Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 85–89 Contents lists available at ScienceDirect Bulletin of Faculty of Pharmacy, Cairo University journal homepage: www.sciencedirect.com Original Article Antifungal and cytotoxic constituents from the endophytic fungus Penicillium sp. Ehab S. Elkhayat a,⇑, Asmaa Mohamed Goda b a b Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt Departments of Medical Microbiology and Immunology, Faculty of Medicine, Sohag University, Sohag, Egypt a r t i c l e i n f o Article history: Received 20 January 2017 Received in revised form 28 February 2017 Accepted 3 March 2017 Available online 9 March 2017 Keywords: Endophyte Pencillium sp. Senecio flavus 4-Methylmellein Cytotoxicity Antifungal activity a b s t r a c t New mellein derivative; 4-methylmellein (1) along with five known compounds: 4-hydroxymellein (2) and 6-hydroxymellein (3), tyrosol (4), cervesterol (5), and stigmast-4-ene-3-one (6) were isolated from the ethyl acetate extract of the endophytic fungus Penicillium sp. isolated from the leaf of Senecio flavus (Asteraceae). Structures were established by 1D and 2D NMR, in addition to UV, IR and MS spectrometry. Compounds (3, 4 and 6) were isolated for the first time from the genus Penicillium. Compounds 1–4 showed antifungal activity against F. oxysporum and variable activities against A. flavus and the yeast C. albicans. Compounds 1–3 showed cytotoxic activity against MCF-7 cell line. Ó 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/). 1. Introduction Since the inspiring isolation of taxol from Pestalotiopsis microspora, the endophytic fungus of the yew tree Taxus wallichiana, many researches have been constructed to explore plant endophytes as a source of novel and biologically active natural products [1]. During the period from 1993 to 2001, literatures have reported 1500 compounds, more than half these compounds showed antibacterial, antifungal and antitumor activities [2]. Although the function of these products is not precisely known, they thought to play a role in the chemical defense and communication [3]. Many of them have been suggested to act as pheromones or repellents [4]. Penicillium is a large fungal genus, widely spread in most terrestrial and marine environments as well as food contaminants or food ingredients as cheese or sausage, it comprises more than 200 described species [5]. Vast array of active secondary metabolites have been isolated from Penicillium species, including naphthalenoids6, alkaloids, mycotoxins, polyketides [7], steroids [8,9]. These metabolites have been reported to be allied with antioxidant, antiviral [10–12], antibacterial [13,14], antifungal [15], immune-suppressants, cholesterol-lowering activities [16]. New bioactive metabolites continue to be discovered from this genus Peer review under responsibility of Faculty of Pharmacy, Cairo University. ⇑ Corresponding author. E-mail address: khayat71@yahoo.com (E.S. Elkhayat). nowadays, reflecting its current importance as a source of novel bioactive molecules [6]. The present work was constructed to investigate the metabolites of Pencillium sp. the endophyte secluded from the inner tissue of the leaf of Senecio flavus (Decne.) (Asteraceae), where the new 4methylmellein have been isolated, as well as 4-hydroxymellein, 6hydroxymellein, tyrosol, cervesterol, and stigmast-4-ene-3-one (Fig. 1). 2. Experimental 2.1. General A Perkin-Elmer polarimeter 341 LC Model was utilized for the measurement of optical rotation (Perkin-Elmer, Waltham, MA, USA). UV spectra measurement was performed on a 1601 UV/VIS Shimadzu spectrophotometer using MeOH (Shimadzu, Kyoto, Japan). Infrared-400 Shimadzu spectrophotometer was utilized for IR measurement (Shimadzu, Kyoto, Japan). HRESIMS was carried out on an Orbitrap LTQ (ThermoFinnigan, Bremen, Germany). ESIMS spectra were achieved using LCQ DECA spectrometer (ThermoFinnigan, Bremen, Germany). A Bruker Avance DRX 400 MHz spectrometer was used for measuring 1D and 2D NMR spectra (Bruker BioSpin, Billerica, MA, USA). Chromatographic separations were achieved on sephadex LH-20 (0.25–0.1 mm) and silica gel 60 (0.04–0.063 mm). TLC analysis was carried out on silica http://dx.doi.org/10.1016/j.bfopcu.2017.03.001 1110-0931/Ó 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 86 E.S. Elkhayat, A.M. Goda / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 85–89 10 OH 5 9 4 4a 6 HO HO 1 3 7 2\ O 8a 8 O O 4 1 OH 1\ OH O OH 1 O OH 2 O 4 3 O HO OH 6 OH 5 Fig. 1. Structure of the isolated compounds 1–6. 60 F254 TLC pre-coated plates (0.2 mm) (Merck, Darmstadt, Germany). added to 100 g commercially available rice and kept overnight prior autoclaving) at room temperature under static conditions for 30 days. 2.2. Plant material Senecio flavus (Decne) (Asteraceae) was collected in April 2015 from the plant wildly growing at campus of Al-Azhar University. The plant sample was authenticated by Prof. Dr. A.A. Fayed, Prof. of Plant Taxonomy, Faculty of Science, Assiut University, Assiut, Egypt. A herbarium specimen was kept at the Department of Pharmacognosy herbarium, Al-Azhar University (SF/4/2015). 2.3. Isolation of fungal material The endophytic fungus Penicillium sp. was isolated from the healthy surface sterilized leaves of S. flavus. The leaves were first washed with water, then immersed in 70% aqueous ethanol for 1 min, and rinsed in sterile water. Finally they were sliced into 2  2 cm pieces, with 3 to 4 pieces deposited on petri dish containing potato dextrose agar plates (PDA, Difco), having gentamicin sulfate (200 mg/L) as antibacterial agent to prohibit bacterial growth and plates were incubated at room temperature. The fungus under investigation was found to grow exclusively out of the leaf tissue. From the growing cultures pure strains of Penicillium sp. were isolated by repeated re-inoculation on PDA plates [8]. 2.4. Identification of the fungal strain The fungus was identified according to the characteristics of the culture morphology and spores features using light microscopy (CX31RBSF, Olympus). The fungi were identified by Ph.D. Mohamad Taha, Department of Microbiology, Al-Azhar University, Assiut, Egypt, and deposited with the culture code (PES/4/2015). 2.5. Cultivation Mass growth of the fungus for the isolation and identification of metabolites was carried out in 12 Erlenmeyer flasks (1 L). The fungus was grown on rice solid medium (100 mL of distilled water 2.6. Metabolites isolation The culture was extracted using EtOAc and concentrated under vacuum. The obtained extract was suspended in distilled water (200 mL) and fractionated between n-hexane and 90% MeOH. The MeOH extract (6.1 g) was separated by VLC (silica gel 60), eluted successively with n-hexane, CHCl3, EtOAc and MeOH, which were independently concentrated to get FV-1 (0.6 g), FV-2 (1.3 g), FV-3 (2.4 g) and FV-4 (1.4 g). FV-2 fraction was chromatographed over silica using n-hexane:EtOAc gradient, 100 mL fractions were gathered and checked by TLC to get seven sub-fractions: FV2-1 to FV27. Subfraction FV2-2 (70 mg) was chromatographed on silica gel using n-hexane:EtOAc (9:1) as an eluent to yield compounds (5) and (6) (2.8 and 2.4 mg, respectively). Fraction FV-3 was chromatographed on sephadex eluted with MeOH to get 5 subfractions FV3-1 to FV3-5. The FV3-2 (23 mg) was chromatographed on silica gel eluted with CHCl3:MeOH gradient, where compound (4) (3.6 mg) was obtained. FV3-3 (30 mg) was chromatographed on silica gel eluted with CHCl3:MeOH gradient to yield compounds (2) and (3) which were individually purified on silica (CHCl3: MeOH, 8:2) to yield (2) (2.7 mg) and (3) (3.1 mg). FV3-4 (21 mg), was handled similar to FV3-3 to yield (1) (2.6 mg). The remaining fractions were kept for further study. 4-Methylmellein (1): White amorphous solid. [a]20 15.6 D (MeOH). UV (MeOH): kmax 218, 245, 315 nm. IR(KBr): 3436, 2978, 1729, 1651, 1056 cm 1. NMR data (CDCl3, 400 and 100 MHz) see Tables 1 and 2. HRESIMS m/z 193.0861 (calcd for 193.0865 [M+H]+, C11H12O3). 4-Hydroxymellein (2): White amorphous solid. [a]20 39.6 D (MeOH). EIMS m/z 195 [M+H]+. UV (MeOH): kmax 215, 265, 320 nm. IR(KBr): 3465, 2981, 1731, 1665, 1610, 1516 cm 1. NMR data (CDCl3, 400 and 100 MHz), Tables 1 and 2. 6-Hydroxymellein (3): White amorphous solid. [a]20 18.4 D (MeOH). EIMS m/z 194 [M]+, 176, 150, 148 and 130. UV (MeOH): 87 E.S. Elkhayat, A.M. Goda / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 85–89 Table 1 H NMR of compounds 1–3 (CDCl3, 400 MHz). 1 No. 1 dH (m, J in Hz) 2 dH (m, J in Hz) 3 dH (m, J in Hz) 1 3 4a 4b 4a 5 6 7 8 8a Me-9 Me-10 8-OH – 4.53 (m, 1 H) 3.18 (m, 1 H) – 4.61 (m, 1 H) 4.47 (br.s, 1 H) – 6.53 (dd, J = 7.7, 2.1 Hz, 1 H) 7.26 (dd, J = 7.7, 7.5 Hz, 1 H) 6.21 (dd, J = 7.5, 2.1 Hz, 1 H) – – 1.39 (d, J = 6.5 Hz, 3 H) 1.28 (d, J = 6.1 Hz, 3 H) 11.22 (br.s) – 6.85 (d, J = 7.3 Hz, 1 H) 7.43 (dd, J = 7.3,7.5 Hz, 1 H) 6.95 (d, J = 7.5 Hz, 1 H) – – 1.50 (d, J = 6.5 Hz, 3 H) – 11.10 (br.s) – 4.63 (m, 1 H) 2.79 (br.s, 1 H) 2.81 (m, 1 H) – 6.27 (d, J = 2.1 Hz, 1 H) – 6.10 (d, J = 2.1 Hz, 1 H) – – 1.49 (d, J = 6.6 Hz, 3 H) – 11.30 (br.s) Table 2 C NMR of compounds 1–3 (CDCl3, 100 MHz). 13 No. dC 1 dC 2 dC 3 1 3 4 4a 5 6 7 8 8a Me-9 Me-10 170.1 80.1 37.6 144.2 117.1 136.1 113.7 161.4 108.1 19.2 17.2 169.2 78.3 67.3 140.4 118.7 136.8 101.3 162.3 107.0 16.1 – 169.5 76.1 34.1 142.3 106.7 164.6 101.5 164.3 101.0 19.7 – kmax 218, 270, 305 nm. IR(KBr): 3430, 1655, 1587, 1475 cm data (CDCl3, 400 and 100 MHz), Tables 1 and 2. 1 . NMR 2.7. Antifungal activity Sterile filter paper discs (6 mm diameter), were impregnated with 50 mg of the samples using methanol as the carrier solvent. The impregnated discs were then placed on agar plates previously inoculated with Aspergillus flavus, Fusarium oxysporum and Candida albicans. Solvent controls were run against each organism. After incubation at 37 °C for 24 h, the antifungal activity was recorded as clear zones of inhibition surrounding the disc (mm). Nystatin (10 mg/disc) was used as standard antifungal. All experiments were performed in triplicate and the activity was expressed as the mean of inhibition zone diameters (Table 3). Fungal strains were secured by Assiut University Mycology Center, Assiut, Egypt. 2.8. Cytotoxicity assay The cytotoxicity was evaluated by the [3H] thymidine assay using breast cancer (MCF-7) and colon cancer (COLO-205) cell lines [17]. Doxorubicin (10 mg), was used as a positive control. The cell lines were obtained from Al-Azhar University Centre for Viral Research, Cairo, Egypt. 3. Results and discussion Compound 1 was isolated as white amorphous solid. Its molecular formula was defined as C11H12O3 by HRESIMS pseudomolecular ion peak at m/z 193.0861 [M+H]+ (calcd. for 193.0865), requiring 6 degrees of unsaturation, four of which were attributed to aromatic ring. The IR spectrum showed absorption bands at mmax cm 1; 3436 (hydrogen bonded phenolic hydroxyl), 2978 (aromatic) and 1651 (ester/lactone carbonyl). The 1H NMR spectrum (Table 1), suggested the presence of trisubstituted aromatic ring as three ABX proton signals at dH 6.53 (dd, J = 7.7, 2.1 Hz, H-5), 7.26 (dd, J = 7.7, 7.5 Hz, H-6) and 6.21 (dd, J = 7.5, 2.1 Hz, H-7), and two secondary methyls at dH 1.39 (d, J = 6.5 Hz, CH3-9) and 1.28 (d, J = 6.1 Hz, CH3-10), in addition to a hydrogen bonded phenolic hydroxyl at dH 11.22 (br.s, 8-OH). The COSY (Fig. 2) and HSQC experiments have identified two spin systems; the first is the aromatic ABX protons, and (CH3)CH–CH(CH3), as the two methyls dH 1.39/dC 19.2 and 1.28/17.2, being connected to the two multiplet methines at dH 4.53/dC 80.1 and 3.18/37.6, respectively. The 13CNMR/HSQC (Table 1), exhibited presence of 11 carbons assigned to four quaternary, two methyls, five methines. Further analysis of the 13C spectrum identified a carbonyl dc 170.1 (C-1), six phenyls dc 108.1 (C-8a), 161.4 (C-8), 113.7 (C-7), 136.1 (C-6), 117.1 (C-5) and 144.2 (C-4a), in addition to an oxymethine dC 80.1 (C-3) and aliphatic methine dC 37.6 (C-4). The HMBC (Fig. 2), correlations of CH3-10/C-3, H-3/C-1 and C-4, CH3-9/C-4, H-4/C-4a, H-5/C-4a, C-4 and C-8a identified a lactone moiety connected with the aromatic ring at the quaternary carbons C-4a and 8a. HMBC correlations of the aromatic proton at dH 7.26 (H-6) with the oxygenated C-8 confirmed its position. These findings identified (1) as 4methylmellein. The sign of optical rotation of 1 was the same as 3R-mellein [18], implying the stereochemistry at C-3 to be R Table 3 Antifungal and cytotoxic activities of the isolated compounds (1–4). Comp. 1 2 3 4 Nystatin Doxorubicin Antifungal activity (Zone of inhibition in mm) Cytotoxicity assay against MCF-7 (ED50) A. flavus F. oxysporum C. albicans – 7 8 – 12 – 6 7 6 6 17 – 5 6 – – – – >10 mg/mL 6.1 mg/mL 8.3 mg/mL – – 1.8 mg/mL 88 E.S. Elkhayat, A.M. Goda / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 85–89 10 OH 5 9 4a HO 6 3 7 1 O O O 8a 8 OH O OH 1 O OH 2 : COSY O 3 : HMBC Fig. 2. Diagnostic COSY and HMBC correlations of compounds (1–3). configuration. Absence of NEOSY correlations between CH3-3 and CH3-4, indicated that these methyls were in opposite directions to each other, which indicated that the relative configurations to be 3R,4S [19]. Comparison with the published data of gamahorin, previously isolated from the phytopathogenic fungus Epichloe typhina [20], revealed that both compounds only differed in the substituent at C-7, where 1 is lacking the hydroxymethylene moiety at C-7. Accordingly, compound 1 was identified as (3R,4S) 4methylmellein, it was previously described as a catalytic hydrogenation derivative of oospolactone (3,4-dimethyl-8-hydroxyiso coumarin), the fungal metabolite isolated from the mycelium of Oospora sp. [21], but this is the first isolation as a natural product. Compound 2 was isolated as white amorphous solid and its EIMS analysis revealed molecular ion peak at m/z 195 [M+H]+. Except absence of the aliphatic methine (dH 3.18) and the presence of two oxymethines at dH 4.61 and 4.47, the 1H NMR (Table 1) spectrum was comparable to 1. The mellein nucleus was evident as presence of the ABX three doublets protons at dH 6.85 (d, J = 7.3 Hz, H-5), 6.95 (d, J = 7.5 Hz, H-7) and 7.43 (dd, J = 7.5, 7.3 Hz, H-6), phenolic hydroxy at dH 11.16 and the secondary methyl at dH 1.50 (d, J = 6.5 Hz, CH3-9). The COSY experiment (Fig. 2), confirmed a spin system of OHCH-CH-CH3 as the cross peaks of the CH3-9/CH-3, which in turn coupled to a hydroxymethine (HOCH-4), identifying 1-methyl-2-hydroxy-ethyl moiety. The 13C NMR/HSQC (Table 2) showed resonance of ten carbons: one methyl, two oxymethines and six aromatic carbons, and a carbonyl. The HMBC experiment (Fig. 2), confirmed the mellein structure as the cross peaks between H-3 (dH 4.61) with the carbonyl (dC 169.2, C-1), the cross peaks between the hydroxymethine (H-4) with the 2 aromatic quaternary carbons dC 107.0 (C-8a) and 140.4 (C-4a), and cross peak of the doublet proton at dH 6.95 (H7) with the oxygenated aromatic carbon dC 162.3 (C-8). Based on the previous data and comparison of the spectral data with literatures, 2 was identified as (3R,4R)-4-hydroxymellein [22]. It was previously isolated from Pencillium sp. secluded from Alibertia macrophylla (Rubiaceae) [23], and from the marine fungus Microsphaeropsis sp. secluded from the marine sponge Myxilla incrustans (H) [24]. Compound 3 was isolated as white amorphous powder, its ESIMS analysis revealed pseudomolecular ion peak at m/z 195 [M +H]+. The 13C NMR (Table 2)/HSQC spectra showed the presence of ten carbons associated with the mellein nucleus. The 1H NMR spectrum (Table 1) was similar to 2 except the absence of the hydroxymethine at dH 4.47, instead there was an additional aliphatic methylene at dH 2.79 (br.s, CH2-4a) and 2.81 (1H, m, CH2-4b). In addition to the presence of two meta-coupled aromatic protons at dH 6.10 and 6.27 (1H, d, J = 2.1 Hz, each) suggesting a hydroxyl group in between. These data proposed 3 to be 6-hydroxymellein. The COSY and HMBC experiments (Fig. 2), confirmed 6-hydroxymellein structure. Comparing the spectral data with literatures, confirmed compound 3 to be 6-hydroxymellein, that was previously isolated from Aspergillus terreus secluded from Arabidopsis thaliana [25], this is the first isolation from the genus Penicillium. Compound 4 was isolated as yellowish oily residue. The IR showed absorption band assigned to a hydroxyl group at 3675 cm 1. Its ESIMS spectrum showed pseudomolecular peak at m/z 139 [M+H]+. The 13C NMR spectrum indicated the presence of an oxygenated aromatic carbon, four aromatic methines, quaternary aromatic, aliphatic methylene, in addition to aliphatic oxygenated meythylene. The 1H NMR spectrum confirmed the presence of p-substituted aromatic ring [two doublets at dH 6.91 (2H, m) and 6.65 (2H, m)], and a hydroxyl-ethyl moiety as the methylene 2.68 (2H, m) and oxymethylene dH 3.68 (2H, m). Comparison of the spectroscopic data with literatures has identified 4 as tyrosol, which was isolated from the marine fungus Penicillium brevicompactum [26], and it is widespread in fungi [27], but this is the first report from terrestrial Penicillium sp. According to the spectral data and comparison with literatures compound 5 was identified as cerevisterol [28], which was previously isolated from Penicillium sp. secluded from Mauritia flexuosa [8]. Compound 6 was identified as stigmast-4-ene-3-one [29], which was previously isolated from Aspergillus terrus [30], but this is the first isolation from genus Penicillium. Evaluating the antifungal activity of the compounds was carried out against A. flavus, F. oxysporum and C. albicans at concentration of 50 mg per disc, compounds 1–4 showed inhibition zones ranged from 6 to 8 mm (Table 3). Compound 2 was active against all the tested organisms, 1 showed activity against F. oxysporum and C. albicans. 3 was active against A. flavus and F. oxysporum. Finally, 4 was only active against F. oxysporum, while 5 and 6 were inactive against all the tested organisms. On evaluating the cytotoxic activity towards MCF-7 and COLO-205 cells, none of the tested compounds were active against COLO-205. Meanwhile 1–3 were active towards MCF-7, with the highest activity attributed for 2 followed by 3, while 1 was the least active (Table 3). 4. Conclusions From the rice solid culture medium of the endophyte Penicillium sp. secluded from the Senecio flavus (Asteraceae) fresh healthy leaf; three mellein derivatives (1–3) were isolated, of which 4methylmellein 1 was a new natural product, in addition to three other compounds. Compounds 3, 4 and 6 were isolated for the first time from the genus Penicillium. Structures were elucidated by extensive spectroscopic analysis. Biological evaluation conferred antifungal activity for compounds 1–4, and cytotoxic activity for compounds 1–3. E.S. Elkhayat, A.M. Goda / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 85–89 Conflict of interest None. References [1] E.S. Elkhayat, S.R. Ibrahim, G.A. Mohamed, Plant endophytes, renewable source of new natural products, Nat. Prod. J. 2 (2012) 225–234. [2] M.N. Fátima, C.F. Maria, M.C. Arriaga, T.L. Lemos, M. Andrade-Neto, M.C. de Mattos, J. Mafezoli, F.M. Viana, V.M. Ferreira, E. Rodrigues-Filhod, A.G. Ferreirad, A new Eremophilane-type sesquiterpene from the phytopathogen fungus Lasiodiplodia theobromae (Sphaeropsidaceae), J. Braz. Chem. Soc. 19 (2008) 478–482. [3] M.A. Tajick, S.M. Hamideh, B. Valiollah, Identification of biological secondary metabolites in three Penicillium species, P. goditanum, P. moldavicum and P. corylophilum, Progr. Biol. Sci. 4 (2014) 53–61. [4] B.H. Daisy, G.A. Strobel, U. Castillo, D. Ezra, J. Sears, D.K. Weaver, J.B. Runyon, Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel endophytic fungus, Microbiology 148 (2002) 3737–3744. [5] J.I. Pitt, A Laboratory Guide to Common Penicillium Species, 3rd ed., Food Science Australia Publishers, Australia, 2000. [6] P. Petit, E.M. Lucas, L.M. Abreu, L.H. Pfenning, J.A. Takahashi, Novel antimicrobial secondary metabolites from a Penicillium sp. isolated from Brazilian cerrado soil, Electron. J. Biotechnol. 12 (2009) 1–9. [7] V.P. Zhelifonova, T.V. Antipova, A.G. Kozlovsky, Secondary metabolites in taxonomy of the Penicillium fungi, Microbiology 79 (2010) 277–286. [8] H.H. Koolen, E.R. Soares, F.M. da Silva, R.A. de Almeida, A.D. de Souza, L.S. de Medeiros, E.R. Filho, A.Q. de Souza, An antimicrobial alkaloid and other metabolites produced by Penicillium sp. an endophytic fungus isolated from Mauritia flexuosa L. f., Quim. Nova 35 (2012) 771–774. [9] A.M. Marinho, P.S. Marinho, E.R. Filho, Esteroides Produzidos Por Penicillium herquei, Um Fungo Endofítico Isolado Dos Frutos De Melia azedarach (Meliaceae), Quim. Nova 32 (2009) 1710–1712. [10] V.L. Challinor, H.B. Bode, Bioactive natural products from novel microbial sources, Ann. NY. Acad. Sci. 1354 (2015) 82–97. [11] R.M. Gutierrez, A.M. Gonzalez, A.M. Ramirez, Compounds derived from endophytes: a review of phytochemistry and pharmacology, Curr. Med. Chem. 19 (2012) 2992–3030. [12] M. Koul, S. Meena, A. Kumar, P.R. Sharma, V. Singamaneni, S.R. Hassan, A. Hamid, A. Chaubey, A. Prabhakar, P. Gupta, S. Singh, Secondary metabolites from endophytic fungus Penicillium pinophilum induce ROS-mediated apoptosis through mitochondrial pathway in pancreatic cancer cells, Planta Med. 82 (2016) 344–355. [13] A. Rancic, M. Sokovic, A. Karioti, J. Vukojevic, H. Skaltsa, Isolation and structural elucidation of two secondary metabolites from the filamentous fungus Penicillium ochrochloron with antimicrobial activity, Environ. Toxicol. Pharmacol. 22 (2006) 80–84. [14] E.M. Lucas, M.C. Castro, A.J. Takahashi, Antimicrobial properties of sclerotiorin, isochromophilone VI and pencolide, metabolites from a Brazilian cerrado isolate of Penicillium sclerotiorum Van Beyma, Braz. J. Microbiol. 38 (2007) 785–789. 89 [15] R. Nicoletti, L. Gresa, M. Pilar, E. Manzo, A. Carella, M.L. Ciavatta, Production and fungitoxic activity of Sch 642305, a secondary metabolite of Penicillium canescens, Mycopathologia 163 (2007) 295–301. [16] O.E. Kwon, M.C. Rho, H.Y. Song, S.W. Lee, M.Y. Chung, J.H. Lee, Y.H. Kim, H.S. Lee, Y.K. Kim, Phenylpyropene A and B, new inhibitors of acyl-CoA: cholesterol acyltransferase produced by Penicillium griseofulvum F1959, J. Antibiot. 55 (2002) 1004–1008. [17] N.M. Al-Musayeib, G.A. Mohamed, S.R. Ibrahim, S.A. Ross, Lupeol-3-Odecanoate. A new triterpene ester from Cadaba farinosa Forsk. growing in Saudi Arabia, Med. Chem. Res. 22 (2013) 5297–5302. [18] M.I. Choudhary, S.G. Musharraf, T. Mukhmoor, F. Shaheen, S. Ali, Atta-urRahman, Isolation of bioactive compounds from Aspergillus terreus, Z. Naturforsch. 59b (2004) 324–328. [19] M. Kuramata, S. Fujioka, A. Shimada, T. Kawano, Y. Kimura, Citrinolactones A, B and C and sclerotinin C, plant growth regulators from Penicillium citrinum, Biosci. Biotechnol. Biochem. 71 (2007) 499–503. [20] H. Koshino, T. Yoshihara, M. Okuno, S. Sakamura, A. Tajlmi, T. Shimanuki, Gamahonolides A, B, and Gamahorin, novel antifungal compounds from stromata of Epichloe typhina on Phleum pretense, Biosci. Biotechnol. Biochem. 56 (1992) 1096–1099. [21] K. Nozawa, M. Yamada, Y. Tsuda, K. Kawai, S. Nakajima, Antifungal activity of oosponol, oospolactone, phylludulcin, and other related compounds, Chem. Pharm. Bull. 29 (1971) 2689–2691. [22] Y. Izawa, T. Hirose, T. Shimizu, K. Koyama, S. Natori, Six new 10-pheynl-[11]cytochalasans, cytochalasins N-S from Phomopsis sp., Tetrahedron 45 (1989) 2323–2335. [23] C.M. Oliveira, G.H. Silva, I.O. Regasini, L.M. Zanardi, A.H. Evangelista, M.C. Young, V.S. Bolzani, A.R. Araujo, Bioactive metabolites produced by Penicillium sp. 1 and sp. 2, two endophytes associated with Alibertia macrophylla (Rubiaceae), Z. Naturforsch. 64c (2009) 824–830. [24] U. Höller, G.M. König, A.D. Wright, Three new metabolites from marinederived fungi of the genera Coniothyrium and Microsphaeropsis, J. Nat. Prod. 62 (1999) 114–118. [25] A. Shimada, M. Kusano, S. Takeuchi, S. Fujioka, T. Inokuchi, Y. Kimura, Aspterric acid and 6-hydroxymellein, inhibitors of pollen development in Arabidopsis thaliana, produced by Aspergillus terreus, Z. Naturforsch. 57c (2002) 459–464. [26] J. Rovirosa, A. Diaz-Marrero, J. Darias, K. Painemal, A. san Martin, Secondary metabolites from marine Penicillium brevicompactum, J. Chil. Chem. Soc. 51 (2006) 775–778. [27] H. Chen, M. Fujita, Q. Feng, J. Clardy, G.R. Fink, Tyrosol is a quorum-sensing molecule in Candida albicans, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 5048– 5052. [28] H. Kawagishi, R. Katsumi, T. Sazawa, T. Mizuno, T. Hagiwara, T. Nakamura, Cytotoxic steroids from the mushroom Agaricus blazei, Phytochemistry 27 (1988) 2777–2779. [29] A. Barla, B.H. Hüsniye, S. Kültür, S. Oksüz, Secondary metabolites from Euphorbia helioscopia and their vasodepressor activity, Turk. J. Chem. 30 (2006) 325–332. [30] E.S. Elkhayat, S.R. Ibrahim, G.A. Mohamed, S.A. Ross, Terrenolide S, a new antileishmanial butenolide from the endophytic fungus Aspergillus terreus, Nat. Prod. Res. 30 (2016) 814–820.