Four butyrolactones and diverse bioactive secondary metabolites from terrestrial Aspergillus flavipes MM2: isolation and structure determination

The chemical constituents and biological activities of the terrestrial Aspergillus flavipes MM2 isolated from Egyptian rice hulls are reported. Seven bioactive compounds were obtained, of which one sterol: ergosterol (1), four butyrolactones: butyrolactone I (2), aspulvinone H (3), butyrolactone-V (6) and 4,4'-diydroxypulvinone (7), along with 6-methylsalicylic acid (4) and the cyclopentenone analogue; terrien (5). Structures of the isolated compounds were deduced by intensive studies of their 1D & 2D NMR, MS data and comparison with related structures. The strain extract and the isolated compounds (1-7) were biologically studied against number of microbial strains, and brine shrimp for cytotoxicity. In this article, the taxonomical characterization of A. flavipes MM2 along with its upscale fermentation, isolation and structural assignment of the obtained bioactive metabolites, and evaluate their antimicrobial and cytotoxic activities were described.


Background
In recent years, numerous metabolites possessing uncommon structures and potent bioactivity have been isolated from strains of bacteria and fungi collected from diverse environments, such as soils, animals, plants and sediments [1,2]. It was noted until Alexander Fleming discovered penicillin G from Penicillium notatum almost 83 years ago (1928) that fungal microorganisms suddenly became a hunting ground for novel drug leads [3,4]. Therefore, many pharmaceutical companies and research groups were motivated to start sampling and screening large collections of fungal strains for antibiotics [3,5,6]. Antimycotics [7,8], antivirals [9], anticancers [10] and pharmacologically active agents [11]. The Aspergilli represents a large diverse genus, containing ca. 180 filamentous fungal species, of substantial pharmaceutical and commercial values [12]. In the research program to explore promising bioactive secondary metabolites from fungi, the terrestrial fungi, Aspergillus flavipes sp. isolate MM2 obtained from rice hulls, was investigated. The strain extract revealed the presence of promising antimicrobial activity against some pathogenic test organisms. Chemical screening (TLC investigation) of the strain extract showed numerous characteristic bands. Therefore, the strain was applied to large-scale fermentation by using Czapeck-Dox medium [13]. Working up of the strain cells produced ergosterol (1), while the filtrate extract afforded six diverse metabolic compounds: butyrolactone-I (2), aspulvinone H (3), 6-methylsalicylic acid (4), terrien (5), butyrolactone-V (6) and 4,4'-diydroxypulvinone (7). The chemical structures of the isolated compounds (1)(2)(3)(4)(5)(6)(7) were identified with the help of NMR (1D & 2D) and mass spectrometry (ESI, EI, HRESIMS) ( Figure 1). The antimicrobial activity was tested against some microorganisms and cytotoxicity was examined by using brine shrimp.

Taxonomical characterization of the fungal strain
The grown colonies of the fungal strain on Czapek-Dox medium showed bright whit-faint yellow colonies on the agar plate with a brown staining background [13]. The colonies are growing rather slowly, showing whitish from conidial masses, with brownish conidiophores shining through, reverse yellow-brown to red brown conidial heads spas, loosely columnar, conidiophores smooth-walled, pale yellow to light brown 2.4-3.2 μm in diameter. According to its morphological and microscopic characteristics and comparison with the taxonomical keys of Raper and Fennel [14], the strain was assigned as A. flavipes MM2.

Fermentation, working up and isolation
Based on the pre-screening study, the fungal strain A. flavipes MM2, cultivated on Czapeks-Dox for 10 days at 28°C, was shown to exhibit biological and chemical interest results. Therefore, the fungal strain was scaled up as 10 L culture using the same cultivating conditions applied for screening studies. After harvesting, both supernatant and mycelial cake phases were individually worked up. Purification of the mycelial extract using silica gel column, followed by washing the afforded major fraction by methanol and purification with Sephadex LH-20, yielded ergosterol (1). An application of the culture filtrate extract of A. flavipes MM2 to silica gel column chromatography, followed by diverse chromatographic techniques, resulted in the isolation of six compounds: butyrolactone-I (2), aspulvinone H (3), methylsalicylic acid (4), terrine (5), butyrolactone-V (6) and 4,4'-diydroxypulvinone (7).

Chemical characterization
Ergosterol (1) was obtained as colourless solid, showing UV activity during TLC, which turned violet on spraying with anisaldehyde/sulphuric acid and changed latter to blue. Structure of 1 was confirmed by different spectroscopic means (EI MS, 1 H, 13 C/APT NMR), chromatographic and comparison with literature [15,16]. Ergosterol plays an important role as inhibitor of lipid per-oxidation and showed strong DPPH radical scavenging activity as well [17,18], along with its cytotoxicity against HL-60 cells [19], MCF-7 cell line [20].

Butyrolactone-I (2)
The molecular weight of 2 was established as 424 Dalton by ESI MS, having the corresponding molecular formula C 24 H 24 O 7 and 13 unsaturation bonds. 1 H/H,H COSY NMR spectra of 2 showed two o-doublets (J8 .8 Hz) each of 2H at δ 7.57 and 6.86, being for 1,4-disubstituted aromatic residue, along with three signals at δ 6.50, 6.48 and 6.40 representing 1,3,4-trisubstituted aromatic ring. A triplet signal of 1H was at δ 5.05, representing an olefinic methine linked to a doublet methylene signal appeared at δ 3.06. A 3H methoxy group (3.76); doublet of an AB methylene group (δ 3.42) attached to sp 2 system; and further two singlet methyls were visible at δ 1.65 and 1.56, representing a prenyl system.

Aspulvinone H (3)
Based on the ESI MS, the molecular weight of 3 was deduced as 432 Dalton, and the corresponding molecular formula as C 27 H 28 O 5 , containing 14 unsaturation bonds, as closely related to butyrolactone-I (2). 1 H NMR spectrum of 3 showed six confused doublets each of 1H between δ 7.81 and 6.73, being of two unsymmetrical tri-substituted aromatic residues, and singlet methine at δ 6.22. A multiplet of 2H (δ 5.36), 4H of two attached sp 2 -bounded methylenes (δ 3.40-3.00) and multiplet signal (δ 1.75, 12H) of four sp 2 -linked methyls, assigning two prenyl systems. Based on the revealed NMR data and molecular formula, and search in Anti-Base [2], structure of 3 was fixed as aspulvinone H [31].

Biological activities
Activity patterns of the mycelial and supernatant extracts produced by fungal strain A. flavipes MM2 against set of microorganisms namely, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger were carried out using agar disc method (25 μg/disc, ∅ 5 mm). In accordance, both extracts showed high antibacterial (16-14 mm) and moderate anti-yeast and antifungal (10-14 mm) activities (Table 1).

Isolation and taxonomy of the producing strain
The terrestrial A. flavipes MM2, which was identified according to the Raper and Fennel [14], has been isolated from rice hulls sample by placing the rice hulls over water agar medium (g/L): Agar-agar (20) and water (100%) with incubation at 28°C for 7 days the developing colony was transferred to Czapeks agar with incubation at 28°C for 10 days. Bright whit-faint yellow colonies with a brown straining background of the fungal strain were grown. The colonies are growing rather slowly, showing whitish from conidial masses, with brownish conidiophores shining through, reverse yellowbrown to red brown Conidial heads spas, loosely columnar, conidiophores smooth-walled, pale yellow to light brown 2.4-3.2 μm diameter. According to its morphological and microscopic characteristics and comparison with literature, the fungal strain was assigned as A. flavipes MM2 [14]. The strain is deposited in Dr Mohammad Magdy El Metwally collection in Microbiology Department, Soil & Water and Environment Research Institute, ARC, Giza, Egypt.

Fermentation, extraction and isolation
Small pieces (1 cm 2 ) of well grown sub-cultures of A. flavipes MM2 were inoculated into thirty 1-L Erlenmeyer flasks, each containing 300 mL of sterilized Czapeck-Dox medium (g/L): Sucrose (30), NaNO 3 (3), K 2 HPO 4 (1), KCl (0.5), MgSO 4 (0.5), FeSO 4 (0.01) and distilled water (1 L) at pH = (7.3). The inoculated flasks were incubated for 10 days at 28°C and 100 rpm. After harvesting, the fungal mate and supernatant were separated by filtration. The fungal mat was then applied to maceration in methanol (3 × 0.5 L). The methanol extract was concentrated in vacuum and the remaining aqueous solution was re-extracted by ethyl acetate followed by concentration to yield 845 mg as brown crude extract. The supernatant was passed through XAD-16 column (4 × 120 cm). After washing with water, the absorbed organic extract was eluted by methanol, followed by concentration under vacuum, and the aqueous residue was re-extracted by ethyl acetate, followed by concentration in vacuo to afford 818 mg as brown crude extract.

Brine shrimp microwell cytotoxicity assay
The cytotoxic assay was performed according to Sajid et al.'s screening [48].