Morphomolecular identication, metabolic prole, anticancer, and antioxidant capacities of Penicillium sp. NRC F1 and Penicillium sp. NRC F16 isolated from Egyptian remote cave

Searching for remote locations to screen for microorganisms, identify their metabolites, and investigate their bioactivities against lethal diseases such as cancer is of critical importance. In the current study, two fungal strains where isolated from a remote cave in Asyut governorate, Egypt. These isolates were morphologically and molecularly identied through sequencing their ITS region as Penicillium sp. NRC F1, and Penicillium sp. NRC F16. Investigating the metabolic proles of the silylated ethyl acetate extracts of these fungi through conducting GC-Ms analysis revealed presence of 114 compounds belonging to different chemical classes. On the other hand, studying the in vitro bioactivity of both extracts showed moderate antioxidant activities. Penicillium sp. NRC F1 extract exhibited higher DPPH scavenging activity (74.41 ± 0.59%) at concentration of 200 µg/ml, in comparison with that exerted by Penicillium sp. NRC F16 extract at the same concentration (65.58 ± 1.55%). Moreover, studying the cytotoxicity of extracts against human colon cancer (HCT116), and human breast cancer (MCF7) cell lines revealed that cytotoxicity of both extracts was dose dependent. Promising cytotoxic effect was achieved against human colon cancer HCT116 using 200 µg/ml of Penicillium sp. NRC F1 extract (95.72 ± 1.13 % cytotoxicity), while Penicillium sp. NRC F16 ethyl acetate extract caused a cytotoxicity of 95.43 ± 1.4 %. Similarly, investigating the in vitro cytotoxicity of the extracts against human breast cancer MCF7 cell line resulted in observing promising activity of Penicillium sp. NRC F1 and Penicillium sp. NRC F16 extracts, and they exhibited 97.29 ± 0.61 %; and 97.08 ± 1.07 % cytotoxicity, respectively. Results from this study nominate those strains as promising isolates and encourage for conducting further in vivo investigations to evaluate their potency.


Introduction
Spreading of fatal diseases as cancer, as well as the reported side effects and shrinking repertoire of effective drugs have directed researches toward screening for new sources for potent compounds having anticancer activities. Cancer diseases are responsible for a considerable number of mortalities worldwide.
According to world health organization reports, breast cancer is ranked as the second cause of deaths among the most common cancers (accounting for about 2. Hence, isolation of new fungal isolates from novel remote sources is critically important in order to fortify and refresh arsenal of secondary metabolites in a trial to nd promising compounds with potent biological activities. In this study, a remote cave located in Asyut governorate, Egypt was used as a source to screen for new fungal isolates. Moreover, two selected isolates were morphmolecularly identi ed through sequencing of their nuclear ribosomal internal transcribed spacer ITS1-5.8S-ITS2 regions. Furthermore, a GC-Ms chemical analysis was performed on the silylated extracts of those fungi in order to identify their metabolic pro les. Finally, the in vitro antioxidant and anticancer activities of both extracts was investigated against the HCT116 colorectal carcinoma, and MCF7 breast carcinoma human tumor cell lines.  Figure 1). The soil samples were from different sites inside the cave including soil from the entrance of the cave, rhizosphere soil at the entrance of the cave; soil from the middle of the cave (transition zone); soil from the wall of the cave; and soil from deep inside the cave (25 m inside the cave). Samples were kept in sterilized bags and transferred in cool box (4°C) and processed within 24h.

Isolation and Puri cation of fungi
One gram of soil samples from each site was placed into 9 ml of sterile distilled water. Ten-fold serial dilutions were prepared from the mixed solution. Isolation was conducted from suitable dilution of the soil samples by spreading over the surface of agar plates of potato dextrose medium (PDA, Sigma-Aldrich, USA). After incubation for seven days at 30±2°C, the plates were checked for colonies growth and single colonies were picked-up and streaked onto the surface of agar plate of the same isolation medium, and allowed to grow for seven days. A touch of the terminal colonial growth of a single separate colony was transferred to pure slants of PDA medium to be preserved in refrigerator by regular sub-culturing every two months.

Morphological Identi cation
Isolates were preliminary identi ed under microscope following the description of (DomschGamsandAnderson 1980) and (Moubasher 1993).

Data analysis
Gel documentation system (Geldoc-it, UVP, England), was applied for data analysis using Totallab analysis software, ww.totallab.com, (Ver.1.0.1). Aligned sequences were analyzed on NCBI website (http://www.ncbi.nlm.nih.gov/webcite) using BLAST to con rm their identity. Genetic distances and MultiAlignments were computed by Pairwise Distance method using ClusteralW software analysis (www.ClusteralW.com). The nucleotide sequences were also compared with Penicillium isolates sequences available in the GenBank.

Fermentation and Extraction of secondary metabolites
Erlenmeyer asks containing potato dextrose broth medium (one liter each) were inoculated with 10 ml fungal spore suspension. The asks were then incubated aerobically at 30±2°C and 150 rpm for 7 days.
Extraction of metabolites was conducted as described by (LiZhouZhuChangYuanGaoZhangZhaoandLou 2015) with some modi cations. Brie y, the whole contents of each ak was transferred to Erlenmeyer asks (of 2 liter capacity) and extracted twice by mixing with ethyl acetate (AnalR, UK) (1:1, v/v), sonicated for 10 min. with gentle warming, and the mixture was kept overnight at room temp. Then, the organic layer was separated and the process was repeated till exhaustion. The ethyl acetate layers were collected and evaporated using rotatory evaporator (heidolph rotary evaporator, Schwabach, Germany) under reduced pressure at 45 о C. The crude extract Penicillium sp. NRC F1 (0.089 g) Penicillium sp. NRC F16 (0.136 g) were kept in the fridge for analysis.

Mass spectrometer
A Finnigan MAT SSQ 7000 mass spectrometer coupled with a Varian 3400 gas chromatograph. DB-5 column, 30 m x 0.32 mm (internal diameter), was employed with helium as carrier gas (He pressure, 20 Mpa/cm2) and GC temperature program, 85 -310 ºC at 3 ºC/ min (10 min. intial hold). The injector temperature was kept at 310ºC. The mass spectra were recorded at 70 eV in electron ionization mode (Li et al., 2015). The scan repetition rate was 0.5 s over a mass range of 39 -650 atomic mass units (amu).
To identify compounds in the ethyl acetate extract of the two fungal isolates, GCMs analyses were conducted, and compounds were identi ed by comparing their retention times and mass fragmentation patterns with those of the database libraries [Wiley (Wiley Int. USA) and NIST (Nat. Inst. St. Technol., USA)]. Moreover, peaks were examined by single-ion chromatographic reconstruction to con rm their homogeneity.

Cytotoxicity assay
Acid phosphatase assay was conducted to evaluate cytotoxicity as described previously (YangSinaiandKain 1996). Brie y, human colon cancer cell line (HCT116), and human breast carcinoma cell line (MCF7) were used by seeding 10000 cell per well in 96 well plates, left overnight till attach, and then treated with different extracts for three days. For one plate, a substrate solution was prepared where 20 mg tablet of pNPP (Sigma; cat. no. N2765) was dissolved in 10 ml buffer solution (0.1 M sodium acetate, 0.1% triton X-100, pH 5.0). Cell monolayers were washed with 250 μl PBS. 100 μl of pNPP substrate solution were added per well, then plates were incubated for 4 hours at 37°C. 10 μl of 1N sodium hydroxide stop solution were added per well. Absorbance was measured directly at wavelength 405 nm. All samples were tested in triplicates, and 0.5% DMSO was used as negative control and 50 μM cisplatin was used as positive control. Extracts were tested at serial dilutions with nal concentration of 200, 100, 50, and 25 μg/ml. Percent cytotoxicity = [1-(D/S)] × 100, where D and S denote the optical density of drug and solvent treated wells, respectively.

Antioxidant activity of extracts
The free radical scavenging activity of extracts was evaluated by using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay described previously (Blois 1958). Extracts were tested at nal concentrations of 200, 100, 50, and 25 μg/ml using 0.1mM DPPH dissolved in methanol. After incubation for 30 min in dark at room temperature, the absorbance was measured at 517 nm. Ascorbic acid (vitamin C) was used as positive control at nal concentrations of 20 µg/ml. The DPPH solutions treated with 0.5% DMSO used as a negative control. The DPPH scavenging activity of extracts was calculated according to the equation: Percentage reduction = (1-(X/ av(NC)) ×100 Where x indicates the absorbance of fraction and av(NC) indicates the average absorbance of the negative control. EC50 values were calculated using probit analysis utilizing the SPSS computer program (SPSS for windows, statistical analysis software package / version 9 / 1989 SPSS Inc., Chicago, USA).

Isolation and morphological and molecular identi cation of fungi
Soil samples were collected from different sites inside El Shekh Sayed cave, Asyut governorate, Egypt. Different fungal strains belonging to speci c genera were morphologically identi ed from samples collected from all sites. As shown in table 1, Aspergillus niger and A. avus were predominantly isolated from all sites. Rhizopusstoloniferand Mucor circinelloides came in the second place and were isolated 4 times each. Alternaria alternatawas isolated 3 times, while A. fumigatus, and A. versicolor appear twice. The richest site in fungal isolates was the rhizosphere soil at entrance of the cave where all isolates were isolated except for A. fumigatus. The most interesting isolates were two different Penicillium species which were isolated from rhizosphere soil at entrance of the cave and showed characteristic antagonistic growth which encouraged for studying both isolates.
The colonies surface of Penicillium sp. NRC F1 appeared in bluish-green color and were velvety sulcate. Colonies reverse was brownish orange in color, and conidiophores appeared two-stage branched under microscope. Conidia were bluish-green, smooth-walled, and appeared globose to subglobose. On the other hand, the morphological and microscopic appearance of the second isolate suggest that it is also a Penicillium species. Colonies appeared velvety with whitish margin, green conidial heads, and colonies revers on PDA was yellowish in color. Conidiophores appeared under microscope smooth-walled, and asymmetrically terverticillate. Conidia were smooth-walled, elliptical, globose to subglobose and arranged in irregular columns. Molecular identi cation of both isolates through sequencing of their nuclear ribosomal internal transcribed spacer ITS1-5.8S-ITS2 regions came in accordance with morphological identi cation. Sequences showed high similarities to those of Penicillium sp., and sequences were deposited in the international Gene Bank as Penicillium sp. NRC F1, and Penicillium sp. NRC F16 under accession numbers MN382318; and MN382317, respectively. Phylogenetic tree was constructed based on nuclear ribosomal ITS1-5.8S-ITS2 region related to Penicillium sp. was shown in gure 2.      Fig. 3 (a, and b) (Fig 3 a). On the other hand, investigating the in vitro cytotoxicity of the extracts against human breast cancer MCF7 cell line (Fig. 3 Fig. 3 (a, and b).

Conclusion
Finding novel sources to screen for microbes having promising biological activities is of critical need, and cave environment is an attractive source for such microbes. Penicillium species recovered in this study from a remote cave in Asyut governorate, Egypt showed promising in vitro bioactivities as antioxidant, and anticancer activities against tested human colon cancer, and human breast cancer cell lines. Further studies are encouraged to investigate the in vivo potential of these promising strains and evaluate the possibility of employing such fungi as sources of bioactive compounds.