B.; Silveira, E. R.; Biochem. Syst. Ecol. 2004, 32, 1227; Militão, G. C. G.; Pessoa, C.; Costa-Lotufo, L. V.; Moraes, M. E. A.; Moraes, M. O.; Luciano, J. H. S.; Lima, M. A. S.; Silveira, E. R.; Pharm. Biol. 2005, 43, 480.

3. Bitar, H. E.; Nguyen, V. H.; Gramain, A.; Sévenet, T.; Bodo, B.; Tetrahedron Lett. 2004, 45, 515.

4. Tanaka, R.; Matsunaga, S.; Phytochemistry 1992, 31, 3535.

5. Ahmad, V. U.; Rahman, A.; Handbook of Natural Products Data. Pentacyclic Ttriterpenoids, Elsevier: New York, 1994.

6. Stevens, J. F.; Wollenweber, E.; Ivancic, M.; Hsu, V. L.; Sundberg, S.; Deinzer, M. L.; Phytochemistry 1999, 51, 771.

7. Bolzani, V. S.; Trevisan, L. M.; Young, M. C. M.; Phytochemistry 1991, 30, 2089.

8. Chu, K. T.; Xia, L.; Ng, T. B.; Peptides 2005, 26, 2098.

Recebido em 12/2/09; aceito em 30/7/09; publicado na web em 21/1/10

MATERIAL SUPLEMENTAR

Figure 2S - Clck to enlarge

Figure 3S - Click to enlarge

Figure 7S - Click to enlarge

Figure 12S - Click to enlarge

Figure 13S - Click to enlarge

Publication Dates

• Publication in this collection
  12 Mar 2010
• Date of issue
  2010

History

• Received
  12 Feb 2009
• Accepted
  30 July 2009