The issue of misidentification of kojic acid with flufuran in aspergillus flavus

Article abstract of DOI:10.3390/molecules24091709

In the course of investigations on the complex phenomenon of bee decline, Aspergillus flavus was isolated from the haemocoel of worker bees. Observations on the metabolomic profile of this strain showed kojic acid to be the dominant product in cultures on Czapek-Dox broth. However, an accurate review of papers documenting secondary metabolite production in A. flavus also showed that an isomer of kojic acid, identified as 5-(hydroxymethyl)-furan-3-carboxylic acid and named flufuran is reported from this species. The spectroscopic data of kojic acid were almost identical to those reported in the literature for flufuran. This motivated a comparative study of commercial kojic acid and 5-(hydroxymethyl)-furan-3-carboxylic acid, highlighting some differences, for example in the ¹³C-NMR and UV spectra for the two compounds, indicating that misidentification of the kojic acid as 5-(hydroxymethyl)-furan-3-carboxylic acid has occurred in the past.

Full text of DOI:10.3390/molecules24091709

molecules
Article
The Issue of Misidentification of Kojic Acid with
Flufuran in Aspergillus flavus
Marina DellaGreca 1 , Gaetano De Tommaso 1, Maria Michela Salvatore 1
,
Rosario Nicoletti 2,3 , Andrea Becchimanzi 3 , Mauro Iuliano 1 and Anna Andolfi 1,
*
1
Department of Chemical Sciences, University of Naples ‘Federico II’, 80126 Naples, Italy;
dellagre@unina.it (M.D.); gaetano.detommaso@unina.it (G.D.T.); mariamichela.salvatore@unina.it (M.M.S.);
mauro.iuliano@unina.it (M.I.)
Council for Agricultural Research and Economics, Research Centre for Olive, Citrus and Tree Fruit,
81100 Caserta, Italy; rosario.nicoletti@crea.gov.it
Department of Agriculture, University of Naples ‘Federico II’, 80055 Portici, Italy;
andrea.becchimanzi@unina.it
2
3
*
Correspondence: andolfi@unina.it; Tel.: +39-081-2539179
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Francesco Vinale and Maria Luisa Balestrieri
Received: 11 April 2019; Accepted: 30 April 2019; Published: 2 May 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: In the course of investigations on the complex phenomenon of bee decline, Aspergillus flavus
was isolated from the haemocoel of worker bees. Observations on the metabolomic profile of this
strain showed kojic acid to be the dominant product in cultures on Czapek-Dox broth. However,
an accurate review of papers documenting secondary metabolite production in A. flavus also showed
that an isomer of kojic acid, identified as 5-(hydroxymethyl)-furan-3-carboxylic acid and named
flufuran is reported from this species. The spectroscopic data of kojic acid were almost identical to
those reported in the literature for flufuran. This motivated a comparative study of commercial kojic
acid and 5-(hydroxymethyl)-furan-3-carboxylic acid, highlighting some differences, for example in
the 13C-NMR and UV spectra for the two compounds, indicating that misidentification of the kojic
acid as 5-(hydroxymethyl)-furan-3-carboxylic acid has occurred in the past.
Keywords: kojic acid; flufuran; 5-(hydroxymethyl)-furan-3-carboxylic acid; Aspergillus flavus;
bees; immunomodulation
1. Introduction
Fungi have evolved the capability to produce a great number of secondary metabolites involved
in the improvement of their ecological fitness, and many of them play important biological roles as
virulence factors, chemical defense agents, developmental regulators, insect attractants, and chemical
signals for communication with other organisms. On these properties is founded the pharmacological
exploitation of many products as antibiotic, antiviral, antitumor, antihypercholesterolemic, and
immunosuppressant agents [1–5]. In this respect, fungi are prime targets of a vigorous investigational
activity, based on the employment of the most advanced analytical and structure elucidation
techniques [6,7].
A wide array of fungal secondary metabolites has been ascribed to the ascomycete genus Aspergillus,
well-known for its ubiquity and cosmopolitan distribution [
well-known as a foodstuff contaminant and a mycotoxin producer, and in this respect its metabolomic
profile has been quite well characterized [ 10]. However, this fungus has been also reported in
association with plants and animals in many different environments [11 12]. Particularly, it has been
7,8]. The species Aspergillus flavus is
9
,
,
directly isolated from bee (Apis mellifera) individuals in different developmental stages and health
conditions [13]. Although reported as the agent of stonebrood disease, further elucidation is required if
Molecules 2019, 24, 1709; doi:10.3390/molecules24091709
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1709
2 of 11
A. flavus basically behaves like an entomopathogen, or if its relationships with bees are more complex
and eventually involve modulation of the immunitary response to other pathogens and parasites of
these insects. Generally, the fungus is considered an opportunistic pathogen of immunocompromised
individuals, gaining access to the host through ingestion, or taking advantage from the interaction of
bees with other pathogens and parasites which negatively affect host immunocompetence and cuticle
integrity [13–16].
During investigations on the honeybee colony collapse, a multifactorial syndrome mainly related
to the compresence of immunosuppressive viruses and Varroa destructor, a parasitic mite which is also
known as a possible vector [16–18], strains of A. flavus were repeatedly isolated from the haemocoel of
worker bees.
The present paper reports the identification of kojic acid (KA) as the main metabolite obtained
from this source. Furthermore, revision of the structure of flufuran, previously characterized as
5-(hydroxymethyl)furan-3-carboxylic acid from A. flavus and other fungal species is proposed, based
on a comparison of spectroscopic data of commercially available compounds and some derivatives.
2. Results
2.1. Comparison of NMR Data Obtained from KA (1) and 5-(Hydroxymethyl)furan-3-carboxylic Acid (2)
A white solid was obtained from the extraction with ethyl acetate (EtOAc) of the culture
filtrate of strain AB1EET of A. flavus, which consisted in a main metabolite, as deduced by its
1H and 13C-NMR spectra. NMR data collected for the extracted compound showed significant
similarities with proton and carbon chemical shifts reported in literature for both KA (
1) [19] and
5-(hydroxymethyl)furan-3-carboxylic acid ( ) [20], a compound characterized from A. flavus and other
2
fungal species (Table 1). A full analysis of the previous reports revealed discrepancy between the carbon
chemical shifts of furan compounds [21] and those assigned for 5-(hydroxymethyl)furan-3-carboxylic
acid. In order to clarify this issue, commercially available KA and 5-(hydroxymethyl)furan-3-carboxylic
acid were submitted to spectroscopic and potentiometric investigations.
Table 1. Previous reports dealing with flufuran identification.
Source
Spectroscopic Data
Activity
Ref.
Aspergillus flavus
A. flavus
1H and 13C-NMR, MS, IR, UV
—
Antifungal
Cytotoxic against human cancer cell lines
[22]
[23]
[24] 1
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[20]
A. flavus
Bacteriostatic (Escherichia coli)
Antibacterial, antioxidant
A. flavus 2
1H and 13C-NMR
MS
A. flavus
—
—
Aspergillus oryzae
A. oryzae
—
1
Antioxidant, antimicrobial
3
Fusarium sp.
1H and 13C-NMR
1H and 13C-NMR
Antimicrobial
Penicillium dipodomyicola
Penicillium polonicum
Pestalotiopsis sp.
Polyporus ciliatus
1
—
—
Monoamine oxidase inhibitor
Cytotoxic against HepG2 cells
1H and 13C-NMR
P.f., MS, 1H and 13C-NMR, MS, IR, UV
—
2
3
This paper is in Chinese and could not be examined in detail. 7-O-Acetyl derivative was also isolated. Methyl
ester was also isolated.
1
The comparison of H-NMR spectra, recorded in CD3OD, showed no significant differences
between 5-(hydroxymethyl)furan-3-carboxylic acid and KA (Figures 1 and 2). Notwithstanding, the
1H-NMR spectra recorded in DMSOd6 for
2
revealed the coupling between the proton of the hydroxyl
group in C-7 and protons of the methylene group which resonate as triplet and doublet, respectively,
at 5.31 and 4.40 (J = 5.8 Hz) and the presence of the signal of proton of the carboxylic group at 12.60
(Figure 3).
δ
δ

Molecules 2019, 24, 1709
3 of 11
Figure 1. 1H-NMR spectra of KA (
recorded at 400 MHz in CD3OD.
1, red) and 5-(hydroxymetyhyl)-furan 3-carboxylic acid (2, green)
Figure 2. Proton and carbon chemical shifts of KA (
1) and 5-(hydroxymethyl)furan-3-carboxylic acid
(2). Data reported in red were erroneously assigned to flufuran. The spectra were recorded in CD3OD.
Figure 3. 1H-NMR spectra of KA (
recorded at 400 MHz in DMSOd6.
1, red) and 5-(hydroxymetyhyl)-furan 3-carboxylic acid (2, green)

Molecules 2019, 24, 1709
4 of 11
The main differences between
C-5 and C-6 carbons of resonate at
1
δ
and
2
were observed, as expected, in 13C-NMR spectra. The C-3,
2
120.1, 156.4 e 165.1 (Figure 2 and Figure S1), respectively, assigned
by 2D-NMR spectra (Figures S2 and S3). However, the 13C-NMR spectrum of flufuran showed chemical
shift values identical to KA (Figure 2), indicating a misinterpretation of the flufuran structure.
2.2. Comparison of Potentiometric, UV, and MS Spectrophotometric Measurements
The acid-base behavior of KA and 5-(hydroxymethyl)furan-3-carboxylic acid was determined by
potentiometric and spectrophotometric methods. The calculated protonation constants cologarithm
values are 7.68
reported in Figure 4.
±
0.05 and 4.03
±
0.05, for compound 1 and 2 respectively. Potentiometric data are
Figure 4. ZH(pH) function in 0.1 M NaClO4 for KA (squares) and 5-(hydroxymethyl)furan-3-carboxylic
acid (circles). The solid lines are calculated with protonation constants cologarithm value 7.68
(on squared) and 4.03 (on circles).
In particular KA constant is very similar to that in 0.1 M KCl (7.7) [33]. While no value on the
protonation of 5-(hydroxymethyl)furan-3-carboxylic acid had been previously reported.
UV spectra of the pure compounds, recorded in water in the interval 200–400 nm, show
λmax at
216 and 270 nm for KA, and at 240 nm for
recorded in methanol.
2. Similar values were observed when the spectra were
In agreement with acid-base behavior of both compounds, measurements conducted in pH
function confirmed that KA spectra are highly influenced by pH [34]. In fact, absorbance value at
315 nm increases from 3 to 9 pH range, instead the peak at 270 nm decreases in the same interval.
Conversely, UV spectra of compound
2 present minimal variation in function of pH (Figure 5). Finally
with pH increasing, KA shows a isosbestic point at 285 nm, while 5-(hydroxymethyl)furan-3-carboxylic
acid presents one at 240 nm.
B)
A)
2.5
2.5
pH= 2.5
pH= 3.0
pH= 3.0
pH= 5.0
2
pH= 4.5
2
pH= 8.0
pH= 5.0
pH= 9.0
1.5
1.5
1
1
0.5
0
0.5
0
200
250
300
350
400
200
250
300
350
400
Wavelenght (nm)
Wavelenght (nm)
Figure 5.
(
A
) UV spectra of 2.0
×
10−3 M in 0.1 M NaClO4 for KA. (
B
) UV spectra of 2.0
×
10−3 M in
0.1 M NaClO4 for 5-(hydroxymethyl)furan-3-carboxylic acid (optical path 0.2 cm).
The fragmentation pattern observed in MALDI TOF/MS spectra evidenced relevant analogies
between the two compounds, as to be expected from considering the nature of the respective functional

Molecules 2019, 24, 1709
5 of 11
groups. In fact, from peaks corresponding to the protonated ions [M+H]+ at m/z 143 of
1 and 2,
fragments deriving from the loss of OH, CHO, and COOH groups were observed in both cases.
2.3. Comparison of KA and 5-(Hydroxymethyl)furan-3-carboxylic Acid Derivatives
Considering reports concerning the presumptive isolation of the 7-O-acetyl derivative of
flufuran [25] and of its methyl ester [29], in this work the differences between derivatives of
and 2 obtained from common acetylation and methylation reaction were also evaluated.
1
The proton spectrum of 5-(acetoxymethyl)furan 3-carboxylic acid (
acetylation of , showed the down shift of ∆δ 0.55 of the signal assigned to the methylene group C-7
which resonates as singlet at 5.08, and the presence of a further singlet at 2.07 assigned to the
CH3 of the acetyl group (Figure 6 and Figure S4). As discussed in relation to and the differences
and 7-O-acetylflufuran [25] are not so marked compared to the ones
4, Figure S4), produced after
2
δ
δ
1
2
between the 1H-NMR spectra of
4
of 13C-NMR spectra (Figure 6 and Figure S5).
61.1
19.2
164.8
4.99
2.03
163.1
HOOC
O
140.0
8.00
107.6
6.62
120.6
H3COCO
7
170.0
57.2
5.08
19.7
2.07
146.3
111.6
6.50
148.3
8.12
OH
CH2OCOCH3
175.1
151.4
O
7
170.8
O
4
3
60.8
5.04
19.1
2.15
164.3
O
149.9
8.30
H3COCO
7
18.7
2.31
170.3
114.1
6.58
5
140.8
OCOCH3
173.8
168.2
O
50.6
3.83
59.3
4.29
5
163.9
168.0
O
139.0
8.08
H3COOC
HO
7
6
106.5
6.64
119.5
147.9
55.7
5
110.7
6.29
4.53
OCH3
56.1
3.64
147.7
8.11
172.8
CH2OH
156.5
O
O
6
7
50.7
3.84
61.8
5.01
19.7
2.15
163.4
166.3
142.6
6.50
O
H3COOC
H3COCO
7
6
109.6
6.78
175.7
119.8
148.5
19.1
2.07
57.1
5.08
5
112.5
6.50
OCH3
148.3
8.15
171.9
CH2OCOCH3
151.6
56.7
3.80
O
7
170.7
O
8
9
Figure 6. Proton and carbon chemical shifts of 7-O-acetylKA, 5,7-O,O’-diacetylKA, 5-O-methylKA,
5-O-methyl-7-O’-acetylKA ( , and ); 5-(acetoxymethyl)furan-3-carboxylic acid, methyl
3,
5,
6
8
5-(hydroxymethyl)furan-3-carboxylate, methyl 5-(acetoxymethyl)furan-3-carboxylate (4, 7, and 9).
From the data reported on the diacetylated flufuran [22], it is possible to exclude its formation
from 2; most likely the described compound is 5,7-O,O0-diacetylKA (5, Figure 6), derived from 1.
The treatment of
methyl 5-(hydroxymethyl)furan-3-carboxylate (
1
and
2
with diazomethane led to the production of 5-O-methylKA (
6) and
7
, Figure 6) as deduced from 1H and 13C-NMR spectra.

Molecules 2019, 24, 1709
6 of 11
In fact, NMR spectra of
7
(Figures S6 and S7) showed further singlets, respectively, at
δ
3.83 and
at
δ 50.6 attributed to methoxy group. As observed for the other compounds under examination,
the comparison of 13C-NMR spectra of methyl derivatives of
(6 and 7, Figure 6).
1
and
2 revealed significant differences
Finally, the preparation of 5-O-methyl-7-O’-acetylKA (8) and methyl 5-(acetoxymethyl)furan-
3-carboxylate (9) and the interpretation of their one-dimensional NMR spectra (Figures S8 and S9)
allowed to confirm the derivative structures, and to establish that the derivative i.e., methyl
5-(acetoxymethyl)furan-3-carboxylate) reported by Evidente et al. [22] is undoubtedly 8.
3. Discussion
The furan compound 5-(hydroxymethyl)furan-3-carboxylic acid was identified for the first time as
a natural product in cultures of Polyporus ciliatus [20], and afterwards reported from other fungi (Table 1).
Although furan derivatives are not an extensive class of fungal compounds [35], over the past few years
the literature concerning these natural products has been enriched by new reports [36–40]. According
to their biosynthetic origin, the structure of natural furan derivatives shows the presence of furan rings
substituted in position 2 and 5 [35]. Flufuran represented an exception to such a general model.
In the present paper, we produce evidence that in most of the previous reports the product
identified as flufuran was instead KA. In addition to being known as a typical secondary metabolite of
both A. flavus and A. oryzae, KA has been reported from several Penicillium spp. [41]. Other fungal
species are claimed as producers of some derivatives [42], indicating that biosynthetic ability for this
compound might be more widespread. To the best of our knowledge, our amendments to previous
findings represents the first evidence of its production in important fungal genera, such as Fusarium and
Pestalotiopsis, which deserve further confirmation also with reference to correct taxonomic identification
of producing strains. In view of the present revision, the bioactivities erroneously assigned to flufuran
should be instead referred to KA, integrating the known profile of biological properties of this γ-pyrone
compound [43]. Actually, these data show that KA and 5-(hydroxymethyl)furan-3-carboxylic acid are
susceptible of misidentification. Particularly, some instrumental techniques commonly used for the
identification of secondary metabolites in fungal extracts (i.e., 1H-NMR and ESI MS data) are not able
to distinguish between these two compounds. However, huge differences can be observed between
and in the UV spectra obtained with different solvents and pH. The acid-base properties are also
valid to differentiate between and . Finally, observations concerning acetyl and methyl derivatives
1
2
1
2
also confirmed the misidentification of KA with flufuran.
4. Materials and Methods
4.1. General Experimental Procedures
1H and 13C-NMR spectra were recorded at 400 and at 100 MHz, respectively, in CD3OD unless
otherwise noted, on a Bruker spectrometer (AscendTM400) (Bremen, Germany); the same solvent was
used as internal standard. Potentiometric titrations were performed in an air-bath thermostat kept
at (25.00
±
0.05) ◦C. A programmable computer-controlled Data Acquisition Unit 34970A, (Agilent
Tecnologies Inc., Santa Clara, CA, USA) was used to perform the potentiometric measurements.
The glass electrodes were Metrohm (Herisau, Switzerland) of 60102-100 type, and Ag/AgCl electrode
was utilized as reference. The EMF values were measured with a precision of
±
0.01 mV using a
Keithley 642 type Digital Electrometer (Tektronix Inc., Beaveron, OR, USA). UV–VIS spectra were
recorded by model Cary 5000 Spectrophotometer by Varian (Palo Alto, CA, USA) from 200 to 600 nm
(optical path 0.2 cm) at 25.0 ◦C, under a constant flow of nitrogen.
MALDI-TOF/MS spectra were acquired by a 4700 Proteomics Analyzer (Applied Biosystems,
Framingham, MA, USA). Compounds were detected in reflector mode using
4-hydroxycinnamic acid (CHCA) as matrix.
α-cyano-

Molecules 2019, 24, 1709
7 of 11
Analytical and preparative TLC were performed on silica gel plates (Kieselgel 60, F254, 0.25 mm)
(Merck, Darmstadt, Germany). The spots were visualized by exposure to UV radiation (253
nm), or by spraying with 10% H2SO4 in MeOH followed by heating at 110 ◦C for 10 min.
5-(hydroxymethyl)furan-3-carboxylic acid and KA were purchased from Enamine Ltd. (Kyiv, Ukraine)
and Alfa Aesar (Karlsruhe, Germany), respectively.
4.2. Isolation of A. flavus from Bees
Worker bee individuals were collected from experimental apiaries at the Department of Agriculture,
University of Naples Federico II (Portici, Italy) in October–November 2018. Freshly emerged bees
(<48 h) were anaesthetized by chilling on ice for 20 min, and surface-disinfected by dipping in 70%
ethanol for 30 s. The intersegmental membrane between head and thorax was cut using a sterile
scalpel. Ten microliters of the liquid matter exuding from haemocoel were aseptically collected
using a micropipette, and spotted on potato-dextrose agar (PDA, Oxoid) amended with 50 mg/L
chloramphenicol. Plates were incubated at 32 ◦C in the dark. Hyphal tips from the emerging fungal
colonies were transferred to fresh PDA plates to obtain pure cultures for morphological identification
and storage. The strains obtained were readily identified as belonging to the Aspergillus section Flavi,
based on the formation of colonies producing a dense felt of yellow-green rough conidiophores with
radiate heads, and subglobose echinulate conidia. An orange reverse could be observed in cultures on
a specific medium (AFPA) [44]. Unequivocal ascription to the species A. flavus resulted after rDNA-ITS
and calmodulin gene sequencing. To this purpose, total genomic DNA was extracted from fresh
mycelium taken from pure culture of strain AB1EET using CTAB protocol [45]. According to reported
methodology, primers ITS1F and ITS4 were used to amplify rDNA-ITS, while primers CF1M and
CF4 were used to amplify the calmodulin gene [45,46]. The original DNA sequences obtained in this
study have been deposited in GenBank under the codes MK611561 (ITS) and MK611938 (calmodulin).
The calmodulin gene sequence proved to be more respondent for unequivocal species identification,
yielding 100% homology with sequences from 60 strains of A. flavus available in GenBank.
4.3. Production and Extraction of KA
Strain AB1EET was cultured in Czapek-Dox broth (Oxoid) following a previously reported
procedure [47]. The freeze-dried culture filtrates (750 mL) were dissolved in 100 mL, acidified with
HCl 2 N at pH 3, and extracted three times with an equal volume of EtOAc. Organic phases were
combined, dried with Na2SO4, and evaporated under reduced pressure originating a white solid
residue (350.7 mg) identified as KA.
4.4. Determination of Protonation Constants
The evaluation of the protolysis constants was conducted through spectrophotometric and
potentiometric titration, at 25 ◦C in 0.1 M NaClO4 as ionic medium [48]. By experimental data the
average number (ZH) of protons released per molecule was assessed through the equation:
Z
H = ([H+] − CH − Kw/[H+])/CL
(1)
where [H+] is hydrogenionic concentration, CH is the total acid concentration, CL is the compound
concentration and Kw is ionic product (10−13.7, in 0.1 M NaClO4). Experimental data were processed
with Hyperquad softare [49].
4.5. UV and MS Data of KA and 5-(Hydroxymethyl)furan-3-carboxylic Acid
Kojic acid (1). UV
λ
max
nm (log ε): (H2O) 216 (4.12), 270 (3.93); (MeOH) 225 (3.99), 270 (3.99);
(pH 3.0) 215 (3.47), 270 (3.30); (pH 5.0) 216 (3.51), 268 (3.17), 318 (2.63); (pH 8.0) 227 (3.66), 315 (3.5);
(pH 9.0) 227 (3.72), 315 (3.15). MALDI TOF/MS: m/z 143 [M+H]+, 125 [M-OH]+, 113 [M-CHO]+,
97 [M-COOH]+, 69 [M-CH2OH-COO]+.

Molecules 2019, 24, 1709
8 of 11
5-(Hydroxymethyl)furan-3-carboxylic acid (
2). UV
λmax nm (log
ε
): (H2O) 240 (3.39); (MeOH) 240
(3.38); (pH 2.5) 243 (2.97); (pH 3.0) 243 (3.07); (pH 4.0) 243 (3.07); (pH 5.0) 243 (3.12). MALDI TOF/MS:
m/z 143 [M+H]+, 125 [M-OH]+, 113 [M-CHO]+, 97 [M-COOH]+.
4.6. Sample Methylation
Fifteen milligrams of samples [KA, 5-(hydroxymethyl)furan-3-carboxylic acid,
5-(acetoxymethyl)furan-3-carboxylic acid (1, 2 and 4)] were dissolved in MeOH (1.5 mL);
an ethereal solution of CH2N2 was slowly added until a yellow color became persistent. The reaction
mixtures were stirred at room temperature for 4 h. The solvent was evaporated under a N2 stream at
room temperature. Residues of each reaction were analyzed by TLC on silica gel;
Rf 0.37 by eluting with EtOAc-MeOH (9:1), while Rf 0.54 and 0.82 corresponded to
as eluted with CHCl3-i-PrOH (92:8).
6
7
, was evidenced at
and respectively,
9
4.7. Sample Acetylation
Ten mg of samples [KA, 5-(hydroxymethyl)furan-3-carboxylic acid, 5-O-methylkojic acid (1, 2, 6)],
dissolved in pyridine (30
acetylation with Ac2O (30
µ
L), were converted into the corresponding acetyl derivatives (5, 4, 7) by
L) at room temperature overnight. The reaction was stopped by addition
µ
of MeOH, and the azeotrope formed by addition of benzene was evaporated in a N2 stream at 40 ◦C.
Residues of each reaction were analyzed by TLC on silica gel;
with CHCl3-i-PrOH (95:5), while Rf 0.54 and 0.82 corresponded to
CHCl3-i-PrOH (92:8).
5
was evidenced at Rf 0.44 by eluting
and respectively, as eluted with
7
9
Supplementary Materials: The following are available online, Figures S1–S3. NMR spectra of KA (
1
) and
5-(hydroxymethyl)furan-3-carboxylic acid (
2
); Figures S4–S9. 1H and 13C-NMR spectra of acetyl and methyl
derivatives of 2.
Author Contributions: M.D., R.N., and A.A. conceived and organized the manuscript, and wrote the text; R.N.
and A.B. isolated and cultivated the fungal strain; M.M.S. and A.A. extracted the culture filtrate and prepared
sample derivatives; M.D. and A.A. performed the NMR analysis; G.D.T. and M.I. performed potentiometric and
spectrophotometric measurements; M.D., M.M.S, R.N., and A.A edited and reviewed the manuscript.
Funding: This research was funded by Finanziamento delle Attivit
à Base della Ricerca (FFABR) 2017 of Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR, Italy).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Chambergo, F.S.; Valencia, E.Y. Fungal biodiversity to biotechnology. Appl. Microbiol. Biotechnol. 2016, 100,
2567–2577. [CrossRef] [PubMed]
2.
Meyer, V.; Andersen, M.R.; Brakhage, A.A.; Braus, G.H.; Caddick, M.X.; Cairns, T.C.; de Vries, R.P.;
Haarmann, T.; Hansen, K.; Hertz-Fowler, C.; et al. Current challenges of research on filamentous fungi in
relation to human welfare and a sustainable bio-economy: A white paper. Fungal Biol. Biotechnol. 2016, 3, 6.
Nicoletti, R.; Salvatore, M.M.; Andolfi, A. Secondary metabolites of mangrove-associated strains of Talaromyces.
Mar. Drugs 2018, 16, 12. [CrossRef] [PubMed]
3.
4.
Félix, C.; Salvatore, M.M.; DellaGreca, M.; Meneses, R.; Duarte, A.S.; Salvatore, F.; Naviglio, D.; Gallo, M.;
Jorrino-Novo, J.V.; Alves, A.; et al. Production of toxic metabolites by two strains of Lasiodiplodia theobromae,
isolated from a coconut tree and a human patient. Mycologia 2018, 110, 642–653. [CrossRef] [PubMed]
Marra, R.; Nicoletti, R.; Pagano, E.; DellaGreca, M.; Salvatore, M.M.; Borrelli, F.; Lombardi, N.; Vinale, F.;
Woo, S.L.; Andolfi, A. Inhibitory effect of trichodermanone C, a sorbicillinoid produced by Trichoderma
citrinoviride associated to the green alga Cladophora sp., on nitrite production in LPS-stimulated macrophages.
Nat. Prod. Res. 2018. [CrossRef]
5.
6.
Keller, N.P.; Turner, G.; Bennett, J.W. Fungal secondary metabolism—from biochemistry to genomics. Nat. Rev.
Microbiol. 2005, 3, 937. [CrossRef] [PubMed]

Molecules 2019, 24, 1709
9 of 11
7.
8.
Salvatore, M.M.; Nicoletti, R.; Salvatore, F.; Naviglio, D.; Andolfi, A. GC–MS approaches for the screening of
metabolites produced by marine-derived Aspergillus. Mar. Chem. 2018, 206, 19–33. [CrossRef]
Samson, R.A.; Visagie, C.M.; Houbraken, J.; Hong, S.B.; Hubka, V.; Klaassen, C.H.W.; Perrone, G.; Seifert, K.A.;
Susca, A.; Tanney, J.B.; et al. Taxonomy, identification and nomenclature of the genus Aspergillus. Stud. Mycol.
2014, 78, 141–173. [CrossRef]
9.
Rank, C.; Klejnstrup, M.L.; Petersen, L.M.; Kildgaard, S.; Frisvad, J.C.; Held Gotfredsen, C.; Ostenfeld
Larsen, T. Comparative chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites 2012, 2,
39–56. [CrossRef]
10. Cary, J.W.; Gilbert, M.K.; Lebar, M.D.; Majumdar, R.; Calvo, A.M. Aspergillus flavus secondary metabolites:
More than just aflatoxins. Food Safety 2018, 6, 7–32. [CrossRef]
11. Nicoletti, R.; Fiorentino, A. Plant bioactive metabolites and drugs produced by endophytic fungi of
Spermatophyta. Agriculture 2015, 5, 918–970. [CrossRef]
12. Ramírez-Camejo, L.A.; Zuluaga-Montero, A.; Lázaro-Escudero, M.; Hernández-Kendall, V.; Bayman, P.
Phylogeography of the cosmopolitan fungus Aspergillus flavus: Is everything everywhere? Fungal Biol. 2012
116, 452–463. [CrossRef] [PubMed]
,
13. Foley, K.; Fazio, G.; Jensen, A.B.; Hughes, W.O. The distribution of Aspergillus spp. opportunistic parasites in
hives and their pathogenicity to honey bees. Veter. Microbiol. 2014, 169, 203–210. [CrossRef] [PubMed]
14. Vojvodic, S.; Jensen, A.B.; James, R.R.; Boomsma, J.J.; Eilenberg, J. Temperature dependent virulence of
obligate and facultative fungal pathogens of honeybee brood. Veter. Microbiol. 2011, 149, 200–205. [CrossRef]
15. Ilyasov, R.; Gaifullina, L.; Saltykova, E.; Poskryakov, A.; Nikolenko, A. Review of the expression of
antimicrobial peptide defensin in honey bees Apis mellifera L. J. Apicultural Sci. 2012, 56, 115–124. [CrossRef]
16. Di Prisco, G.; Annoscia, D.; Margiotta, M.; Ferrara, R.; Varricchio, P.; Zanni, V.; Caprio, E.; Nazzi, F.;
Pennacchio, F. A mutualistic symbiosis between a parasitic mite and a pathogenic virus undermines honey
bee immunity and health. Proc. Natl. Acad. Sci. 2016, 113, 3203–3208. [CrossRef] [PubMed]
17. Benoit, J.B.; Yoder, J.A.; Sammataro, D.; Zettler, L.W. Mycoflora and fungal vector capacity of the parasitic
mite Varroa destructor (Mesostigmata: Varroidae) in honey bee (Hymenoptera: Apidae) colonies. Int. J.
Acarology 2004, 30, 103–106. [CrossRef]
18. Yang, X.; Cox-Foster, D.L. Impact of an ectoparasite on the immunity and pathology of an invertebrate:
Evidence for host immunosuppression and viral amplification. Proc. Natl. Acad. Sci. 2005, 102, 7470–7475.
[CrossRef]
19. Li, X.; Jeong, J.H.; Lee, K.T.; Rho, J.R.; Choi, H.D.; Kang, J.S.; Son, B.W. γ-Pyrone derivatives, kojic acid methyl
ethers from a marine-derived fungus Altenaria sp. Arch. Pharm. Res. 2003, 26, 532–534. [CrossRef]
20. Cabrera, G.M.; Roberti, M.J.; Wright, J.E.; Seldes, A.M. Cryptoporic and isocryptoporic acids from the fungal
cultures of Polyporus arcularius and P. ciliatus. Phytochemistry 2002, 61, 189–193. [CrossRef]
21. Alvarez-Ibarra, C.; Quiroga-Feijó
o, M.L.; Toledano, E. An analysis of substituent effects on 1H and 13C-NMR
parameters of substituted furans. Linear free energy relationships and PM3 semiempirical calculations.
J. Chem. Soc., Perkin Trans. 2 1998, 3, 679–690. [CrossRef]
22. Evidente, A.; Cristinzio, G.; Punzo, B.; Andolfi, A.; Testa, A.; Melck, D. Flufuran, an antifungal
3,5-disubstituted furan produced by Aspergillus flavus Link. Chem. Biodiver. 2009, 6, 328–334. [CrossRef]
[PubMed]
23. Balde, E.S.; Andolfi, A.; Bruyère, C.; Cimmino, A.; Lamoral-Theys, D.; Vurro, M.; Damme, M.V.; Altomare, C.;
Mathieu, V.; Kiss, R.; et al. Investigations of fungal secondary metabolites with potential anticancer activity.
J. Nat. Prod. 2010, 73, 969–971. [CrossRef] [PubMed]
24. Ma, C.; Ma, Y.M.; Li, T.; Wang, J. Structure and activity of secondary metabolites of endophytic fungus S19
strain in Cephalotaxus fortune. Guizhou Nongye Kexue 2014, 42, 152–156.
25. Ma, Y.M.; Ma, C.C.; Li, T.; Wang, J. A new furan derivative from an endophytic Aspergillus flavus of
Cephalotaxus fortunei. Nat. Prod. Res. 2016, 30, 79–84. [CrossRef] [PubMed]
26. Saldan, N.C.; Almeida, R.T.; Avíncola, A.; Porto, C.; Galuch, M.B.; Magon, T.F.; Pilau, E.J.; Svidzinski, T.I.E.;
Oliveira, C.C. Development of an analytical method for identification of Aspergillus flavus based on chemical
markers using HPLC-MS. Food Chem. 2018, 241, 113–121. [CrossRef]
27. Lee, M.; Cho, J.Y.; Lee, Y.G.; Lee, H.J.; Lim, S.I.; Lee, S.Y.; Nam, Y.D.; Moon, J.H. Furan, phenolic, and heptelidic
acid derivatives produced by Aspergillus oryzae. Food Sci. Biotechnol. 2016, 25, 1259–1264. [CrossRef]

Molecules 2019, 24, 1709
10 of 11
28. Yang, X.; Wang, P.; Ma, Y.; Jia, Q. Metabolites of Aspergillus oryzae, an endophytic fungus associated with
Lycium ruthenicum Murr. Tianran Chanwu Yanjiu Yu Kaifa 2015, 27, 1554–1557.
29. Zhang, H.C.; Ma, Y.M.; Liu, R. Antimicrobial materials derived from the endophytic fungus Fusarium sp. of
Eucommia ulmoides. Advan. Mater. Res. 2012, 531, 346–349. [CrossRef]
30. He, S.; Yan, X.; Wang, T. Marine Penicillium dipodomyicola for manufacture of flufuran. 2014, CN 104031845 A
20140910. CN 104031845 A 20140910, 2014.
31. Elsunni, M.A.; Yang, Z.-D. Secondary metabolites of the endophytic fungi Penicillium polonicum and their
monoamine oxidase inhibitory activity. Chem. Nat. Comp. 2018, 54, 1018–1019. [CrossRef]
32. Zhou, J.; Li, G.; Deng, Q.; Zheng, D.; Yang, X.; Xu, J. Cytotoxic constituents from the mangrove endophytic
Pestalotiopsis sp. induce G0/G1 cell cycle arrest and apoptosis in human cancer cells. Nat. Prod. Res. 2018, 32,
2968–2972. [CrossRef]
33. Nurchi, V.M.; Crisponi, G.; Lachowicz, J.I.; Murgia, S.; Pivetta, T.; Remelli, M.; Rescigno, A.; Nicl
ós-Gutìerrez, J.;
Gonz lez-P rez, J.M.; Dom nguez-Mart n, A.; et al. Iron (III) and aluminum (III) complexes with
á
é
ì
ì
hydroxypyrone ligands aimed to design kojic acid derivatives with new perspectives. J. Inorg. Biochem. 2010
,
104, 560–569. [CrossRef] [PubMed]
34. Paterson, R.R.M.; Kemmelmeier, C. Neutral, alkaline and difference ultraviolet spectra of secondary
metabolites from Penicillium and other fungi, and comparisons to published maxima from gradient
high-performance liquid chromatography with diode-array detection. J. Chromatogr. A 1990, 511, 195–221.
[CrossRef]
35. Klaiklay, S.; Rukachaisirikul, V.; Phongpaichit, S.; Buatong, J.; Preedanon, S.; Sakayaroj, J. Flavodonfuran:
A new difuranylmethane derivative from the mangrove endophytic fungus Flavodon flavus PSU-MA201.
Nat. Prod. Res. 2013, 27, 1722–1726. [CrossRef] [PubMed]
36. Kobori, H.; Sekiya, A.; Yasuda, N.; Noguchi, K.; Suzuki, T.; Choi, J.H.; Hirai, H.; Kawagishi, H. Armillariols
A to C from the culture broth of Armillaria sp. Tetrahedron Lett. 2013, 54, 5481–5483. [CrossRef]
37. Ding, L.J.; Gu, B.B.; Jiao, W.H.; Yuan, W.; Li, Y.X.; Tang, W.Z.; Yu, H.B.; Liao, X.J.; Han, B.N.; Li, Z.Y.; et al. New
furan and cyclopentenone derivatives from the sponge-associated fungus Hypocrea koningii PF04. Mar. Drugs
2015, 13, 5579–5592. [CrossRef]
38. Chen, L.L.; Wang, P.; Chen, H.Q.; Guo, Z.K.; Wang, H.; Dai, H.F.; Mei, W.L. New furan derivatives from a
mangrove-derived endophytic fungus Coriolopsis sp. J5. Molecules 2017, 22, 261. [CrossRef] [PubMed]
39. Uchoa, P.K.S.; Pimenta, A.T.; Braz-Filho, R.; de Oliveira, M.D.C.F.; Saraiva, N.N.; Rodrigues, B.S.;
Pfenning, L.H.; Abreu, L.M.; Wilke, D.V.; Florêncio, K.G.D.; et al. New cytotoxic furan from the marine
sediment-derived fungi Aspergillus niger. Nat. Prod. Res. 2017, 31, 2599–2603. [CrossRef] [PubMed]
40. Wu, X.; Pang, X.J.; Xu, L.L.; Zhao, T.; Long, X.Y.; Zhang, Q.Y.; Qin, H.L.; Yang, D.F.; Yang, X.L. Two new
alkylated furan derivatives with antifungal and antibacterial activities from the plant endophytic fungus
Emericella sp. XL029. Nat. Prod. Res. 2018, 32, 2625–2631. [CrossRef] [PubMed]
41. Bentley, R. From miso, sake and shoyu to cosmetics: A century of science for kojic acid. Nat. Prod. Rep. 2006
23, 1046–1062. [CrossRef] [PubMed]
,
42. Dembitsky, V.M.; Kilimnik, A. Anti-melanoma agents derived from fungal species. Mathews, J. Pharm. Sci.
2016, 1, 002.
43. Mohamad, R.; Mohamed, M.S.; Suhaili, N.; Salleh, M.M.; Ariff, A.B. Kojic acid: Applications and development
of fermentation process for production. Biotechnol. Mol. Biol. Rev. 2010, 5, 24–37.
44. Pitt, J.I.; Hocking, A.D.; Glenn, D.R. An improved medium for the detection of Aspergillus flavus and A.
parasiticus. J. Appl. Bacteriol. 1983, 54, 109–114. [CrossRef] [PubMed]
45. Cubero, O.F.; Crespo, A.; Fatehi, J.; Bridge, P.D. DNA extraction and PCR amplification method suitable for
fresh, herbarium-stored, lichenized, and other fungi. Plant Syst. Evol. 1999, 216, 243–249. [CrossRef]
46. Peterson, S.W. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia
2008, 100, 205–226. [CrossRef] [PubMed]
47. Nicoletti, R.; Buommino, E.; De Filippis, A.; Lopez-Gresa, M.P.; Manzo, E.; Carella, A.; Petrazzuolo, M.;
Tufano, M.A. Bioprospecting for antagonistic Penicillium strains as a resource of new antitumor compounds.
World J. Microbiol. Biotechnol. 2008, 24, 189–195. [CrossRef]

Molecules 2019, 24, 1709
11 of 11
48. Meloun, M.; Havel, J.; Högfeldt, E. Computation of Solution Equilibria: A Guide to Methods in Potentiometry,
Extraction and Spectrophotometry; Ellis Horwood Ltd.: Chichester, UK, 1988.
49. Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination of equilibrium constants
with the HYPERQUAD suite of programs. Talanta 1996, 43, 1739–1753. [CrossRef]
Sample Availability: Samples of the compounds 1-9 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).