Purpureone, an antileishmanial ergochrome from the endophytic fungus Purpureocillium lilacinum

1 Introduction

Endophytic fungi are organisms ubiquitously found in plants and residing intercellularly or intracellularly, at least for a portion of their lives without causing apparent symptoms of infection. They play important physiological and ecological roles in their host life. Research has shown that the symbiosis of many fungi with host plants produce bioactive metabolites in chemical defenses against pathogenic organisms. In the past two decades, many valuable bioactive compounds including terpenoids, steroids, xanthones, quinones, phenols, isocoumarines, benzopyranones, tetralones, cytochaslasins, and enniatins with antimicrobial, insecticidal, cytotoxic, antiparasitic, and anticancer activities have been successfully discovered from the endophytic fungi and may have potential for use in modern medicine and plant protection [1]. Considering that 6 of 20 of the most commonly prescribed medications are of fungal origin [2], [3] and that only 5% of the endophytic fungi have been described [4], [5] they may play a key role in the search for drugs against parasitic diseases such as malaria, leishmaniasis, and trypanosomiasis where conventional therapy faces problems due to resistance of the parasites to existing drugs. In the case of leishmaniasis and trypanosomiasis, drugs are outdated, complicated to administer, and can cause severe adverse reactions [6], [7], [8]. Consequently, newer and efficient drugs are needed. One of the rational approaches in the search for new drugs is the exploration of plants endophytes. They offer an enormous potential for new products if selected plants are from unique environmental settings such as Rauvolfia macrophylla (Apocynaceae), a Cameroonian medicinal plant used in traditional medicine to treat malaria and other parasitic diseases [9]. Previous investigation of this plant reported the isolation of several alkaloids [10]. However, to the best of our knowledge, no report has been published on its endophytic fungi. In a continuing search for antiparasitic compounds from Cameroonian medicinal plants and their endophytes, we have investigated the ethyl acetate extract of the mycelium of Purpureocillium lilacinum, which was found to possess potent antileishmanial activity against Leishmania donovani with IC₅₀ value of 0.174 μg mL⁻¹. We report here the isolation and the structure elucidation of a new ergochromone derivative (1), isolated from an active fraction of the extract together with the evaluation of its antiparasitic and antimicrobial activities.

2 Results and discussion

Three endophytic fungi were isolated from R. macrophylla (Apocynaceae). The taxonomic classification of these endophytes and their internal transcribed spacer (ITS) sequences were amplified by applying polymerase chain reaction (PCR), sequenced and analyzed in a BLAST-based approach. All obtained sequences showed hits against the databases with high identities (99%–100%) and sequence coverage (99%–100%). The strain T26 was classified on species level as P. lilacinum, whereas T23 belonged to the genus Fusarium and T18 was classified as Aspergillus strain (Table 1). The ITS sequences in these two genera were 100% equal on the DNA level for quite many species. Therefore a classification on species level was here not possible.

The ethyl acetate extracts prepared from the mycelia of these three endophytic fungi, P. lilacinum, Aspergillus sp., and Fusarium sp., isolated from the roots of R. macrophylla (Apocynaceae) were screened for their antiprotozoal activity against Plasmodium falciparum (NF54), L. donovani, Trypanosoma brucei rhodesiense, and Trypanosoma cruzi. The extract from the mycelium of P. lilacinum showed potent antileishmanial activity in vitro against L. donovani (IC₅₀ value of 0.174 μg mL⁻¹) with good selectivity (SI=94.9) toward the L6 cell line. It was subjected to successive column chromatography over silica gel, which yielded a new ergochromone derivative (1), together with six known compounds: (22E,24R)-stigmasta-5,7,22-trien-3-β-ol (2) [11] (22E,24R)-stigmasta-4,6,8(14),22-tetraen-3-one (3) [11], emodin (4) [12] chrysophanol (5) [12]), aloe-emodin (6) [12] (Fig. 1), and palmitic acid [13].

Fig. 1:

Structures of compounds 1–6 and neosartorin (1a).

Compound 1 was obtained as a yellow amorphous powder. It gave a positive ferric chloride test, indicating its phenolic nature. The molecular formula C₃₂H₃₀O₁₄, corresponding to 18 double-bond equivalents, was deduced from the electrospray ionization (ESI)-high resolution mass spectrometry (HRMS) which showed a pseudo-molecular ion peak [M+Na]⁺ at m/z=661.1528 (calcd. 661.15333 for C₃₂H₃₀O₁₄Na, [M+Na]⁺). The IR spectrum of 1 indicated the presence of characteristic functional groups at 3337 (OH), 2948 (C–H), 1720 and 1613 (C=O), 1570 (C=C), 1230 and 1073 (C–O–C) cm⁻¹.

The broad band-decoupled ¹³C NMR spectrum of compound 1 (Table 2) displayed signals corresponding to 32 carbon atoms, which were assigned using distortionless enhancement by polarization transfer and heteronuclear single-quantum coherence spectra to three CH₂ groups (δ_C=23.1, 24.4, and 32.6 ppm); five CH groups, among them two aliphatic oxymethines (δ_C=67.0 and 71.3 ppm) and three aromatic CH (δ_C=107.7, 109.0, 140.1 ppm); two methyl groups including one aromatic CH₃ (δ_C=21.2 ppm) and another aliphatic (δ_C=17.5 ppm); two methoxy groups (δ_C=53.4 and 53.5 ppm); and 20 quaternary carbons of which two C atoms of ester groups (δ_C=171.4 and 171.5 ppm) and two carbonyl of xanthones hydrogen bounded to two adjacent hydroxyl groups (δ_C=187.0 and 187.5 ppm). In the ¹H NMR spectrum (Table 2), signals of four chelated hydroxyl groups at δ_H=11.63, 11.71, 13.98, 13.99 ppm, and two broad singlets at δ_H=2.79 and 2.67 ppm exchangeable by D₂O, were observed. The ¹H NMR spectrum also exhibited resonances of two ortho-coupled aromatic protons at δ_H=7.25 ppm (1H, d, J=8.4 Hz) and 6.60 ppm (1H, d, J=8.4 Hz); one aromatic singlet at δ_H=6.50 ppm (1H, s); two oxymethines at δ_H=4.13 ppm (brd, J=1.7 Hz) and 4.34 ppm (dd, J=4.3 Hz, J=2.0 Hz); two methoxy groups at δ_H=3.74 ppm (3H, s) and 3.75 ppm (3H, s); two signals of methyl groups at δ_H=2.13 ppm (3H, s) and 1.19 ppm (3H, d, J=6.7 Hz); and other aliphatic H atoms between δ_H=1.90–2.95 ppm. The presence of two conjugated carbonyl groups at δ_C=187.0 and 187.5 ppm hydrogen bonded to two adjacent hydroxyl groups at δ_H=11.71, 13.98, 11.63, and 13.99 ppm (characteristic shifts of a keto-enol structure), as well as the analysis of the long-range correlations of the heteronuclear multiple-bond correlation (HMBC) experiments, suggested the presence of two tetrahydroxanthone rings [14] as shown in Fig. 2. Analysis of these data revealed some similarities with that of neosartorin (1a) [15]. The difference between these two compounds was indeed the presence of the signal of two nonchelated hydroxyl groups (δ_H=2.67 and 2.79 ppm) in compound 1and the lack of the signals of the acetyl group of compound 1a. Therefore, the locations of the hydroxyl groups were unambiguously deduced from the correlations observed in the HMBC spectrum between the hydroxyl protons at δ_H=2.67 and 2.79 ppm and the carbons C-5′ (δ_C=71.3 ppm) and C-5 (δ_C=67.0 ppm), respectively. The relative stereochemistry of compound 1 was determined on the basis of ¹H–¹H correlation spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) correlations. The correlations observed in the NOESY spectrum between H-5 and the two protons of the C-6 methylene group and the coupling constant of 4.25 Hz and 2.0 Hz are consistent with the equatorial orientation of H-5. The pseudoaxial orientation of the methylene hydrogen H-7′ (δ_H=2.55 ppm, dd, J=12.1, 19.0 Hz) was suggested by the vicinal coupling constant J_6′,7′=12.1 Hz, and consequently suggested the pseudoequatorial orientation of another diastereotopic hydrogen H-7′ (δ_H=2.42 ppm, m). The small coupling constant of H-5′ (br d, J=1.7 Hz) and correlation observed in the NOESY spectrum between H-5′ and H-6′ suggest the equatorial orientation of H-5′. Methoxycarbonyl moieties at C-10 and C-10′ are in a cis position with respect to protons H-5 and H-5′, respectively, as shown by the presence observation of nuclear Overhauser effect crosspeaks between the CH₃ protons of these groups and protons H-5 and H-5′. These data clearly indicated that compound 1is a new ergochrome from P. lilacinum with the structure as shown.

Fig. 2:

Selected HMBC, and COSY, and NOESY correlations observed in compound 1.

The antiprotozoal activities of the ethyl acetate extracts from the mycelia of P. lilacinum, Aspergillus sp., and Fusarium sp., and compound 1 were evaluated in vitro against four parasitic protozoa: P. falciparum, L. donovani, T. brucei rhodesiense, and T. cruzi. The cytotoxic potential of these samples on mammalian L6 cells was also assessed and the selectivity index (SI: IC₅₀ L6 cells/IC₅₀ parasite) was calculated (Table 3).

All the tested samples exhibited growth inhibiting activity against L. donovani. In fact, the extract from the mycelium of P. lilacinum, and the new compound 1 exhibited potent antileishmanial activity with IC₅₀ values below 0.29 μg mL⁻¹ and good selectivity (SI>49). The activity of the extract (IC₅₀=0.174 μg mL⁻¹) was comparable with that of the reference miltefosine (IC₅₀=0.145 μg mL⁻¹) in vitro, whereas the tetrahydroxanthone 1 was almost potent as miltefosine with an SI value of 49.5. The pure compound was less selective than the crude extract where it was isolated. This might be due to the synergism of the extract constituents. The extracts from the Aspergillus sp. and the Fusarium sp. exhibited only moderate activity.

Apart from the extract of the Aspergillus strain, other tested samples were active against P. falciparum but showed poor selectivity toward L6 cell lines. The ethyl acetate extracts from Fusarium sp. and P. lilacinum were the most active ones with IC₅₀ values of 3.96 μg mL⁻¹ and 5.53 μg mL⁻¹, respectively.

Concerning the antitrypanosomal assays, all samples exhibited moderate growth inhibitory activity against both T. brucei rhodesiense and T. cruzi albeit with limited selectivity (SI<10). A previous study indicated that the ethanol extract of P. lilacinum UFMGCB 1510 from the soil of Antarctic Peninsula displayed high trypanocidal against T. cruzi and moderate activity against L. amazonensis [16].

The extract, sub-fractions T2 and T3 from P. lilacinum, and compound 1 were also evaluated for their antibacterial activity with respect to the measurement of the zone of inhibition and minimum inhibitory concentration (MIC) against Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Escherichia coli, Providencia stuartii, Klebsiella pneumoniae, and Pseudomonas aeruginosa in liquid medium (Tables 4 and 5). All the tested samples exhibited antibacterial activity at least against one strain of the tested microorganisms with compound 1showing the best potency (62.5 ≤ MIC ≤ 150 μg mL⁻¹). The ratio MIC/minimum bactericidal concentration (MBC) below 2 indicated their bactericidal properties. Although these activities are lower than those of the reference molecule gentamicin on the same strains, these results are significant because the tested strains are resistant to antibiotics.

The results obtained from this study, both the new ergochromone and the biological activities, are of interest. So far ergochromes were detected as secondary metabolites of Claviceps Spp., Penicillium oxalicum, Aspergillus ochraceus, A. fumigatiaffinis, Neosartorya fischeri, and various lichen species [17], [18]. A few bioactive metabolites have been isolated from P. lilacinum, including paecilotoxins, pyridone alkaloid paecilomide, and acremoxanthone [19]. This is the first isolation of an ergochrome from the mycelium of P. lilacinum. In addition, the low toxicity of compound 1 against a mammalian (L6) cell line is interesting and indicated that it could be considered as lead compound to be investigated for the formulation of new antileishmanial drugs. Xanthones have been reported to possess antileishmanial and cytotoxic activity [20]. Previous studies showed that the antileishmanial activity of xanthones involves complexation of heme, inhibiting the hemozoin formation [21], [22].

The interesting antibacterial activity of compound 1 confirmed the antimicrobial potential of ergochromes, a class of secondary metabolites that have shown good activity even on clinical resistant strains of bacteria [17].

All these results highlight the bioactive potency of ergochromones and justify the interest of the investigation of endophytic fungi as potential sources of new antiparasitic agents.

3 Materials and methods