Diketopiperazines and arylethylamides produced by Schizophyllum commune, an endophytic fungus in Alchornea glandulosa

36 Eclética Química Journal, vol. 44, n. 3, 2019, 36-42 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v44.3.2019.p36-42 Diketopiperazines and arylethylamides produced by Schizophyllum commune, an endophytic fungus in Alchornea glandulosa Carolina Rabal Biasetto1 , Andressa Somensi1 , Fernanda Sales Figueiro2 , Luiz Alberto Beraldo de Moraes2 , Geraldo Humberto Silva3 , Maria Claudia Marx Young4 , Vanderlan da Silva Bolzani1 , Angela Regina Araújo1+


Introduction
Microorganisms as the endophytic fungi (endon = inside; phyton = plant) living asymptomatically in the intercellular space of host plants and establish a mutualistic relationship with its host during a part or all their life. These endophytes are found in parts of plants such as leaves, stems, fruits, seeds, and roots, and reside inside the plant all its life, being transmitted in some cases to future generations through seeds of the host [1][2][3][4] . The interactions established by endophytes may diversify from mutualism (endophyte and host plant obtain advantages) to parasitism (only the endophyte is benefited) 5,6 . Most natural products isolated from endophytes showed antimicrobial activity, and in many cases, these have been implicated in protecting the host plant against phytopathogenic microorganisms. 1,7,8 Studies in 2012 showed trichodermin and cercosporamide with strong antifungal and antitumor activities, which were isolated from endophyte belonging to the genus Phoma associated with Arisaema erubescens. 9 Alchornea glandulosa (Euphorbiaceae) belongs to Brazilian Cerrado, which is well-known as sources of bioactive secondary metabolites [10][11][12][13] . Several chemical and pharmacological studies were performed with extracts and pure substances from this plant and presented promising results [14][15][16] . Several endophytic fungi were isolated from this plant, including S. commune, which were chemically and biologically investigated. S. commune is a filamentous fungus belonging to the class of Basiodiomicetos, known as a producer of pigments, antiviral, anticancer and immunomodulatory compounds 17 . From this endophyte were isolated phenolic compounds as gallic acid and L-ascorbic acid, both with antioxidant activity 18 . Diketopiperazines present biological activities such as antiviral, antifungal, antibacterial, insecticidal, antihyperglycemic agents, receptor affinities of calcium channels and serotonergic [19][20][21] . Recent studies have demonstrated that diketopiperazine trans-cyclo(D-tryptophanyl-L-tyrosyl) presented low antiproliferative and cytotoxic effect with L-929 mouse fibroblast cells, K-562 leukemia cells and human HeLa Human cervix carcinoma 22 . Several arylethylamides present phytotoxic potential 23 . Thus, this work reports isolation, structural elucidation and biological activities of these compounds.

General
NMR spectra were carried out one-dimensional 1 H Varian INOVA-300 spectrometer operating at 300 MHz for 1 H nucleus and TMS as internal reference. 1 H NMR spectra and 13 C 1D and 2D and NOESY 1D were performed on Varian INOVA-500 spectrometer operating at 500 MHz for 1 H nucleus and the nucleus 125 MHz for 13 C and TMS as internal reference. Mass spectra of high and low resolution were obtained on a spectrometer of UltrOTOFQ -ESI-TOF Mass Spectrometer (Bruker Daltonics, Billerica, MA, USA). Analyses were performed on a mass spectrometer with Varian 1200L LC autosampler, and triplequadrupole electrospray ionization source (ESI). The mass spectra of low resolution were obtained on a LCQ Fleet spectrometer (Thermo Scientific) with electrospray ionization source (ESI). TLC was performed using Merck silica gel 60 G F254. Spots on TLC plates were visualized under UV light and by spraying with anisaldehyde-H2SO4 reagent followed by heating at 120 °C. Analytical HPLC was performed on a Shimadzu (Shimadzu SPD-M20A) with diode array ultraviolet (DAD) detector, using a Phenomenex Gemini analytical column (C-18) (250 x 4.60 mm, 5 µm). Preparative HPLC was performed on Varian ProStar device coupled to the ProStar UV-Vis detector, using Phenomenex Luna C-18 silica semi-preparative column (150 x 21.20 mm; 5 µm). Optical rotation values were obtained at JASCO P-1020 polarimeter with sodium lamp, and 1.0 mL of cell Jasco Spectra Manager software.

Fungal isolation and identification
The traditional methodology 24 was used for the isolation of the endophytic fungus S. commune from healthy leaves of Alchornea glandulosa. S. commune was identified by sequencing and phylogenetic analysis of ribosomal operon gene fragments (CPQBA-UNICAMP-Multidisciplinary Center for Agricultural and Biological Chemistry Research, Campinas, Brazil). DNA sequence of the ITS region was analyzed using the GenBank BLAST routine and the CBS database (Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre).
The phylogenetic tree constructed from the sequences retrieved in GenBank and analyzes based on molecular data allowed the identification of this fungus as Schizophyllum commune (Filo Basidiomycota, order Agaricales and family Schizophyllaceae). The sequence of the isolate showed 99-98% similarity to sequences from different S. commune isolates, including S. commune BCC22128 and DSM 1025. In addition, in the phylogenetic tree, the isolate formed a cluster with the S. commune PRJ21 and 1-84 isolates supported by a bootstrap value of 94%. This endophytic fungus was stored in the NuBBE fungi collection in Araraquara, Brazil (kept in sterile water at 25 ℃) encoded as Ag-Sc-02 and in CPQBA as CPQBA 154-09 DRM.

Different culture media (small scale)
S. commune was grown in Petri plates containing PDA (potato starch 4 g, dextrose 20 g, agar 15 g, H2O 1000 mL) and incubated during ten days. After this period, this endophyte was cultivated in small scale in 2 Erlenmeyer flasks (500 mL) containing 300 mL of each liquid medium and these were autoclaved at 121 ℃ for 20 min. S. commune was cultivated in different liquid culture media (PBD, YM, Nutrient, Czapek, Malt Extract) under agitation at 120 rpm, for 28 days at 25 ℃. At the end of the incubation period, the broth was separated from the mycelium by filtration and the crude extracts were obtained by extraction with EtOAc (3 x 50% of the broth volume each) and dried in rotatory evaporator, affording 49.2, 54.0, 26.7, 26.5 and 38.2 mg, respectively. The cultivation on solid medium was performed: rice and corn inoculated in 2 Erlenmeyer flasks (500 mL each), containing each one: 90 g of rice or corn in 75 mL of Milli-Q H2O. The culture media were autoclaved three times (three consecutive days) at 121 ℃ for 20 min. The cultures were incubated at 25 ± 2 ℃ for 21 days. At the end of the growth period, the solid cultures were ground and extracted with EtOAc (5x200 mL). The EtOAc fraction was subjected to liquidliquid partition with H2O. The EtOAc extract concentrated was dissolved in CH3CN and then subjected to liquid-liquid partition with C6H14. The CH3CN fractions were evaporated to give CH3CNdeffated extracts, affording 279.7 (rice) and 335.1 (corn) mg.

PDB (large scale)
S. commune was inoculated in 52 Erlenmeyer flasks of 500 mL (large scale), containing each one: 1.2 g of potato starch, 6.0 g dextrose (PDB) in 300 mL of Milli-Q H2O, which were autoclaved at 121 °C for 20 min. Approximately 5 small pieces (1x1 cm) of PDA medium, from the Petri dish containing biomass of the S. commune isolated were inoculated into Erlenmeyer flasks, sealed with cotton, to permit aerobic growth, and incubated at 25 ± 2 ℃ for 28 days. At the end of the incubation period, the cultures were combined, filtered, extracted with EtOAc (3x0.5 l). The solvent was evaporated, yielding a crude EtOAc extract (960 mg).

Identification of diketopiperazines in different crude extracts
Analyses were performed on a mass spectrometer with Varian 1200L LC, direct insertion (ID) in positive mode. After the acquisition of information about protonated molecules by direct insertion, conducted experiments using MS-MS. Malt extract, Nutrient and YM (small scale) were subjected to MS-MS analysis in positive mode with application of 15 V collision energy. The crude extracts (20 μg uL -1 ) were eluted in TLC, being CHCl3:CH3OH (88:12) to the crude extract in liquid medium, Hexane: EtOAc (70: 30) for the extracts in solid media. Compounds 7 and 8 (5 μg μL -1 ) were applied on TLC layers, followed by elution with Hexane:EtOAc (70:30, v/v) and subsequent drying. The plates were then sprayed with enzyme solution (6.66 U mL -1 ), thoroughly dried and incubated at 37 ℃ for 20 min (in a moist atmosphere). Enzyme activity was detected by spraying with a solution consisting of 0.25% 1naphthyl acetate in CH3CH2OH containing 0.25% Fast Blue B salt (in aqueous solution). Potential acetylcholinesterase inhibitors appeared as clear zones on a purple-colored background. Electric eel AChE type V (product no. C 2888, 1000 U) and the other reagents were purchased from Sigma-Aldrich. The positive standard for comparison was physostigmine (0,05 µg mL -1 ) 25 .

Results and discussion
The AcOEt extract of S. commune (PDB and corn) afforded compounds 1-8 (Figure 1) by using a combination of reversed (C-18) and normal phase (silica gel) chromatography. 1 H NMR and LC-MS analysis allowed to elucidate and identify the pure substances or in mixtures.
The substance 7 showed low potential to inhibit the enzyme acetylcholinesterase and strong activity against C. cladosporioides. The substance 8 showed moderate potential to inhibit the enzyme acetylcholinesterase and weak activity against C. cladosporioides and C. sphaerospermum.
The crude extracts showed promising results for biological assays as antioxidant, highlighting the Malt Extract with a significant DPPH˙ sequestration rate of 68.36% and IC50= 28.7 µg mL -1 , compared to IC50= 7.49 µg L -1 standard quercetin. The majority these extracts exhibited strong antifungal activity against the phytopathogenic fungi C. cladosporioides and C. sphaerospermum. The crude extracts displayed moderate AChE inhibitory activity. Unfortunately, the substances responsible by these bioactivities were not isolated.
The diketopiperazines 1, 3, 5 and 6 were detected in PDB extract (small scale) by MS. The crude extracts obtained from YM, Nutrient and Malt were subjected to MS-MS analysis and indicated the presence of diketopiperazines 1, 5 and 6. Analysis of crude extracts by direct insertion (ID), showed the protonated diketopiperazines and their fragments in five crude extracts by MS-MS experiment, which corroborate with the fragmentation characteristic of diketopiperazines isolated or identified in mixture.
In the crude extracts Czapek, rice (CH3CN) and corn (CH3CN) (small scale) the diketopiperazines were not identified, suggesting that the enzymes responsible by the biosynthesis of these compounds were not expressed by S. commune, in these media. With these observations was possible to confirm that the culture media influences the microorganisms metabolic production.

Conclusions
The present study was designed to be the most comprehensive investigation of chemical constituents in S. commune. This is the first report of the identification and isolation of diketopiperazines, arylethylamides, phydroxybenzoic acid in endophytic fungus of the Schizophyllum genus. Diketopiperazines was observed by mass spectrometry in the different crude extracts in small scale and PDB large scale, indicating this endophyte as a prolific producer of class of compounds by variation of nutrients of culture media. The biological activities reported by the compounds produced by S. commune suggest an ecological interaction, as well as in the plant protection as a defense mechanism of other organisms that inhabit the plant species.
These results reinforce the potential of these microorganisms as sources of secondary metabolites and collaborating with understanding the endophytic interaction and its host plant.
First attempts made to correlate variations in bands assigned as carbonyl stretching frequencies in the infrared region for solid metal ion complexes with the stability constants of these same complexes in solution date back to early 1950s.