Antifungal ether diglycosides from
Matayba guianensis
Aublet
Polyana A. de Assis
a, Phellipe N. E. T. Theodoro
a, José E. de Paula
b, Ana J. Araújo
c, Letícia V. Costa-Lotufo
c,
Sylvie Michel
d, Raphaël Grougnet
d, Marina Kritsanida
d,⇑, Laila S. Espindola
a,⇑aLaboratório de Farmacognosia, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, 70910-900 Brasília, DF, Brazil bLaboratório de Anatomia Vegetal, Universidade de Brasília, Brasília, Brazil
cDepartamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, Brazil
dLaboratoire de Pharmacognosie, U.M.R./C.N.R.S. 8638, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, 4 Avenue de
l’Observatoire, 75006 Paris, France
a r t i c l e
i n f o
Article history:
Received 31 October 2013 Revised 6 January 2014 Accepted 8 January 2014 Available online 21 January 2014
Keywords:
Antifungal activity Ether diglycosides Matayosides E and F
Matayba guianensis
Brazilian Cerrado
a b s t r a c t
Since the 1960s, fungal infections have become a major worldwide public health problem. Antifungal treatments have many limitations, such as toxicity and resistance.Matayba guianensisAublet (Sapinda-ceae) was chemically investigated as part of our ongoing search for lead molecules against fungi in the Brazilian Cerrado biome. The ethanolic extract ofM. guianensisroot bark revealed the presence of two previously unreported ether diglycosides: matayoside E (1) and F (2) with antiCandidaactivity, along with two known compounds: cupanioside (3) and stigmasterol (4).
Ó2014 Elsevier Ltd. All rights reserved.
Since the end of the 60s, an important increase of the incidence of fungal infections is observed, regarding especially immunocom-promised or hospitalized patients. Current therapy is limited by the short arsenal of antifungal drugs, toxicity problems and the emergence of resistance to most classes of antifungals.1
The opportunistic pathogenic fungus Candida albicans is the main etiological agent of candidiasis.2However, infections due to
Candidanon-albicansare also raising up.3Thus, 10-years-long mul-ticentric studies in Brazilian hospitals revealed high incidence of these species, especially ofCandida parapsilosis.4
This work is incorporated into our research group program for the conservation of Brazilian Cerrado biome plant secondary metabolites. One of the axes of this program concerns the search for lead molecules against fungi. Cerrado is considered to be one of most threatened regions on Earth and is designated as a biodi-versity hotspot.5 Comparative studies concluded that socioeco-nomic, environmental and ecological factors present in hotspots
are correlated to the origin of emerging infectious diseases.6 In this context, our interest focused on Matayba guianensis
Aublet (Sapindaceae), a typical endemic tree of Cerrado with a characteristic rough trunk bark. It is 8–18 m high with white
flowers of 4 mm diameter and a 2.5 cm edible fruit locally known as ‘camboatá’.7
The root bark powder ofM. guianensiswas submitted to extrac-tion process by maceraextrac-tion in hexane. Thereafter, the residue was air-dried and exhaustively extracted with ethanol.
Four new ether diglycosides, named matayosides A–D, were isolated from the hexanic extract and previously published by our research group.8
The ethanolic root bark extract showed inhibitory activity against yeasts:Candida albicans ATCC 10231 (MIC = 1.95
lg/mL)
andCandida parapsilosisATCC 22019 (MIC = 0.97lg/mL); and
der-matophytes:Trichophyton mentagrophytesLMGO 09 (MIC = 15.62lg/mL) and
Trichophyton rubrumLMGO 06 (31.25lg/mL). LMGO
(Laboratório de Micologia de Goiás) strains are clinical isolates from patients at the Federal University of Goiás Hospital, Brazil. In contrast, the hexanic root bark extract was only active againstT. rubrumLMGO 06 with MIC value of 125
lg/mL.
The ethanolic extract ofM. guianensisroot bark was submitted to a liquid–liquid partition (EtOAc/H2O). The organic phase was
fractionated successively by silica gel column chromatography and Medium Pressure Liquid Chromatography (MPLC), leading to the isolation of two previously unreported ether diglycosides: matayoside E (1) and matayoside F (2), along with the known com-pounds: cupanioside (3)9 and stigmasterol (4)10 (Fig. 1). All the structures were established using spectroscopic techniques, including 1D and 2D NMR analysis, HRMS and comparison with previously reported data.
0960-894X/$ - see front matterÓ2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.022
⇑Corresponding authors. Tel.: +33 (0)153739805; fax: +33 (0)140469658 (M.K.); tel.: +55 61 31072016; fax: +55 61 31071943 (L.S.E.).
E-mail addresses: marina.kritsanida@parisdescartes.fr (M. Kritsanida),
darvenne@unb.br(L.S. Espindola).
Bioorganic & Medicinal Chemistry Letters 24 (2014) 1414–1416
Contents lists available atScienceDirect
Bioorganic & Medicinal Chemistry Letters
Compound1was isolated as a colorless amorphous solid, opti-cally active½
a
20D 47.8 (c0.14, CHCl3). Its molecular formula was
established as C32H58O12 by the HR-ESIMS positive ion at m/z
657.3820 [M+Na]+ (calcd 657.3826) (Supplementary data,
Figs. S1–S8), indicating four degrees of unsaturation. The IR
absorption bands at 3401 and 1728 cm1
implied the presence of hydroxyl and carbonyl functionalities.
1H NMR spectrum (Table 1) showed similarities with previously
published matayosides.8Indeed, a doublet (J= 6 Hz) integrating for 3 protons atdH1.17, a broad singlet (1H) at dH5.28, a doublet (J= 7.5 Hz) atdH4.34, together with signals of osidic protons be-tweendH3.31 anddH4.20 are characteristic of a glucose–rhamnose type diglycoside.8 In addition, two singlets integrating for three protons each at dH2.12 anddH2.16 and the resonances of two
deshielded protons, a broad doublet (J= 3.5 Hz) atdH5.09 and a triplet (J= 10 Hz) atdH4.85 are in good agreement with two acet-ylated osidic position. A triplet (J= 7 Hz) integrating for 3 protons at dH0.87, a large signal centered at dH 1.25 integrating for 22 (400to 1400) protons, and a multiplet (2H) atd
H1.59 are compatible
with a C16fatty alcohol. These data were confirmed by the13C and
HSQCed NMR spectra with four methyls, two oxygenated methyl-enes, eight oxygenated and two dioxygenated methines, two carbonyls, and a set of methylenes corresponding to the fatty part. Examination of the 2D COSY spectrum was informative as it permitted to determine the acetylated position. The correlation of the anomeric proton H-10 atd
H5.28 with the deshielded one
atdH5.09, of this proton with another one atdH4.02, then with this latter with the second deshielded proton at dH4.85 allowed to determine a 2,4-diacetylrhamnose moiety. This was confirmed by correlations through a 3J coupling constant on the 2D HMBC
spectrum of the protons atdH5.09 and atdH4.85 with carbonyls atdC171.9. The linkage of the two sugar units was established as C-2glc–O–C-1rhaby observation of3Jcoupling constant correlations
between H-10 with C-2 and H-2 with C-10, while correlations between H-1 with C-100and H-100with C-1 determined the position of the fatty alcohol on the anomeric glucose carbone (Fig. 2). Cor-relations on the 2D NOESY spectrum between H-1 with H-3, H5 and H-100and between H-10with H-20and H-100(Fig. 2) confirmed the structure of1as hexadecyl-[O-2,4-di-O-acetyl-a-L -rhamnopyr-anosyl-(1?2)]-b-D-glucopyranoside, named matayoside E.
Compound2, also isolated as colorless amorphous solid (opti-cally active:½
a
20D 45.1 (c0.15, CHCl3) with the same molecular
formula C32H58O12 established by the HR-ESIMS positive ion at m/z 657.3820 [M+Na]+ (calcd 657.3826) (Supplementary data,
Figs. S9–S16), showed many spectroscopic similarities with 1.
The IR absorption bands at 3390 and 1740 cm1implied the
pres-ence of hydroxyl and carbonyl functionalities.
1H and 13C NMR spectra (Table 1) were also characteristic of
glucose–rhamnose diglycoside with a fatty alcohol and two acety-lated osidic carbons. As in the case of1, the linkage between the sugar units C-2glc–O–C-1rhaand the position of the fatty alcohol
on C-1 was determined by observation of 3Jcoupling constants appropriate correlations on the HMBC spectrum (Fig. 2). Thorough examination of the 2D COSY spectrum, especially on the sequence of correlations H-10atd
H5.24, H-20atdH4.20, H-30atdH5.09 and
H-40atd
H5.10, determines C-30and C-40as the acetylated position.
This difference with1was further confirmed by the correlations on the HMBC spectrum with a3Jcoupling constant of H-30and H-40 with acetyl groups at dC 170.5 and dC 171.2 respectively. Consequently, 2 could be defined as hexadecyl-[O-3,4-di-O -acetyl-a-L-rhamnopyranosyl-(1?2)]-b-D-glucopyranoside, and was attributed the name of matayoside F.
Proposed stereochemistry of both compounds (1 and 2) was attributed after comparison with the literature data and taking into account the biosynthetic pathways.
Compounds1,2and3were tested for their antifungal activity by microdilution11,12and compared to positive controls amphoter-icin B, itraconazole and fluconazole (Table 2). 1 and 2 showed strong activity againstC. parapsilosisATCC 22019, with MIC values of 6.31 and 3.15
lM, respectively, on the same range to that
observed for amphotericin B (4.33lM) (
Table 2).C. parapsilosisis regarded as a reference yeast by CLSI.11In order to investigate the selectivity of compounds1–3toward a normal proliferating cell, the MTT assay was performed with peripheral blood mononuclear cells (PBMC) after 72 h drug exposure. Doxorubicin (0.02–8.62
lM) was used as a positive
control (Table 2).13Matayosides E (1) and F (2) bear two acetyl groups on the rham-nose moiety. Positions 20and 40are acetylated on1, 30and 40on2. Compounds 1and 2showed potential antifungal effect, without O
HO
HO
H O
O OH
O
R3O
R2O
OR1 H
1: R1=R3=Ac, R2=H
2: R2=R3=Ac, R1=H 3: R1=R2=R3=Ac
1
1'
(CH2)9
1''
16''
Figure 1.Molecular structure of compounds1–3.
Table 1
1H (400 MHz) and13C (75 MHz) NMR data assignments for the compounds1and2in CDCl3
1 2
Position dC dH, mult (Jin Hz) dC dH, mult (Jin Hz)
1 102.0 4.34, d (7.5) 102.0 4.35, d (7.5)
2 77.5 3.47, ov 77.7 3.47, ov
3 77.9 3.63, t (9) 78.2 3.61, ov
4 70.7 3.53, t (9) 70.2 3.57, ov
5 75.4 3.31, m 75.5 3.28, m
6 62.5 3.81, dd (12, 4.5) 62.0 3.85, ov
3.89, ov 3.85, ov
10 97.5 5.28, br s 100.4 5.24, br s
20 73.3 5.09, br d (3.5) 69.1 4.20, ov
30 68.6 4.02, dd (10, 3.5) 71.9 5.09, ov
40 75.0 4.85, t (10) 71.9 5.10, ov
50 66.4 4.20, dd (10, 6) 66.9 4.24, ov
60 17.5 1.17, d (6) 17.5 1.15, d (6)
20-CO 171.9
30-CO 170.5
40-CO 171.9 171.2
20-CH
3CO 21.4* 2.12* 30-CH
3CO 21.2 2.02
40-CH
3CO 21.6* 2.16* 21.4 2.08
100 70.7 3.47, ov 70.9 3.49, ov
3.85, ov 3.85, ov
200 30.0 1.58, m 30.1 1.59, m
300 26.4 1.34, ov 26.7 1.31, m
400–1400 30.0–30.1 1.23–1.27 30.0–30.1 1.22–1.27
1500 23.1 1.29, ov 23.2 1.26, ov
1600 14.5 0.87, t (7) 14.8 0.87, t (7)
Ov: overlapped, coupling constants were not directly measurable. Chemical shifts (d, ppm) and coupling constants (J, Hz).
*The discrimination of the two methyls of the acetates in positions 20and 40is not possible as the carbon values of the carbonyl of these positions are identical (dC
171.9).
cytotoxicity on PBMC in the time and concentrations tested. The strongest activity with the highest selectivity index (12.5) was ob-served for2.
Cupanioside (3) differs from 1 and2 by an additional acetyl group on the rhamnose. Thus, position 20, 30and 40are acetylated. This slight structural difference looks responsible for the lack of antifungal activity against yeast with MIC 30 to 60 times lower than those of1and2, associated to a cytotoxicity towards PBMC (IC50= 25.45
lM after 72 h of incubation) (
Table 2). MatayosidesA–D,8which are also triacetylated on Rha-20, Rha-30 and Rha-40, did not cause haemolysis of human erythrocytes, maybe due to the presence of acetyl groups on the glucose moiety. These com-pounds A–D exhibited antiplasmodial activity, with IC50values of
4.7, 7.1, 13.0, and 15.7
lg/mL on parasite growth, respectively.
They were considered as the active principles of the hexanic root bark extract of M. guianensis. This latter demonstrated activity against a chloroquine resistant strain (FcB1) ofPlasmodium falcipa-rum (IC50= 6.1lg/mL) without obvious cytotoxicity to L-6 (rat
myoblast-derived) and MRC-5 (human diploid embryonic lung) cells (IC50= 61.1 and >100
lg/mL).
7A 400.0 g portion of the EtOH extract ofM. guianensiswas sub-mitted to a liquid–liquid partition with EtOAc and H2O. A 27.0 g
portion of the EtOAc fraction was chromatographed by silica gel column (70–230 mesh, Merck) with cyclohexane, dichloromethane and MeOH as binary mixtures of increasing polarity, yielding 210 fractions analyzed by thin layer chromatography (TLC Silica gel 60 F254, Merck). Fractions 167–174 (502.9 mg) were pooled and
chromatographed on MPLC column (Interchim PuriFlash™ 25 g– 22 bars P/N: IR 50 SI/25 g Upti—prep sílica technology™ 50
lm),
eluted by a gradient of increasing polarity of MeOH in CH2Cl2ata flux of 15 mL/min to furnish 144 fractions analyzed by TLC.
Fractions 56–60 (70.7 mg) were identified as cupanioside (3); 74–76 (5.6 mg) as matayoside E (1) and 91–93 (12.2 mg) as matayoside F (2). Flash chromatography of fractions 80–138 (350 mg) led to the isolation of stigmasterol (4) (54.9 mg).
The strong antifungal activities without cytotoxicity of matayo-side E (1) and F (2) indicated that these compounds could inspire the search of new antifungal agents. Our investigations support conservation of Cerrado against ever increasing anthropogenic activity by safeguarding biodiversity information for thishotspot
biome.
Acknowledgments
The authors wish to thank the CNPq and CAPES Brazilian Agencies, and the ‘ChemBioFight Project’—PEOPLE MARIE CURIE ACTIONS—FP7-PEOPLE-2010-IRSES for their financial support. We would also like to extend special thanks to Dr. Maria do Rosário Rodrigues Silva for providing the fungal strains.
Supplementary data
Supplementary data (1H and13C NMR, HSQC, HMBC and1H–1H
COSY spectra; HRESIMS spectra and IR spectra of the matayoside E and matayoside F) associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.bmcl.2014.01.022.
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O
HO
HO
O O OH
O
R3O
R2O
OR1
1: R1=R3=Ac, R2=H
2: R2=R3=Ac, R1=H 1
1'
(CH2)9 1''
16''
H
H H H
H H
H
H
H H
HMBC correlations
NOESY correlations
Figure 2.Important HMBC and NOESY correlations of matayoside E (1) and F (2).
Table 2
MIC values (lM) againstCandida parapsilosisATCC 22019 and IC50values (lM) on peripheral blood mononuclear cells (PBMC)
Compound Candida parapsilosis
ATCC 22019 MIC (lM)
PBMC IC50(lM)
Matayoside E (1) 6.31 >39.43
Matayoside F (2) 3.15 >39.43
Cupanioside (3) 189.35 25.45
Amphotericin B 4.33 >5.41
Itraconazole 0.71 1.53
Fluconazole 0.2 >16.32
Doxorubicin — 1.7 (0.9–3.1)
—: Not tested.
