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Article

Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea

by
Hani Z. Asfour
1,
Zuhier A. Awan
2,
Alaa A. Bagalagel
3,
Mahmoud A. Elfaky
4,
Reda F. A. Abdelhameed
5 and
Sameh S. Elhady
4,6,*
1
Department of Medical Microbiology and Parasitology, Faculty of Medicine, Princess Al-Jawhara Center of Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah 21589, Saudi Arabia
2
Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia
3
Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
4
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
5
Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
6
Department of Pharmacognosy, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
*
Author to whom correspondence should be addressed.
Biomolecules 2019, 9(9), 480; https://doi.org/10.3390/biom9090480
Submission received: 20 August 2019 / Revised: 8 September 2019 / Accepted: 9 September 2019 / Published: 12 September 2019
(This article belongs to the Section Natural and Bio-derived Molecules)
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Versions Notes

Abstract

:
The diversity of symbiotic fungi derived from two marine sponges and sediment collected off Obhur, Jeddah (Saudi Arabia), was investigated in the current study. A total of 23 isolates were purified using a culture-dependent approach. Using the morphological properties combined with internal transcribed spacer-rDNA (ITS-rDNA) sequences, 23 fungal strains (in the majority Penicillium and Aspergillus) were identified from these samples. The biological screening (cytotoxic and antimicrobial activities) of small-scale cultures of these fungi yielded several target fungal strains which produced bioactive secondary metabolites. Amongst these isolates, the crude extract of Aspergillus terreus strain S020, which was cultured in fermentation static broth, 21 L, for 40 days at room temperature on potato dextrose broth, displayed strong antimicrobial activities against Pseudomonas aeruginosa and Staphylococcus aureus and significant antiproliferative effects on human carcinoma cells. Chromatographic separation of the crude extract by silica gel column chromatography indicated that the S020 isolate could produce a series of chemical compounds. Among these, pure crystalline terrein was separated with a high yield of 537.26 ± 23.42 g/kg extract, which represents the highest fermentation production of terrein to date. Its chemical structure was elucidated on the basis of high-resolution electrospray ionization mass spectrometry (HRESIMS) or high-resolution mass spectrometry (HRMS), 1D, and 2D NMR spectroscopic analyses and by comparison with reported data. The compound showed strong cytotoxic activity against colorectal carcinoma cells (HCT-116) and hepatocellular carcinoma cells (HepG2), with IC50 values of 12.13 and 22.53 µM, respectively. Our study highlights the potential of A. terreus strain S020 for the industrial production of bioactive terrein on a large scale and the importance of future investigations of these strains to identify the bioactive leads in these fungal extracts.

Graphical Abstract

1. Introduction

The marine habitat is an attractive source of both biological and chemical diversity. It has been reported that oceans contain nearly 291,000 described species, representing only a small number of the total species that have yet to be discovered [1,2,3]. Almost all macro-organisms in marine habitats, e.g., sediment, fish, algae, sponges, corals, ascidians, have been investigated for their content of natural product entities [4,5]. A bioactive compound with unique structures has been isolated and was shown to possess novel anti-inflammatory, antitumor, and antimicrobial properties [6,7,8,9,10,11,12,13,14]. As interests have turned to marine symbionts, fungi have now begun to be recognized as a likely source of bioactive natural products after having received little attention from natural products chemists [15]. Recently, marine-associated fungi have proved to be a gorgeous source of pharmacologically active natural compounds [14,16,17,18,19,20,21,22,23,24,25,26,27]. Most of these micro-organisms grow in a unique, extreme, and stressful environment; therefore, they have the ability to yield unusual and unique secondary metabolites [15].
Fungi belonging to the Aspergillus genus are one of the major contributors to the secondary metabolites of fungal origin [14]. Marine-derived isolates of Aspergillus terreus are well known for the production of structurally diverse and biologically active natural products [28,29]. Terrein, a fungal metabolite isolated from A. terreus, has been proven to perform diverse biological activities [30,31,32,33,34,35]. Although terrein has application value in the fields of medicine, cosmetology, and agriculture, large-scale production of pure (+)-terrein cannot be achieved easily. The production of terrein by chemical synthesis is difficult due to its stereoselectivity [36,37,38,39] and high-cost chemical reagents [40,41]. In this study, we aimed to use fungal fermentation as a method of low-cost production of terrein in high yield. To our knowledge, the present study describes the highest fermentation production of terrein to date [29,42,43,44,45,46,47,48,49].
The static culture broth and mycelia of A. terreus S020 fungus were extracted, and the combined extracts were partitioned by silica gel column chromatography. The pure compound terrein was finally purified using a C18 semi-preparative HPLC column.
As part of our ongoing study to isolate and identify compounds from a marine host [50,51,52,53,54], we cultured and identified fungi from a marine source, assessed bioactive activity against Pseudomona aeruginosa ATCC27853, Bacillus subtilis ATCC6633, Staphylococcus aureus, ATCC25923 and Candida albicansATCC76615, and cytotoxic effects on breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116) cells, using crude extracts of the cultured fungal isolates. Further separation, structure determination, and bioactive assessment of the yielded metabolites of selected target strains were performed. A compound, terrein, was separated and purified from the static culture broth of A. terreus strain S020 (Figures S1 and S2). Its chemical structure was elucidated on the basis of HRMS, 1D, and 2D NMR spectroscopic analysie and by comparison with reported data. The isolated compound was tested for its antiproliferative activity.

2. Experimental Section

2.1. Biological Materials

The deep-sea sediment and the marine sponge specimens used in this study were collected from the Red Sea at Obhur, Jeddah, Saudi Arabia, in January 2016 by SCUBA diving at depths of 17 and 27 m. After collection, the materials were kept at −25 °C until investigation.

2.2. Fungi Isolation

2.2.1. Sponge Samples

Surface sterilization of the sponge samples was performed. The sponge samples were disinfected with 5% sodium hypochlorite, followed by 70% ethanol [55]. The inner tissues of sponge materials were cut into pieces of approximately 2 cm3 and homogenized aseptically with sterile, artificial seawater. Three dilutions (1:10, 1:100, and 1:1000) of the resulting homogenate were made with sterile seawater.

2.2.2. Sediment Samples

The deep-sea sediment sample was homogenized aseptically with 15 mL of sterile water, and the resulting solution was diluted by a serial dilution method.
For fungal cultivation, 90 μL of each diluted homogenate was transferred onto plates of each of the following media in triplicate (HiMedia Laboratories, Mumbai, India): Czapek–Dox yeast agar medium (CYE), malt agar medium (ME), and Sabouraud dextrose agar medium (SD). All media were supplemented with an antibiotic (0.25% chloramphenicol) and 2% NaCl. According to Wei’s morphological criteria [56], the fungal isolates were identified morphologically on agar plates after 7–14 days incubation at 29 °C. A series of purification and subculture steps were carried out to obtain purified fungal isolates, and photos were taken of each pure isolate (Figure 1).

2.3. DNA Genome Extraction from Pure Subcultured Fungal Isolates

The distinct, pure fungal isolates described above were subcultured in Sabouraud dextrose liquid medium for 3–7 days at 29 °C. The mycelia were separated by filtration and dried using a freeze dryer. According to the manufacturer’s instructions, the fungal DNA extraction of the resulting tissues was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). The integrity of the extracted DNA was checked and confirmed by gel electrophoresis.

2.4. Internal Transcribed Spacer-rDNA (ITS-rDNA) Fragments Amplification, Sequencing, and Phylogenetic Analysis of Fungal Isolates

Using the primers ITS1 (5′-TCCGTAGGTGAACCTGCG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [57], the genomic DNA of the fungal isolates was used as the template to amplify fungal ITS-rDNA fragments. For preliminary identification, the sequences of fungal ITS-rDNA regions were compared with related sequences at the National Center for Biotechnology Information (NCBI) as previously described [58,59,60].

2.5. Fermentation and Preparation of Extracts of the Fungal Isolate S020

A. terreus strain S020 was cultured under static conditions at room temperature in 2 L Erlenmeyer flasks containing 500 mL of potato dextrose liquid culture medium (PDB). After 40 days of cultivation, 21 L of whole broth was filtered through cheesecloth to separate the culture broth from the mycelia. The broth was extracted three times with ethyl acetate (EtOAc), while the mycelia were extracted three times with methanol (MeOH). Both EtOAc and MeOH extracts showed a similar TLC pattern and so were combined and concentrated to generate a crude extract (17.49 g) for further separation.

2.6. Isolation and Purification of Terrein

The total crude extract (17.49 g) of A. terreus strain S020 was subjected to silica gel column chromatography (CC) using gradient elution of n-hexane, CHCl3, and MeOH at a flow rate of about 20 mL/min. Fractions of 100 mL were collected and examined by TLC; similar fractions were combined and evaporated under reduced pressure to obtain fractions 1–10. Fraction 5 (CHCl3 fraction, 13.50 g) was subjected to silica gel CC with CHCl3–MeOH gradient elution to afford seven further fractions. Of these, the bioactive fractions eluted with CHCl3–MeOH (20:1) were concentrated to yield a terrein precipitate (9.20 g, purity 85%) which was finally purified by HPLC (XDB-C18 Zorbax, 5 µm, 250 mm × 4.6 mm) using 20% CH3CN/H2O at a flow rate of 1 mL/min and UV detection at 281 nm.
Terrein was characterized as follows: Colorless crystal needles (10.3 mg); α D 25 + 151.0 (c 0.5, MeOH); UV (λmax, MeOH) (log ε): 227 (4.31), 281 (2.51) nm; spectroscopic NMR data: (see Supplementary Materials); ESI-MS: m/z 155.07 [M + H]⁺; high-resolution electrospray ionization mass spectrometry (HRESIMS): m/z 155.0698 (calculated for C8H11O3 [M + H]⁺, 155.0708).

2.7. Biological Activity

2.7.1. Preparation of the Extracts of Isolates S001–S023

The fungal strains S001–S023 were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of the corresponding liquid media (Table 1), incubated at 29 °C, and continuously shaken at 150 rounds per minute (rpm) in an orbital shaker for 14 days. After incubation, 50 mL of EtOAc was added to each flask and left overnight to stop cell growth. The mycelia were separated by filtration, and the filtrate was extracted three times (3 × 50 mL) with EtOAc. The organic portion (combined extracts) was evaporated under vacuum, and the residues obtained were washed with water and then taken to dryness to obtain colored crude extracts (broth extract). Other crude extracts (mycelia extract) were obtained by extraction of mycelia with MeOH and evaporation of the solvent under a vacuum. The resulting EtOAc and MeOH extracts were lyophilized and stored for biological screening.

2.7.2. Antimicrobial and Cytotoxic Activities of Fungal Extracts of Isolates S001–S023

The crude extracts of the broth and mycelial biomass were subjected to antimicrobial activity assessment against four pathogenic microorganisms: P. aeruginosa (ATCC27853), B. subtilis (ATCC6633), S. aureus (ATCC25923) and C. albicans (ATCC76615). Assays were performed by placing 50 µL of the test extract solution (2 mg/mL, DMSO) into each hole on the plates and allowing solutions to stand overnight into an incubator at an appropriate temperature. Activity is indicated by the presence of a clear zone of growth inhibition surrounding the holes. Inhibition zones were measured in mm, and the results are reported in Table 1.
The cytotoxic activity of the test extracts on MCF-7, HCT-116, and HepG2 carcinoma cells was tested using the sulforhodamine B (SRB) assay, as previously described in our studies [54], and the results are presented in Table 2.

3. Results and Discussion

3.1. Diversity of Culturable Fungal Strains Derived from the Marine Samples

The cultivation of fungal strains from two marine sponges (Stylissa carteri and Hyrtios erectus) and deep-sea sediment of the Red Sea yielded a total of 36 isolates. Based on morphological traits as well as DNA analysis of the ITS regions [57,61], the redundant strains were excluded, and 23 distinct, pure isolates, were identified (S001–S023; Table 3, Figure 1). The strains from Penicillium spp. and Aspergillus spp. accounted for a large proportion of the total isolates. Twenty-three isolates were identified on the basis of morphological traits at the genus and species levels via genomic DNA extraction and sequencing analysis.
The phylogenetic tree of fungal strains (Figure 2) represents those fungi that are easily cultivable and could be recovered when culture-dependent techniques are applied [62]. Representative fungal isolates of these strains that have been previously cultured from marine samples include sponges, algae, cnidarians, and sea grasses [63]. The marine invertebrate symbiotic fungi have been reported as a rich source of bioactive secondary metabolites, such as polyketides, with antimicrobial and/or antitumor activities [14,62,63,64]. Our study revealed that the diversity of symbiotic fungal isolates (Penicillium, 6 strains; Aspergillus, 17 strains; Pleosporaceae, 1 strain) obtained from the marine samples was high.

3.2. Large-Scale Production and Purification of Terrein from A. terreus Strain S020

The culture broth and mycelia of A. terreus strain S020 were extracted using organic solvents, and the combined extracts were fractionated using silica gel column chromatography. The compound terrein was finally purified using a C18 semi-preparative HPLC column.

3.3. Structure Elucidation of the Isolated Terrein

Terrein (Figure 3) was isolated and purified as pale yellow crystal needles. The molecular formula, C8H10O3, was determined to be m/z 154.06 by HRESIMS at m/z 155.0698 [M + H]⁺ and m/z 177.0529 [M + Na]⁺. Interpretation of NMR and HRMS data (Figures S4–S8) suggested that the compound isolated was terrein, as previously reported [28,42].

3.4. Biological Activities

3.4.1. Antimicrobial Activities of the Fungal Extracts

All 23 fungal isolates were cultured on a small scale, and the crude extracts of their broth and mycelia were tested for antimicrobial activity (against P. aeruginosa ATCC27853, B. subtilis ATCC6633, S. aureus ATCC25923 and C. albicans ATCC76615) using the agar diffusion assay [65]. Fungal isolate extracts displayed different levels of antimicrobial activities against at least one pathogen (Table 1). It is worth pointing out that the extracts of most fungal strains displayed exceptionally high antibacterial activities against P. aeruginosa and S. aureus (inhibition diameters more than 15 mm). Amongst these, the extract of fungal strain S013 exhibited high activity against C. albicans, while other extracts were inactive (Table 1).
From our investigation in this study, we found that extracts of some fungal broths and/or mycelia have antimicrobial effects and that different fungal strains could secrete intracellular and extracellular bioactive metabolic products [66]. Because of the efficacy of these extracts against P. aeruginosa and S. aureus, these extracts might have the potential to serve as drug leads to treat a wide variety of diseases. The results of the antimicrobial assay revealed that fungi derived from the marine source isolated in this study might be a prolific source of active compounds, which may hold potential as antibacterial and antifungal natural compounds.

3.4.2. Antiproliferative Activities of the Fungal Extracts

All 23 fungal isolates were cultured on a small scale, and the crude extracts of their fungal broth and mycelia were tested for antiproliferative activity against MCF-7, HepG2, and HCT-116 cell lines using the SRB-U assay [67]. The extracts showed variable antiproliferative activity against the cell lines under investigation. Amongst these, extracts of S004, S006, S016, S017, and S020 presented the most promising antiproliferative profile (IC50 values of < 50 µg/mL) (Table 2). The present study revealed the diversity of the antiproliferative potential of marine fungal extracts and, hence, demonstrated their strong potential to produce cytotoxic compounds. Marine fungi and their purified extracts have been shown to be good producers of antiproliferative and cytotoxic compounds. This property could be attributed to different classes of secondary metabolites, as reported previously [20,25,26,27,68,69,70,71].

3.4.3. Cytotoxic Activity of the Isolated Compound Terrein

The cytotoxic effect of terrein (Table 4) against HCT-116 and HepG2 cancer cell lines (concentration range 0.01–100 μM) was assessed using the SRB-U assay [67]. The compound displayed strong antiproliferative activity against the two cell lines under investigation, with IC50 values of 12.13 μM and 22.53 μM for HCT-116 and HepG2 cells, respectively. Doxorubicin was used as a standard cytotoxic control.

4. Conclusions

In conclusion, a total of 23 symbiotic fungi distributed among 3 genera were identified and isolated from marine sponges and sediment collected off Obhur (Saudi Arabia). The biological screening of small-scale cultures of these fungi yielded several target fungal strains which produced secondary bioactive metabolites. Amongst these isolates, the chromatographic separation of the crude extract of A. terreus strain S020 by silica gel column chromatography led to the isolation of pure crystalline terrain, with a high yield of 537.26 ± 23.42 g/kg dried crude extract; this represents the highest fermentation production of terrein to date. The chemical structure was elucidated on the basis of HRMS, 1D, and 2D NMR spectroscopic analyses, and by comparison with reported data. The isolated compound, terrein, showed strong cytotoxic activity against colorectal carcinoma cells and hepatocellular carcinoma cells, with IC50 values of 12.13 and 22.53 µM, respectively. Our study contributes to the understanding of fungal diversity and provides the basis for future investigations of these symbionts with respect to identifying and purifying the bioactive leads in these fungal extracts. This study also describes an efficient approach for producing bioactive terrein in a very high yield. Currently, there is great demand for the development of new drugs to combat the emergence of bacterial resistance to traditional antibiotics.

Supplementary Materials

The following are available online at https://www.mdpi.com/2218-273X/9/9/480/s1, Figure S1: HPLC chromatograms of terrein (A) and Aspergillus terreus S020 extract (B), Figure S2: UV-Vis spectra (200-400 nm) of crude extract of A. terreus S020 and terrein, Figure S3: The standard curve for quantifying terrein, Figure S4 (a,b): HRESIMS spectrum of terrein, Figure S5: ESIMS spectrum of terrein, Figure S6: 1H-NMR spectrum of terrein (CD3OD), Figure S7: 13C-NMR spectrum of terrein (CD3OD), Figure S8: HMBC spectrum of terrein (CD3OD).

Author Contributions

H.Z.A. and S.S.E. designed the experiments; S.S.E. collected the marine specimen; M.A.E. performed the experiments; Z.A.A. performed the biological activity; S.S.E., H.Z.A., and A.A.B. analyzed the data; and R.F.A.A. and S.S.E. wrote and edited the manuscript.

Funding

This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant No. G-328-140-38. The authors, therefore, acknowledge with thanks DSR for technical and financial support.

Conflicts of Interest

All contributing authors declare no conflict of interest to disclose, whether financial or of any other nature.

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Biomolecules 09 00480 g001a 550Biomolecules 09 00480 g001b 550
Figure 1. Morphological photos of the 23 derived fungal isolates (S001–S023).
Figure 1. Morphological photos of the 23 derived fungal isolates (S001–S023).
Biomolecules 09 00480 g001aBiomolecules 09 00480 g001b
Biomolecules 09 00480 g002 550
Figure 2. Phylogenetic tree of isolated fungal strains S001–S023.
Figure 2. Phylogenetic tree of isolated fungal strains S001–S023.
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Biomolecules 09 00480 g003 550
Figure 3. The structure of terrein.
Figure 3. The structure of terrein.
Biomolecules 09 00480 g003
Table
Table 1. Antimicrobial activities of isolated fungal strains’ (S001–S023) crude extracts of both fermentation broth and mycelia.
Table 1. Antimicrobial activities of isolated fungal strains’ (S001–S023) crude extracts of both fermentation broth and mycelia.
Marine.
Host		Fungal StrainCulture MediaPseudomonas aeruginosaBacillus subtilisStaphylococcus aureusCandida albicans
GenusStrainBrothMyceliaBrothMyceliaBrothMyceliaBrothMycelia
Stylissa carteriPenicilliumS001CZYB−+++−−++++++++−−
AspergillusS002CZYB−+++−−++++++++−−
SedimentPenicilliumS003CZYB−++++−−+++++++−−
AspergillusS004CZYB−++++−−−+++−−
AspergillusS005CZYB+++++++−−++++++++−−
AspergillusS006CZYB−++++−−−++++−−
AspergillusS007CZYB−++++−−−++++−−
PleosporaceaeS008CZYB−++++−−−++++−−
Hyrtios erectusAspergillusS009MEB−−−−−+++−−
PenicilliumS010MEB−−−−−+++−−
PenicilliumS011MEB−+++−−−++++−−
SedimentAspergillusS012MEB−+++++++−+++++++−−
SedimentAspergillusS013MEB−++++−−−++++++++−
H. erectusPenicilliumS014SDB−+++−−++++++−−
S. carteriPenicilliumS015SDB+++++++−−++++++++−−
Deep SeaAspergillusS016SDB−+++++++−−++++−−
AspergillusS017SDB++++++++++−+++++++−−
AspergillusS018SDB−++++−−−++++−−
SedimentAspergillusS019SDB−++++++−+++++++−−
AspergillusS020SDB−++++−−−++++−−
AspergillusS021SDB−++++−−+++++++−−
AspergillusS022SDB++++++−−++++++−−
AspergillusS023SDB−+++−−++++++−−
Sabouraud dextrose broth (SDB); malt extract broth (MEB); Czapek–Dox broth (CZYB). Extracts tested at concentrations of 2 mg/mL; inhibition zone in mm including disc. Inhibition diameters were used to describe the groups of microbial growth inhibition: growth inhibition diameter more than 15 mm (++++); between 15 and 10 mm (+++); and less than 7 mm (+); no inhibition noticed (−).
Table
Table 2. In vitro antiproliferative activities (IC50, μg/mL) of isolated fungal strains’ (S001–S023) crude extracts of both fermentation broth and mycelia against human carcinoma cells (MCF-7, HCT-116, and HepG2).
Table 2. In vitro antiproliferative activities (IC50, μg/mL) of isolated fungal strains’ (S001–S023) crude extracts of both fermentation broth and mycelia against human carcinoma cells (MCF-7, HCT-116, and HepG2).
Marine
Host		Fungal StrainCulture MediaCell Lines
GenusStrain Breast
MCF-7		Hepatocellular HepG2Colorectal
HCT-116
BrothMyceliaBrothMyceliaBrothMycelia
S. carteriPenicilliumS001CZYB75.44˃10079.26˃100˃10092.60
AspergillusS002CZYB73.28˃10061.96˃100˃10048.00
SedimentPenicilliumS003CZYB55.16˃10048.96˃100˃10068.40
AspergillusS004CZYB22.7281.9513.55˃10042.7551.40
AspergillusS005CZYB70.29˃10060.22˃100˃10070.66
AspergillusS006CZYB34.2792.1540.20˃10062.20˃100
AspergillusS007CZYB60.50˃10065.63˃10087.0080.00
PleosporaceaeS008CZYB92.00˃100˃100˃100˃100˃100
H. erectusAspergillusS009MEB˃100˃100˃100˃100˃100˃100
PenicilliumS010MEB73.60˃100˃100˃100˃100˃100
PenicilliumS011MEB57.67˃10083.20˃100˃100˃100
SedimentAspergillusS012MEB˃10092.0092.00˃100˃10097.00
SedimentAspergillusS013MEB39.0079.9254.8186.7673.6092.60
H. erectusPenicilliumS014SDB79.53˃10092.80˃100˃100˃100
S. carteriPenicilliumS015SDB64.70˃10066.26˃10074.2015.00
Deep SeaAspergillusS016SDB23.2793.5041.7067.4462.0081.00
AspergillusS017SDB42.26˃10054.4099.0853.16˃100
AspergillusS018SDB60.48˃10097.70˃10090.00˃100
SedimentAspergillusS019SDB51.00˃10086.72˃10049.80˃100
AspergillusS020SDB44.0081.9557.00˃10047.8367.00
AspergillusS021SDB89.9368.1563.29˃10074.6089.66
AspergillusS022SDB65.1287.0091.80˃10081.60˃100
AspergillusS023SDB˃100˃100˃100˃100˃100˃100
Doxorubicin 0.41 ± 0.10.85 ± 0.10.11 ± 0.04
Doxorubicin positive cytotoxic control, presented as the mean ± SD; n = 3.
Table
Table 3. Identification of the isolated fungal strains (S001–S023). The closest relatives to fungal strains according to a BLAST search are presented.
Table 3. Identification of the isolated fungal strains (S001–S023). The closest relatives to fungal strains according to a BLAST search are presented.
IsolateGenus IdentificationSequence Length (bp)Related Strain
(BLAST)		Access No.Similarity (%)
S001Penicillium sp.522Penicillium chrysogenumKU55630199%
S002Aspergillus sp.490Aspergillus cristatusKT15156598%
S003Penicillium sp.524P. chrysogenumKX26683198%
S004Aspergillus sp.531Aspergillus nigerKJ88137795%
S005Aspergillus sp.530A. nigerKU90448096%
S006Aspergillus sp.532Aspergillus flavusHQ64594099%
S007Aspergillus sp.530Aspergillus tubingensisKX01598897%
S008Pleosporaceae sp505Pleosporaceae sp.KJ44326497%
S009Aspergillus sp.629Aspergillus sp.HQ02322798%
S010Penicillium sp.534Penicillium polonicumKF49414899%
S011Penicillium sp.525P. polonicumKP79407498%
S012Aspergillus sp.527Aspergillus sp.KX01599497%
S013Aspergillus sp.502Aspergillus foveolatusKU86661599%
S014Penicillium sp.519P.chrysogenumKX01599898%
S015Penicillium sp.526P. chrysogenumKU74390098%
S016Aspergillus sp.523A. flavusFJ21638898%
S017Aspergillus sp.529A. flavusKX01598695%
S018Aspergillus sp.644A. nigerKT37725099%
S019Aspergillus sp.438A. nigerLC19500398%
S020Aspergillus sp.538Aspergillus terreusKX69414898%
S021Aspergillus sp.330Aspergillus sp.KX00026994%
S022Aspergillus sp.536Aspergillus fumigatusKU74388998%
S023Aspergillus sp.531Aspergillus sp.KU84242699%
Table
Table 4. In vitro cytotoxic activity (IC50, µM) of terrein against human carcinoma cells (HCT-116 and HepG2).
Table 4. In vitro cytotoxic activity (IC50, µM) of terrein against human carcinoma cells (HCT-116 and HepG2).
Cell TypeCell LineDoxorubicin Terrein
ColorectalHCT-1160.11 ± 0.0412.13
HepatocellularHepG20.85 ± 0.122.53
Doxorubicin positive cytotoxic control, presented as the mean ± SD; n = 3.

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Asfour, H.Z.; Awan, Z.A.; Bagalagel, A.A.; Elfaky, M.A.; Abdelhameed, R.F.A.; Elhady, S.S. Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea. Biomolecules 2019, 9, 480. https://doi.org/10.3390/biom9090480

AMA Style

Asfour HZ, Awan ZA, Bagalagel AA, Elfaky MA, Abdelhameed RFA, Elhady SS. Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea. Biomolecules. 2019; 9(9):480. https://doi.org/10.3390/biom9090480

Chicago/Turabian Style

Asfour, Hani Z., Zuhier A. Awan, Alaa A. Bagalagel, Mahmoud A. Elfaky, Reda F. A. Abdelhameed, and Sameh S. Elhady. 2019. "Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea" Biomolecules 9, no. 9: 480. https://doi.org/10.3390/biom9090480

APA Style

Asfour, H. Z., Awan, Z. A., Bagalagel, A. A., Elfaky, M. A., Abdelhameed, R. F. A., & Elhady, S. S. (2019). Large-Scale Production of Bioactive Terrein by Aspergillus terreus Strain S020 Isolated from the Saudi Coast of the Red Sea. Biomolecules, 9(9), 480. https://doi.org/10.3390/biom9090480

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