Hypocrol A, a new tyrosol derivative from a sponge-derived strain of the fungus Hypocrea koningii

Abstract In continuation of our search for new antibacterial and antioxidant metabolites from sponge-derived fungi, one new tyrosol derivative, hypocrol A (1), together with four known congeners, trichodenol B (2), 4-hydroxyphenethyl acetate (3), 4-hydroxyphenethyl tetradecanoate (4) and 1-oleyltyrosol (5), was isolated from the strain Hypocrea koningii PF04. Their planar structures were unequivocally elucidated by spectroscopic methods and comparison with the literature data. All the compounds displayed weak antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus and Escherichia coli, whereas compounds 1 and 2 exhibited a moderate antioxidant efficacy in the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay with IC50 values of 48.5 and 97.4 μg/mL, respectively. Graphical abstract


Introduction
Naturally occurring tyrosol (4-hydroxyphenethyl alcohol) and its derivatives are one class of the most representative phenolic compounds which are extensively present in plants and microbes, such as Olea europaea and Fusarium solani (Bianco et al. 2004;Tadpetch et al. 2015). Due to versatile biological functions including antioxidant (Barontini et al. 2014), antimicrobial (Aissa et al. 2012), cytotoxic (Ahn et al. 2008, antileishmanial (Aissa et al. 2012) activities and excellent surface-active properties (Lucas et al. 2010), these tyrosol derivatives have captured ever-increasing attention of organic chemists, biologists and pharmacologists. Hypocrea spp. produced a plethora of new chemicals (Liu et al. 2011), and as part of our ongoing quest for new antibacterial and antioxidant agents, the chemical investigation of the etOAc-soluble fraction from the fungus Hypocrea koningii PF04 residing in the sponge Phakellia fusca resulted in the isolation of five tyrosol derivatives that were composed of one new member, hypocrol A (1), together with trichodenol B (2), 4-hydroxyphenethyl acetate (3), 4-hydroxyphenethyl tetradecanoate (4) and 1-oleyltyrosol (5). Herein, the detailed descriptions of the isolation, structure elucidation and biological activities of compounds 1-5 were reported.

Results and discussion
The etOAc extract of rice culture of the fungus H. koningii PF04 was subjected to CC (silica gel, ODS and HPLC) to afford five tyrosol derivatives. Their planar structures were elucidated by a combination of spectroscopic methods (NMR, MS, UV and IR).
Compounds 1-5 were tested for antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus and Escherichia coli as well as the antioxidant activity. All the compounds displayed weak antibacterial activities against three pathogenic strains with minimum inhibitory concentration values ranging from 64 to 128 μg/mL. In addition, compounds 1-2 had a modest antioxidant effect with IC 50 values of 48.5 and 97.4 μg/mL, respectively, whereas compounds 3-5 did not exhibit activity (IC 50 values above 300 μg/mL).

Fungal material and fermentation
The fungus H. koningii PF04 isolated from the inner tissue of the marine sponge P. fusca collected off Yongxing Island in the South China Sea, was identified by ITS gene sequencing (genBank accession No. FJ941853). Initially, the fungal strain was cultivated on PDA medium in a Petri dish for 7 days and then inoculated into 50 mL of seed medium composed of 20% potato, 2% dextrose dissolved in seawater in 250-mL erlenmeyer flasks which were placed on an orbital shaker (180 rpm, 25 °C) for 48 h. Subsequently, the large-scale fermentation was performed in 50 × 250 mL erlenmeyer flasks each containing 80 g of rice and 120 mL of seawater under static conditions at 25 °C for 40 days after being inoculated with 10 mL of seed culture.

Antibacterial activity assay
The antibacterial activities against S. aureus, methicillin-resistant S. aureus and E. coli were evaluated in 96-well microplates as described previously with some modifications (Correa et al. 2011). In this procedure, each compound was diluted to 256,128,64,32,16,8,4 and 2 μg/mL as the final concentrations, and afterwards transferred to the corresponding wells. Chloramphenicol was employed as a positive control.

Antioxidant activity assay
The antioxidant activity was examined by the DPPH radical scavenging assay according to the previously described method (Ma et al. 2014). Briefly, a mixture of appropriate dilutions of the tested sample (100 μL) and 100 μL of DPPH solution (methanol, 0.2 mM) in 96-well microplates were incubated at 37 °C for 30 min, the absorbance of which were measured at 517 nm on a microplate reader. IC 50 values denoted the concentration of a sample required to scavenge 50% of DPPH radicals. Ascorbic acid was used as a positive control.

Conclusion
Five tyrosol derivatives (1-5) including a new compound, hypocrol A (1) were isolated and characterised from the fermentation of the sponge-derived fungus H. koningii PF04. Unlike the known tyrosol derivatives (2−5), hypocrol A (1) was constructed through the esterification between the tyrosol moiety and the substrate containing an aromatic nucleus. Thus, in the case of H. koningii PF04, the enzymatic machinery involves the coupling reaction of the tyrosol with a very broad substrate scope, ranging from the aliphatic to aromatic-containing molecules, thereby giving rise to diverse tyrosol derivatives. Furthermore, all the compounds possessed weak antibacterial activities against S. aureus, methicillin-resistant S. aureus and E. coli, whereas compounds 1 and 2 displayed a moderate antioxidant capacity.

Supplementary material
Supplementary material related to this article is available online alongside Figures S1-S8.

Disclosure statement
No potential conflict of interest was reported by the authors.