Chemical Composition of Aspergillus creber Extract and Evaluation of its Antimicrobial and Antioxidant Activities

Fungal secondary metabolites represent a diverse group of bioactive natural products often produced at a restricted part of the life cycle (Keller et al. 2005). Aspergillus species are among the major contributors to the secondary metabolites of fungal origin (Dewi et al. 2012; Bai et al. 2014). Although the functions of these compounds for the producing fungi are obscure or unknown, they confer important benefits to humankind as many of them have antibiotic and pharmaceutical activities (Yu and Keller 2005; Siddiquee et al. 2015). Isolation of a new fungal strain is often conducted to the identification of new natural products (Brakhage and Schroeckh 2011).

Aspergillus species assigned to section Versicolores are among the most ubiquitous fungi, frequently isolated from environmental samples (Gautier et al. 2016). The important characteristics that render these species of interest to the scientific community are their prevalent in indoor environments, their capacity to produce sterigmatocystin (STC), a carcinogenic mycotoxin, and their diverse biotechnological applications (Siqueira et al. 2016). Before using molecular methods for species identification, isolates belonging to section Versicolores were commonly reported as A. versicolor (Despot et al. 2016). In 2012, Jurjevic et al. revised the section Versicolores and accepted 13 species; among them, Aspergillus creber was described for the first time as a new species. Currently, the section Versicolores comprises 17 distinct species (Despot et al. 2017); among them, A. versicolor and A. sydowii are still the most commonly reported and studied species (Siqueira et al. 2016). However, very few reports are available concerning the species A. creber and those reports were only concerned with contamination by A. creber and its ability to produce STC. A. creber was reported as the most prevalent species in indoor air environments in USA (Jurjevic et al. 2012) and in Italian libraries (Micheluz et al. 2015). A. creber was also reported as among the lower producers of STC (Jurjevic et al. 2013).

Therefore, the lack of knowledge of this species recently assigned to section Versicolores has attracted our attention to more explore and study this species. In our research A. creber was assayed for its secondary metabolites diversity using UHPLC-MS/MS technique. Then, the antimicrobial activity of A. creber against some human pathogens and its ability to scavenge different free radicals, and to produce antioxidant compounds were also evaluated to improve the knowledge of this species, which has never been tested before for its biological activities.

Fungal strain.Aspergillus isolate showed the typical macro- and micro-morphological features described for section Versicolores. For species designation, sequencing of the ITS rDNA and CaM gene followed by a BLAST search revealed that the isolated strain had a high identity (99%) with A. creber. Thus, according to these results, the strain was identified as A. creber and its sequences data were submitted at GenBank with accession numbers: MH796366 and MH796367.

UHPLC-MS/MS analysis. After two weeks of growth in the fermentation broth, the secondary metabolites of A. creber were extracted, through EtOAc, filtered and concentrated in vacuo to give EAF, which was subjected to UHPLC-MS/MS analysis. On the basis of the fungal metabolites list established by (Lehner et al. 2011), molecular mass data, MS/MS analysis and references, five known fungal metabolites were tentatively identified as asperlactone (1), emodin (5), sterigmatocystin (7), deoxybrevianamide E (9) and norsolorinic acid (10) (Table I) whereas five metabolites remained unidentifiable. The Total Ion Current (TIC) chromatograms of the ten peaks of detected compounds are shown in Fig. 1.

Fig. 1.

Antimicrobial activity. Results obtained for the antimicrobial activity of EAF are shown in Table II. In the case of bacteria, the highest zone of inhibition was displayed against Gram-positive DRB, including MRSE and SABL, followed by Gram-negative bacteria K. pneumoniae and S. typhi with an inhibition zone ranged from 8.5 ± 0.6 to 14.0 ± 0.2 mm. No activity was observed against E. coli and P. aeruginosa at the concentration used. In the case of yeasts, the EAF inhibited the growth of all tested yeasts (C. albicans and C. glabrata) with maximum inhibitory activity against C. albicans, which displayed an important zone of inhibition (20.6 ± 0.8 mm).

Table II showed also the MIC values of A. creber extract against the tested organisms, which showed an inhibition zone in the disc diffusion assay. The EAF of A. creber showed MIC values ranging from 0.325 to 5 mg/ml. The higher MIC values were recorded against C. albicans (0.325 mg/ml) and Gram-positive DRB (0.625 mg/ml) whereas the lower MIC values were recorded against C. glabrata (1.25 mg/ml) and Gram-negative bacteria K. pneumoniae (2.5 mg/ml) and S. typhi (5 mg/ml).

Antioxidant analysis. In the DPPH assay, the EAF of A. creber extract demonstrated a dose-dependent scavenging activity and the highest decolorization was recorded at 400 μg/ml (Fig. 2a). In the ABTS assay, like the DPPH assay, the EAF exhibited a rich scavenging effect and the highest decolorization was also recorded at a concentration of 400 μg/ml (Fig. 2b). In both assays, the scavenging activity of EAF was slightly lower than that of ascorbic acid. In the Folin-Ciocalateu assay, the EAF showed a high level of total phenols (Table III).

Fig. 2.

Fungi are major producers of secondary metabolites with different biological activities and various chemical structures (Abo-Elmagd 2014). Until today, except for A. versicolor and A. sydowii, there is no thorough study on metabolic profiles for the Aspergillus species belonging to section Versicolores (Despot et al. 2017). Moreover, to our knowledge, there are no published reports recorded the antimicrobial and the antioxidant activities of A. creber.

Our study demonstrated that A. creber is able to produce a variety of secondary metabolites previously reported as potent biologically active compounds. The biological activity of asperlactone, a polyketide metabolite, had been established for the first time by (Balcells et al. 1995). It was the first fungal compound that exhibited insect growth regulating activity in in vivo tests. Asperlactone has also been reported as a potent antibacterial and antifungal metabolite (Chen et al. 2014). Emodin is the most studied anthraquinone derivative for its diverse biological activities, including antibacterial (Hatano et al. 1999), antifungal (Kim et al. 2004), antioxidant (Izhaki 2002), and anticancer properties (Cheshmi et al. 2017; Zhao et al. 2017). STC is a polyketide mycotoxin and a precursor of Aflatoxin B1 (AFB1), however, its toxicity is lower than that of AFB1 (Piontek et al. 2016). Deoxybrevianamide E is a prenylated indole alkaloid that belongs to the family of brevianamides. Although a variety of biological activities were exhibited by many brevianamides including anti-bacterial, anti-insect pests and antitubercular properties (Xu et al. 2017) there are no reports in the literature concerning the bioactivities of deoxybrevianamide E. Norsolorinic acid is an anthraquinone derivative fungal metabolite. (Wang et al. 2008) reported that norsolorinic acid has antiproliferative activity on T24 human bladder cancer cells.

The ability of A. creber to produce STC is in accordance with previous studies demonstrated that most of Aspergillus section Versicolores are STC producers (Jurjević et al. 2013, Despot et al. 2016). Moreover, our findings are in agreement with the study of (Micheluz et al. 2016) reported that A. creber is able to produce emodin, STC, deoxybrevianamide E and norsolorinic acid. However, to our knowledge, this is the first report concerning the production of asperlactone from the species A. creber.

In the antimicrobial assays, our study indicated that A. creber exhibited more potent antibacterial activity against Gram-positive than Gram-negative bacteria. This variation of susceptibility could be attributed to the morphologic difference in the composition of their cellular membranes, which influences their reaction to antibacterial compounds (Valle Jr et al. 2015). In addition to its antibacterial activity, the EAF of A. creber exhibited higher anticandidal activity against Candida species. In the case of Candida species, ergosterol is the main target of antifungal drugs (Martins et al. 2015). These results are in contrast to the previous studies reported that the metabolites produced by A. versicolor, the most studied species of Aspergillus section Versicolores, exhibited lower or no antibacterial and anticandidal activities (Zhuang et al. 2011; Song et al. 2012; Ebada et al. 2018). Another attractive finding on A. creber was the higher MIC values of its ethyl acetate extract against C. albicans (0.312 mg/ml) and Grampositive DRB (0.625 mg/ml) since these species are the most common opportunistic pathogens (Martins et al. 2015; Knafl et al. 2017). Moreover, our findings can be considered very promising since the extract was crude and obtained by using non-optimized fermentation, which usually produces a poor yield of active compounds (Noor Ifatul et al. 2016).

Our study also brings additional data on A. creber, since the antioxidant activity and the TPC of this species were evaluated for the first time. The antioxidant ability of A. creber was tested using two different procedures, DPPH and ABTS assays. The DPPH assay is a widely used method that based on the reduction of the purple DPPH free radical to a yellow colored compound 1,1-diphenyl-2-picryl hydrazine (Floegel et al. 2011) whereas the ABTS assay is based on the reduction of the blue-green pre-formed ABTS^·+ to the colorless ABTS form (Re et al. 1999). Thus, the higher scavenging activities of the EAF indicated its bioactive potential to neutralize the DPPH free radicals and ABTS^·+. These results are in agreement with many other studies reported the antioxidant potency of filamentous fungi from different sources (Abo-Elmagd 2014; Kumaresan et al. 2015; Sharma 2015; Smith et al. 2015; Sugiharto et al. 2016). The slight difference between the antioxidant capacities of EAF and ascorbic acid may be explained either by the purity of ascorbic acid, whereas the extract of A. creber is a mixture of multiple compounds or by the capacity of some compounds in the fungal extract to neutralize or to inhibit the effect of active compounds (Dhankhar et al. 2012). Moreover, our data revealed that A. creber is able to produce much higher content of phenols than the other filamentous fungi in the literature (Jakovljević et al. 2014; Smith et al. 2015; Sugiharto et al. 2016). Many previous studies attributed the antioxidant activity of filamentous fungi to their TPC (Abdel-Monem et al. 2013; Smith et al. 2015, Nwobodo et al. 2017). In accordance with this, the higher percentages of inhibition of DPPH radical and ABTS^·+ in our fungal extract might contribute to the high level of phenolic compounds.

In conclusion, UHPLC-MS/MS analysis revealed that A. creber could produce a variety of secondary metabolites including polyketides, anthraquinones, and alkaloids. Our findings suggest that A. creber might represent a novel source of natural bioactive products since we have demonstrated its ability to inhibit the growth of many pathogen microorganisms, mainly C. albicans and Gram-positive DRB, and its high antioxidant activity. Our work will increase the knowledge about the species A. creber newly described and serve as a prelude to a better understanding of the biology and the chemistry of its metabolites.