A new antifungal phenolic glycoside derivative, iridoids and lignans from Alibertia sessilis (vell.) k. schum. (Rubiaceae)

Abstracts

A new antifungal phenolic glycoside, 3,4,5-trimethoxyphenyl-1-O-beta-D-(5-O-syringoyl)apiofuranosyl-(1->6)-beta-D-glucopyranoside (1), together with four known iridoids, geniposidic acid (2), geniposide (3), 6alpha-hydroxygeniposide (4) and 6beta-hydroxygeniposide (5); two lignans, (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside (6), (-)-lyoniresinol-3alpha-O-beta-D-glucopyranoside (7); and two phenolic acids, chlorogenic (8) and salicylic acids (9) and D-manitol (10), were isolated from the ethanolic extract of the stems of Alibertia sessilis. Structures of 1 and of the known compounds were determined by spectroscopic analysis. All compounds isolated were evaluated for their antifungal activities against two phytopathogenic fungi strains Cladosporium cladosporioides and C. sphaerospermum by direct bioautography.

Rubiaceae; Alibertia sessilis; phenolics; iridoids; antifungal

Um novo derivado fenólico glucosilado antifúngico, 3,4,5-trimetoxifenil-1-O-beta-D-(5-Osiringoila)-apiofuranosil-(1->6)-beta-D-glicopiranosideo (1), juntamente com quatro iridóides conhecidos, ácido geniposídico (2), geniposídeo (3), 6alfa-hidroxigeniposideo (4) e 6betahidroxigeniposideo (5); duas lignanas, (+)-lioniresinol-3alfa-O-beta-D-glicopiranosideo (6), (-)lioniresinol-3alfa-O-beta-D-glicopiranosideo (7); e dois ácidos fenólicos, ácidos clorogênico (8) e salicílico (9) e D-manitol (10), foram isolados do extrato etanólico dos galhos de Alibertia sessilis. As estruturas de 1 e dos compostos conhecidos foram determinadas por análise espectroscópica. Todos os compostos isolados foram avaliados quanto à atividade antifúngica frente aos dois fungos fitopatogênicos Cladosporium cladosporioides e C. sphaerospermum por bioautografia direta.

ARTICLE

A new antifungal phenolic glycoside derivative, iridoids and lignans from Alibertia sessilis (vell.) k. schum. (Rubiaceae)

Viviane C. da Silva^I; Vanderlan da S. Bolzani^I; Maria C. M. Young^II; Márcia N. Lopes^I,*

^IDepartamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista, CP 355, 14801-970 Araraquara-SP, Brazil

^IISeção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, CP 4005, 01061-970 São Paulo-SP, Brazil

ABSTRACT

A new antifungal phenolic glycoside, 3,4,5-trimethoxyphenyl-1-O-b-D-(5-O-syringoyl)apiofuranosyl-(1®6)-b-D-glucopyranoside (1), together with four known iridoids, geniposidic acid (2), geniposide (3), 6a-hydroxygeniposide (4) and 6b-hydroxygeniposide (5); two lignans, (+)-lyoniresinol-3a-O-b-D-glucopyranoside (6), (-)-lyoniresinol-3a-O-b-D-glucopyranoside (7); and two phenolic acids, chlorogenic (8) and salicylic acids (9) and D-manitol (10), were isolated from the ethanolic extract of the stems of Alibertia sessilis. Structures of 1 and of the known compounds were determined by spectroscopic analysis. All compounds isolated were evaluated for their antifungal activities against two phytopathogenic fungi strains Cladosporium cladosporioides and C. sphaerospermum by direct bioautography.

Keywords: Rubiaceae, Alibertia sessilis, phenolics, iridoids, antifungal

RESUMO

Um novo derivado fenólico glucosilado antifúngico, 3,4,5-trimetoxifenil-1-O-b-D-(5-Osiringoila)-apiofuranosil-(1®6)-b-D-glicopiranosideo (1), juntamente com quatro iridóides conhecidos, ácido geniposídico (2), geniposídeo (3), 6a-hidroxigeniposideo (4) e 6bhidroxigeniposideo (5); duas lignanas, (+)-lioniresinol-3a-O-b-D-glicopiranosideo (6), (-)lioniresinol-3a-O-b-D-glicopiranosideo (7); e dois ácidos fenólicos, ácidos clorogênico (8) e salicílico (9) e D-manitol (10), foram isolados do extrato etanólico dos galhos de Alibertia sessilis. As estruturas de 1 e dos compostos conhecidos foram determinadas por análise espectroscópica. Todos os compostos isolados foram avaliados quanto à atividade antifúngica frente aos dois fungos fitopatogênicos Cladosporium cladosporioides e C. sphaerospermum por bioautografia direta.

Introduction

In our continuing search for antifungal agents from plants from the Cerrado and Atlantic Forest in the State of São Paulo, Brazil, we screened several species of the Rubiaceae family, which is recognized as a rich source of bioactive metabolites.^1-5 We particularly studied plants of the Alibertia genus, since relatively few chemical studies have been reported. Our previous investigation with the leaves of A. macrophylla resulted in the isolation of antifungal iridoid aglycones, and caffeic acid ester derivatives.^6,7 Additionally, flavones and coumarin have been reported for A. myrciifolia.⁸

This paper reports the bioassay-guided isolation and structure elucidation of the new 3,4,5-trimethoxyphenyl-1- O-ß-D-(5-O-syringoyl)-apiofuranosyl-(1?6)-ß-D-glucopyranoside (1) (Figure 1), and the known lignans (+)- lyoniresinol-3a-O-ß-D-glucopyranoside (6), (-)-lyoniresinol-3a-O-ß-D-glucopyranoside (7), which are being described for the first time for Rubiaceae. This paper also reports the isolation of iridoids geniposidic acid (2), geniposide (3), 6a- hydroxygeniposide (4) and 6ß-hydroxygeniposide (5), phenolic acids, chlorogenic (8) and salicylic acid (9) and D-manitol (10). The antifungal activity of compound 1 against Cladosporium sphaerospermum and C. cladosporioides was detected by using direct bioautography.

Results and Discussion

The antifungal ethanol extract from Alibertia sessilis stems was partitioned with hexane, ethyl acetate and n-butanol. These sub-extracts were tested against Cladosporium sphaerospermum and C. cladosporioides using direct bioautography showing that only the ethyl acetate fraction was active. This sub-extract was bioassay-guide fractioned to afford a new phenolic derivative 1 along with several known compounds.

Compound 1 was isolated as a brown amorphous powder. The molecular formula was determined as being C₂₉H₃₈O₁₇ from the HRESIMS at m/z 657.2065 [M-H]^–. The UV spectrum showed an absorption maximum at 279 nm, which is compatible with a phenolic structure. The IR spectrum showed a characteristic absorption band attributable to the following groups: carbonyl (1708 cm^-1), hydroxyl (3418 cm^-1), and aromatic (1605 and 1509 cm^-1), as well as a glycosidic linkage (1063 cm^-1). The ¹H and ¹³C NMR spectra (Table 1) revealed signals for two 1,3,4,5-tetrasubstituted symmetrical aromatic rings. The ¹H NMR exhibited singlet signals corresponding to five aromatic methoxyls at d_H 3.77 (6H, OCH₃-3,5), 3.68 (3H, OCH₃-4), and 3.87 (6H, OCH₃-3''',5''') and two sets of aromatic proton signals at d_H 6.41 (2H, H2,6) and 7.34 (2H, H-2''',6''') due to a 3,4,5-trimethoxy phenyl derivative and symmetrical 3,5-dimethoxy-4hydroxy-benzoyl moieties, respectively.⁹ This was supported by the gHMBC spectrum, which showed cross-peaks corresponding to a long-range coupling of hydrogen at d_H 6.41 and carbon signals at d_C 155.9 (C-1), 154.7 (C-3,5), 134.7 (C-4) and 96.5 (C-2,6) and also between hydrogen at d_H 7.34 and carbon signals at d_C 167.9 (C=O), 149.1 (C-3''',5'''), 143.1 (C-4'''), 120.6 (C-1''') and 108.5 (C-2''',6'''). Others signals observed in ¹H and ¹³C NMR spectra are consistent with glycosyl moieties. According to literature,¹⁰ the comparison of the ¹³C NMR data of the glycosidic moiety of various other analogous compounds, the structure of 1 seems to be composed of a glucosyl [d_C 103.2 (C-1'), 74.9 (C-2'), 77.9 (C-3'), 71.5 (C-4'), 76.9 (C-5') and 68.2 (C-6')] and an apiosyl [d_C 110.4 (C1''), 78.7 (C-2''), 79.0 (C-3''), 75.0 (C-4'') and 67.9 (C-6'')] moieties. The ¹H, ¹³C NMR and TOCSY spectra confirmed the presence of these two sugar moieties due to the presence of two anomeric signals at d_H 4.79 (d, J 7.5 Hz)/d_C 103.2 (CH) for glucose and d_H 5.01 (d, J 2.3 Hz)/d_C 110.4 (CH) for apiose. Furthermore, the coupling constants J 7.5 Hz of the anomeric proton signal of the D-glucosyl moiety as well as the chemical shift (d_C 110.4) of the anomeric carbon of the D-apiosyl moiety, demonstrated that both sugar moieties have b-anomeric configurations. The gHMBC spectrum of 1 revealed cross-peaks corresponding to long-range couplings of hydrogens H-6' of b-glucose at d_H 3.63 (d, J 10.0 Hz) and 4.06 (d, J 10.0 Hz) and carbon C1'' of apiose at d_C 110.4; in this way, the b-D-apiosyl moiety was attached to C-6' of the glucose. The chemical shift of the oxymethylene carbon C-6' (d_C 68.2) was used to confirm the disaccharide linkage as apiofuranosyl-(1®6)-glucopyranoside.¹⁰ In addition, long-range correlations between H-5'' signals of b-Dapiose [d_H 4.31 (d, J 11.4 Hz) and 4.39 (d, J 11.4 Hz)] and the syringoyl carbonyl group (d_C 167.9) indicated that the 3,5-dimethoxy-4-hydroxy-benzoyl group was attached to C-5'' of the b-D-apiose. Finally, the proton at d_H 4.79 (H-1' of glucose) was correlated with C-1 of the 3,4,5-trimethoxy phenyl group (d_C 155.9), indicating that the b-D-glucosyl moiety was located at C-1. Therefore, the structure of compound 1 was established as being 3,4,5-trimethoxyphenyl-1-O-b-D(5-O-syringoyl)-apiofuranosyl-(1®6)-b-D-glucopyranoside.

The known compounds 2-10 were identified by comparing their ¹H and ¹³C NMR data with those previously published in literature.^11-16

The antifungal activity of compounds 1, 2, 6-10 against Cladosporium cladosporioides and C. sphaerospermum was evaluated by direct bioautography on a TLC plate.^17,18 Only compound 1 exhibited moderate activity (MIC of 100 µg), when compared with the standard nystatin (1.0 µg).

Experimental

General experimental procedures

1D- (¹H, ¹³C, TOCSY) and 2D- (gHMQC and gHMBC) NMR experiments were recorded on a Varian INOVA 500 spectrometer (11.7 T) at 500 MHz (¹H) and 126 MHz (¹³C), using adequate solvent with TMS as internal standard. Accurate-mass measurements were performed on a quadrupole-time of flight instrument (UltrOTOF-Q, Bruker Daltonics). IR spectrum was obtained on a Nicolet730 FT-IR spectrometer using KBr discs. UV absorption was measured on a Varian ProStar 330 diode array spectrophotometer. Optical rotation was measured on a Polamat A Carl Zeiss Jena. Preparative HPLC was carried out in a Varian HPLC, using a Phenomenex Luna RP18 (2) column (250 × 21.20 mm i.d. × 10 µm).

Plant material

Alibertia sessilis was collected in the Estação Ecológica e Experimental de Mogi-Guaçu (Ecological and Experimental Reserve of Mogi-Guaçu), São Paulo, Brazil in November 2003 by Dr M.C.M. Young and identified by Dr I. Cordeiro. The voucher specimen (SP 370.914) was deposited at the Botanical Institute Herbarium, São Paulo, Brazil.

Extraction and isolation of constituents

The dried and powdered stems (50.0 g) of A. sessilis were extracted exhaustively with ethanol, at room temperature, to give a crude material (4.15 g), which was partitioned in hexane, EtOAc and n-BuOH. After evaporation, the EtOAc extract (1.30 g) was submitted to column chromatography on Sephadex LH-20 using MeOH as eluent to give 15 fractions after TLC analysis. Fraction 1 (116.2 mg) was composed of a precipitate, which was recrystalized with H₂O to give D-manitol (10) (63.7 mg). Fraction 2 (146.4 mg) was subjected to column chromatography on RP-18 using a gradient mixture of H₂O/MeOH and MeOH/EtOAc as eluent to give 11 subfractions. Subfraction 2.1 (80.0 mg) was rechromatographed on RP-18, eluted with H₂O with increasing amounts of MeOH to afford geniposidic acid (2) (11.8 mg). Subfraction 2.7 (29.6 mg) was also submitted to column chromatography on RP-18, using a gradient solvent system of H₂O/MeOH and MeOH/ EtOAc as eluent to yield 7 subfractions after TLC analysis. Subfraction 2.7.6 (18.8 mg) was chromatographed by preparative HPLC (C₁₈ column; mobile phase: MeOH/H₂O (3:7 v/v); flow rate: 10 mL min^-1; UV detector: 238 nm) to separate the mixture of the diastereoisomeric lignans (+)-lyoniresinol 3a-O-b-Dglucopyranoside (6) (6.5 mg) and (-)-lyoniresinol 3aO-b-D-glucopyranoside (7) (5.8 mg).¹⁹ Subfraction 2.7.7 (6.7 mg) was identified as 3,4,5-trimethoxyphenyl-1-Ob-D-(5-O-syringoyl)-apiofuranosyl-(1®6)-b-D-glucopyranoside (1). Fraction 4 (274.2 mg) was fractioned on RP-18 using H₂O with increasing amounts of MeOH as mobile phase to give salicylic acid (9) (6.4 mg). Fractions 5 and 6 (104.9 mg) were combined and submitted to column chromatography on silica gel eluting with hexane/EtOAc and EtOAc/MeOH to give 6.9 mg of chlorogenic acid (8).

The n-butanol extract (0.55 g) was submitted to column chromatography on Sephadex LH-20 using MeOH as eluent to give 7 fractions after TLC analysis. Fraction 3 (180.0 mg) was subjected to column chromatography on RP-18 using a gradient mixture of H₂O/MeOH to give 9 subfractions. Subfraction 3.4 (10.2 mg) was identified as an iridoid mixture 3-5 and subfraction 3.6 (8.6 mg) as a lignan mixture 6-7.

3,4,5-trimethoxyphenyl-1-O-b-D-(5-O-syringoyl)apiofuranosyl-(1®6)-b-D-glucopyranoside (1)

Brown amorphous powder (6.7 mg, 0.16%). [a]²_D⁵ –2.36 (c 0.021, MeOH). UV l_max /nm (MeOH) 279. IR (KBr) n_max/cm^-1: 3418, 2931, 1708, 1605, 1509, 1461, 1427, 1338, 1228, 1125, 1063, 816, 764. HRESIMS m/z 657.2065 [M - H]^– (calcd for C₂₉H₃₇O₁₇, m/z 657.2031 [M -H]^–).¹H and ¹³C NMR: see Table 1.

Known compounds isolated

The structures of known compounds were established by ¹H and ¹³C NMR and gHMBC data and by comparing their spectroscopy data with those reported in literature.

Geniposidic acid (2) (0.28%)

The ¹H and ¹³C NMR data, in CD₃OD, were in accordance with those reported in reference 11.

Geniposide (3); 6a-hydroxygeniposide (4); 6bhydroxygeniposide (5) (0.24%)

The ¹H and ¹³C NMR data of the mixture, in CD₃OD, were in accordance with those reported in references 12 and 13. The compounds were identified in mixture mainly on the basis on their TOCSY spectra.

(+)-Lyoniresinol-3a-O-b-D-glucopyranoside (6) (0.15%) and (-)-lyoniresinol-3a-O-b-D-glucopyranoside (7) (0.14%)

The ¹H and ¹³C NMR data, in CD₃OD, were in accordance with those reported in reference 14.

Chlorogenic acid (8) (0.16%)

The ¹H and ¹³C NMR data, in DMSO-d₆, were in accordance with those reported in reference 15.

Salicylic acid (9) (0.15%)

The ¹H and ¹³C NMR data, in CD₃OD, were in accordance with those reported in reference 16.

D-manitol (10) (1.53%)

The ¹H and ¹³C NMR data, in D₂O, were in accordance with those reported in reference 16.

Antifungal Assay

The microorganisms used in the antifungal assays Cladosporium sphaerospermum (Penzig) SPC 491 and C. cladosporioides (Fresen) SPC 140 had been kept at the Botanical Institute of São Paulo, Brazil. For the antifungal assay, solutions containing 100, 50, 25, 10, 5 and 1 µg of the test compound were prepared. 10.0 µL of each solution was applied to pre-coated TLC plates eluted with EtOAc/ MeOH (7:3 v/v) and dried to completely remove all solvents. The chromatograms were sprayed with a spore suspension of Cladosporium sphaerospermum or C. cladosporioides in glucose and salt solution and incubated for 72 h in darkness in a moistened chamber at 25 ºC. A clear inhibition zone appeared against a dark background indicating the minimal amount of the compound required to eliminate the fungus (detection limits - minimum amount required for the inhibition of fungal growth on TLC plates). Nystatin was used as the positive control (1 µg).^17,18

Conclusions

Lignans 6 and 7 are here described for the first time as being found in Rubiaceae and the antifungal phenolic glucoside 1 is being reported for the first time in literature. Compound 10 was previously isolated from Alibertia myrciifolia. Phenolics 8 and 9 are here reported for the first time as being found in genus Alibertia. Iridoids 2 and 3-5 are in agreement with chemosystematic correlations and botanical occurrence in the Rubiaceae family. This study will contribute significantly to improve knowledge about secondary metabolites and biological activity for one more species from the Brazilian Cerrado.

Acknowledgments

The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support and also to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a fellowship granted to V.C.S.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br, as PDF file.

Received: May 23, 2007

Web Release Date: November 14, 2007

Supplementary Information

Table S1 - click to extend

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Publication Dates

• Publication in this collection
  08 Jan 2008
• Date of issue
  2007

History

• Accepted
  14 Nov 2007
• Received
  23 May 2007