Preliminary report on antifungal activity of a Solanum nigrum extract against five mycotoxin-producing fungi

*Corresponding author: Mauro Musto, University of Basilicata, School of Agricultural, Forestry, Food and Environmental Sciences, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy, E-mail: mauro.musto@gmail.com Received: 04 August 2015; Revised: 13 October 2015; Accepted: 20 October 2015; Published Online: 20 October 2015 Musto: Solanum nigrum extract and Mycotoxigenic Fungi 826 Emir. J. Food Agric ● Vol 27 ● Issue 11 ● 2015 Previous studies (e.g. Juglal et al., 2002; Rasooli & Abyaneh, 2004; Razzaghi-Abyaneh et al., 2008; Nguefack et al., 2009; Morcia et al., 2012) have found that essential oils from some aromatic plants were able to inhibit the growth of some Aspergillus, Fusarium, and Penicillium strains, all responsible of mycotoxin production. Phenolic extracts from edible plants were also active against A. flavus and aflatoxin production (Oliveira & Furlong, 2008). Weedy plants may also be used to prepare active extracts against mycotoxic fungi. Thembo et al. (2010) evaluated the antifungal activity of some weeds against some Aspergillus and Fusarium isolates. The authors reported that all organic extracts showed growth inhibitory activity against most isolates of the Fusarium spp., whereas no inhibition of the Aspergillus spp. was observed. A common weed of arable land and gardens in most parts of Europe and the African continent is Solanum nigrum, a member of the Solanaceae family. The medicinal values of this plant as hepatoprotective, antimicrobial, antitumor, etc., are well documented (Atanu et al., 2011). Methanolic and ethanolic extracts of S. nigrum have been previously tested in vitro for their antifungal activity against some Aspergillus and Fusarium species with ambiguous results (Mohamed et al., 1996; Harisankar et al., 2011; Zubaır et al., 2011; Abbas at al., 2014). In the present study, the potential of an aqueous crude extract from S. nigrum lyophilized leaves for inhibiting the growth of some mycotoxigenic fungi was evaluated by using in vitro techniques. The aqueous extract was prepared by following the procedures described by Musto et al. (2014), who reported that the extract was active against Penicillium digitatum non only in vitro, but also in vivo preinfection conditions on lemons wound-inoculated with P. digitatum. Moreover, the authors highlighted that their important results were obtained by using water, which is not toxic as other solvents (e.g. methanol or ethanol) and does not interfere with the bioassays. MATERIALS AND METHODS Extract preparation Fresh leaves of S. nigrum were washed, lyophilized (-53°C, 0.250 mbar pressure) for 24 h by using a freeze dry system (Model Freezone 18, Labconco, Kansas City, MI, USA), and then powdered with a laboratory blender (Model 7010S, Waring Laboratory, New Hartford, CT, USA) for 30 s. To prepare the extract, 10 grams of lyophilized powder were poured into 150 ml of boiling double-distilled water (ddH2O). After 24 h in the dark under agitation, the mixture was clarified by centrifugation (at 20,000 rpm for 30 min) and the supernatant filtered through Whatman No. 4 paper. The obtained extract was finally sterilized through filter membrane (0.22 μm) before use. Phytochemical screening The presence of some phytochemical compounds was evaluated by using the protocols of Trease et al. (1989) and Pradhan et al. (2010), and modified by Musto et al. (2014). Alkaloids. 5 ml of the extract was treated first with 2 ml of HCl, and then with 1 ml of Dragendroff ’s reagent. An orange or red precipitate indicated the presence of alkaloids. Flavonoids. 1 ml of the extract was treated with few drops of 2% NaOH to produce an intense yellow colour. After adding few drops of dilute HCl, the extract became colourless if it contained flavonoids. Glycosides. 1 ml of the extract was treated first with 2 ml of CH3COOH mixed with few drops of FeCl3, and then with 1 ml of H2SO4. Formation of a reddish brown colour at the junction of two layers and the bluish green colour in the upper layer indicated the presence of glycosides. Saponins. 1 ml extract was first diluted with 5 ml of dd H2O, and then agitated for 15 min. The formation of at least 1 cm layer of foam indicated the presence of saponins. Steroids. 1 ml of the extracts was treated first with 10 ml of CHCl3, and then with 10 ml of H2SO4. A red colour in the upper layer and a yellow colour in H2SO4 layer indicated the presence of steroids. Tannins. 1 ml of the extract was first diluted with 4 ml of ddH2O, and then treated with few drops of 10% FeCl3. Formation of a blue/green colour indicated the presence of tannins. Terpenoids. 5 ml of the extract was treated first with 2 ml of (CH3CO)2O, and then with 2 ml of CHCl3; finally, 2 ml of H2SO4 were added. Formation of reddish violet colour indicated their presence in the extract. Reducing sugars. 1 ml of the extract was first treated with 5–8 drops of Fehling’s solutions (A and B), and then heated in a water-bath. Formation of a red precipitate indicated the presence of sugars. Antifungal activity evaluation Fungal cultures. Five mycotoxin-producing fungi were used in this study (Table 1). The spores of each fungus were harvested by flooding the surface of 10 days-old cultures with sterile 0.85% saline containing 0.1% Tween 80, and scraping the surface of colonies with glass rods. Spore suspensions were filtered through double-layered sterile cheesecloth, and the concentration was then adjusted to 106 spores/ml. Musto: Solanum nigrum extract and Mycotoxigenic Fungi Emir. J. Food Agric ● Vol 27 ● Issue 11 ● 2015 827 In vitro antifungal activity assays. Two kinds of agar diffusion tests were performed, the well diffusion (WD) and the paper disk diffusion (PD) test. In both tests, about 20 ml of the Saboraud Dextrose Agar (SDA) was poured into Petri plates (9 cm) and allowed to solidify. For each test and for each fungus, ten SDA plates were prepared by spreading 0.1 ml of the spore suspension (104 spores·ml-1) on each plate. In the WD test, 3 wells (6 mm diameter) were aseptically punched at equidistant points into the agar medium of each plate, and then filled with 100 μl of the crude extract. In the PD test, 3 sterile papers disks (Whatman No. 4 paper, 5 mm diameter), previously immersed in the crude extract and allowed to dry, were placed at equidistant points on agar surface of each plate. In both tests, Amphotericin B and sterile ddH2O were used as positive and negative controls, respectively. After 72 h at 25°C, the antifungal activity of the extract was evaluated by measuring the diameters of the growth inhibition zones around each well and disk. The experiments were carried out in triplicate. RESULTS AND DISCUSSION As shown in Table 2, seven out of eight tested phytochemical compounds were found in the extract. In particular, the extract contained alkaloids, flavonoids, glycosides, saponins, steroids, tannins, and terpenoids. These results are consistent from the previous studies on S. nigrum extracts (Gogoi & Islam 2012; Musto et al., 2014; Modilal et al., 2015; Singh & Vidyasagar, 2015a, b). According to the authors, thanks to the wide range of biological activities of these compounds, the extract may be exploited for the development of novel biopesticides or phytopharmaceuticals. The results from the in vitro antifungal activity assays (Table 3) showed that the aqueous extract was active, in both agar diffusion tests, against A. carbonarius (WD: 23.6 mm; PD: 15.3 mm) and P. expansum (WD: 22.1 mm; PD: 15.1 mm). The inhibitory activity of the extract against these two fungi was slightly lower than that of positive control: for A. carbonarius the difference between the inhibition zones ranged from 6.1 (WD) to 8.4 (PD) mm, whereas for P. expansum the range was 4.7 (WD) 6.9 (PD) mm. A higher concentration of the extract would have been as effective as the positive control. In fact, previous studies (e.g. Ekwenye and Elegalam, 2005; Okigbo and Igwe, 2007; Okigbo et al., 2012) on antimicrobial activity of different plant extracts have shown that the inhibitory effects are highly dependent on their concentrations, with stronger inhibition occurring at higher concentrations. No inhibition was instead observed against A. flavus, F. graminearum, and F. verticillioides. A previous study revealed that a methanolic extract of S. nigrum inhibited the growth of A. flavus (Harisankar et al., 2011). The authors also found a weak activity against F. verticillioides. However, Zubaır et al. (2011) reported that a methanolic extract of S. nigrum leaves did not inhibit the growth of A. flavus and A. niger. Similarly, Mohamed et al. (1996) found an ethanolic extract of S. nigrum to be not active against A. niger. More recently, Abbas at al. (2014) reported that the growth of against A. niger, A. flavus, and A. fumigatus was inhibited by a methanolic extract of the dried powdered fruit of S. nigrum. Compared to our results, the different antifungal activity against some mycotoxigenic fungi described in the aforementioned studies, are probably related to the different type of solvent used in the extraction procedures (methanol vs. water), as well as to the part of the plant used as starting vegetal material (leaf vs. fruit). These aspects can play a crucial role in preparing plant extracts (Ncube et al., 2008; Das et al., 2010). Overall, our results further confirm the antifungal activity of S. nigrum (Mohamed et al., 1996; Mutu et al., 2006; Atanu et al., 2011; Yanar et al., 2011; Lin et al., 2011; Sun et al., 2012; Singh et al., 2015a, b), and suggest Table 1: List of mycotoxin‐producing fungi used in this study


INTRODUCTION
In past decades, there has been an increasing interest toward some fungi belonging to the genera Aspergillus, Penicillium, Fusarium, and Alternaria. This interest is motivated by the fact that the occurrence of these fungi in foods and feeds may lead not only to significant economic losses, but may represent a health hazard to both humans and animals. This is mainly due to the production of some compounds from secondary metabolism process known as mycotoxins. It has been reported that more than 300 mycotoxins are currently known (Zain, 2011;Didwania, 2014), of which aflatoxins, ochratoxins, fumonisins, trichothecenes, and zearalenone are the most important ones (Pitt, 2000;CAST, 2003). These compounds can exert several toxic effects when ingested, from allergic responses to death. For instance, aflatoxins, which are produced by some Aspergillus species (Peraica et al., 1999;Richard, 2007;Didwania, 2014) have been defined as 'acutely toxic, immunosuppressive, mutagenic, teratogenic and carcinogenic compounds' (Peraica et al., 1999;p. 756). Ochratoxins, which are produced by some Aspergillus and Penicillium strains, are no less dangerous than aflatoxins. In particular, ochratoxin A is the most toxic one, due to its nephrotoxic, immunosuppressive, carcinogenic and teratogenic activity (Peraica et al., 1999;Richard, 2007).
The presence of mycotoxins is strictly related to some aspects related to storage, environmental, and ecological conditions of foods and feeds. Therefore, mycotoxin contamination can occur at various stages (e.g. in the field, during storage and processing) in the food chain (Bennett & Klich, 2003). Several biological, chemical and physical approaches have been proposed to reduce contamination levels in food and feeds, and, hence, to minimize human and animal exposure to mycotoxins (Varga & Tóth, 2005;Kabak et al., 2006;Zain, 2011). Approaches like physical separation of contaminated commodities, microwave or sonic drying, or the use of chemical antifungal agents, are undoubtedly effective, although they have some inherent disadvantages related to the sustainability of the equipment and chemicals, reduction of the nutritional value of the foods, the presence of toxic derivatives in the treated product, etc. (Juglal et al., 2002;Varga & Tóth, 2005;Kabak et al., 2006;Thembo et al., 2010).
A promising alternative approach is the use of plant extracts or plant phytochemical constituents for inhibiting fungal growth and, hence, to control mycotoxin contamination.
Weedy plants may also be used to prepare active extracts against mycotoxic fungi. Thembo et al. (2010) evaluated the antifungal activity of some weeds against some Aspergillus and Fusarium isolates. The authors reported that all organic extracts showed growth inhibitory activity against most isolates of the Fusarium spp., whereas no inhibition of the Aspergillus spp. was observed. A common weed of arable land and gardens in most parts of Europe and the African continent is Solanum nigrum, a member of the Solanaceae family. The medicinal values of this plant as hepatoprotective, antimicrobial, antitumor, etc., are well documented (Atanu et al., 2011). Methanolic and ethanolic extracts of S. nigrum have been previously tested in vitro for their antifungal activity against some Aspergillus and Fusarium species with ambiguous results (Mohamed et al., 1996;Harisankar et al., 2011;Zubaır et al., 2011;Abbas at al., 2014). In the present study, the potential of an aqueous crude extract from S. nigrum lyophilized leaves for inhibiting the growth of some mycotoxigenic fungi was evaluated by using in vitro techniques. The aqueous extract was prepared by following the procedures described by Musto et al. (2014), who reported that the extract was active against Penicillium digitatum non only in vitro, but also in vivo preinfection conditions on lemons wound-inoculated with P. digitatum. Moreover, the authors highlighted that their important results were obtained by using water, which is not toxic as other solvents (e.g. methanol or ethanol) and does not interfere with the bioassays.

Extract preparation
Fresh leaves of S. nigrum were washed, lyophilized (-53°C, 0.250 mbar pressure) for 24 h by using a freeze dry system (Model Freezone 18, Labconco, Kansas City, MI, USA), and then powdered with a laboratory blender (Model 7010S, Waring Laboratory, New Hartford, CT, USA) for 30 s. To prepare the extract, 10 grams of lyophilized powder were poured into 150 ml of boiling double-distilled water (ddH 2 O). After 24 h in the dark under agitation, the mixture was clarified by centrifugation (at 20,000 rpm for 30 min) and the supernatant filtered through Whatman No. 4 paper. The obtained extract was finally sterilized through filter membrane (0.22 μm) before use.

Phytochemical screening
The presence of some phytochemical compounds was evaluated by using the protocols of Trease et al. (1989) and Pradhan et al. (2010), and modified by Musto et al. (2014).
Alkaloids. 5 ml of the extract was treated first with 2 ml of HCl, and then with 1 ml of Dragendroff 's reagent. An orange or red precipitate indicated the presence of alkaloids.
Flavonoids. 1 ml of the extract was treated with few drops of 2% NaOH to produce an intense yellow colour. After adding few drops of dilute HCl, the extract became colourless if it contained flavonoids.
Glycosides. 1 ml of the extract was treated first with 2 ml of CH 3 COOH mixed with few drops of FeCl 3 , and then with 1 ml of H 2 SO 4 . Formation of a reddish brown colour at the junction of two layers and the bluish green colour in the upper layer indicated the presence of glycosides.
Saponins. 1 ml extract was first diluted with 5 ml of dd H 2 O, and then agitated for 15 min. The formation of at least 1 cm layer of foam indicated the presence of saponins.
Steroids. 1 ml of the extracts was treated first with 10 ml of CHCl 3 , and then with 10 ml of H 2 SO 4 . A red colour in the upper layer and a yellow colour in H 2 SO 4 layer indicated the presence of steroids.
Tannins. 1 ml of the extract was first diluted with 4 ml of ddH 2 O, and then treated with few drops of 10% FeCl 3 . Formation of a blue/green colour indicated the presence of tannins.
Terpenoids. 5 ml of the extract was treated first with 2 ml of (CH 3 CO) 2 O, and then with 2 ml of CHCl 3 ; finally, 2 ml of H 2 SO4 were added. Formation of reddish violet colour indicated their presence in the extract.
Reducing sugars. 1 ml of the extract was first treated with 5-8 drops of Fehling's solutions (A and B), and then heated in a water-bath. Formation of a red precipitate indicated the presence of sugars.

Antifungal activity evaluation
Fungal cultures. Five mycotoxin-producing fungi were used in this study (Table 1). The spores of each fungus were harvested by flooding the surface of 10 days-old cultures with sterile 0.85% saline containing 0.1% Tween 80, and scraping the surface of colonies with glass rods. Spore suspensions were filtered through double-layered sterile cheesecloth, and the concentration was then adjusted to 10 6 spores/ml.
In vitro antifungal activity assays. Two kinds of agar diffusion tests were performed, the well diffusion (WD) and the paper disk diffusion (PD) test. In both tests, about 20 ml of the Saboraud Dextrose Agar (SDA) was poured into Petri plates (9 cm) and allowed to solidify. For each test and for each fungus, ten SDA plates were prepared by spreading 0.1 ml of the spore suspension (10 4 spores·ml -1 ) on each plate. In the WD test, 3 wells (6 mm diameter) were aseptically punched at equidistant points into the agar medium of each plate, and then filled with 100 μl of the crude extract. In the PD test, 3 sterile papers disks (Whatman No. 4 paper, 5 mm diameter), previously immersed in the crude extract and allowed to dry, were placed at equidistant points on agar surface of each plate. In both tests, Amphotericin B and sterile ddH 2 O were used as positive and negative controls, respectively. After 72 h at 25°C, the antifungal activity of the extract was evaluated by measuring the diameters of the growth inhibition zones around each well and disk. The experiments were carried out in triplicate.

RESULTS AND DISCUSSION
As shown in Table 2, seven out of eight tested phytochemical compounds were found in the extract. In particular, the extract contained alkaloids, flavonoids, glycosides, saponins, steroids, tannins, and terpenoids. These results are consistent from the previous studies on S. nigrum extracts (Gogoi & Islam 2012;Musto et al., 2014;Modilal et al., 2015;Singh & Vidyasagar, 2015a, b). According to the authors, thanks to the wide range of biological activities of these compounds, the extract may be exploited for the development of novel biopesticides or phytopharmaceuticals.
The results from the in vitro antifungal activity assays (Table 3) showed that the aqueous extract was active, in both agar diffusion tests, against A. carbonarius (WD: 23.6 mm; PD: 15.3 mm) and P. expansum (WD: 22.1 mm; PD: 15.1 mm). The inhibitory activity of the extract against these two fungi was slightly lower than that of positive control: for A. carbonarius the difference between the inhibition zones ranged from 6.1 (WD) to 8.4 (PD) mm, whereas for P. expansum the range was 4.7 (WD) -6.9 (PD) mm. A higher concentration of the extract would have been as effective as the positive control. In fact, previous studies (e.g. Ekwenye and Elegalam, 2005;Okigbo and Igwe, 2007;Okigbo et al., 2012) on antimicrobial activity of different plant extracts have shown that the inhibitory effects are highly dependent on their concentrations, with stronger inhibition occurring at higher concentrations.
No inhibition was instead observed against A. flavus, F. graminearum, and F. verticillioides. A previous study revealed that a methanolic extract of S. nigrum inhibited the growth of A. flavus (Harisankar et al., 2011). The authors also found a weak activity against F. verticillioides. However, Zubaır et al. (2011) reported that a methanolic extract of S. nigrum leaves did not inhibit the growth of A. flavus and A. niger. Similarly, Mohamed et al. (1996) found an ethanolic extract of S. nigrum to be not active against A. niger. More recently, Abbas at al. (2014) reported that the growth of against A. niger, A. flavus, and A. fumigatus was inhibited by a methanolic extract of the dried powdered fruit of S. nigrum. Compared to our results, the different antifungal activity against some mycotoxigenic fungi described in the aforementioned studies, are probably related to the different type of solvent used in the extraction procedures (methanol vs. water), as well as to the part of the plant used as starting vegetal material (leaf vs. fruit). These aspects can play a crucial role in preparing plant extracts (Ncube et al., 2008;Das et al., 2010). Overall, our results further confirm the antifungal activity of S. nigrum (Mohamed et al., 1996;Mutu et al., 2006;Atanu et al., 2011;Yanar et al., 2011;Lin et al., 2011;Sun et al., 2012;Singh et al., 2015a, b), and suggest    that the obtained aqueous extract is as effective as other plant antimicrobial compounds (Soliman & Badeaa, 2002;Selvi et al., 2003;Rasooli & Abyaneh 2004;Bansod & Rai 2008;Oliveira & Furlong, 2008;Nguefack et al., 2009;Vilela et al., 2009;Thembo et al., 2010;Morcia et al., 2012;Philippe et al., 2012) for inhibiting the growth of some mycotoxigenic fungi.
The antifungal activity exhibited by our aqueous extract is strictly related to the presence of its phytochemical constituents (Table 2). Among them, some steroidal alkaloids (e.g. solamargine, solasonine, solanine, and saponin) have been identified as active compounds exhibiting a strong antifungal activity (Muto et al., 2006;Zhou et al., 2006;Al-Fatimi et al., 2007;Lin et al., 2011). For instance, Lin et al. (2011) found degalactotigonin, a steroidal saponin, to be active against Alternaria brassicicola, the causative agent of cabbage black leaf spot disease. The antifungal activity of the extract may be also due to the other phytochemical constituents, such as naturally occurring phenolic compounds. In other plants, these compounds have been found important for inhibiting growth and toxin production by toxigenic fungi (Guiraud et al., 1995;Beekrum et al. 2003;Bisogno et al., 2007;Palumbo et al. 2007;Romero et al., 2009). For instance, the growth of A. carbonarius and OTA production may be inhibited by some phenolic compounds (e.g. caffeic acid, quercetin, and rutin), especially at higher concentrations (Romero et al., 2009).
The precise mechanisms by which the phytochemical constituents of the extract can inhibit fungal growth are not fully understood. da Cruz Cabral et al. (2013) have recently highlighted some aspects concerning the mode of action of plant-derived extracts on which most authors agree. With regard to saponins, Nishikawa et al. (1984) and Keukens et al. (1995) reported that these compounds are capable to complex with sterols in fungal cell membranes, causing a loss of membrane integrity. Similarly, phenolic compounds are thought to affect the permeability of cell walls, the release of intracellular constituents, and to interfere with membrane function (Al-Reza et al., 2010). Moreover, phenolic acids can inhibit mycotoxin formation, as they may counter the oxidative stress that triggers or enhances toxin production (Mahoney & Molyneux 2004).

CONCLUSIONS
Results from the present study confirmed that S. nigrum is a good source of antimicrobial compounds. In particular, a crude aqueous extract from lyophilized leaves of S. nigrum is active against two out of five tested mycotoxin-producing fungi. These results are encouraging, since the extract was obtained by using water, a solvent characterized by low toxicity and capable of extracting several bioactive compounds. This confirms the effectiveness of the procedures (i.e. leaf lyophilisation and maceration with water) used to obtain the extract. However, the choice of adopting these procedures should be also considered as a limitation of this study, since the research was not specifically designed to evaluate the whole potential of S. nigrum for inhibiting the growth of mycotoxigenic fungi. In this regard, further studies are needed to evaluate some factors related to extraction procedures (e.g. solvents, extract concentrations, etc.), as well as to clarify how the compound(s) found in the extract acts against the fungi. Further research might also explore wheatear these compounds affect mycotoxin production.