Mohamed Shaaban^1,2*, Hamdi Nasr^1,3, Amal Z. Hassan², Mohsen S. Asker⁴

¹ Institute of Organic and Biomolecular Chemistry, University of Gottingen, Tammannstrasse 2, D-37077 Gottingen, Germany.

² Chemistry of Natural Compounds Department, Division of Pharmaceutical Industries, National Research Centre, El-Behoos st. 33, Dokki-Cairo 12622, Egypt.

³ Department of Organic Chemistry, Faculty of Science, Alazhar University, Assuit-branch, Assuit, Egypt.

⁴ Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Division, National Research Centre, ElBehoos st., Dokki-Cairo 12622, Egypt.

Received November 2012.
Accepted February 2013.

During the research for bioactive secondary metabolites from microorganisms, the endophytic fungi Aspergillus fumigatus sp. isolate R7 was found to produce a set of promising bioactive compounds (1-10) after its large scale fermentation, working up and purification using a series of chromatographic techniques. Structural elucidation of the yielded compounds using intensive studies of their NMR (¹H, ¹³C & 2D NMR) and mass (EI MS, ESI MS) spectrometry confirmed them as linoleic acid (1), R(-)-glycerol monolinoleate (2), bis-dethio-(bis-methyl-thio)-gliotoxin (3), fumiquinazoline-F (4), fumiquinazoline-D (5), (Z,Z)-N,N'-[1-[(4-Hydroxy-phenyl)-methylene]-2-[(4-methoxy-phenyl)-methylene]-1,2-ethanediyl]-bis-formamide (6), pyrazoline-3-one trimer (7), Tricho-9-ene-2α,3α,11α,16-tetraol (8), 2'-deoxy-thy-midine (9), and cerebroside A (10). In this article, taxonomical characterization, fermentation, structural characterization of the obtained metabolites were reported together with their antimicrobial and cytotoxic activities.

Keywords: Endophytic Fungi; Taxonomy; Secondary Metabolites; Structural Elucidation; Bioactivity.

RESUMEN

En la investigación de metabolitos secundarios bioactivos de microorganismos, después de la fermentación a gran escala, el procesamiento y la purificación, utilizando una serie de técnicas cromatográficas, del hongo endofito Aspergilius fimigatus sp. aislado R7 se encontró la producción de una serie de compuestos bioactivos prometedores (1-10). La elucidación estructural de los compuestos producidos, utilizando estudios de RMN (¹H, ¹³C y 2D RMN) y espectrometría de masas (EM IE, EM ESI) permitió identificarlos como ácido linoléico (1), R(-)-monolinoleato de glycerol (2), bis-destio-(bis-metil-tio)-gliotoxina (3), fumiquinazolina-F (4), fumiquinazolina-D (5), (Z,Z)-N,N'-[1-[(4-Hidroxifenil)-metilen]-2-[(4-metoxi-fenil)-metilen]-1,2-etanediil]-bis-formamida (6), trimero de pyrazolin-3-ona (7),Tricho-9-ene-2α,3α,11α,16-tetraol(8), 2'-desoxi-timidina (9), and cerebrosida A (10). En este artículo se reporta la caracterización taxonómica, fermentación, caracterización estructural de los metabolitos obtenidos junto con su actividad antimicrobiana y citotóxica.

Palabras clave: Hongos endófitos; Taxonomía; Metabolitos secundarios; Elucidación estructural; Bioactividad.

1 INTRODUCTION

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 (Faulkner, 2000; Laatsch, 2006; Laatsch, 2010). Therefore, many pharmaceutical companies and research groups were motivated to start sampling and screening large collections of fungal strains for antibiotics (Butler, 2004), antimycotics (Li & Strobel, 2001), antivirals (Singh et al., 2003), anticancers (Zhang et al., 2006) and pharmacologically active agents (Song et al., 2004). Even though more than 30000 diseases are clinically described today, less than one-third of these can be treated symptomatically, and even a fewer can be cured. The increasing occurrence of multiresistant pathogenic strains has limited the effect of traditional antimicrobial treatment. Hence, there is an urgent need for new therapeutic agents with infectious disease control (Larsen et al., 2005). Endophytic fungi were originally defined as all fungi that live asymptomatically within living plant tissues (Saikkonen et al., 1998). They were generally considered as com-mensalistic symbionts, receiving nutrients and habitat from their hosts, which mostly provided the host with chemical protection from insects and browsers (Stierle et al., 2000). In the past ten years, the biology of endophytic fungi in aerial plant tissues has become an important area for study, however, the chemistry of these organisms is only beginning to be explored (Stierle et al., 2000). There is growing evidence that bioactive substances produced by microbial endophytes may not only be involved in the host-endophyte relationship, but may also ultimately have applicability in medicine, agriculture and industry (Strobel, 2002). According to recent reported literatures, endophytic fungi were confirmed as unusual productive sources of bioactive metabolites (Hassan, 2007; Chen, 2011; Shiono, 2011), which might be helpful to treat some of the recently explored diseases.

Imagen (1-10)

2 RESULTS AND DISCUSSION

2.1 TAXONOMICAL CHARACTERIZATION AND PRE-SCREENING

The endophytic Aspergillus fumigatus sp. R7 was isolated from the leaves of sweat potato; Ipomoea batatas using the reported methods of Petrini (Petrini, 1986) and Khan (Khan et al., 2007). The fungus was shown to produce surficial and submerged hyphae on potato dextrose agar (PDA) medium. The growth was being most prominent on czapek-dox agar (CDA) at 25-30°C, showing good rate of growth even up to 45°C on CDA. The colony on CDA is typically bluish-green. The mycelium is colourless and inconspicuous. Microscopic studies of the fungus (Fig. 1) has shown the conidiophores as smooth to finely rough walled, 200-300µm long, up to 7µm in diameter, enlarging gradually into vesicles of 18-20µm diameter. Metulae is absent; phialides are ampulliform, 7-9µm long, with a short neck. The conidia are mostly subglobose-globose to ellipsoidal, 2.5-3µm in length, echinulate, adhering in long compact columns. Based on these typical features, the fungus has been identified as Aspergillus fumigatus (Klich, 2002; Raper & Fennell, 1965).

According to a carried out pre-screening, the endophytic isolate Aspergillus fumigatus R7 showed high antibacterial activity against Gram positive (B. subtilis [16 mm], and St. aureus [15 mm]) and Gram negative bacteria (P. aeruginosa [19 mm], and E. coli [16 mm]). In contrast, the strain extract showed no activity against pathogenic fungi; A. niger, A. flavus, C. albicans. Alternatively, the strain extract showed numerous bands during TLC of different polarities, some of them were UV in active, while the others are UV absorbing during TLC, which were mostly turned pink-orange on spraying with anisaldehyde/sulphuric acid and heating.

2.2. ISOLATION AND STRUCTURE ELUCIDATION

Large scale fermentation of the strain was carried out on M₂ medium showing yellow culture broth. After harvesting and working up, the afforded crude extract was applied to purification using a series of chromatographic techniques to deliver the mentioned ten secondary metabolites (1-10). Structures of the afforded compounds were confirmed on the bases of different spectroscopic means (NMR, and MS) and comparison, and identified as linoleic acid (1) (Shaaban, 2004), R(-)-glycerol monolinoleate (2) (Laatsch, 2010), bis-de-thio-(bis-methyl-thio)-gliotoxin; FR-49175 (3) (Zhang, 2011; Abdel Rahim, 2011), fumiquinazoline-F (4) (Takahashi et al., 1995; Larsen et al., 1998; Silva et al., 2004; Abdel Rahim, 2011), fumiquinazoline-D (5) (Zhang, 2011; Abdel Rahim, 2011), (Z,Z)- N,N'-[1-[(4-Hydroxy-phenyl)-methylene]-2- [(4-methoxy-phenyl)-methylene]-1,2-ethanediyl]-bis-formamide (6) (Breinhold et al., 1996; Abdel Rahim, 2011), pyrazoline-3- one trimer (7) (Davidson& Schumacher, 1993; Ridley& Simpson, 1981; Fotso et al., 2006; Abdel Rahim, 2011), Tricho-9-ene-2a,3a,11 a,16-tetraol (8) (McCormick et al., 1989), 2'-deoxy-thymidine (9) (Shaaban, 2004) and cerebroside A (10) (Koga et al., 1998; Sitrin, 1988; Mohamed, 2010).

Bis-dethio-(bis-methyl-thio)-gliotoxin ( 3) was isolated recently from endophytic Penicillium sp. BCC16054 (Intaraudom et al., 2013), showing a very strong antitubercular activity against Mycobacterium tuberculosis with MIC value of 48.8 ng/mL (0.14 lM) (Intaraudom et al., 2013). These results supported the fact that gliotoxin has been considered as a potential antitubercular drug (McMahon et al., 2011).

Fumiquinazoline F (4) had been previously isolated from the fungus Aspergillus fumigatus, which was isolated from the marine fish Pseudolabrus japonicus (Takahashi et al., 1995). Fumiquinazoline F (4) was also isolated from Aspergillus lentulus (Larsen et al., 2007), Penicillium thymicola (Larsen et al., 1998) and Penicillium corylophilum (Silva et al., 2004). Biologically, fumiquinazoline F (4) was reported to show high antitumor activity (Han et al., 2007; Zhang et al., 2007). Similarly, fumiquinazoline-D (5) was produced previously by the fungus Aspergillus fumigatus, which was isolated from the marine fish Pseudolabrus japonicus, exhibiting moderate cytotoxicity against cultured P388 cells (Takahashi et al., 1995). Fumiquinaozolines were as well reported recently from the pathogenic Aspergillus sydowii and other Aspergillus species (Cai & Lu, 2012). Alternatively, (Z,Z)-N,N'-[1-[(4-Hydroxy-phenyl)-methylene]-2-[(4-methoxy-phenyl)-methylene]-1,2-ethanediyl]-bis-for-mamide (6) was previously isolated from the fungus Hamigera avellanea and reported to exhibit a marginal activity against a variety of pathogenic fungi and bacteria (Breinhold et al., 1996; Abdel Rahim, 2011).

Tricho-9-ene-2α,3α,11α,16-tetraol (8) is belonging to trichothecenes, sesquiterpene metabolites, possessing an olefinic bond and an epoxide group, which are produced by several genera of fungi, including Fuisarium (McCormick et al., 1989), Trichothecium, Trichoderma, Myrothecium, Cephalosporium, Stachybotrys, Verticimonosporium, and Cylindocarpon (Matsumoto et al., 1977; Minato et al., 1975; Mirocha et al., 1977; Ishii & Ueno, 1981). The biochemical basis of the toxicity of the trichothecenes is their inhibition of protein biosynthesis (Apsimon et al., 1985; Bennett et al., 1980).

Finally, cerebroside A (10) is an anti-fungal agent against Candida albicans. Ce-rebrosides; glycosphingolipids, were reported in several phytopathogens as elicitors that induce the disease resistance in e.g. rice plants (Umemura et al., 2002). Cerebrosides are a kind of important bioactive substances isolated mainly from sea cucumber (Xu et al., 2011). Cerebrosides are mostly composed of three different structural units; a polar head group (monosaccarides, such as glucose or galactose), an amide-linked fatty acid, and a long-chain base (LCB) which is also called a sphingoid base (Xu et al, 2011). Recent studies indicated that cerebrosides have antitumor, immunomodulatory, antibacterial and cytotoxic activities (Xu et al., 2011). Due to their moderate bioactivities and smaller side-effects, cerebrosides have been developed to prevent and cure chronic diseases.

2.3 BIOLOGICAL ACTIVITIES

Diverse antimicrobial activity testing for the crude extract of endophytic fungi Aspergi-llus fumigatus sp. isolate R7 was carried out in comparison with the whole isolated compounds (1--10) against eleven microbial tests on the bases of agar diffusion method (40 µg/disc). The crude extract showed high cytotoxic activity (100%) which was attributed reasonably to fumiquinazoline-F (4, 85%) and fumiquinazoline-D (5, 85%). The strain extract showed high antibacterial activity against the Gram-positive Bacillus subtilis (18 mm) and Streptomyces viridochromogenes (Tü 57, 18 mm), and the microalgae Chlorella vulgaris (15 mm), Chlorella sorokiniana (13 mm) and Scene-desmus subspicatus (15 mm). Compounds 4 and 5 exhibited further activity against the Gram-positive Bacillus subtilis (12, 15 mm), Staphylococcus aureus (12, 15 mm) and fungi (C. albicans [11, 11 mm] and M. miehi [12, 13 mm]). Except compounds 4 and 5, cytotoxic examination of the remaining compounds (1-3, 6-10) against the brine shrimp, confirmed their activities to be ranged between moderate and weak. Activity of the whole compounds (1-10) are listed in Table 1.

3. EXPERIMENTAL

The NMR spectra were measured on Varian Unity 300 (300.145 MHz) and Varian Inova 600 (150.820 MHz) spectrometers. ESI MS was recorded on a Finnigan LCQ with quaternary pump Rheos 4000 (Flux Instrument). EI mass spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV). Flash chromatography was carried out on silica gel (230-400 mesh). R_f values were measured on Polygram SIL G/UV₂₅₄ TLC cards (Macherey-Nagel & Co.). Size exclusion chromatography was done on Sephadex LH-20 (Lipophilic Sephadex, Amersham Biosciences Ltd; purchased from Sigma-Aldrich Chemie, Steinheim, Germany).

3.1 ENDOPHYTIC FUNGUS ASPERGILLUS FUMIGATUS R7 3.1.2 ISOLATION

The endophytic fungi Aspergillus fumigatus sp. R7 was isolated from the red leaves of sweat potato; Ipomoea batatas, collected from the herbarium of National Research Centre for Agriculture, Cairo. Leaves of sweat potato were cut into small segments and surface-sterilized by sequential washes in 95% ethanol (30 s), 5% sodium hypo-chlorite (5 min), 95% ethanol (30 s) and rinsed with sterile water. The strain was cultivated on Potato dextrose agar (PDA) medium [Potato infusion 200 g; Dextrose 20 g; Agar 20 g, Distilled water 1 liter], Antibiotic, ampicillin and streptomycin 200 | g/L of the medium was added to the media to inhibit the bacterial growth until the mycelium or colony originating from the newly formed surface of the segments appeared (Phongpaichit et al., 2006). Plates were incubated at 28°C for 1 week. Furthermore, the endophytic nature of the isolated strain was checked daily until within 21 growing days. Individual fungal colonies were transferred onto other plates with PDA. Fungal spore formation was encouraged by placing the endophytes onto autoclaved carnation leaves. The plates were continuously monitored for spore formation by stereo and light microscopy.

3.1.2 FERMENTATION AND WORKING UP

3.1.3 ISOLATION

The crude extract (8.3 g) was applied to column chromatography on silica gel (40x10 cm) and eluted with cyclohexane-CH₂Cl₂-MeOH gradient. According to TLC, four fractions were obtained; FI (1.6 g), FII (3.2 g), FIII (2.2 g) and FIV (0.6 g). An application of the fast fraction to a further silica gel column (2 x 60 cm) followed by Sephadex LH-20 (DCM/40% MeOH) delivered two colourless oils of linoleic acid (1 , 45 mg) and R(-)-glycerol monolinoleate (2, 120 mg). FII was purified by PTLC chro-matogram (20 x 40 cm, DCM/7% MeOH, double elution) and then by Sephadex LH-20 (MeOH) yielding three colourless oils of bis-dethio-(bis-methyl-thio)-gliotoxin (3, 25 mg), fumiquinazoline-F (4, 8 mg) and fumi-quinazoline-D (5, 12 mg). Fraction III was purified by PTLC chromatogram (20 x 40 cm, DCM/7% MeOH, double elution) and then by Sephadex LH-20 (MeOH) delivering two colourless solids of (Z,Z)-N,N'-[1-[(4-Hydroxy-phenyl)-methylene]-2-[(4-metho-xy-phenyl)-methylene]-1,2-ethanediyl]-bis-formamide (6, 11 mg) and pyrazoline-3-one trimer (7, 9 mg). A final purification of fraction FIV using PTLC chromatogram (20 x 20 cm, DCM/15% MeOH) and Sephadex LH-20 (MeOH) gave three colourless solids of Tricho-9-ene-2α,3α,11α,16-tetraol (8, 7 mg), 2'-deoxy-thymidine (9, 23 mg) and cerebroside A (10, 18 mg). Spectroscopic data of the isolated compounds (1-10) are present in attached file "Supplementary Data".

3.2 BIOLOGICAL ACTIVITY

3.2.1 ANTIMICROBIAL ACTIVITY

Antimicrobial assays were conducted utilizing the discagar method (Burkholder et al., 1960) against diverse sets of microorganisms. The fungal extract was dissolved in CH₂Cl₂/10% MeOH at a concentration of 1 mg/mL. Aliquots of 40 µl were soaked on filter paper discs (9 mm Ø, no. 2668, Schleicher & Schüll, Germany) and dried for 1 h at room temperature under sterilized conditions. The paper discs were placed on inoculated agar plats and incubated for 24 h at 38 °C for bacterial and 48 h (30°C) for the fungal isolates, while the algal test strains were incubated at ~ 22°C in day light for 8~10 days. The fungal extract was examined against the following test microorganisms: Bacillus subtilis, Staphylococcus aureus, Streptomyces viridochromogenes (Tü 57), Escherichia coli, Candida albicans, Mucor miehi, Chlorella vulgaris, Chlorella sorokiniana, Scenedesmus subspicatus, Rhizoctonia solani and Pythium ultimum.

For the fungal extract examination, representative test microbes; Aspergillus niger, Aspergillus flavus, Bacillus subtilis, Candida albicans, Escherichia coli, Pseu-domonas aeruginosa and Staphylococcus aureus were served. Both bacterial and fungal strains were grown on nutrient agar medium (g/l): Beef extract 3; peptone,10; and agar, 20. The pH was adjusted to 7.2.

The fungal strain was grown on Czapek-Dox medium (g/l): Sucrose, 30; NaNO₃, 3; MgSO₄.7H₂O, 0.5l; KCl, 0.5; FeSO₄, 0.01; K₂HPO₄, 1; and agar, 20. The pH was maintained at 6.0. The disc diffusion test has been done according to Collins (Collins et al. , 1985). Filter paper discs (5 mm diameter) were saturated with 200 mg from the culture extract, and located on the surface of the agar plates (150 mm diameter containing 50ml of solidified media). The paper discs were placed on inoculated agar plats and incubated for 24 h at 38 °C (bacteria and yeast) and 48 h at 30°C (fungi).

3.2.2 CYTOTOXICITY

The cytotoxic assay was performed according to Takahashi method (Takahashi et al. , 1989) and Sajid et al. screening (Sajid et al., 2009).

ACKNOWLEDGMENT

The authors are grateful to Prof. Laatsch, Institute of Organic and biomolecular chemistry, Goettingen for lab facilities, providing spectral and analytical data for the synthesized compounds. We are indebted to Mrs. F. Lissy for cytotoxicity testing and Mr. A. Kohl for technical assistance. This work has been financed by the German Egyptian Scientific Projects (GESP), project No. 7.

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