Identification of some secondary metabolites produced by four Penicillium species

INTRODUCTION

Fungi produce primary metabolites (amino acids, proteins, carbohydrates, vitamins, acetone, ethanol, etc.) and secondary metabolites (antibiotics, toxins, alkaloids, fatty acids, ketones, alcohols, etc.) during active cell growth (Devi et al. 2009). However, these components play no role in primary metabolism of fungi. Hawksworth (2001) studied fungal biodiversity and suggested that nearly 1.5 million fungal species exist on earth, from which just 5% are identified so far. Soil fungi are substantially prolific sources of highly bioactive secondary metabolites, which is interesting as a complex aspect of fungal development (Bok & Keller 2004). Identification of a number of new metabolites and evaluation of their biological activities have been studied (Gao et al. 2010).

Penicillium is an anamorphic ascomycete and comprises more than 200 species, many of which are common soil inhabitants. Penicillium species are important because of their widespread occurrence and ability to produce a wide range of bioactive secondary metabolites, including antibacterial, antifungal, immune suppressants, cholesterol-lowering agents and mycotoxins (Petit et al. 2009).

The fungi producing secondary metabolites have industrial application. Thus, identification of these components and optimization of the fungal growth conditions can help us manage optimum production of secondary metabolites. In this study, we aimed to identify some secondary metabolites produced by four Penicillium species, including P. jenseii, P.pusillum, P. purpurogenum and P. canescens, using gas chromatography combined with mass spectrometry (GC-MS). The top biological activities of major metabolites were described according to the related references.

MATERIALS AND METHODS

Growth conditions

Penicillium species were prepared of living cultures from laboratory of mycology, department of plant pathology at the Sari Agricultural Sciences and Natural ResourcesUniversity. Then, spores were suspended in potato dextrose broth (PDB) and incubated at 25^ºC on a shaker for 14 days at 130 rpm (Siddiquee et al. 2012).

Extraction procedure

The metabolites were determined using gas chromatography according to the method of Siddiquee et al. (2012), with some modifications. Extraction was performed by adding 10 ml Ethyl acetate (C₄H₈O₂) to 50 ml liquid culture in an Erlenmeyer flask, and the mixture was incubated at 4^ºC for 10 min. Then, flasks were shaken for 20 min at 130 rpm. The supernatant (metabolites and C₄H₈O₂) was separated from the liquid culture and evaporated, using a rotary evaporator at 45^ºC. The residue was dissolved in one  ml methanol (CH₄OH), filtered through a 0.2 μm syringe filter (Millipore) and stored at 4 ºC for 24h before injection to GC-MS.

GC-MS conditions

An Agilent technologies 7890 A gas chromatograph connected to a 5975 Cinert MSD was used for identification of secondary metabolites from the crude extract of Penicillium species. In addition, an HP-5MS fused silica capillary column (Hewlett-Packard, 30 m* 0.25 mm i.d., 0.25 μm film, cross-linked to 5% phenyl methyl siloxane stationary phase) was applied. The entire system was controlled by Chemstation software (Hewlett-Packard, version A.01.01). Electron impact mass spectra were recorded at 70 electron voltage and ultra-high pure He (99.999%) gas was used as the carrier gas at flow rate of 1 mL/min. The injection volume was 1 μL and all the injections were performed in a split-less mode. Temperature of the injector and detector was 250 and 280^°C, respectively. Column oven temperature was initially set at 50^°C for 5 min, then increased to 260^°C (ramp: 4^°C/min) and held for 5 min.

RESULTS AND DISCUSSION

The retention time and abundance of the compounds under the described conditions in GC-MS section are shown in tables 1, 2, 3 and 4.

In this study, various secondary metabolites were detected within Penicillium species. We have described the biological activity of certain metabolites and compared the species based on the produced metabolites.

Many of the important compounds were produced by Penicillium jenseii. Some of them were not detected in any of the other species, for instance 3-oxoquinuclidine. This compound is a derivative of quinuclidine (1-azabicyclo [2.2.2] octane), which has a wide variety of biological activities and has a substantial importance as a structural fragment of a synthetic pharmaceutical compound (Odzak et al. 2007).

Quinoline is an alkaloid mycotoxin (Kozlovsky 1990). Qunnoline and isoquinoline compounds have antiprotozoal activity, so that malaria infections are treated by several drugs, including quinoline (Osorio et al. 2008). Quinoline and 5-phenylisoquinoline were detected only in P. pusillum. Moreover,formamidine and uvidin B were detected in P. pusillum, but not in the other Penicillium species. There have already been many reports concerning the various biological activities and properties of formamidines, including acaricidal, bactericidal, antiprotozoal, antihelminthic, fungicidal and herbicidal effects (Hollingworth 1976). Uvidin B which is one of the drimane-type sesquiterpenes, was isolated from Lactarius uvidus by Bernardi et al. (1980).

Limonene, the terpenoid compound was detected only in P. purpurogenum. This compound was reported to be the major component in the extract prepared from cultivated Trichoderma sp. (Khethr et al. 2008). They also investigated the antibacterial and antifungal activities of limonene against five pathogenic gram positive and gram negative bacterial strains and five pathogenic fungi, and reported that the compounds have positive antibacterial, but no antifungal activity. Limonene has been reported as one of the secondary metabolites in P. olsonii, P. roqueforti and P. vulpinum (Frisvad et al. 2004).

Thymol is an essential oil with strong antifungal activity (Sokovic et al. 2009). Previous investigations by Sokovic & Griensven (2006) show that thymol has a very high activity against three major pathogens of the button mushroom, Agaricus bisporus, including Verticillium fungicola , Trichoderma harzianum and Pseudomonas tolaasii. This compound has also showed a very strong antibacterial activity against food spoilage bacteria (Sokovic et al. 2007) and was detected only in P. purpurogenum in the present study.

Table 1. Compounds produced by Penicillium pusillum identified by GC-MS.

Table 2. Compounds produced by Penicillium jenseii identified by GC-MS.

Unique metabolites detected in P. canescens include orcinol, 1,3,8-p-menthatriene and aphthosin. Orcinol is a member of fumigatins family, one of the secondary metabolites (Frisvad et al. 2008). Fumigatins were reported as active antibiotics against some gram-negative and gram-positive bacteria (Waksman & Geiger 1944). Frisvad et al. (2008) reported the production of at least 226 potentially bioactive secondary metabolites by Aspergillus fumigatus, including orcinol.

Taran et al. (2010) demonstrated the antimicrobial activity of 1, 3, 8-p-menthatriene and some other essential oils of Ferulago angulata subsp. carduchorum. Staphylococcus aureus (MIC=15μg/ml) and Listeria monocytogenesis (MIC=137μg/ml) showed a high sensitivity to essential oils of aerial parts and seeds, respectively. Ferulago species have sedative, tonic, digestive and antiparasitic effects (Baser et al. 2002). Aphthosin, the first example of tetradepsides was isolated from Peltigera aphtosa by Bachelor & King (1970).

Phenanthrenes are from a rather uncommon class of aromatic metabolites. A large number of different phenanthrenes has been reported to occur in plants and studied for their cytotoxicity, phytotoxicity, antimicrobial, spasmolytic, anti-inflammatory, antiplatelet aggregation and antiallergic activities (Kovacs et al. 2008). Phenanthrene was produced by allthe Penicillium species in this study,except P. pusillum.

Anthracene is a polycyclic aromatic hydrocarbon, consisting of three fused benzene rings. Krivobok et al. (1998) studied the toxicity of anthracene on 39 micromycetes. There are also some other reports demonstrating that white rot fungi (Bezalel et al. 1996) and some other fungi, including Cunninghamella elegans and Rhizoctonia solani (Sutherland 1992) are able to metabolize anthracene. In this study, we found anthracene in the culture of P. jenseii, P. purpurogenum and 9,10-anthracenedione inP. canescens. Also, thioxanthene was produced by all the species, except P. pusillum. Kristiansen and Vergmann (1986) investigated the antibacterial effect of various thioxanthene derivatives on mycobacteria in vitro and mentioned that the antibacterial capacity against the slow-growing mycobacteria is the same as stereo-isomeric analogs of thioxanthene derivatives. Ford et al. (1990) studied the structure-activity relati-onship of a series of thioxanthene isomers in a multidrug resistant (MDR) human breast cancer cell line.

Sarcocapnidine, the isocularine alkaloid was produced by P. purpurogenum and P. jenseii. This compound was reported to be the major alkaloidscomponent in the genus Sarcocapnos by Suau et al. (2005).

Dibenzothiophene (DBT) is the organic sulphur that was produced by P. purpurogenum, P. jenseii and P. pusillum. Many of the bacteria, such as Brevibacterium and Pseudomonas utilize DBT as the source of carbon, sulfur and energy (Acharya et al. 2005).

Table 3. Compounds produced by Penicillium purpurogenum identified by GC-MS.

Table 4. Compounds produced by Penicillium canescens identified by GC-MS.

There have been several reports about the production of isolongifolene derivatives in the fungi and plants. Hang et al. (2012) analyzed the isolongifolen-5-one and other volatile components from Dictyophorarubrovolota. This compound was found in P. pusillum in this study.Neoisolongifolene reported as one of the essential oils of the eaglewood tree (Aquilaria agallocha) (Bhuiyan et al. 2008), was detected in P. jenseiiin the present study.

Oxalic acid is a secondary metabolite produced by several organisms. It can be found extensively in nature and secreted to the environment by many fungi. Production of oxalic acid is beneficial to the producing organisms, to some extent (Blumenthal 2004). Cessna et al. (2000) reported that oxalic acid seems to be the pathogenicity factor in Sclerotinia sclerotiorum. Dutton and Evans (1996) studied the production of oxalic acid in fungi. They expressed that it affects the pathogenicity through acidification of the host tissues and causes the sequestration of calcium from the host cell. Jarosz-Wilkolazka and Gadd (2003) reported that the wood-rotting fungi produce oxalic acid as a major metabolite in response to toxic stress. In the present study, this compound was produced by P. canescens and P. jenseii.

Camazulene and Azetidine were produced by P. pusillum and P. purpurogenum. Camazulene is an essential oil with antifungal activity (Liu et al. 2001). Azetidine is an alkaloid compound possessing potent actomyocin ATP ase-activating properties (Pinder 1992).

Commonly, the fatty acids and hydrocarbonsareproducedby Penicillium species. Fatty acids are organic acids with antibacterial, antimalarial and antifungal activities (Pohl et al. 2011). Fatty acids are present anywhere in nature and are also physiologically important class of molecules involved in cell energy storage, membrane structure and in various signaling pathways (Liu et al. 2008).Pohl et al. (2011) reported that fatty acids and their derivatives are very important as antifungal agents and also are used as novel antifungal compounds. They concluded thatthe most important target of antifungal fatty acids is the cell membrane, and reported that augmentation of the membrane fluidity is partly because of the presence of fatty acids, which results in discharge of the intracellular components and cell death. Similarly, Helander et al. (1998) reported that the outer membrane disintegrating properties may be the cause of the action of the oil. Some studies suggest that these compounds permeate inside the cell, where they are inhibited by cellular metabolism (Marino et al. 2001). Other studies explain the deterioration of cellular membrane structure and reaction of the active sites of enzymes or their act as a H+ carrier, exhausting the adenosine triphosphate (ATP) pool (Farag et al. 1989). Dodecanoic (lauric) acid, as an unsaturated fatty acid was produced by P. pusillum. On the other hand, the fatty acids reported by Pohl et al. (2011), including hexadecanoic (palmitic) acid, octadecanoic (stearic) acid and decanedioic acid were produced by P. canescens and P. jenseii, P. jenseii and P. purpurogenum, and P. pusillum, respectively.

The hydrocarbons, such as decane, dodecane, tridecane, heptadecane, eicosane, hexadecane and undecane were produced by all the tested Penicillium species.

Some secondary metabolites are involved in the complex interactions between fungi and their living environment. Thus, analytical methods for the identification of compounds are useful to study their functions in biological interactions. The GC–MS technique is a powerful tool for the study of the dynamic range of secondary metabolites. Based on the specific criteria like mass-spectral factor, the application of this method shows a wide variety of major metabolites in Penicillium species. According to the trait of the compounds described in this study, we suggest the usage of complementary methods for purification of Penicillium species compounds, due to their application in agriculture as biological control agents of plant pathogens, or in medicine as sources of antimicrobial substances.