A new dimeric naphtho-γ-pyrone from an endophytic fungus Aspergillus niger AKRN associated with the roots of Entandrophragma congoënse collected in Cameroon

1 Introduction

Endophytic fungi are eukaryotic organisms living inside the tissues of the host plants without causing any visible manifestations of disease, and live in mutualistic association with plant for at least a part of their life cycle [1]. These microorganisms have recently been recognized as a rich source of structurally novel natural products with high level of biodiversity, interesting biological activities, and may directly or indirectly be used as therapeutic agents or prodrugs against several diseases [1–3]. The genus Aspergillus from the Trichocomaceae family consists of a set of filamentous saprophytic fungi that can be found in almost all aerobic environments and is among the most common endophytic fungal genera in plants. This genus with over 180 species has been found to produce a wide range of secondary metabolites including alkaloids, terpenoids, cytochalasins, butyrolactones, and lignans, which showed antibacterial, antifungal, antifeedant, cytotoxicity, and anticancer activities [4–12]. Aspergillus niger is commonly isolated from plant tissues, and it produces α-pyrones, γ-pyrones, and naphtho-γ-pyrones as the most widely encountered classes of bioactive metabolites. Examples include campyrones A–C [13], nigerapyrones A–H, asnipyrones A–B [14], asperpyrones A–C [15], aurasperones A–E, rubrofusarin, flavasperone, and nigerone [16]. In addition, harmful mycotoxins such as malformin C [17], ochratoxin A, 3-nitropropionic acid, and oxalic acid have also been isolated [18]. As part of our ongoing search for bioactive compounds from a natural source, we investigated the chemical constituents of the fungal strain A. niger, isolated from the inner tissues of Entandrophragmacongoënse collected in Nkomokui near Yaounde, Cameroon, and fermented in static culture on solid rice. We report herein the isolation and characterization of a new dimeric naphtho-γ-pyrone 2-hydroxydihydronigerone (1).

2 Results and discussion

The fungal strain AKRN, isolated from the roots of E. congoënse (De Wild) A. chev. (Meliaceae), was identified as A. niger. The strain was cultured on a solid rice medium for 30 days and then extracted with ethyl acetate. Column chromatography of the crude extract led to the isolation of a new dimer of naphtho-γ-pyrone named 2-hydroxydihydronigerone (1) together with five known compounds: nigerone (2) [19], pyrophen (3) [20], kojic acid (4) [21], 4-(hydroxymethyl)-5-hydroxy-2H-pyran-2-one (5) [22], and p-hydroxyphenylacetic acid (6) [23].

Compound 1 was obtained as a yellow and optically active powder ([α]D20 = −56°). Its molecular formula was established as C₃₂H₂₉O₁₁ from the [M + H]⁺ peak at m/z = 589.1733 (calcd. 589.1709 for C₃₂H₂₉O₁₁) in the positive high resolution electronspray ionization mass spectroscopy (HRESIMS), indicating 20° of unsaturation. The IR spectrum exhibited strong absorptions at 1765 and 1653 cm⁻¹ due to two carbonyl groups which were confirmed in the ¹³C NMR spectrum with signals at δ_C = 185.0 and 197.1 ppm. The UV spectrum showed absorption bands at λ_max = 230, 278, and 330 nm, suggesting the presence of an extended conjugated ring system, characteristic of a heminigerone derivative [19]. The ¹H NMR spectrum (Table 1) exhibited signals for two chelated hydroxyl groups at δ_H = 15.23 and 14.60 ppm, two meta-coupled aromatic protons at δ_H = 6.40 and 6.20 ppm (1H each, J = 2.0 Hz), one olefinic proton at δ_H = 5.98 ppm (1H, s), one olefinic methyl group at δ_H = 2.09 ppm (3H, s), and four methoxyl groups at δ_H = 3.48 (3H, s), 3.49 (3H, s), 3.54 (3H, s), and 4.06 ppm (3H, s). In the ¹³C NMR spectrum, signals at δ_C = 21.1, 55.5, 55.6, and 56.1 ppm confirmed the presence of the olefinic methyl group and methoxyl groups. The ¹³C NMR spectrum (Table 1) also displayed signals including those of five methine groups (δ_C = 96.2, 96.8, 97.4 [× 2], and 107.6 ppm), and 30 quaternary sp² carbons. In the heteronuclear multiple-bond correlation (HMBC) spectrum (Fig. 1), the olefinic proton signal at δ_H = 5.98 ppm displayed cross peaks with carbon signals at δ_C = 185.0 ppm (C-4) and 104.7 ppm (C-4a), which in turn showed correlations to the deshielded proton signal at δ_H = 15.23 ppm. The latter also showed correlations with carbon signals at δ_C = 151.9 ppm (C-5) and 107.1 ppm (C-5a), which also revealed an intense cross peak with the aromatic proton signal at δ_H = 6.40 ppm (H-7). Furthermore, the HMBC spectrum revealed important cross peaks due to ²J and ³J correlations of the aromatic proton signal at δ_H = 6.20 ppm (H-9) with the carbon signals at δ_C = 141.2 ppm (C-9a) and 109.0 ppm (C-10). These findings established the heminigerone moiety in the structure of 1. In addition, signals were observed at δ_H = 11.88 ppm (hydroxyl group) and δ_H = 2.90, 2.97 ppm (methylene group) in the ¹H NMR spectrum, as well as signals at δ_C = 100.3 ppm (acetal group) and 47.2 ppm (methylene group) in its ¹³C NMR spectrum (Table 1). In the HMBC experiment, correlations were observed between the proton signal at δ_H = 1.41 ppm (methyl group) and carbon signals at δ_C = 100.3 ppm (acetal group) and 47.2 ppm (methylene group), suggesting the oxidation of one heminigerone moiety across one of the double bond at C-2 or C-2′ in the structure of 2. Furthermore, cross peaks were observed between the proton signal at δ_H = 14.60 ppm (OH-5′) and carbon signals at δ_C = 103.8 ppm (C-4a′), 151.9 ppm (C-5′), and 104.6 ppm (C-5a′), confirming the presence of the suggested hydrated heminigerone feature. Compound 1 is then a derivative of nigerone (2), consisting of two sub-structures including a heminigerone and a 2-hydroxydihydroheminigerone. Chemical shifts of different atoms in 1 were assigned on the basis of heteronuclear single-quantum coherence and HMBC spectra and by comparison of the NMR data with those of nigerone already published in the literature. The comparison of the calculated CD spectra (Fig. 2) for compound 1 and nigerone (2), as well as the reported data of the natural (–)-nigerone and related compounds [19, 24, 25], revealed the same respective conformers in compounds 1 and 2, as established in (–)-nigerone [24]. Consequently, the absolute configurations of compound 1 and nigerone (2) were assigned to be M [24]. However, the configuration of the chiral center C-2′ remained unknown as reported for similar 2,3 (or 2′,3′)-hydrated naphtho-γ-pyrones [15, 16, 26]. On the basis of this evidence, the structure of 1 was determined to be that of an asymmetrical dimeric naphtho-γ-pyrone consisting of heminigerone and 2′-hydroxydihydroheminigerone linked at C-10/C-10′ as shown in Fig. 3.

Fig. 1:

Important HMBC correlations of 1.

Fig. 2:

CD spectra of compound 1 and nigerone (2).

Fig. 3:

Structures of isolated compounds 1–6.

The structures of the known compounds were established via spectroscopic data and/or by comparison with literature data.

The isolated compounds 1–5 which were obtained in sufficient quantity were evaluated against five bacterial strains, namely Enterobacter aerogenes (CM64), Enterobacter cloacae (BM67), Klebsiella pneumonia (K2), and Escherichia coli (ATCC8739 and ATCC10536). These tested secondary metabolites have shown a very weak inhibition on the bacterial growth, even at the highest concentration of 500 μg mL⁻¹.

3 Chemotaxonomic significance

The genus Aspergillus has been reported as a rich source of several classes of secondary metabolites including pyrones, naphthopyrones, terpenoids, or steroids. The present study involved the investigation of the fungal endophyte Aspergillus niger AKRN strain fermented in static culture on solid rice, which yielded a mixture of four steroids as well as six pure compounds allocated into two naphtho-γ-pyrones (1, 2), one γ-pyrone (4), two α-pyrones (3, 5), and one acetic acid derivative (6). The occurrence of nigerone (2) and pyrophen (3) from A. niger has been reported [19, 20], whereas kojic acid (4) and 4-(hydroxymethyl)-5-hydroxy-2H-pyran-2-one (5) have been previously produced by A. oryzae and A. flavus, respectively [21, 22]. However, p-hydroxyphenylacetic acid (6) has been noted previously from the fungal strain Stachylidium sp. [23], and the naphtho-γ-pyrone, namely 2-hydroxydihydronigerone (1), was found to be a new compound. The obtained results from our chemical investigation of A. niger AKRN have revealed constituents which are in agreement with the identification, the classification, and the chemotaxonomy of this fungal strain.

4 Experimental section